SP.01 What we Have Learned from Structures of the Ribosome Venki Ramakrishnan MRC, Cambridge, United Kingdom Ever since its discovery in the 1950s, the ribosome has been the object of study by labs world-wide, because of its central role in the translation of genetic information into proteins. However, its large size meant a long delay in the determination of its structure, despite the fact that the crystals of the ribosome were first obtained around 1980. I shall discuss our determination of the structure of the 30S subunit, and more recent work on high-resolution functional complexes of the entire ribosome, as well as insights into ribosome function from these structures. In particular, the structural basis of decoding, in which the tRNA corresponding to a codon on mRNA is accurately selected, will be discussed. AW.01 Crystallography - is the Gold Standard Getting Tarnished? David Watkin University of Oxford, Oxford, United Kingdom X-rays were discovered by Roentgen in 1895, diffraction of X-rays by von Laue in 1912, and the crystal structure of sodium chloride was elucidated by Bragg in 1913. Initially structures were determined by trail and error methods, but in 1936 Patterson published his interpretation 2 of the F synthesis - the Patterson function. After that, there was no looking back, and the determination of molecular structures using X-ray diffraction developed rapidly. By the end of the 1960's most of the theoretical background which we now use had been worked out. The technique had been almost completely explored in just 30 years, so that many of the senior practitioners had experienced all the stages in its development. Because they had done much of the work by hand, from estimating intensities through to laborious calculations, they appreciated the significance of all the different kinds of data they were working with. They understood the risks in taking short cuts, and were able to make informed decisions about the advantages and disadvantages in alternative procedures. Because these people really understood what they were doing, their results (within the limits of the instrumentation available) were soundly based and secure - they were The Gold Standard of analytical techniques. I trained as a student in the 1960's and so had a reasonably formal education in crystallography. I experienced the transition from cameras to analogue diffractometers, to serial diffractometers, TV and image plate diffractometers, through to ccd and now pixel array detectors. Computers have reduced calculation times by perhaps four orders of magnitude (one could now do in one year what would have taken 10,000 years). Most of the theories developed by the pioneers are routinely available in brilliant programs. While crystallography in the broad sense still has many exciting frontiers, much molecular structure analysis has become routine, and some would say of a deteriorating quality. The IUCr, in trying to maintain standards, has the unenviable tasks of trying to distinguish good work on poor samples from poor work on good samples. 02.01.1 How Low Can You Go: Investigation of the Effects of Redundancy on Absorption Correction Michael Takase Massachusetts Institute of Technology, Cambridge, MA, United States Semi-empirical absorption correction using the multi-scan approach [1] is based on the comparison of equivalent reflections. It can be expected that this method would work best if the number of equivalent reflections in a given dataset is high. Similarly, semi-empirical absorption correction should not work very well for datasets with low or very low redundancies. In an attempt to determine the critical value of minimum redundancy (if, in fact, there is such a value) below which effective semi-empirical absorption correction is no longer possible, we are analyzing a number of datasets from a variety of different samples. Variables to be examined are crystal size, presence or absence of heavy atoms, Laue symmetry and wavelength of X-radiation [1] R.H. Blessing, Acta Crystallogr., Sect A 1995, 51, 33-38. 02.01.2 How are we doing? A Review of Small Molecule Crystallography based upon Data Mined from the Cambridge Structural Database. Joseph Reibenspies Texas A & M University, College Station, Texas, United States It is human nature for one to stand back from time to time and ask themselves: “how am I doing?” The question is of course very subjective; however it is fair and reasonable to ask such a question of a group as a whole. To measure progress and thus provide an answer to the title question one can examine a representative instrument, such as the Cambridge 1 Structural Database . The database consists of crystallographic information from 1923 to the present and encompasses organic and inorganic molecular compounds. Information such as 2 structure counts, residual factors, volumes, percent errors, total disorder and bond distance precision provides statistical data that can be used to measure progress in terms of quantity, quality and complexity. With this information in hand one can also be so bold as to predict the direction the science of crystallography is taking and what the future holds. At the end of the discussion we may ask ourselves “How are we doing?” and the answer may come as a bit of a surprise. 1 Allen, F. R. (2002). Acta Cryst. B58, 380–388. 2 Flippen-Anderson, J. L., J. R. Deschamps, Gilarid, R.D., George, C. (2001) Crystal Engineering 4, 131-139. OQMOPMR r ‒·¦ ·‒¡? \ `‹ ⁄›‹„?k ‹ ¡‹ h‹ · ¡?›¢?n‒£\‹ ¦?b⁄¡« ‒„K?t‹ ¡‒ „?›¢?y·‒ ¦⁄K?y·‒ ¦⁄K?r• ¡‒ \‹ \ ›‹Y?\· ›«\ ›‹K? £ \‹¦¡K?‹¡•? ›› The validation of crystal structures via the checkCIF software began in late 1997. Before then, it was up to the practitioner to check carefully that all was well with each structure determination; a manageable task when only a few dozen or less structures were determined per year and a wise professional crystallographer was at hand for guidance. Nowadays, diffractometers, in some cases in do-it-yourself labs, have such high throughput that mere mortals are overwhelmed by the amount of data being accumulated. To cope with the number of structures being determined, intelligent tools to automate the routine parts of the experiment are welcome and new automatic structure determination software is appearing. Can we now sit back, push a button and enjoy seeing a finished (routine) structure appear before our eyes together with a clean validation report, then assume all is okay? There may be aspects in any structure determination that the validation tools cannot evaluate or give appropriate feedback about before it is too late. It is still necessary to keep a watchful eye on the entire experiment and to think carefully and critically about the results. Every crystal has its own peculiarities, some of which may need special treatment, so even at the data collection stage one should be watchful of what is unfolding and be prepared to take appropriate action. A recent addition to the checkCIF suite is the ability to validate the structure factor listing against the corresponding CIF. This enables users to confirm that their archived structure factor file corresponds with the refinement run used to generate the CIF. The tests may also give feedback about, among other things, overlooked twinning and other inconsistencies within the CIF that might arise, for example, from incorrect editing of an existing CIF after a new refinement. Access to the service is at: http://journals.iucr.org/services/cif/checking/checkcifhkl.html 02.01.4 High Pressure Cryocooling at MacCHESS Chae Un Kim , Irina Kriksunov , William A. Miller , Mike Cook , Doletha M. E. Szebenyi , Sol 2,3 M. Gruner 1 1 1 1 1 1 MacCHESS (Macromolecular diffraction at CHESS), Ithaca, NY, United States, Cornell High 3 Energy Synchrotron Source, Ithaca, NY, United States, Physics Department, Cornell University, Ithaca, NY, United States A novel high-pressure cryocooling technique for macromolecular crystallography has been developed and explored at the Macromolecular Diffraction Facility at the Cornell High Energy Synchrotron Source (MacCHESS) [1]. The method involves cooling macromolecular crystals to cryogenic temperatures (~ 100 K) in high-pressure (up to 200 MPa) helium gas. Applications include successful cryocooling with little or no penetrating cryoprotectant. The method has been extended to Kr/Xe single-wavelength anomalous dispersion (SAD) phasing, native sulfur SAD phasing, and preparation of cryocooled crystal samples in capillaries. A mechanism involving high-density amorphous (HDA) ice is used to explain why the method works. Surprising results include visualization of ligands which could not be seen using other methods [2, 3], and insight into the phases of water in a protein crystal [4]. The high pressure cryocooling method is available to researchers with suitable crystals: See [http://www.macchess.cornell.edu/MacCHESS/about_macchess.html#Pressur e]. References [1] C. U. Kim, R. Kapfer, and S. M. Gruner (2005), Acta Cryst. D61, 881-890. [2] R. A. Albright, J. -L. V. Ibar, C. U. Kim, S. M. Gruner, and J. H. Morais-Cabral (2006), Cell 126, 1147-1159. [3] J. F. Domsic, B.S. Avvaru, C. U. Kim, S. M. Gruner, M. Agbandje-McKenna, D. N. Silverman, and R. McKenna (2008), J. Biol. Chem. 283, 30766-30771. 2 [4] C. U. Kim, B. Barstow, M. W. Tate, and S. M. Gruner (2009), Proc. Natl. Acad. Sci., 106, 4596-4600. 02.01.5 Progress in Using Short Wavelength Radiation for Chemical Crystallography Juergen Graf , Bernd Hasse , Francesca Fabbiani , Thomas Schulz , Dietmar Stalke , Holger 3 1 Ott , Carsten Michaelsen 1 3 1 1 2 2 2 Incoatec GmbH, Geesthacht, Germany, University of Goettingen, Goettingen, Germany, Bruker AXS GmbH, Karlsruhe, Germany 2 Combining synthetic multilayer mirrors with microfocus X-ray sources (rotating or stationary target) has become a standard with in-house X-ray sources for single crystal diffraction as well as a number of applications in powder diffraction. The maximum angle of incidence at which a multilayer mirror reflects is significantly smaller for higher energy radiation, such as Mo-Kα or Ag-Kα radiation than it is for Cu-Kα radiation. This is why synthetic multilayer mirrors traditionally have been used for Cu-Kα radiation or softer wavelengths. Modern deposition technology, however, allows for the reproducible production of high quality multilayer mirrors with smaller d-spacing. In consequence these mirrors reflect higher energy radiation at larger angles of incidence. Combined with the latest generation of microfocus sealed tubes this provides new high-performance low-power X-ray sources for shorter wavelengths. We will present selected results on the use of these low-power consumption, highperformance sources in small molecule and high-pressure crystallography. 02.01.6 A Hybrid Pixel Detector in the Home Laboratory: Prospects for Better Data Joseph Ferrara, Colin Acheson, Angela Criswell, Pierre Le Magueres, James Pflugrath, Katsunari Sasaki Rigaku Americas Corp, The Woodlands, TX, United States We have begun using a hybrid pixel detector (HPD), specifically the Dectris Pilatus 100K, in home lab single crystal X-ray diffraction experiments. In order to assess the utility of such a device for the home lab, we have studied the performance of this device for both small molecule and protein data collection experiments with copper radiation. We will present results comparing HPD data collection to conventional CCD data collection as well as results comparing conventional data collection to “shutterless” data collection in terms of data quality and increased throughput. 07.01.1 First Results of Femtosecond Protein Nanocrystallography Mark Hunter , Petra Fromme , Rick Kirian , Uwe Weierstall , Bruce Doak , Henry Chapman , 1 John Spence Arizona State University, Tempe, AZ, United States, Hamburg, Germany 1 2 1 1 1 1 1 2 CFEL/University of Hamburg, Serial crystallography has been used to show the first proof-of-principle for femtosecond nanocrystallography at the Linac Coherent Light Source, a 2keV pulsed X-ray laser at SLAC -15 which provided 3-300 femtosecond (10 s) pulses. The intensity of the X-ray pulses exceeds rd 3 generation sources by 12-orders of magnitude, yet the pulses are so short that X-ray diffraction data are collected before the sample is destroyed. In serial crystallography, X-ray diffraction is collected from a stream of fully hydrated protein nanocrystals in their mother liquor. The jet introduced nanocrystals of Photosystem I, a complex membrane protein with a mass of 1056000 Da consisting of 36 protein subunits and 381 cofactors, to the femtosecond X-ray beam produced at the LCLS. Individual diffraction patterns, read out at 30 Hz, could then be indexed and assembled into a working data set. Over six million diffraction patterns from Photosystem I nanocrystals were collected, and diffraction was recorded to the detectorlimited resolution of 9Å. The experiments indicate that in this diffract-and-destroy mode, even a 70 fs pulse may terminate before detectable radiation damage or spot fading occur, to the available resolution. The impact and potential of the LCLS for future structural determinations of membrane proteins will be discussed. This project is a large international collaboration, involving the CAMP group from three Max Plank Institutes and ASU physics. PIs include H. Chapman, P. Fromme, I. Schlichting, B. Doak, U. Weierstall, J. Uhlich, A. Barty, L. Struder, D. Rolles, the LCLS staff and the ASG team. 07.01.2 Crystal structure of the membrane fusion protein CusB from Escherichia coli Edward Yu Iowa State Universiry, Ames, United States Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes belonging to the resistance-nodulation-division family to expel diverse toxic compounds from the cell. These systems contain a periplasmic membrane fusion protein that is critical for substrate transport. We here present the x-ray structures of the CusB membrane fusion protein from the copper/silver efflux system of E. coli. This is the first structure of any membrane fusion proteins associated with heavy-metal efflux transporters. CusB bridges the inner membrane efflux pump CusA and outer membrane channel CusC to mediate resistance + + to Cu and Ag ions. Two distinct structures of the elongated molecules of CusB were found in the asymmetric unit of a single crystal, which suggests the flexible nature of this protein. Each protomer of CusB can be divided into four different domains, whereby the first three domains are mostly -strands and the last domain adopts an entirely helical architecture. Unlike other known structures of membrane fusion proteins, the -helical domain of CusB is folded into a three-helix bundle. This three-helix bundle presumably interacts with the periplasmic domain of CusC. The N and C-termini of CusB form the first -strand domain, which is found to interact with the periplasmic domain of the CusA efflux pump. Atomic + + details of how this efflux protein binds Cu and Ag were revealed by the crystals of the CusBCu(I) and CusB-Ag(I) complexes. The structures indicate that CusB consists of multiple binding sites for these metal ions. These findings reveal novel structural features of a membrane fusion protein in the resistance-nodulation-division efflux system, and provide evidence that this protein specifically interacts with transported substrates. 07.01.3 Crystal structure of the ectodomain complex of the CGRP receptor, a Class-B GPCR, reveals the site of drug antagonism Ernst ter Haar, Christopher Koth, Norzehan Abdul-Manan, Lora Swenson, Joyce Coll, Judith Lippke, Christopher Lepre, Miguel Garcia-Guzman, Jonathan Moore Vertex Pharmaceuticals Incorporated, Cambridge, MA, United States The calcitonin gene-related peptide (CGRP) is a potent vasodilator directly implicated in the pathogenesis of migraine. Its receptor (CGRP-R) is a heterodimer containing the calcitonin receptor-like receptor (CLR), a class B GPCR, and RAMP1, a receptor activity-modifying protein. We have solved the crystal structure of the CLR/RAMP1 N-terminal ectodomain heterodimer, revealing how RAMPs bind to and modulate the activities of the CLR GPCR subfamily. We have also determined the structures of CLR/RAMP1 in complex with antagonists olcegepant (BIBN4096BS) and telcagepant (MK0974). Both drugs act by blocking access to the CGRP binding cleft at the interface of CLR and RAMP1. These structures reveal how small molecules bind to and modulate the activity of a class B GPCR, and highlight the challenges of designing potent receptor antagonists for the treatment of migraine and other class B GPCR-related diseases. 07.01.4 Resolving the Structures of Membrane Pores Formed by Antimicrobial Peptides Huey Huang Rice University, Houston, Texas, United States Antimicrobial peptides (AMPs) are ubiquitous components of the innate immune systems found in all plants and animals. Soon after their discovery in the 80’s, they were found to kill microbes by forming pores in the microbial membranes. Since the conventional antibiotics have been facing the serious issue of resistance, this new type of antimicrobials has attracted a great deal of interest. However their molecular mechanisms as well as the structures of their pores have been controversial. In this talk I will describe how we used neutron scattering and X-ray diffraction to resolve the structural and mechanism issues. In particular we have developed a new MAD procedure to resolve the phase problem of diffraction. 07.01.5 Interaction of Lipid Monolayers and Single Supported Bilayers with Cholera Toxin: X-ray and Neutron Reflectometry and Grazing Incidence X-Ray Diffraction Studies Jaroslaw Majewski , Tonya Kuhl , Chad Miller , Erik Watkins 1 1 2 2,1 1,2 2 Los Alamos National Laboratory, Los Alamos, NM, United States, University of California Davis, Davis, CA, United States Biological membranes are critical components of functioning cells and many bacterial toxins bind to and gain entrance to target cells through specific interactions with membrane components. Using surface sensitive neutron and x-ray reflectometry and grazing-incidence diffraction we were able to follow the process of cholera toxin attack on a model lipid mono- and bi-layer. In-plane and out-of-plane changes in 2-D packing of cholera toxin molecules and the lipid membrane were investigated. We followed the process of the toxins assault on the monolayer in time. A firm understanding of the molecular mechanisms by which cholera toxin penetrates and translocates across a membrane will stimulate the design of possible interventional therapies to prevent infections that use the same mechanism to enter the cell. Furthermore, a similar mechanism could be employed to transfect cells with a desired therapy. 07.01.6 Lipid membrane-mediated 2D assembly of proteins and viruses at liquid interfaces Masafumi Fukuto , Suntao Wang , Sumit Kewalramani , Matthew Lohr , Zhongwei Niu , 2 2 1 Giang Nguyen , Qian Wang , Lin Yang Brookhaven National Laboratory, Upton, NY, United States, University of South Carolina, Columbia, SC, United States Biomolecular nanoparticles (BNPs), such as proteins and viruses, are ideal nanoscale building blocks because of the intrinsic monodispersity in their size, shape, and surface properties. In particular, BNPs bound to a lipid monolayer at a solution-vapor or solution-substrate interface are well suited for investigating ordered 2D assembly of nanoscale objects. Using in situ grazing-incidence x-ray scattering, we have recently studied density-driven 2D crystallization of BNPs for two types of BNPmembrane interactions, one based on specific ligand binding and the other based on electrostatic interactions. For the first system, the 2D assembly of the protein streptavidin (SA) on a biotinbearing lipid monolayer was studied as a function of the surface density of biotin, a protein-binding ligand. The results of detailed x-ray scattering and optical Brewster-angle microscopy measurements reveal that the 2D crystallization of the lipid-bound SA occurs as a density-driven first-order phase transition. Significantly, the threshold biotin density for inducing the 2D crystallization is found to be roughly equal to the density of the ligand-binding sites in the SA crystal. Moreover, the extracted protein adsorption isotherm indicates that the fully bound state of SA, corresponding to two biotinlipids per protein, is achieved already below the threshold biotin density. These results demonstrate that in addition to a well-defined molecular orientation, high lateral packing density is essential to the 2D crystallization of proteins. For the second system, the electrostatic 2D assembly of cowpea mosaic virus (CPMV) on a mixed cationic-zwitterionic (DMTAP/DMPC) lipid monolayer was studied as a function of the subphase pH and the membrane charge density. GISAXS data show that 2D crystals of CPMV are formed above a threshold membrane charge density and only in a narrow pH range just above CPMV's isoelectric point, where the charge on CPMV is expected to be weakly negative. The particle density for the 2D crystals is similar to that for the densest lattice plane in the 3D crystals of CPMV. The results demonstrate that the 2D crystallization is achieved in the part of the phase space where the electrostatic interactions are expected to maximize the adsorption of CPMV onto the lipid membrane. 1 2 1 1 1 1 2 01.01.1 LOUIS DELBAERE and the Early Days of Crystallography in Edmonton, Alberta. Title: Michael James University of Alberta, Edmonton, Alberta, Canada Louis Delbaere made many seminal contributions to the advancement of Canadian Crystallography during his days in Edmonton. He solved several structures of monosaccharides while he was a post doctoral fellow in Ray Lemieux’s laboratory. He also made major contributions to the understanding of the structures of the blood-group determinants. Louis also made major contributions to protein crystallography and to solving the first protein structure done in my laboratory in Edmonton. He was one of the main motivating forces in solving the crystal structures of the A and B peptidases from Streptomyces griseus. He also collaborated with Gary Brayer on the structure of the alphalytic protease. He worked with I-Nan Hsu and Theo Hofmann on the structure of the first aspartic peptidase to be solved, that of Penicillopepsin. 01.01.2 Louis Delbaere: Friend and Colleague J. Wilson Quail Department of Chemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada For years to come, many Canadians who never knew Louis will benefit from his efforts. He left a mark on many people and organizations. 01.01.3 Louis Delbaere and the International Union of Crystallography Sine Larsen International Union of Crystallography, Chester, United Kingdom, University of Copenhagen, Copenhagen, Denmark When Louis too early passed away in 2009 he had served as a member of the IUCr Executive Committee (EC) for 14 months. Both his election to the EC and the selection of Montreal as the venue for the 2014 IUCr congress demonstrated the respect and appreciation that the international crystallographic community held for Louis. Having known Louis from the time we both started our crystallographic career it was a personal pleasure to work with him in the EC the short time we had together. This presentation will focus on the role Louis played in the international crystallographic society and the impact of his work for the IUCr. 1 2 01.01.5 Surface Constrained Protein Nanotubes for Bionanoelectronics Gerald Audette, Stephanie Lombardo, Agnesa Shala York University, Toronto, ON, Canada Powering the next generation of implantable devices will rely systems that are more biologically accessible. To achieve this, we must harness the power of proteins and interface them with scaffolds, creating unique bio-nanosystems. Bionanoelectronics are interface a protein system to a conducting surface, thereby connecting them to micro fuel sources such as biofuel cells. One system we are currently investigating is the type IV pilus from Pseudomonas aeruginosa, a nanofibre composed of multiple copies of a single protein subunit, the type IV pilin. In the presence of a hydrophobic surface or solution, engineered pilin monomers oligomerize into soluble, high molecular weight structures – protein nanotubes (PNTs). P. aeruginosa pilins, pili and pilin-derived PNTs have been shown to bind both biotic surfaces (cells) as well as abiotic surfaces such as stainless steel. We have recently examined the oligomerization of PNTs onto alkylthiol functionalized gold surfaces. PNTs oligomerized from surface constrained alkylthiols have been observed to be several micrometers in length with an average diameter of 36 ± 3 nm. In comparison with reported values for the diameter of native type IV pili and PNTs in solution (~6 nm), the average observed diameter of surface oligomerized PNTs suggests a multiple PNT clustered filament on the gold surface. Current research targets the directed engineering of the pilin monomer to facilitate metal ion binding for bionanowire development and patterned PNT oligomerization on gold surfaces via differential functionalization of the gold surface with a variety of alkylthiols. 01.01.6 AlgK and AlgE form the outer membrane secretin of a novel bacterial expolysaccharide secretion system Lynne Howell , John Whitney , Carrie Keiski , Maria Amaya , Mirela Neculai , Patrick 1 1 1 1 1 4 Yip , Laura Riley , Joel Weadge , Francis Wolfram , Yura Lobsanov , Howard Robinson , Lori 3 5 Burrows , Dennis Ohman The Hospital for Sick Children, Toronto, ON, Canada, University of Toronto, Toronto, ON, 3 4 Canada, McMaster University, Hamilton, ON, Canada, Brookhaven National Laboratory, 5 Upton, NY, United States, Virginia Commonwealth University Medical Center, Richmond, VA, United States Pseudomonas aeruginosa is the predominant pathogen associated with chronic lung infections in Cystic Fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by the overproduction of the exo-polysaccharide alginate. Ten proteins, encoded by the algD operon, have been implicated in alginate biosynthesis. Since polymerization requires protein components in both the inner and outer membrane, these proteins are believed to form a large multi-protein complex that spans the cell envelope and facilitates export of the polymer. To understand how alginate is exported we have undertaken structure-function studies on two outer membrane components of this system, the lipoprotein, AlgK, and the -barrel porin, AlgE, both of which are essential for the production of high molecular weight alginate. Complementation and subcellular fractionation studies have shown that AlgE is required for alginate secretion, and that AlgK plays an important role in localizing AlgE to the outer membrane; a finding that suggests that the two proteins may interact directly. Structures of AlgK and AlgE have been determined at 2.5 and 2.3Å resolution, respectively, and reveal that AlgK is an all -helical solenoid protein with at least 9.5 tetratricopeptide-like (TPR) protein-protein interaction motifs. This topology suggests that AlgK may play a role in assembly of the alginate biosynthetic complex. AlgE is a monomeric 18-stranded -barrel that shows remarkable structural similarity to members of the substrate-specific OprD-family of outer membrane channels. The occlusion of AlgE’ s electropositive conduit by extracellular loop L2 and periplasmic loop PL8, suggests that, unlike in other channels, conformational changes may be required to facilitate alginate export. Bioinformatic analyses have also revealed that the proteins BcsA, PgaA and PelB, involved in the production and export of cellulose, poly- -1,6-N-Acetyl-D-glucosamine and Pel exopolysaccharide, respectively, share the same topology as AlgK/E. Together our data provide a model for alginate export and suggest that AlgK/E represent the periplasmic and outer membrane components of a new type of outer membrane secretin that differs from canonical bacterial capsular polysaccharide secretion systems. 1 2 1,2 1,2 1,2 1 1 01.01.7 Dynamics of Siderophore Reception at the Staphylococcus aureus Cell Surface Jason Grigg , John Cooper , Johnson Cheung , David Heinrichs , Michael Murphy 1 2 1 2 2 2 1 University of British Columbia, Vancouver, BC, Canada, University of Western Ontario, London, ON, Canada Staphylococcus aureus is a devastating bacterial pathogen. It thrives in the human body by scavenging iron with secreted small molecule chelators called siderophores. S. aureus synthesizes two polycarboxylate-type siderophores, staphyloferrin A (SA) and staphyloferrin B (SB). Siderophore-iron complexes are bound at the cell surface by lipoprotein receptors (HtsA and SirA) and shuttled to membrane permease for import. We have determined the crystal structures of both receptors in their apo and siderophore-bound complexes. The SA receptor, HtsA, binds a single SA-Fe complex with nM affinity. Structures have been determined of apo-HtsA (1.35 Å resolution), HtsA-SA-Fe in an open conformation (1.30 Å resolution) and closed conformation (2.2 Å resolution). The closed ternary complex structure was obtained from a twinned crystal (P21 with operator l,-k,h). Together these structures reveal that instead of more typical domain movement, significant local conformational changes isolated to three loops in the C-terminal domain coincide with SA-Fe binding. A ~12 Å C shift of Tyr239 brings it into H-bonding distance of SA, enclosing the SA-Fe complex in the pocket. The conformational change also results in a ~ 2.8 Å shift of Glu250, located at the apex of the domain where it likely forms a salt-bridge with the permease, allowing discrimination between apo and holo receptors without large scale domain movement. Importantly, the structure of SA-Fe is also well defined in the binding pocket allowing assignment of chiral centers, one of which differs from previous predictions based on model compound properties. The apo-SirA (2.2 Å resolution) and SirA-SB-Fe ternary complex (1.7 Å resolution) structures have been determined, again allowing definition of the SB-Fe structure. SirA belongs to the same structural family as HtsA and also undergoes localized conformational changes following ligand binding. However, the large structural change occurs on the opposite side of the binding pocket to HtsA, highlighted by a ~13 Å shift of Asp263 C to enclose SB-Fe. Localized conformational changes bring two arginine residues in H-bonding distance of SB as well as a reorientation of Glu245 by ~1.6 Å, which analogous HtsA is predicted to allow discrimination of ligand bound and apo-SirA. 07.02.1 Ultrafast synchrotron and laboratory in situ powder diffraction studies on the synthesis of functional materials John Evans Durham University, Durham, United Kingdom Traditionally the vast majority of functional inorganic materials have been synthesised under conditions of thermodynamic control - the "heat and beat" method of solid state synthesis. Such methods are, of course, unsuitable for the preparation of metastable materials. In this presentation I'll discuss how in-situ powder diffraction methods have been crucial in allowing the preparation of several metastable inorganic framework materials, and in suggesting the topotactic mechanisms that allow their isolation. Examples will include the preparation of new molybdate and phosphate materials followed using laboratory based techniques.[1] I'll also discuss new ultrafast powder diffraction experiments performed at beamline ID11 at the ESRF in which full quantitative Rietveld refinement is possible on reacting ceramic systems with a time resolution down to 0.1 seconds. These experiments have allowed us to identify conditions under which we can prepare the negative thermal expansion material ZrMo2O8 directly from its constituent oxides. The ability to rapidly scan temperature and composition space on a reaction that is complete in time periods of ~10 seconds has allowed us to discover a small temperature window at around 1500 K in which this material is thermodynamically stable, and to optimise conditions for its synthesis. I'll also discuss data analysis methodologies which allow one to extract the maximum information from large bodies of diffraction data collected during in-situ experiments. [1] Lister, S.E., Evans, J.S.O., unpublished results. [2] Readman, J.E., S.E. Lister, L. Peters, J. Wright, and J.S.O. Evans, Direct Synthesis of Cubic ZrMo2O8 Followed by Ultrafast In Situ Powder Diffraction. Journal of the American Chemical Society, 2009. 131(48): p. 17560-17562. 07.02.2 Structural behaviour of (Mg,Fe )(AlSi)O3 perovskite at pressures of the Earth’ s lower mantle Tiziana Boffa Ballaran , Alexander Kurnosov , Konstantin Glazyrin , Marco Merlini , Daniel J. 1 Frost Bayerisches Geoinstitut, Bayreuth, Germany, Dept. Earth Science, University of Milano, Milano, Italy Throughout the bulk of the Earth’s lower mantle, MgSiO3-perovskite is expected to contain significant proportions of both Al and Fe, with Fe potentially in both 2 and 3+ oxidation states. Several studies have reported that ferrous iron exists in an intermediate spin state throughout the Earth’s lower mantle whereas ferric Fe may be involved in a high-spin low-spin transition. Changes in spin states in silicate perovskite can be expected to affect the coordination polyhedra around the iron atoms and influence therefore the elastic properties of this mineral. In order to quantify the effect of such spin transitions on the structural behaviour of perovskite, we have synthesised at the Bayerisches Geoinstitut single-crystals of a very Fe,Al-rich perovskite using a multi-anvil technique. The crystals are of an excellent quality as indicated by their sharp diffraction profiles and lack of twinning. High-pressure single-crystal X-ray diffraction has been performed at the beam line ID09 at the European Synchrotron Radiation 3+ Facility (ESRF). A single crystal of (Mg,Fe )(AlSi)O3 (longest dimension 25 microns) has been loaded in a diamond anvil cell with ruby as pressure standard and He as pressure transmitting medium. Intensity data have been collected at different pressures up to 75 GPa and isotropic structural refinements always converged with discrepancy factors smaller than 4%. Octahedral and dodecahedral sites have similar compressibility and the orthorhombic distortion only sligthly increases with pressure. However, the octahedral tilting along the c axis shows a change in behaviour above 50 GPa. 1 2 1 1 1 2 3+ 07.02.3 Probing the high-pressure behaviour of H2SO4 and MgSO4 hydrates with neutrons A. Dominic Fortes , Ian G. Wood , Matthew G. Tucker 1 1,2 1,2 3 2 Department of Earth Sciences, University College London, London, United Kingdom, Centre 3 for Planetary Sciences at UCL/Birkbeck, London, United Kingdom, ISIS Facility, Rutherford Appleton Laboratory, Chilton, Oxfordshire, United Kingdom Hydrates of sulfuric acid, and of magnesium sulfate, have been reported on the surfaces of the Galilean satellites of Jupiter [1], and models suggest that these hydrates will be abundant in their deep interiors [2], consequently experiencing modest hydrostatic pressures. Investigation of their high-pressure behaviour is important since there are likely to be changes in both the hydrogen bond network as well as possible changes in ion speciation, and pressure-induced dehydration. Such phase changes may influence heat transport inside icy planetary bodies, and hence control their overall structure and evolution. Using neutron powder diffraction, we have carried out studies upon a range of hydrates relevant to the internal structure and dynamics of icy satellites, including deuterated isotopologues of sulfuric acid 8-, 6½-, and 4-hydrate, and magnesium sulfate 11-, and 7-hydrate, at pressures up to 4 GPa in the Paris-Edinburgh press. Much of our earlier work is summarised in reference [3]. In sulfuric acid tetrahydrate, we have identified two monoclinic high-pressure polymorphs, SAT-II and SAT-III, in addition to the low-pressure tetragonal phase. SAT-II is formed by warming SAT-I above 235 K at 550 MPa, and this has been successfully recovered to atmospheric pressure at 50 K. SAT-III has been observed over the range 1.6—3.9 GPa, melting at 380 K at 3.9 GPa. However, sulfuric acid tetrahydrate is extremely difficult to crystallise at high-pressure, requiring deep undercooling. In the MgSO4-hydrates we have now observed a reproducible sequence of phase transitions, in agreement with ultrasonic wave-velocity observations reported elsewhere [4], in which pressure-induced dehydration occurs. At 295 K, MgSO4· 7D2O (synthetic epsomite) undergoes its first transition at 1.2 GPa to a lower hydrate + aqueous solution, this phase being stable over only a narrow pressure range (0.2 GPa), before the onset of the next phase transition and the growth of ice VII. Similarly, MgSO4· 11D2O (synthetic meridianiite), when compressed at 240 K, breaks down to a lower hydrate + ice VI at ~ 0.9 GPa. We report the status of our work to interpret the high-pressure behaviour of these materials, including recent attempts to recover the products of various phase transitions to ambient pressure for better characterisation. References [1] Orlando et al. (2005) Icarus 177, 528-533: McCord et al. (2001) Science 292, 1523-1525. [2] Kargel (1991) Icarus 94, 369-390. [3] Fortes & Choukroun (2010) Space Sci. Rev. 10.1007/s11214-010-9633-3. [4] Gromnitskaya et al. (2010) High Press. Res. 30, 51-54. 07.02.4 Crystallographic studies of medical gases adsorbed in metal-organic frameworks Russell Morris University of St Andrews, St Andrews, United Kingdom Metal-organic frameworks comprise one of the most exciting classes of solids in current science. These highly porous solids have been of particular interest as gas sorbent materials. Many of these studies have concentrated on adsorbing hydrogen, carbon dioxide, methane and other gases of interest for energy and environmental applications. However, metalorganic frameworks are also exceptional materials for the adsorption, storage and delivery of medically important gases, such as nitric oxide (NO). In this presentation I will describe our crystallographic studies of NO adsorbed into several different framework solids, and how this information helps us to understand the particular properties of the materials in question. To complete these studies we primarily use single crystal X-ray diffraction studies at synchrotron sources (Daresbury, UK and the ALS, USA). The experiments are completed using a specially designed environmental gas cell that allows the single crystals to be thermally activated and then loaded with gas while it is on the diffractometer. References. 1. Xiao B et al. Chemically blockable transformation and ultraselective low-pressure gas adsorption in a non-porous metal organic framework Nature Chemistry 289-294 (2009). 2. McKinlay AC et al. Exceptional behavior over the whole adsorption-storage-delivery cycle for NO in porous metal organic frameworks J. Am. Chem. Soc. 130, 10440-10444 (2008). 3. Warren JE et al A prototype environmental gas cell for in situ small-molecule X-ray diffraction J. Appl. Crystallogr. 42, 457-460 (2009). 07.02.6 High pressure behavior of a family of 2+ 2+ 2+ 2+ [DMA][M(CHO2)3], M = Mg , Co , Mn and Zn 1 2 perovskite-related 3 metal 1,4 formates, Lauren A. Borkowski , Hyunsoo Park , Stephen Moggach , John B. Parise 1 2 Mineral Physics Institute, Stony Brook University, Stony Brook, NY, United States, 3 Department of Chemistry, Indiana University, Bloomington, IN, United States, School of 4 Chemistry, The University of Edinburgh, Edinburgh, Scotland, United Kingdom, Departments of Chemistry and Geosciences, Stony Brook University, Stony Brook, NY, United States 2+ The M formates crystallize in a perovskite-related structure, ABX3, where the A cation is dimethylamine (DMA), and some display multi-ferroic behavior at low temperatures. The inorganic perovskites display remarkable structural flexibility, often dominated by polyhedral distortion and tilting. We are interested in exploring the high pressure behavior of this new class of materials and comparing this to known perovskite compression mechanisms. Previous work at low temperature shows [DMA][Mg(CHO2)3] transforms from R-3c to C2/c. 2+ 2+ Single crystal and powder high pressure experiments on the Mg and Co analogs show that the high pressure behavior of these materials consists of tilting of the metal octahedra as well as a partial ordering of the DMA molecules on the A site. 07.02.5 The XIPHOS diffraction facility for experiments at ultra low temperatures 1 1 extreme 1 sample environments: 1 In-house 2 Craig Robertson , Michael Probert , Jonathan Coome , Andres Goeta , Brian Michell , Judith 1 Howard 1 Durham University, Durham, United Kingdom, Bruker AXS Inc., Madison, WI, United States 2 The XIPHOS (X-ray – Interface for Photo-Induced High pressure lOw temperature Structural studies) diffraction facility has been developed to collect diffraction data within a range of 1 sample environments. The system couples a Bruker direct drive Mo rotating anode generator operating at 5.4 kW with the latest Helios focusing optics. This source is mounted on a four circle Huber goniometer equipped with an APEX II CCD detector. To reach ultra rd low temperatures, an APD 202E Displex cryogenic refrigerator with an additional 3 JouleThompson stage has been installed, allowing for temperatures as low as 2 K to be maintained. This new diffraction facility will be presented in detail; furthermore, low temperature calibration, crystal centring and the results of recent experiments will be presented. References: 1) Probert, M. R.; Robertson, C. M.; Coome, J. A.; Howard, J. A. K.; Michell, B. C.; Goeta, A. E. Submitted, J. Appl. Cryst., February 2010 (hx5107). 07.02.7 'The Big Squeeze on Porous Materials.' Stephen Moggach The University of Edinburgh, Edinburgh, United Kingdom Recent interest in gas storage materials has led to a plethora of papers on the synthesis of [1, 2] metal organic framework materials (MOFs). Structural variation in MOFs can be achieved through chemical modification, with accompanying changes in pore size and shape (and therefore internal surface area) giving rise to an increasingly diverse array of sorption properties. Such sorption measurements are performed at pressures up to 0.01GPa, though what effect higher pressures have on the framework is relatively unknown. A sub-family of MOFs are the so called zeolitic imidazolate framework (ZIF) materials. ZIFs, related to zeolites through the 145˚ angle subtended at the bridging imidazolate ligand, are of increasing interest. Their tuneable pore size, chemical robustness and thermal stability combine the most desirable features of conventional MOF and zeolite structures, making them ideal candidates for gas storage applications. Over the last 10 years, developments in highpressure single-crystal diffraction techniques have allowed us to study much larger [3] compounds than was previously possible. The scope for pressure to change material properties has been demonstrated in previous work on amino acids and molecular magnets. This work focussed on tuning hydrogen bonding interactions or magnetism respectively. Here, we present the effect of pressure on porous molecular materials, in particular ZIF-8 (Zn(MeIM)2, MeIM = 2-methylimidazolate). On increasing pressure, we were not only able to [4] tune the pore size, but also the pore content of this material through ‘pressure’ modification. [1]R. Banerjee, A. Phan, B. Wang, C. Knobler, H. Furukawa, M. O’Keeffe, O. M. Yaghi, Science 2008, 319, 939. [2]R. Kitaura, G. Onoyama, H. Sakamoto, R. Matsuda, S.-i. Noro, S. Kitagawa, Angew. Chem., Int. Ed. 2004, 43, 2684. [3]S. A. Moggach, D. R. Allan, S. Parsons, J. E. Warren, J. Appl. Crystallogr. 2008, 41, 249. [4]S. A. Moggach, T. D. Bennett, A. K. Cheetham, Angew. Chem., Int. Ed. 2009, 48, 7087. 07.03.1 Data Collection, Reduction and Semi-automatic Structure Solution with HKL-3000 Wladek Minor , M. Cymborowski , M. Chruszcz , Z. Otwinowski , D. Borek 1 2 1 1 1 2 1 Univ. of Virginia, Charlottesville, VA, United States, UT Southwestern Medical Center, Dallas, TX, United States HKL-3000 integrates data collection, data reduction, phasing, and model building to significantly accelerate the process of structure determination, and on average, minimize the number of data sets and crystals required for structure solution. Execution of the package merges several modules and software applications into the structure determination pipeline. There are modules for experimental control of some beamlines and home instruments, data reduction, phasing by SAD/MAD or molecular replacement, fast model building, and initial refinement. The system is being developed and tested in the high-throughput environment of the Midwest Center for Structural Genomics (MCSG) and Center for Structural Genomics of Infectious Diseases (CSGID). The robustness of HKL-3000 has improved considerably over time and currently over 1000 structures have been determined with it. The continuous advancement of the decision-making procedures within HKL-3000 have made it the system of choice for MCSG and CSGID projects. Transforming raw images into a solved structure (with 70% of the model built) in 10-15 minutes is no longer a surprise, but a routine operation for crystals that diffract to 2.5 or better. Our experience with the determination of hundreds of structures by experimental phasing methods helped us to establish rules for the best approaches when the available data fall into three categories: unsolvable with current data, borderline and easy. Current work concentrates on improving the approach to borderline cases of structure determination rather than optimizing intermediate calculations for easy cases, thus shifting borderline cases into the easy category and unsolvable into borderline. An important implication is that simple experimental protocols are sufficient in most cases and may even be optimal for the most challenging ones. Feedback from fast preliminary structure solution proved to be one of the critical components of success. References Minor W, Cymborowski M, Otwinowski Z, Chruszcz M (2006) Acta Crystallographica Section D: Biological Crystallography62:859-66. Kirillova O, Chruszcz M, Shumilin IA, Skarina T, Gorodichtchenskaia E, Cymborowski M, Savchenko A, Edwards A, Minor W (2007) Acta Crystallographica Section D: Biological Crystallography63:348-54. Otwinowski Z, Borek D, Majewski W, Minor W (2003) Acta Crystallographica. Section A: Foundations of Crystallography59:228-34. Zheng H, Chruszcz M, Lasota P, Lebioda L, Minor W (2008) Journal of Inorganic Biochemistry102(9):1765-76. Chruszcz M, Wlodawer A, Minor W (2008) Biophysical Journal95(1):1-9. Wlodawer A, Minor W, Dauter Z, Jaskolski M (2008) Febs Journal275:1-21. Otwinowski Z, Minor W (1997) Methods in Enzymology275:307-326. 07.03.2 CMDDENZO and CMDXDS: Single-Line-Command-Driven Automated Data Processing at SER-CAT 1,2 1,2 3 User 1,2 Interfaces 1,2 for Zheng-Qing Fu , Zhongmin Jin , Andy Howard , John Chrzas , Jim Fait , Unmesh 1,2 1,2 1,2 1,2 1,2 Chinte , John Gonczy , Rod Salazar , John Rose , Bi-Cheng Wang Department of Biochemistry & Molecular Biology, University of Georgia, Athens, United 2 3 States, SER-CAT, APS, Argonne National Lab, Argonne, United States, BCPS, Illinois Institute of Technology, Chicago, United States As part of ongoing SER-CAT efforts to monitor data quality on-the-fly we have developed command-line-driven user interfaces (UI's) CMDDENZO and CMDXDS, which exploit functions in some of the widely-used data reduction packages such as 1) 2) 3) 4) 5) DENZO/SCALEPACK , D*TREK , SPGR4D, 3DSCALE , XDS , X-GEN for X-ray single crystal diffraction data reduction. These non-graphical UIs are not intended to match the expert use of a particular program, but to provide a means to automatically process and characterize a data set, which includes determining Space Group, Resolution Cutoff, Rmerge, Completeness, Redundancy, I/SigI etc. They also provide a set of handy diagnostic tools to quickly identify problems, if any, which should be helpful for remote and/or quick data collection at synchrotron beamlines. Details of the various UI's and their application to real data will be presented. Work supported by the SER-CAT Member Institutions. References: 1). Otwinowski, Z. & Minor, W. (1997), Met. Enz. 276:307-326. 2). Pflugrath, J.W. (1999), Acta Cryst. D55:1718-1725. 3). Fu, Z.Q. (2005), Acta Cryst. D61:1643-1648. 4). Kabsch, W. (1988), J. Appl. Cryst. 21:916-924. 5). Howard, A.J. (1996), Proc.Macromol. Cryst. Comput. Sch., Oxford University Press,Oxford, UK. 1 07.03.3 DrugSite: A Web-based Platform for Sharing Overlaid Protein:Ligand Complex Structures Barry Finzel, Ramprasad Akavaram, Aravind Ragipindi University of Minnesota College of Pharmacy, Minneapolis, MN, United States The popular drug design techniques of scaffold hopping and target hopping rely heavily on the accurate overlay of experimentally derived protein:ligand complex structures to emphasize both the differences and similarities in diverse examples of ligand binding. We have long believed that optimal superposition of such complexes results from an overlay of select protein substructures surrounding the binding site. The chosen substructure should include key structural elements contributing to complex stabilization, but not include elements subject to conformational change when different ligands bind. Suitable substructures are empirically identified, and the selection can evolve over the course of a project as different classes of ligands are added into the ensemble. Modern software has not evolved to simplify this approach. Rather, the emphasis has been on ready access to algorithms employing sequence and secondary structure matching that seeks to empower users to overlay progressively more divergent protein family members. While these algorithms have their uses, they do not result in the most useful alignments of ligands for drug design applications. We have been working to develop a database of “overlay methods” that captures optimized procedures for aligning important targets in drug-design. A web-based interface provides access to computational tools to apply these methods to either user-contributed complex structures or structures from the PDB, and allows users to share aligned structures with collaborators. The resulting platform provides an excellent means for communicating ligand structural data in a most useable form – already aligned on relevant homolog structures – for use by chemist and biochemist collaborators who might not otherwise have the expertise to devise an optimal superposition of the complexes. 07.03.4 Auto-Rickshaw: A tool for online validation of X-ray diffraction experiment and model completion Santosh Panjikar, Venkataraman Parthasarathy, Manfred Weiss, Victor Lamzin, Paul Tucker EMBL-Hamburg Outstation, Hamburg, Germany The Auto-Rickshaw is an automated structure determination software pipeline and it is based on several distinct computer coded crystallographic decision-makers, which invoke a variety of macromolecular crystallographic programmes/ programme packages during the structure determination process [1]. The primary aim of the pipeline is to validate the crystallographic experiment at the synchrotron site while the crystal is still at, or near, the beamline. The system is optimized for speed, so that structure determination can proceed within minutes after integrated and scaled diffraction data are available. Typically within a few minutes the answer is provided whether the collected data will be of sufficient quality to allow successful structure determination. Currently, the platform offers S/MAD, SIRAS, RIP and MR phasing protocols as well as combination of MR with various experimental phasing methods [2]. The result is an improvement of poor MR, or poor experimental phases and the determination of a larger percentage of the model, from X-ray data better than 2.9 Å resolution, in an automated manner. The platform has been installed on a Linux cluster at EMBL-Hamburg and is remotely accessible to the beamline users via a web-server [3]. It is accessible from most Internet browsers and allows beamline users to validate their X-ray diffraction experiments and model completion. Since 2008, Auto-Rickshaw web server [3] has been made accessible to the worldwide scientific community. References [1] Panjikar et al., (2005). Acta Cryst. D61, 449-457. [2] Panjikar et al., (2009). Acta Cryst. D65, 1089-1097 [3] http://www.embl-hamburg.de/Auto-Rickshaw/ 07.03.5 The CCP4 Software Suite - Current Status and Future Developments Ronan Keegan , Martyn Winn , Eugene Krissinel , Charles Ballard , Natalie Zhao , George 1 Pelios STFC Rutherford Appleton Laboratory, Oxfordshire, United Kingdom, STFC Daresbury Laboratory, Cheshire, United Kingdom CCP4 exists to produce and support a world-leading, integrated suite of programs that allows researchers to determine macromolecular structures by X-ray crystallography. CCP4 aims to develop and support the development of cutting edge approaches to experimental determination and analysis of protein structure, and integrate these approaches into the suite. The current CCP4 software suite is on release series 6.1.x. A particular focus of these releases is the automation of significant parts of the structure solution process, including XIA2 for data processing, Crank for experimental phasing, MrBUMP and Balbes for Molecular Replacement, and Buccaneer for model building. There are also a number of new programs, including Pointless for Laue group and spacegroup determination, the new iMosflm interface, Parrot for density modification, and PISA for identification of protein-protein interfaces. We will give an overview of the additions to the CCP4 suite, as well as an update on established programs. A major overhaul of the CCP4 suite is under development. A new graphical front-end will provide easier control of the suite, and considerable help with interpreting and evaluating the results. At the core, there will be in-built support for automation, making straightforward structures simple to solve, while continuing support for more challenging projects. Finally, usage of the suite will be underpinned by better data management, with support for database back-ends. CCP4 also aims to enhance its functionality related to the maintenance and use of data on small molecules (ligands). Firstly, a considerably larger library of chemical compounds will be provided with the Suite. Extended search functions will be provided to allow for efficient retrieval of known compounds or their close analogs. Secondly, existing functions for generating restraint data for new ligands will be enhanced by the inclusion of relevant software, such as ProDRG, into the Suite, as well as by the development of new methods for structure reconstruction on the basis of partial similarity to structures in the library. Functionality will be available through a graphical front-end application, jLigand. 1 2 1 2 1 1 1 07.03.6 How good is “good enough”? Predicting the success or failure of structure solution from first principles. James Holton University of California, San Francisco, CA, United States, Lawrence Berkeley Laboratory, Berkeley, CA, United States How much x-ray exposure is required to solve a structure? Multiplicity is “good” but how much will add “too much” read-out noise? What about a better detector? What about a perfect detector? Answering these questions requires that damage, noise and signal be placed on a common, absolute scale. To this end, a quantitative simulator of the entire diffraction experiment called "MLFSOM" (MOSFLM in reverse) was created. The input to the simulator is a protein data bank (PDB) file and parameters such as photon flux, crystal size and detector performance characteristics entered in conventional units such as photons/s and millimeters. MLFSOM was used to produce images in SMV format that were subsequently processed with ELVES. The general result of these trials was that one and only one of the many sources of noise in the diffraction experiment will dominate a given data set, and the optimal strategies for MAD/SAD and high-angle data collection are mutually exclusive. Faint, high-angle spots are best collected with exposures long enough to “bury” the detector read out noise under the background-photon noise (but no longer), but the optimal strategy for MAD/SAD was collecting a large number of very brief exposures, or “dose slicing”. 1 2 07.04.1 Radiation damage in macromolecular crystallography: current challenges. Elspeth Garman , Ian Carmichael 1 1 2 2 University of Oxford, Oxford, United Kingdom, Notre Dame Radiation Laboratory, Notre Dame, United States For protein crystals at room temperature, radiation damage during the diffraction experiment is rapid even on a laboratory X-ray source. In the past, the required data had to be collected from several different crystals and merged together. The intense X-ray beams produced by third generation synchrotrons can destroy crystalline order in a matter of seconds. Over the last 20 years, the use of cryo-cooling techniques which allow X-ray data to be collected with the sample held in a stream of cooled nitrogen gas at 100K, has become the norm [1, 2]; at 100K crystals can withstand many times the dose (J/kg=Gy) [3] compared with room temperature (depending on the dose rate [4]), and the necessary data can usually be obtained from a single crystal. However, observations of degradation of crystal diffraction with increasing radiation dose at 100K have now become commonplace at third generation synchrotrons. Researchers are trying to understand the physical and chemical processes involved in this damage (reviewed in [5,6]), which manifests itself in a number of different ways, including: changes in crystal colour, decreasing diffraction power with dose, a small but measurable linear increase in unit cell volume, and specific structural damage to covalent bonds in the amino acids of the protein molecules [7]. Enzyme active sites seem particularly sensitive to damage, so this phenomenon can lead to incorrect conclusions on biological mechanisms being drawn. Thus the issue of radiation damage during diffraction experiments has recently come to the fore as a concern for all structural biologists. Current issues being addressed and the challenges of research into this area will be outlined, informed by the material presented at the Sixth International Workshop on Radiation Damage to Biological Crystalline Samples held at SSRL in March 2010. References: [1] Teng, T-Y (1990) J Appl. Cryst. 23, 387-391 [2] Garman, E.F. & Schneider, TR (1997) J Appl. Cryst. (1997) 30, 211-237. [3] Owen, RL, Rudiño-Piñera, E and Garman, EF (2006) PNAS (2006) 103, 4912-4917. [4] Southworth-Davies, RJ.,Medina, MA.,Carmichael, I, & Garman, EF. Structure (2007) 15, 1341. [5] Ravelli, RGB & Garman, EF (2006) Current Opinion of Structural Biology (2006) 16, 624. [6] Garman, EF (2010) Acta Cryst. D66, 339-351. [7] Weik, M et al. (2000) PNAS 97, 623-628. [8] Burmeister, W.P. (2000) Acta Cryst. D56, 328-341. [9] Ravelli, R.G.B. & McSweeney, S. (2000) Structure 8, 315-328. 07.04.2 Quality Versus Quantity: the Role of Carefully Planned Diffraction Experiments in Highthroughput Crystallography Tobias Krojer, Frank von Delft Structural Genomics Consortium, Oxford, United Kingdom Our experience at SGC Oxford shows that the rate of success in a high-throughput environment does not only depend on the number of proteins going into crystallization, but on careful planning of data collection experiments. SGC-Oxford, is part of a world-wide structural genomics initiative and currently the Oxford site deposits four novel, human structures per month. Because our target list is fixed and the proteins consistently challenging, the emphasis in the crystallography group is on ensuring success even for marginal experiments through best practice data collection, which dramatically lowers the workload on the upstream pipeline. A vital ingredient in this philosophy is frequent access to high-quality beamlines, in our case at SLS and DIAMOND. Although these facilities provide equipment of unprecedented quality, we note that successful data collection still depends equally on experimenters' experience and skills. Here we present results from the numerous data sets that we have collected over the last six years at synchrotron beamlines and come up with suggestions for future software developments. We conclude that (i) beam sizes smaller than the diffracting volume of a crystal are of little benefit, and that (ii) exploratory datasets are of great value for predicting crystal lifetimes but hard to interpret for marginal diffraction. Thus, we still lack tools for routine experiments, specifically for characterizing the intersection of beam and crystal, as well as robust, real-time metrics for monitoring crystal decay. 07.04.3 The minimum crystal size needed to solve a structure. James Holton , Kenneth Frankel 1 1,2 1 2 University of California, San Francisco, CA, United States, Lawrence Berkeley Laboratory, Berkeley, CA, United States The total amount of photons scattered into diffraction spots by a cryo-cooled protein crystal before it is “dead” is fixed because radiation damage and accumulated scattered intensity (photons/spot) are both proportional to fluence (incident photons/area). This means that damage-limited data quality is independent of data collection time, and therefore also independent of flux (photons/s). We calculated the damage-limited spot intensity from a protein crystal at a desired resolution given the molecular weight, crystal volume, solvent content, Wilson B factor and X-ray wavelength using classic scattering formulae and a simple spot-fading model. Theoretically, a perfect lysozyme crystal 1.2 micron in diameter should be sufficient for a complete data set (4 photons/hkl at 2 Å), but background scattering on contemporary equipment pushes this “minimum lysozyme” size up to 8 microns. An easy-touse calculator for other cases is available at http://bl831.als.lbl.gov/xtalsize.html 07.04.4 Matthew Warkentin, Robert Thorne Cornell University, Ithaca, NY, United States We report the temperature dependence of global radiation damage to thaumatin crystals between T=300 and 100 K. The amount of damage for a given dose decreases sharply as the temperature decreases from 300 K to 220 K, and then decreases much more gradually on further cooling below the protein-solvent glass transition. We observe two regimes of temperature-activated behaviour. At temperatures above ~200 K, the activation energy of 4.3 kcal/mol indicates that radiation damage is dominated by diffusive motions: Diffusion of radicals through solvent channels, the diffusive relaxation of protein hydration water, and diffusive motions of loose side chains all have activation energies in this range. At temperatures below ~200 K the activation energy is only 0.24 kcal/mol, on the order of the thermal energy. Similar activation energies describe the temperature dependence of radiation damage to a large variety of small-molecule organic crystals over the temperature range between T=300 K and 80 K. These systems have atomic vibrational spectra and energies that are similar to those of proteins. This suggests that the temperature dependence of radiation damage below T=200 K is associated with the thermal occupation of the first few excited atomic vibrational states, and that diffusive processes do not contribute significantly to global damage. Below ~80 K, vibrational excitations are frozen out, zero point motions dominate, and global radiation damage becomes temperature independent. Using the radiation damage model of Blake and Phillips (1962), we show that radiation damage proceeds sequentially, with native protein first becoming disordered and then amorphous at all temperatures. The ratio of the amorphization rate to the disordering rate is constant below T~200 K but grows above it. Large scale conformational and molecular motions are frozen out below T=200 K, but become increasingly prevalent and make an increasing contribution to overall damage at higher temperatures. Blake, C., and Phillips, D.C. (1962). Effects of X-irradiation on single crystals of myoglobin. In Proceedings of the Symposium on the Biological Effects of Ionising Radiation at the Molecular Level (Vienna: International Atomic Energy Agency), pp. 183–191. 07.04.5 Spatial dependence and mitigation of MX radiation damage by focusing Edward Stern , Yanhui Zou , Yizhak Yacoby , Andrzej Joachimiak , Randy Alkire , Kenneth 4 Evans-Lutterodt 1 1 1 2 3 3 Univ. of Washington, Seattle, WA 98195, United States, Racah Institute of Physics, Hebrew 3 Univ., Jerusalem, Israel, Argonne National Laboratory, Argonne, IL 60439, United States, 4 Brookhaven National Lab, Brookhaven, NY, United States 2 Recently, strategies to reduce primary radiation damage have been proposed which depend on focusing x-rays to dimension smaller than the penetration depth of excited photoelectrons (PE’s). For a line focus as used here the penetration depth is the maximum distance from the irradiated region along the x-ray polarization direction that the PE’s penetrate. Reported here are measurements to determine the penetration depth and magnitude of PE damage excited by 18.6keV photons in a lysozyme crystal. It is found that the x-ray dose has a significant contribution from the crystal’s 9 w% solvent NaCl atoms. The 15.8 keV PE’s of the Cl atoms and their accompanying 2.8 keV localized dose from the decay of the resulting excited atoms more than doubles the dose deposited in the focused region because of a much greater cross section and higher energy of the excited atom, degrading the mitigation of radiation damage. Eliminating heavier atoms from the solvent will significantly improve the mitigation of damage by focusing. The experimental results showed the penetration depth of ~17 keV PE’s is 1.36+/- 0.2 µm, well below previous theory estimates. Such a small penetration depth raises challenging technical issues to mitigate damage by focusing because the optimum requirements are gaussian line focused beams with sigma of 0.15 µm and distance between lines of 1.8 µm to reduced damage by a factor of 2. 07.04.6 Reduced Radiation Damage In Protein Crystals With Micron-Sized X-Ray Beams Robert F. Fischetti , Ruslan Sanishvili , Derek Yoder , Sudhir Pothineni , Janet L. Smith , 3 1 1 1 Gerold Rosenbaum , Shenglan Xu , Oleg Makarov , Sergey Stepanov , Venugopalan 1 1 Nagarajan , Stefan Vogt gm/Ca-Cat, Biosciences Division, Argonne National Laboratory, Argonne, Il, United States, life Scineces Inst., Dept. Of Biological Chemistry, U. Of Michigan, Ann Arbor, Mi, United 3 States, dept. Of Biochemistry And Molecular Biology, U. Of Georgia, Athens, Ga, United 4 States, experimentl Facilities Division, Aps, Argonne National Laboratory, Argonne, Il, United States 2 1 1 1 1 1 1,2 Cryo-cooling of protein crystals significantly reduces X-ray induced radiation damage, but does not eliminate it. The predominant mechanism of interaction of an X-ray with an atom in the crystal is the emission of a photoelectron carrying most of the energy of the incident X-ray and causing damage as it deposits that energy in the crystal. When a photoelectron interacts with an atom, it loses energy slowly at first and then more rapidly as its energy decreases. Thus, if the beam size is small compared to the distance the photoelectron travels from its point of emission, then deposition of photoelectron energy outside the beam footprint may reduce radiation damage inside the beam footprint. Monte-Carlo simulations predict that a photoelectron of typical energy could travel 4 – 5 m from the point of emission before being absorbed. We studied radiation damage to lysozyme crystals by monitoring the diffracted intensity of 18.5-keV X-rays as a function of dose and beam size (0.86 – 20 m) at beamline 23-ID-B at the Advanced Photon Source. We observed a 3-fold reduction of damage per dose absorbed within the footprint of the smallest compared to the largest beam. In addition, the spatial extent of radiation damage was mapped using both 15.1- and 18.5-keV X-rays and a ~1- m beam. The damage profiles displayed spatial anisotropy with greater damage occurring along the direction of the X-ray polarization, as expected. The spatial extent of the damage was limited to about 4 m. GM/CA CAT is supported by the NIH National Institute of General Medical Sciences and National Cancer Institute. The APS is supported by the US Department of Energy. 07.05.1 Probing reactions in real time using pair distribution function analysis Karena Chapman X-ray Science Division, Argonne, IL, United States The pair distribution function (PDF) method provides valuable insights into the local atomic structure in materials independent of crystallinity, heterogeneity or particle size. Recent advances in experimental methods and the advent of dedicated X-ray PDF beamlines, such as 11-ID-B at the Advanced Photon Source, have led to rapid growth in both PDF studies and the associated user community. This growth has occurred in parallel with the increasing interest in nanoscale and disordered materials, for which conventional Bragg crystallographic methods offer limited insight. Current state-of-the-art PDF set ups (with optimized beam intensity, sample environments and detectors) now allow total scattering data suitable for PDF analysis to be collected at up to 30 Hz. This allows for the structural changes during reactions to be probed in-situ to reveal changes in bonding during catalytic reactions and particle nucleation and growth—from the earliest X-ray amorphous multi-atom clusters to nanoparticles and beyond. The insights gained into the reaction kinetics and mechanism can ultimately lead to greater control of structure and functional behavior. 07.05.2 STRUCTURE OF CRYSTALLOGRAPHICALLY STORAGE MATERIALS Hyunjeong Kim Los Alamos National Laboratory, Los Alamos, NM, United States Hydrogen is considered a promising alternative fuel for transportation, provided we can find a way to store a large amount of it in a compact way. The realization of such a storage system can be achieved by developing materials that can easily absorb, safely store, and rapidly release hydrogen. However, there is currently no material to meet all the requirements for on board storage. Great efforts have been made to look for a way to improve properties or to prepare new materials. One of widely adopted ways to prepare new hydrogen storage materials is mechanical alloying or ball milling. Materials prepared by this method are often nano- or amorphous-phases or mixture of both and they exhibit interesting hydrogen storing properties [1]. Alternatively, packing hydrogen storage materials into porous materials [2] leads to great improvement in their properties; such nano-confinement allows materials to release high purity hydrogen at lower temperatures without a significantly long induction period. Despite of favorable changes in properties, little is known about the structure of both types of systems. This is partly because amorphous or nano-sized nature limits the use of conventional crystallographic analysis and, therefore, structural determination becomes very challenging. In this talk, I will present our local structural studies on such crystallographically challenged hydrogen storage materials by using the atomic pair distribution function analysis [3] on total scattering data. The systems of interest are MgxCo100-x alloys prepared by ball milling [4] and nano-phase ammonia borane (NH3BH3) confined in pores of mesoporous silica MCM-41 [5]. [1] S. Orimo and H. Fujii, Appl. Phys. A 72, 167-186 (2001). [2] A. Gutowska et al., Angew. Chem. Int. Ed. 44, 3578-3582 (2005); R. K. Bhakta et al., J. Am. Chem Soc. 131, 13198-13199 (2009). [3] T. Egami & S. J. L. Billinge, Underneath the Bragg Peaks: Structural Analysis of Complex Materials, Pergamon Press Elsevier, Oxford, England, 2003; Th. Proffen & H. J. Kim, J. Mater. Chem. 19, 5078-5088 (2009). [4] Y. Zhang et al., J. Alloys Compd. 393, 147-153 (2005); H. Shao et al., Scripta Materialia 60, 818-821 (2009); J. Matsuda et al., Nanotechnology 20, 204015 (2009). [5] H. J. Kim et al., J. Am. Chem. Soc. 131, 13749-13755 (2009). CHALLENGED HYDROGEN 07.05.3 Local Structure Effects in Magnetoresistance Materials Efrain Rodriguez , Anna Llobet , Thomas Proffen , Katharine Page , Mark Green 1 2 1 2 2 2 1 NIST Center for Neutron Research, Gaithersburg, MD, United States, Lujan Neutron Center, LANL, Los Alamos, NM, United States The magnetoresistance (MR) effect is a technologically important property employed in magnetic hard disks and sensors. In efforts to find new materials with promising MR properties, researchers have focused on the mixed valence manganites Ln1-xAxMnO3 where Ln is a trivalent lanthanide and A is a divalent alkaline earth metal. Another set of materials showing promise in this area is the series of solid solutions with the stoichiometries Zn1xCuxCr2Se4. This talk will focus on how we employed neutron and X-ray powder diffraction to obtain the local and long-range structure of both materials to have a better understanding of the microscopic interactions leading to the MR effect. In particular, we present the case of La0.5Ca0.5MnO3 below the charge-ordering temperature. By combining Rietveld and pair distribution function (PDF) analysis with the total neutron scattering data, we examined two competing models describing the low temperature, charge-ordered/orbital-ordered (CO-OO) 3+/ 4+ phase: 1) the Mn Mn checkerboard model and 2) the Mn-Mn dimer model or so-called Zener polaron model. In the case of the selenides, we use PDF analysis of X-ray data to find how the local environment of the Cu and Cr cations lead to the observed magnetic and transport properties. 07.05.4 Coupling total scattering and density functional theory computations to solve the structure of complex disordered aluminosilicates Claire White , John Provis , Thomas Proffen , Daniel Riley , Jannie van Deventer 1 2 1 1 2 1 1 University of Melbourne, Victoria, Australia, Los Alamos National Laboratory, Los Alamos, NM, United States Understanding the atomic structure of complex metastable materials is of great importance in research and industry, however, such structures resist solution by most standard techniques. Here, a novel synergy between total scattering and density functional modelling is presented to solve the structure of the metastable aluminosilicate material metakaolin. Metakaolin is obtained by calcination of kaolinite, and is used in quantities of millions of tonnes per annum in blending with Portland cement for concretes, as well as being a useful geopolymer precursor, and is a key intermediate in processing of many fired ceramics. The structure is elucidated by two independent methods, both based on the combination of total scattering/pair distribution function analysis (PDF) and density functional theory (DFT). In the first method, the structure is obtained by iteration between least-squares real-space refinement using neutron PDF data, and geometry optimisation using DFT. The resulting structural representation is both energetically feasible and in excellent agreement with experimental data. In the second, the process of kaolinite dehydroxylation is modeled using DFT and a step-wise methodology, where several water molecules at a time are removed from the original kaolinite structure, geometry optimization is carried out, and the process is repeated until the dehydroxylated structure is reached. The structures generated during the dehydroxylation process are then validated by comparison with X-ray and neutron PDF data. This study provides new insight into the local environment of the aluminum atoms in metakaolin, including evidence of the existence of tri-coordinated aluminum. By the availability of this detailed atomic description of its structure, there exists the opportunity to tailor chemical and mechanical processes involving metakaolin and other complex metastable materials at the atomic level to obtain optimal performance at the macro-scale. 07.05.5 Phase progression of alumina nanoparticle catalyst supports as a function of synthetic temperature Stacey Smith , Branton Campbell , Baiyu Huang , Calvin Bartholomew , Brian Woodfield , 2 4 4 5 Juliana Boerio-Goates , Katherine Page , Hyunjeong Kim , Karena Chapman Brigham Young University, Physics & Astronomy, Provo, UT, United States, Brigham Young 3 University, Chemistry & Biochemistry, Provo, UT, United States, Brigham Young University, 4 Chemical Engineering, Provo, UT, United States, Los Alamos National Laboratory, Los 5 Alamos Neutron Science Center, Los Alamos, NM, United States, Argonne National Laboratory, Advanced Photon Source, Chicago, IL, United States We have developed a simple and uniquely cost-effective synthetic method for producing Al2O3 nanoparticles of exceptional size (3-5 nm) and purity. The product shows promise as an improved industrial catalyst support due its enhanced surface area and the mesoporous character of its agglomerates. To establish the temperature range through which we can produce the catalytically-active gamma phase, we must determine the phase progression of our samples as a function of synthetic temperature. This is challenging because the alumina phase diagram includes many closely-related phases that are not readily distinguished from powder-diffraction data due to the extremely particle-size broadened Q-space peaks. In these cases, PDF analysis was able to resolve the distinct local structures of the candidate phases. We will demonstrate that a combination of PDF and Rietveld refinements best resolves our alumina nanoparticle phase progression pathway. 1 2 1,2 1 2 3 2 07.05.6 Ferroelectric-relaxor crossover in Ba(Ti1 xZrx)O3 studied using neutron total scattering measurements and reverse Monte Carlo modeling Ilkyoung Jeong, C. Y. Park, J. S. Ahn, S. Park, D. J. Kim Pusan National University, Busan, Korea, Republic of Comprehensive structural studies on normal ferroelectric to relaxor crossover in Ba(Ti1-xZrx)O3 (BTZ) are performed using neutron total scattering measurements analyzed by reverse Monte Carlo modeling. In BTZ solid solution, we estimated the degree of the displacement correlation between Ti ions and found that it is stronger and extends much longer for ferroelectric state than relaxor state. In addition, we present evidence that the overall o_centering behavior of Ti ion changes from directional to random displacements between ferroelectric and relaxor phases, and thus provide atomistic picture for ferroelectric-relaxor crossover with increasing Zr concentration. 07.05.7 Applications of Single Crystal Diffuse X-ray Scattering for Studies of Polymorphism in Pharmaceuticals. Eric Chan, Darren Goossens, Aidan Heerdegen, Richard Welberry Australian National University, Canberra, Australia Due to the recent advancements in modern computing power, the analysis and interpretation of single crystal X-ray diffuse scattering for molecular crystals now involves the construction of a computer model of a dynamic crystal. The method allows inclusion of structural features on a local level that may be tested against their effect on the observed diffuse scattering. This gives great insight into the dynamic behavior of organic molecules in the solid state and the models can also be used to explain the structural nature of packing defects or lattice strain. In this paper we discuss the analysis of three polymorphic systems; namely, benzocaine, paracetamol and aspirin. For benzocaine, a low temperature phase transition occurs whereby the orthorhombic phase (form II) transforms to a twinned monoclinic phase (form III). The low temperature twinned crystal displays many 'well-defined' Bragg peaks. For the room temperature form II, diffuse scattering features are observed in the absence of 'low temperature' Bragg peaks which, when modeled, show that at a local level the form II crystal has a structure which exhibits precursor effects of the incipient phase transition. All the simulations use Hooke's law springs associated with intermolecular connections to approximate the normal modes of vibration in a molecular crystal. For benzocaine a simplified set of important connections were used and force constants needed to be determined through trial and error. The work on the monoclinic and orthorhombic forms of paracetamol demonstrates that much trial and error is no longer necessary and the force constants in a model can be approximated from knowledge of Van der Waals radii provided that all intermolecular interactions within a certain distance threshold are taken into consideration. The approximation works well and its effect on the simulation is shown quantitatively using a least squares refinement. Results from modeling diffuse data collected from form II of aspirin suggests that the crystal has undergone layer dislocations, during or after the crystallization, that resemble the form I packing. Because these layer dislocations are not perfect within the crystal, a resultant lattice strain is also observed in the diffuse scattering. This strain should affect the solid-state physical properties of the form II crystal. 07.06.1 Precise Absolute Structure Refinement Using Quotient Restraints Simon Parsons , Howard Flack , Oliver Presley , Trixie Wagner , Paul McGovern 1 2 1 2 3 4 1 University of Edinburgh, Edinburgh, Scotland, United Kingdom, University of Geneva, 3 4 Geneva, Switzerland, Oxford Diffraction, Yarnton, Oxfordshire, United Kingdom, Novartis, Basel, Switzerland In an absolute structure determination one absolute structure is refined competitively against the inverted alternative. The result is expressed by the Flack parameter x(u), which for absolute configuration determination can be interpreted as the mole fraction of the alternative enantiomer in the crystal. The physical range of x is 0 to 1 and even if the bulk material is known to be enantiopure the standard uncertainty (u) should be less than 0.1 before any firm conclusions regarding the absolute structure can be drawn. This criterion has proved to be extremely demanding for light-atom structures. We have developed a method for absolute structure determination based on the quantity D(h) I (h) - I (-h) I (h) I (-h) (1- 2 x ) F 2 (h) - F 2 (-h) F 2 (h) F 2 (-h) The term based on I(h) and I(-h) and its standard uncertainty can be calculated from a single 2 2 crystal X-ray diffraction data set. The term based on F (h) and F (-h) can be calculated from the model. It is therefore possible to write out a set of restraints based on observed and calculated values of D(h) and apply these in an absolute structure refinement. Systematic errors in the intensities, such as absorption, tend to cancel out (in an average way) so that measured values of D(h) should be more accurate that the values of the measured intensities. We have found that this method yields significantly more precise values of the Flack parameter than conventional refinement. For example when a data set was collected for Lalanine with Cu-K radiation at 100 K, conventional refinement yielded a Flack parameter equal to 0.12(21), whereas the restrained refinement yielded a value of 0.00(8). The method also carries the advantage that the Flack parameter is allowed to refine along with all the other parameters, so that its standard uncertainty reflects correlations present in the refinement. 07.06.2 Experimental conditions for absolute structure determination using Bayesian statistics Martin Lutz , Rob W. W. Hooft 1 1 2 2 Utrecht University, Utrecht, Netherlands, Netherlands Bioinformatics Centre, Nijmegen, Netherlands The first absolute structure determination of an organic molecule was performed by Bijvoet and coworkers based on intensity differences of 15 pairs of reflections. This approach was followed by many similar studies, often with a slightly different way to select the reflection pairs or with different weighting of the selected pairs. In the 1980’s the original procedure of examining a subset of Bijvoet pairs was superseded by the inclusion of an absolute structure parameter in the least-squares refinement. A renaissance of the Bijvoet method appeared by a contribution of Hooft, Straver and Spek (2008), where likelihood calculations in combination with Bayesian statistics are applied. In contrast to the original Bijvoet method, where only a subset of reflection pairs is considered, the Hooft method takes all Bijvoet pairs into account. The Hooft method appears to be very successful, even if only weak anomalous scatterers (e.g. oxygen) are present. This paper will deal with the experimental conditions, which are necessary for a reliable absolute structure determination. Special emphasis will be on the standard uncertainties of the experimental intensities. Outlier handling will be discussed on the assumption of a normal error distribution. In case the error distribution is non-Gaussian, use is made of the Student tdistribution to increase the robustness of the method. Example data are taken from our own laboratory as well as from Acta Crystallographica, where reflection data are deposited as supplementary material. 07.06.3 Improved Light-Atom Absolute Configuration Determination Using Sources. 1 2 1 1 Microfocus 1 Michael Ruf , Holger Ott , Matthew M. Benning , Bruce C. Noll , Charles F. Campana 1 Bruker AXS Inc., Madison, Wisconsin, United States, Germany 2 Bruker AXS GmbH, Karlsuhe, Determination of absolute configuration for light-atom structures has become central to research in pharmaceuticals and natural products synthesis. In the absence of elements heavier than silicon, it is often problematic to make a significant assignment of absolute configuration. Traditionally, a heavy-atom derivative has been prepared, but this is not always feasible. Making these assignments has become somewhat easier with the advent of highintensity microfocus sources, as the increased flux density can improve the anomalous signal from these samples through improvements in counting statistics. The improvement in data quality from a high-intensity microfocus source will be demonstrated in comparison to data from a conventional sealed-tube source. 07.06.4 Absolute Structure Determination - Interpreting the Flack Parameter Amber L. Thompson, David J. Watkin Chemical Crystallography, Oxford, United Kingdom It is now well established that the different enantiomers of a chiral material can have significantly different physiological properties - for example d and l-limonene. As a consequence of this, drug manufacturers and drug authorisation authorities are increasingly concerned about the absolute configuration of active pharmaceutical ingredients. In appropriate cases, X-ray structure analysis can give very reliable results. The first absolute structure determination, of sodium rubidium tartrate, was carried out in 1951 by Bijvoet, Peerdeman and van Bommel. Until Rogers introduced his eta parameter into the least-squares refinement (1981), direct comparisons of Bijvoet pairs, or the application of the Hamilton R-factor ratio test were the principal crystallographic techniques used to assign absolute structures. Rogers eta parameter was quickly superseded by the Flack "x" parameter, a least-squares parameter which treated the crystal as a mixture of the original enantiomer and its twin by inversion. Flack pointed out that where as the Rogers parameter (which varied between +1 and -1) had no physical meaning as it approached the mid point, zero, the Flack parameter had a physical meaning over its entire range (from 0 to 1). A Flack parameter somewhere near the middle of the range, and with a suitable small e.s.d., indicated that the sample was twinned by inversion. The incorporation of this parameter into most refinement programs, its ease of use, and its apparent robustness to less-than-ideal data collection strategies contributed to its rapid acceptance, and to misunderstandings about its interpretation. In 2000, almost 20 years after the Flack parameter was first described, Flack and Bernardinelli described the statistical interpretation of the parameter. In spite of this, there continued to be a hunch amongst practical crystallographers that Flack's own interpretation of his parameter was unduly pessimistic. The publication of the derivation of the Hooft, Straver and Spek parameter in 2008 encouraged us to carry out a critical analysis of 150 samples of known absolute configuration, only light atoms and measured with molybdenum radiation. 07.06.5 Absolute Configuration of CHON Organics from Mo Radiation: Can You Believe It? Frank Fronczek Dept. of Chemistry, Louisiana State University, Baton Rouge, LA, United States Analysis of Bijvoet pairs using the method of Hooft et al. (J. Appl. Cryst. (2008). 41, 96-103) has greatly enhanced the sensitivity of determining absolute structure from compounds with only light atoms. For oxygen-containing crystals of good quality with Cu radiation, it is quite reliable and relatively easy. For excellent crystals of oxygen-rich compounds, absolute configuration determination appears to be possible using the Hooft method even with Mo radiation. This author (ACA Toronto, P-T076) reported about a dozen such absolute configuration determinations with MoK , all of which agreed with the known configurations. That poster contained the statement “With data from modern instrumentation, I have never seen a Flack parameter near zero with standard uncertainty 0.4 or less fail to yield the correct (known) absolute configuration.” Experiences in the quest to find a counterexample will be described, including several interesting case histories. Oxygen-rich chiral crystals of high quality frequently yield Flack x near zero with standard uncertainties ~ 0.3 - 0.5 and Hooft P2(true) ~ 1.0, if care is taken in data acquisition. Datasets which maximize the probability of success are collected at low temperature to high resolution, with high redundancy and a high completeness of Bijvoet pairs. It also appears that larger molecules (within reason) are easier than smaller ones, presumably because more Bijvoet pairs are available. ð 07.06.6 Absolute Structure without Heavy Atoms: Experimental Tests Raymond P. Scaringe, John D. DiMarco, Mary F. Malley, Michael A. Galella, Marta Dabros . Bristol-Myers Squibb, Princeton, NJ, United States The determination of absolute structure by Flack parameter analysis, although requiring some 1 nd care , is well established for structures containing 2 row or heavier atoms. However, even 2,3 for structures that lack atoms heavier than oxygen, recent work suggests that a Bayesian analysis of the Bijvoet differences can provide a reliable route to the determination of absolute structure. The practical implication of this finding is that a “light atom” material, already suitable for single crystal analysis, may yield an absolute structure without the need of preparing heavy atom salts or solvates. Since salt screening, solvent screening, and crystal growth can be labor and material intensive activities, the method is of considerable practical interest. In comparison to heavy atom materials, the number of examples of light atom absolute structure determinations based on resonance scattering methods is much smaller. In this work we present the results of attempted absolute structure determinations for a number of compounds containing no atoms heavier than oxygen. Results based on Flack 2 parameter refinement will be compared to those based on Bayesian indicators. 1 2 3 Flack, H.D. & Bernardinelli, G. (2008) Chirality 20, 681-690. Hooft, R.W.W., Straver, L.H., Spek, A.L. (2008) J. Appl. Cryst. 41, 96-103. Hooft, R.W.W., Straver, L.H., Spek, A.L. (2009) Acta Cryst. A65, 319–321 07.06.7 Absolute Configuration of Epoxyresibufogenin Jeffrey Deschamps , Terrence Boos , Kejun Cheng , Arthur Jacobson , Kenner Rice 1 2 1 2 2 2 2 Naval Research Laboratory, Washington, DC, United States, Chemical Biology Research Branch, National Institute on Drug Abuse and the National, Bethesda, MD, United States Epoxyresibufogenin is a C-20,21-epoxy relative of the well-known resibufogenin. Resibufogenin is a cytotoxic steroid first isolated in 1952 from the Chinese drug Chan su. Chan su (also refered to as "senso" in Japanese) is a traditional Chinese medicine derived from the venom of various toad species, especially Duttaphrynus melanostictus and Bufo gargarizans. Chan su acts as a stimulant to the central nervous system and exerts cardiopulmonary effects, thus components of Chan su have been lead compounds in numerous drug studies. In this study we report on the absolute configuration of a derivative of resibufogenin, the C20,20 epoxy-resibufogenin, as determined using the method of Hooft et al. (2008). This configuration was verified by formation of a p-bromobenzoate of the hydroxyl on C3. HO (S) (S) (R) (R) (R) O O O (S) (S) (Z) H O (R) S-001 Structure function studies of vaccinia virus host-range protein K1 reveal a novel functional surface for ankyrin-repeat proteins Junpeng Deng , Xiangzhi Meng , Yan Xiang , Yongchao Li 1 2 1 2 2 1 Oklahoma State University, stillwater, United States, University of Texas Health Science Center at San Antonio, San Antonio, United States Poxvirus host tropism at the cellular level is regulated by virus-encoded host-range proteins acting downstream of virus entry. The functioning mechanisms of most host-range proteins are unclear, but many contain multiple ankyrin (ANK) repeats, a motif that is known for ligand interaction through a concave surface. Here, we report the crystal structure of one of the ANK-repeat-containing host-range proteins, the vaccinia virus K1 protein. The structure, at a resolution of 2.3Å, showed that K1 consists entirely of ANK-repeats, including 7 complete ones and two incomplete ones; one each at the N and C-terminus. Interestingly, Phe82 and Ser83, which were previously shown to be critical for K1’ s function, are solvent exposed and locate on a convex surface, opposite to the consensus ANK interaction surface. The importance of this convex surface was further supported by our additional mutagenesis studies. We found that K1’ s host-range function was negatively affected by substitution of either Asn51 or Cys47 and completely abolished by substitution of both residues. Cys47 and Asn51 are also exposed on the convex surface, spatially adjacent to Phe82 and Ser83. Altogether, our data showed that K1 residues on a continuous convex ANK-repeat surface are critical for the host-range function, suggesting that K1 functions through ligand interaction and does so with a novel ANK interaction surface. S-004 Crystal Structures of the Catalytic and Carbohydrate Binding Domains Endoglucanase D in Complex with Cellotriose from Clostridium cellulovorans Christopher Bianchetti , Robert Smith , Thomas Rutkoski1 , George Phillips, Jr 1 1,2 1 1 1 2 of Department of Biochemistry, University of Wisconsin, Madison, WI, United States, Graduate Program in Biophysics, University of Wisconsin, Madison, WI, United States The enzymatic degradation of cellulose by cellulases is a critical step in the conversion of plant biomass into an abundant renewable energy source. An understanding of the structural features that cellulases utilize to bind crystalline cellulose, extract a single cellulose strand, and hydrolyze the -1,4-glycosidic bonds of cellulose to produce fermentable sugars would greatly facilitate the engineering of improved cellulases for the large-scale conversion of plant biomass. Endoglucanase D (EngD) from Clostridium cellulovorans is a modular enzyme composed of a N-terminal catalytic domain and a C-terminal carbohydrate-binding module (CBM) that are attached via a flexible proline/threonine-rich linker. Here, we present the 2.1 Å resolution crystal structures of full-length EngD with and without bound cellotriose. The EngD CBM, a member of the Carbohydrate-Binding Module Family 2, adopts a β -sandwich fold that contains a planer strip of aromatic residues that are involved in the binding and localization of the catalytic domain to the surface of crystalline cellulose. The catalytic domain belongs to the Glycoside Hydrolase family 5 (GH5) and adopts a (β /α )8 fold. An extended active site cleft runs along one face of the catalytic domain and is partially enclosed by Trp162 and Tyr232 near the +1 and +2 glucose binding subsites. Two molecules of cellotriose are observed in the EngD cellotriose-bound structure. One molecule is bound in each monomer at the -3, -2, and -1 subsites while another cellotriose molecule is bound between two non-crystallographic symmetry related monomers at the +3, and +4 subsites of the catalytic domain. The residues located on the active site cleft, in particular the surface-exposed Trp162 and Tyr232, which interact act with the cellotriose molecules, hints at the possible role these residues play in the initial attachment and strand extraction of cellulose. The cellotriose-bound EngD structure presented here describes the residues that form the glucose binding subsites and provides a structurally based mechanism for the binding of the catalytic domain to crystalline cellulose. S-007 Crystal structure of the gH/gL complex – a conserved herpesvirus fusion regulator Tirumala Kumar Chowdary , Tina M Cairns , Doina Atanasiu , Gary H Cohen , Roselyn J 2 1 Eisenberg , Ekaterina E Heldwein Tufts University School of Medicine, Boston, MA, United States, University of Pennsylvania, Philadelphia, PA, United States Herpesviruses enter cells by fusing their viral envelope with the cellular membrane. Unlike most other enveloped viruses, herpesviruses require not one but three surface glycoproteins for fusion. These are the conserved glycoproteins B (gB) and the heterodimeric gH/gL, plus other, non-conserved glycoproteins. gB is a class III viral fusogen that normally functions only in the presence of gH/gL, the role of which is poorly understood. To gain insight into its role in herpesvirus-mediated membrane fusion, we determined the crystal structure of the gH/gL complex from herpes simplex virus 2, at 3-Å resolution. The structure was solved using a single-wavelength anomalous dispersion method and a selenomethionine derivative. The structure revealed an unusually tight complex of a novel architecture that, contrary to previous ideas, does not resemble any known viral fusogen. Instead, we hypothesize that gH/gL activates gB for fusion by binding it directly. A neutralizing monoclonal antibody inhibited interaction of gH/gL with gB and thus membrane fusion. Thus, we propose that a putative gBbinding site on the gH/gL surface overlaps the epitope of the neutralizing monoclonal antibody. 1 2 1 2 2 2 S-010 Fast and automated functional classification with MED-SuMo: An application on purine-binding proteins Olivia Doppelt-Azeroual , Francois Delfaud , Fabrice Moriaud , Alexandre G. de Brevern , 3 Stephane Richard MEDIT SA, Palaiseau, France, INSERM, Paris, France, MEDIT USA, San Diego, CA, United States 1 2 3 1 1 1 2 Ligand–protein interactions are essential for biological processes, and precise characterization of protein binding sites is crucial to understand protein functions. MED-SuMo is a powerful technology to localize similar local regions on protein surfaces. Its heuristic is based on a 3D representation of macromolecules using specific surface chemical features associating chemical characteristics with geometrical properties. MED-SMA is an automated and fast method to classify binding sites. It is based on MED-SuMo technology, which builds a similarity graph, and it uses the Markov Clustering algorithm. Purine binding sites are well studied as drug targets. Here, purine binding sites of the Protein DataBank (PDB) are classified. Proteins potentially inhibited or activated through the same mechanism are gathered. Results are analyzed according to PROSITE annotations and to carefully refined functional annotations extracted from the PDB. As expected, binding sites associated with related mechanisms are gathered, for example, the Small GTPases. Nevertheless, protein kinases from different Kinome families are also found together, for example, Aurora-A and CDK2 proteins which are inhibited by the same drugs. Representative examples of different clusters are presented. The effectiveness of the MED-SMA approach is demonstrated as it gathers binding sites of proteins with similar structure-activity relationships. Moreover, an efficient new protocol associates structures absent of cocrystallized ligands to the purine clusters enabling those structures to be associated with a specific binding mechanism. Applications of this classification by binding mode similarity include target-based drug design and prediction of cross-reactivity and therefore potential toxic side effects. S-013 Use of halide quick soaking method for structure solution of a major pilin, SpaA of gram-positive bacteria group B streptococcus Vengadesan Krishnan , Hung Ton-That , Sthanam V.L. Narayana 1 1 2 1 2 CBSE, University of Alabama at Birmingham, Birmingham, AL, United States, Department of Microbiology & Molecular Genetics, University of Texas-Houston Medical School, Houston, Texas, United States Many pathogens use their cell wall anchored pili to initiate adherence to host cells, which is the key initial step for bacterial colonization. The presence of pili in Gram-positive bacteria is relatively a recent discovery, where the Streptococcus agalactiae or Group B streptococcus (GBS) pili were discovered in 1997. The Gram-negative pili assembly is well understood, however the same is not true for Gram-positive bacteria. It is been speculated, mainly from well studied Corynebacterium diphtheria pili model, that their pili are assembled through covalent linkage of individual protein subunits (pilins) in contrast to Gram-negative pili, where they are held together by non-covalent and hydrophobic interactions. A transpeptidase sortase, conserved all across Gram-positive bacteria, is implicated for such covalent linkage between pilin subunits. The GBS causes pneumonia, septicaemia and meningitis in neonates and is responsible for significant morbidity and mortality in the United States and Europe. Three pilins; SpaA (GBS80), SpaB (GBS52) and SpaC (GBS104) constitute the pili of GBS. It is hypothesized that the major pilin SpaA forms pilus shaft, while minor pilins, SpaB decorated along the shaft and SpaC sitting at the tip of the pili, are essential for adhesive function. We have crystallized 35 kDa fragment of major pilin SpaA containing middle and C-terminal domains. The crystals diffracted to 1.8Å on a Rigaku R-axis IV imaging-plate detector using CuK radiation at home source. The structure was solved by SAD using NaI halide quick soaking method on home source diffractometer. The structure reveals two IgG-like folds with an iso-peptide bond. ¡ S-016 Structural studies of propionyl-coenzyme A carboxylase Christine Huang , Kianoush Sadre-Bazzaz , Yang Shen , Binbin Deng , Z. Hong Zhou , 1 Liang Tong 1 1 1 1 3 2,3 Columbia University, New York, NY, United States, Univ. of California, Los Angeles, Los 3 Angeles, CA, United States, Univ. of Texas Medical School at Houston, Houston, TX, United States Propionyl-CoA carboxylase (PCC) is a highly conserved, biotin-dependent enzyme of 750 kDa that is required for the conversion of propionyl-CoA to yield D-methylmalonyl-CoA, a necessary step following the beta-oxidation of odd-carbon fatty acids. In addition to its key role in the catabolism of fatty acids, cholesterol, and several amino acids, PCC is an essential metabolic enzyme whose abrogated activities result in propionic acidemia, a genetic disorder with an incidence rate of 1:100,000 births in the US and results in developmental delays, neurological deficits, and severe immune deficiencies. 2 The PCC holoenzyme exists as an α 6β 6 dodecamer, with the α and β subunits harbouring active sites for carboxyl group tethering and transfer, respectively. We have solved the crystal structure of a bacterial PCC holoenzyme at 3.2 Å resolution, and present it alongside cryo-EM data at 15 Å to support a similar structure for human PCC. The structures reveal a number of new findings regarding the inner workings of the enzyme, including novel subunit arrangements and active site architecture, and provide a foundation for understanding the molecular basis of disease mutations associated with propionic acidemia. In addition, the structures also provide insight into the activities of other biotin-dependent carboxylases, many of which are fundamental metabolic enzymes. S-019 Structural insights into inositol pyrophosphate synthase regulation and activity Gayane Machkalyan, Gregory J. Miller McGill University, Montreal, QC, Canada Inositol phosphates (IPs) are small molecules that regulate a variety of cellular signaling 2+ pathways. The best characterized IP, inositol-1,4,5-triphosphate (IP3), triggers Ca release from intracellular reservoirs; however, there are >30 differently phosphorylated IPs that perform diverse signaling roles. Diphosphoinositol polyphosphates (diIPs) are the most highly phosphorylated IPs and are structurally distinct from IPs due to pyrophosphate moieties on 1 or more positions of the inositol ring. Of paramount importance for the advancement of our understanding of the signaling roles of diIPs is the exploration of the mechanisms through which diIPs are produced and how their synthesis is controlled. Two classes of IP kinases produce diIPs: inositol pyrophosphate synthetase (IPS) and inositol hexakisphosphate kinases (IP6Ks). IPS is a dual-domain protein: at its N-terminus is a kinase domain belonging to the ATP-grasp family, and at its C-terminus is a histidine phosphatase-like domain. The substrate for the histidine-phosphatase domain has not yet been determined, and in fact, its sequence suggests it may not retain catalytic activity at all, but it may function as a ligand- or protein-binding module. There are no available structures for either the kinase or the histidine phosphatase-like domains, which would reveal the mechanism for the production of specific diIP isomers and would provide clues to the functional roles of the C-terminal domain. Using combinations of X-ray crystallography and solution methods, we explore the structure and mechanism of IPS to answer key functional and mechanistic questions, including: (1) What is the structural basis for the production of distinct diIPs? and (2) How is IPS activity regulated? S-022 Structural and functional analysis of the motor domain regions of the heterodimeric Kar3/Vik1-like kinesin from Candida glabrata Da Duan, Michelle Chan, Darlene Davis, John Allingham Queen's University, Kingston, Ontario, Canada All animal and plant cells rely on nanometre-sized protein motors called kinesins to segregate chromosomes between dividing cells and haul vital cargo-containing sacks to where they are needed. Many kinesins operate as a complex of identical pairs of molecules that cooperate to move along microtubules and perform a single cellular function. However, a few kinesins in certain species and cell types mix-and-match different molecules in ways that allow the motor protein to perform multiple functions. The budding yeast kinesin Kar3, for example, forms heterodimers with two non-catalytic kinesin-like proteins named Vik1 and Cik1 that influence the cellular localization and function of Kar3 during yeast mating and division. The way in which Kar3 and Vik1, or Kar3 and Cik1, operate at a molecular level as a motor complex is not yet known. However, our recent determination of the X-ray crystal structure of the motor domain region of a Vik1 ortholog from Candida glabrata has revealed structurally dynamic regions in this protein that sheds new light on how this protein may work at the atomic level with Kar3. Specifically, our crystals of CgVik1 contained two molecules in the asymmetric unit that exhibit two very different conformations of an alpha-helical segment that is analogous to the ‘neck’ found in the Drosophila kinesin Ncd. The intramolecular interactions of the CgVik1 neck and motor domain core differ in each conformation and are accompanied by subtle movements in elements of the motor domain core that are analogous to the P-loop and part of the microtubule binding surface of catalytic kinesins. In order to better understand the importance of these interactions and displacements, the effects of mutating residues that form conformation-dependent interactions between the neck and motor core were structurally and functionally evaluated. S-025 The Crystal Structure of the RNA helicase Mtr4 reveals a unique and novel arch domain that is required for nuclear 5.8 S rRNA processing Ryan Jackson, Sean Johnson Utah State University, Logan, UT, United States The cell must extensively monitor and correctly process a myriad of RNA species in order to maintain the proper regulation and expression of genes. Exonucleolytic decay is utilized by the cell as a quality control mechanism that eliminates unneeded or erroneous RNA, as well as a tool that processes RNA to proper maturity. Mtr4 is an essential and conserved RNA helicase that is central to the processing and degradation of RNA in the nucleus. Mtr4 activates the multi-subunit nuclear exosome which processes or completely degrades RNA substrates. Many of the molecular details of how Mtr4 recognizes and delivers appropriate RNA substrates to the exosome are currently unknown due to a complete lack of structural data. To enhance the understanding of these mechanisms we have determined the crystal structure of Mtr4. The structure reveals a novel arch-like domain that is unique to Mtr4 and Ski2 (the cytosolic homolog of Mtr4). In vivo and in vitro analyses demonstrate that the Mtr4 arch domain is required for proper 5.8 S rRNA processing, and suggest that the arch functions independently of canonical helicase activity. Additionally, extensive conservation along the face of the putative RNA exit site highlights a potential interface with the exosome. These studies provide a molecular framework for understanding fundamental aspects of helicase function in exosome activation, and more broadly define the molecular architecture of Ski2-like helicases. S-028 Structural characterization of ATPase/Response Regulator pairs involved in type IV pilus biology Aditya Gupta , Ana M. Misic , Katrina T. Forest 1 2 1,2 1,3 1,3 Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, United States, Biophysics Graduate Training Program, University of Wisconsin-Madison, Madison, WI, 3 United States, Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, United States Type IV pili (Tfp) are evolutionarily conserved bacterial appendages utilized for a variety of functions such as host cell adhesion, DNA uptake, and twitching motility. In Pseudomonas aeruginosa, over 40 proteins are involved in the biogenesis and retraction of Tfp. Among these are PilB and PilT/PilU, hexameric P-loop ATPases that play a role in powering pilus assembly and retraction, respectively. PilG and PilH are two single domain response regulators (CheY homologs) that control twitching motility. Recent genetic results indicate that PilG acts upstream of PilB and PilH acts upstream of the pilus retraction process (Bertrand, West, and Engel 2010), although the biochemical pathway for these control processes has not yet been elucidated. We will present progress on overexpression, purification, and crystallization of these response regulator proteins, both alone and as PilG/B and PilT/U/H complexes. We anticipate that this work will shed light on the molecular mechanisms involved in the complex process of P. aeruginosa pilus assembly, retraction, and modulation. S-033 A Uniquely Open Conformation Revealed in the Structure of a Novel Protein Arginine Methyltransferase Yuan Cheng, Monica Frazier, Matthew Redinbo University of North Carolina at Chapel Hill, Chapel Hill, NC, United States Protein arginine methyltransferase 10 (PRMT10) is a type-I arginine methyltransferase that plays a critical role in the regulation of flowering-related genes in Arabidposis thaliana. PRMT10 is only active in the dimeric state and displays unique substrate specificity. In this work, X-ray crystallography is combined with molecular dynamics simulations and biochemical studies to explore the mechanism underlying these functional features. The 2.6 Å resolution crystal structure of PRMT10 reveals a large set of structural features that are unique to PRMT10, including the surface charge distribution, the shape of the substratebinding groove, the active sites’ accessibility, an the mode of dimerization. This information d provides substantial clues to understanding the mechanism governing the substrate specificity of PRMT10. In addition, structure-based molecular dynamics (MD) simulations were conducted to understand why dimerization is essential for the enzymatic activity of PRMT10. Upon dimerization, the motion of the cofactor-binding domain shifted from noncorrelated to highly-correlated, which likely facilitates the binding of cofactor and the subsequent catalytic reaction. Furthermore, dimerization was shown to help the formation of a major substrate-binding groove in PRMT10 by reducing the local fluctuation. Similar results were also observed in MD analyses we conducted on mammalian PRMT1 and PRMT3 structures. Together, these results demonstrate the importance of dynamic motion in the regulation of PRMT activity, and suggest a general model to explain why dimerization is essential for the PRMT family of enzymes. S-035 Crystal Structure of LL-DAP-AT from Chlamydia trachomatis: The open conformation and its implication on the broad substrate specificity Nobuhiko Watanabe , Chenguang Fan , Matthew D. Clay , Marco J. van Belkum , John C. 2 1 Vederas , Michael N. G. James Department of Biochemistry, University of Alberta, Edmonton, Canada, Department of Chemistry, University of Alberta, Edmonton, Canada The lysine biosynthetic pathway is an attractive target for the development of novel antibiotics because it is absent in humans. Previously, three different lysine biosynthetic pathways have been characterized in bacteria. However, none of the previous bacterial lysine biosynthetic pathways were found in Chlamydia or in plants. Recently, LL-DAP-AT was discovered to be the missing piece of the lysine biosynthetic pathways in Chlamydia, plants, and some archaea. Due to the absence of this enzyme in humans, LL-DAP-AT is an attractive target for anti-Chlamydial drugs. Previously, we have determined the crystal structure of Arabidopsis LL-DAP-AT (AtDAP-AT) and elucidated its substrate recognition mechanism. Although the Chlamydial LL-DAP-AT (CtDAP-AT) is 41% identical to AtDAP-AT in its primary sequence, it has a much broader substrate specificity than AtDAP-AT does. In order to understand the differences in the substrate specificity and to assist in the development of novel antibiotics against Chlamydial infections, we have determined the crystal structure of the pyridoxal-5’phosphate-bound CtDAP-AT to 2.3 Å resolution. Unlike our previously determined crystal structures of AtDAP-AT, the small domain (Ala297-Met388) and the N-terminus arm (Met1Gln48) of CtDAP-AT have moved away from the active site significantly with a maximum displacement of 8.9 Å. Now, the active site is exposed to the solvent and the loops lining the active site (A: Gly41-Gln48, C: Thr67-Pro73) are completely disordered. From our previous AtDAP-AT structures, the loop A is determined to be important for substrate entry and binding. Given that the active site residues are almost completely conserved between the two species, and that the active site residues of the “closed” conformation of AtDAP-AT makes very tight interactions with L-Glu and LL-DAP, the “open” conformation with its highly flexible loops may account for the broad substrate specificity of CtDAP-AT. 1 2 1 2 2 2 S-038 Removal of a dynamic Ω -loop: Structural and enzymatic consequences Troy Johnson, Todd Holyoak University of Kansas Medical Center, Kansas City, Kansas, United States Many studies have shown that dynamic motions of individual protein segments can play a role in enzyme function. Structural studies on the metabolic enzyme phosphoenolpyruvate carboxykinase (PEPCK) have revealed a dynamic element in the form of a 10-residue Ω -loop domain which acts as an active site lid adopting an ordered, closed conformation upon the formation of complexes that mimic the Michaelis and enolate intermediate states. Based upon these structural studies and our recent work on the dynamic nature of this loop we have proposed a model for the mechanism of PEPCK catalysis in which the closed conformation of the mobile lid-domain is necessary for correct substrate positioning, sequestering of the reaction intermediate, and protection of the intermediate from alternate chemistries. Furthermore, the ability of the lid to occupy the closed conformation involves a fine energetic balance between the entropic penalty for lid closure and the free energy of ligand binding. To further test this model the flexible Ω -loop was removed and replaced with 1, 2, or 3 glycine residues. Structural and kinetic characterization was carried out on all three of the lid deletion mutants. The kinetic experiments were carried out utilizing multiple steady state assays and the results were as expected: removal of this loop significantly reduces or eliminates the catalytic activity and efficiency of all three mutant PEPCKs. To further investigate the cause of this catalytic deficiency the structures of each mutant were solved in complex with either the physiological substrates or the respective analogs. The structural data revealed an unexpected backbone shift, which resulted in the Cα of R87 (a catalytically important residue for OAA binding) being displaced by 6 Å and the side chain being flipped out occupying space where the Ω -loop would normally reside when in the closed conformation. This suggests that R87 may play a role in the energetic balance of lid dynamics, acting as a conduit for the transfer of free energy from binding to offset the entropic penalty of lid closure. The data presented here supports our model for the role of the Ω -loop lid domain in correctly positioning substrates, sequestering and protecting the intermediate species, and gives further insight into how the transfer of free energy could be mediated through the protein structure to allow the energetically costly lid closed conformation to form so catalysis can occur. S-041 Brandon Goblirsch , Amanda Lee , Bennett Streit , Jennifer DuBois , Carrie Wilmot 1 2 1 2 3 3 1 University of Minnesota, Minneapolis, MN, United States, Purdue University, West 3 Lafayette, IN, United States, University of Notre Dame, South Bend, IN, United States Chlorite dismutase (Cld) is a heme enzyme which rapidly and selectively decomposes chlorite to Cl¯ and O2. The ability of Cld to promote O2 formation from chlorite (ClO2¯ ) is unusual. Heme enzymes generally utilize chlorite as an oxidant for reactions such as oxygen atom transfer to a second substrate. The X-ray crystal structure of Dechloromonas aromatica Cld co-crystallized with the substrate analogue nitrite (NO2¯ ) was determined to investigate features responsible for this novel reactivity. The enzyme active site contains a single b-type heme coordinated by a proximal histidine residue. Structural analysis identified a glutamate residue hydrogen bonded to the heme proximal histidine that may stabilize reactive heme species. A solvent exposed arginine residue likely gates substrate entry to a tightly confined distal pocket. Based on the proposed mechanism of Cld, initial reaction of ClO2¯ within the distal pocket generates hypochlorite (ClO¯ ) and a compound I intermediate. The sterically restrictive distal pocket probably facilitates the rapid rebound of hypochlorite with compound I forming the Cl¯ and O2 products. Common to other heme enzymes, Cld is inactivated after a finite number of turnovers, potentially via the observed formation of an off-pathway tryptophanyl radical species through electron migration to compound I. Three tryptophan residues of Cld have been identified as candidates for this off-pathway radical. Finally, a juxtaposition of hydrophobic residues between the distal pocket and the enzyme surface suggests O2 may have a preferential direction for exiting the active site. S-044 1.75 Å Structure of a Fungal Type III Polyketide Synthase, a Potential Biosynthetic Tool for "Unnatural" Natural Products Anna Lytle, Jia Zeng, Jixun Zhan, Sean Johnson Utah State University, Logan, UT, United States Polyketides, a class of secondary metabolites, comprise a significant portion of structurally diverse and biologically active natural products. These valuable compounds, utilized as antibiotic, anticancer, anti-tumor and anti-cholesterol drugs, are generated by three classical types of polyketide synthases (PKSs). Although type III PKSs were thought to exist only in plants and bacteria, the genes for these iterative homodimeric enzymes were recently discovered in fungi. In the industrially important fungus, Aspergillus oryzae, four similar yet distinct type III PKS genes have been identified, providing an attractive opportunity to understand chemical diversity in fungal secondary metabolites. Here we report the 1.75 Å apo-crystal structure of one of these enzymes, CsyB. The structure reveals a homodimeric protein with a highly conserved Cys-His-Asn triad that catalyzes the condensation of different saturated fatty acyl-CoAs into a variety of aromatic polyketides. The apo structure sets the stage for investigation of complexes with substrate and product bound in the multi-functional active site. The goal is to delineate the structural basis for the specific function and unique products of CsyB. Future work will examine the other PKSs present in A. oryzae. The structural characterization of these enzymes will greatly inform the design of biosynthetic PKSs to produce novel bioactive "unnatural" natural products. S-047 Structural Investigation of the Mechanism and Regulation of PLP Synthase. Amber Smith , David Akey , Markos Koutmos , Janet Smith 1 2 1,2 2 2 1,2 University of Michigan, Ann Arbor, MI, United States, Life Sciences Institute, Ann Arbor, MI, United States PLP synthase (PLPS) from the bacterium G. stearothermophilus generates pyridoxal 5’phosphate (PLP), the active form of vitamin B6. PLPS is a 24-mer complex made up of two subunits, a 32 kDa PdxS synthase subunit and a 25 kDa PdxT glutamine amidotransferase (GAT) subunit. PdxS, possessing the ( / )8 barrel fold, forms a cylindrical dodecamer of two 1 2 hexameric rings . PdxT binds in a 1:1 ratio around the outside of the PdxS stacked rings . PdxT functions as a Triad GAT catalyzing the hydrolysis of glutamine to glutamate and ammonia. The ammonia is then channeled to the synthase active site of PdxS, where PLP is formed from ammonia, ribose 5-phosphate and glyceraldehyde-3-phosphate. The mechanism of this reaction is unknown. A flexible C-terminal tail of PdxS is essential to PLP synthesis and 3 to the cross talk between the active sites of the two subunits . Using an inactive mutant of PdxT, we co-crystallized PLPS with substrates glutamine and ribose 5-phosphate. Initial crystals diffracted to ~4 Å and revealed density for the C-terminal tail. Dehydration experiments improved the diffraction quality of the crystals to 2.75 Å. The higher resolution structure will provide more structural information in order to determine the functional relevance of the C-terminal tail. Supported by NIH grant: DK42303. 1 Zhu, J., Burgner, J. W., Harms, E., Belitsky, B. R. & Smith, J. L. A new arrangement of ( / )8 barrels in the synthase subunit of PLP synthase. J Biol Chem 280, 2791427923, (2005). Zein, F., Zhang, Y., Kang, Y., Burns, K., Begley, T., & Ealick, S. Structural insights into the mechanism of the PLP synthase holoenzyme from Thermotoga maritima. Biochemistry 45, 14609-14620, (2006). Raschle, T., Speziga, D., Kress, W., Moccand, C., Gehrig, P., Amehein, N., WeberBan, E., & Fitzpatrick, T. Intersubunit cross-talk in pyridoxal 5’-phosphate synthase, coordinated by the C terminus of the synthase subunit. J Biol Chem 284, 7706-7718, (2009). 2 3 S-050 Inhibition of Saccharomyces cerevisiae S-formylglutahtione Hydrolase and Activation of the H160I variant by Peroxide Patricia M. Legler, Charles B. Millard Walter Reed Army Institute of Research, Silver Spring, MD, United States S. cerevisiae S-formylglutathione hydrolase (SFGH, homologous to human esteraseD) belongs to a class of serine hydrolases that are resistant to organophosphates but sensitive to classical thio-enzyme inhibitors such as Hg and N-ethylmaleimide. Under oxidizing conditions, inhibition of SFGH activity is attributable to a cysteine (Cys-60) adjacent to the catalytic triad and approximately 8.0 Å away from the O of the active site serine. Cys-60 is oxidized to a sulfenic acid in the structure of the diethylphosphate inhibited W197I variant (PDB 3C6B). While this structural snap-shot captured an unstable reversibly oxidized state, it remained unclear as to whether the oxidation occurred before, during or after the reaction with the organophosphate inhibitor. To determine if the oxidation of Cys-60 was linked to ester hydrolysis we used kinetic experiments and site-directed mutagenesis in combination with X-ray crystallography. In the presence of substrate, the rate of inhibition of the WT SFGH by peroxide increases 14-fold suggesting that oxidation is linked to ester hydrolysis. We also found one variant, H160I, which is activated by peroxide. This variant is activated at comparable rates in the presence and absence of substrate suggesting that the conserved His-160 is involved in the inhibitory mechanism linking ester hydrolysis to the oxidation of Cys-60. Mechanisms for inhibition by both peroxide and CuCl2 are proposed as well as a mechanism for peroxide activation of the H160I variant. A Dali structural similarity search uncovered two other enzymes (B.subtilis RsbQ, 1WOM and C. acetobutylicum lipaseesterase, 3E0X) that contain a similar Cys adjacent to a catalytic triad. We speculate that the regulatory motif uncovered is conserved in some D-type esterases and discuss its structural similarities in the active site of Human Protective Protein (HPP; also known as Cathepsin A). This work was funded by the U.S. Defense Threat Reduction Agency JSTO award 1.D0015_06_WR_C (CBM). The opinions or assertions contained herein belong to the authors and are not necessarily the official views of the U.S. Army or the U.S. Department of Defense S-053 A Short, Strong Hydrogen Bond in the Active Site of Human Carbonic Anhydrase II Balendu Avvaru , Chae Kim , Katherine Sippel , Sol Gruner , Mavis Agbandje-McKenna , 1 1 David Silverman , Robert McKenna University of Florida, Gainesville, FL, United States, CHESS, Cornell University, Ithaca, NY, United States The crystal structure of human carbonic anhydrase II (HCA II) obtained at 0.9 Å resolution reveals that a water molecule, termed deep water, Dw, and bound in a hydrophobic pocket of the active site forms a short, strong hydrogen bond with the zinc-bound solvent molecule, a conclusion based on the observed oxygen-oxygen distance of 2.45 Å. This water structure has similarities with hydrated hydroxide found in crystals of certain inorganic complexes. The energy required to displace Dw contributes in significant part to the weak binding of CO2 in the enzyme-substrate complex, a weak binding that enhances kcat for the conversion of CO2 into bicarbonate. In addition, this short, strong hydrogen bond is expected to contribute to the low pKa of the zinc-bound water and to promote proton transfer in catalysis. 1 2 1 2 1 2 1 S-056 New Insights for the Role of Calcium in Human Calcium Activated Nucleotidase (CAN) Activity and Dimerization Stefanie Ward, Terrence Kirley, Andrew Herr University of Cincinnati, Cincinnati, OH, United States Blood-sucking insects secrete nucleotidases that prevent host blood clotting by hydrolyzing ADP, a platelet agonist, to AMP in the blood. Mammals also express a homologous, soluble calcium-activated nucleotidase (CAN), however, the mammalian ortholog prefers GDP to ADP as a substrate and therefore is not efficient at preventing thrombosis. Calcium is required for CAN catalytic activity of CAN and potentiates CAN’s activity by inducing its dimerization, although the allosteric mechanism is unknown. Therefore, we have introduced a point mutation at a key residue in the dimer interface, Ile170Lys (I170K), to produce an obligate monomer species in order to study calcium’s role in catalysis and dimerization through crystallographic analysis. I170K demonstrates a significant reduction of ADP hydrolysis, which correlates with the loss of dimerization. I170K was crystallized in two crystal forms and the structures were solved at 1.6 and 1.8 Å resolution by molecular replacement. Additionally, the wild-type CAN was co-crystallized with the non-hydrolyzable substrate GMPCP in the presence of calcium. Structural alignments with the CAN wild-type apo structure 2H2N indicate significant main chain and side chain rotamer shifts for residues within the enzymatic active site and dimer interface, suggesting a likely mechanism by which dimerization can modulate activity. In addition, our high resolution data reveal the location of multiple calcium ions within the active site that are likely to play important roles in substrate binding and hydrolysis. These data demonstrate the importance of calcium-induced dimerization for nucleotidase function, which could lay the groundwork for the development of an engineered human CAN with improved ADP cleavage for use as a blood clot inhibitor. S-059 A sulfotransferase and thioesterase working together to produce the terminal alkene in curacin A Jennifer Gehret, David Sherman, Janet Smith University of Michigan, Ann Arbor, MI, United States Curacin A is a small-molecule antimitotic natural product, produced by the marine cyanobacterium Lyngbya mujuscula. Curacin is unique among polyketide natural products because of its many interesting chemical groups, including a terminal double bond, created during offloading of the final product from the polyketide synthase (PKS). The double bond contrasts with the more common cyclized macrolactone or linear carboxylic acid produced by a thioesterase (TE) domain at the end of the PKS. The curacin pathway instead contains both sulfotransferase (ST) and TE domains, which work together to create the terminal 1 alkene . The ST sulfonates a -hydroxyl in the penultimate intermediate, after which the TE hydrolyses, decarboxylates, and desulfates the intermediate to offload curacin A with its terminal double bond. This scheme includes the first observation of biological sulfation for chemical activation as well as novel decarboxylation/desulfation activity in a TE. This system has potential in bioengineering hydrocarbon production for biofuels. The crystal structures of the ST (1.6Å) and TE (1.7Å) were determined as individual domains excised from the PKS module. The TE has the expected / hydrolase fold but differs from other offloading TEs in lid structure, dimer interface position, and an open-cleft active site. Comparison with uncharacterized sequences of putative tandem ST-TE domains with presumably similar activity reveals dense conservation within the cleft. A model of the predicted acyl enzyme intermediate shows a conserved Arg205 which may confer specificity to TE for the -sulfate, a prediction that is supported by site-directed mutagenesis studies. 1 L. Gu et al., J Am Chem Soc 131, 16033 (2009). This work was supported by NIH grant DK42303 S-062 Distal Pocket Effects on Nitrite Binding to Heme Iron Jun Yi, George Richter-Addo University of Oklahoma, Norman, OK, United States The binding of the nitrite anion to metal centers occurs via a number of ways. The "nitro" N-binding mode (metal-NO2) is quite common for synthetic heme models and is present in the crystal structures of the nitrite adducts of cytochrome cd1 nitrite reductase (NiR), E. coli sulfite reductase hemeprotein, and cytochrome c NiR. We reported the X-ray crystal structure of the nitrite adduct of ferric horse heart myoglobin (hh Mb) and showed that the nitrite ligand was bound to heme Fe in an unprecedented O-binding mode. We hypothesized that the distal His64 residue in this Mb(ONO) complex was responsible for directing the O-binding of the nitrite ligand. To test this hypothesis, we prepared and characterized the nitrite adducts of the mutant H64V and the double mutant H64V/V67R. The lack of a distal pocket His64 residue in the H64V-nitrite adduct resulted in the nitrite ligand adopting the more common Nbinding mode. Reintroducing a distal pocket H-bonding side chain (i.e., in the H64V/V67R double mutant) resulted in the restoration of the observed nitrito O-binding mode. These results will be presented and discussed in context of the proposed (by others) nitrite reductase activity of the Mb protein. S-065 Structure Based Insight into the Mechanism of Type I Dehydroquinate Dehydration Samuel Light , George Minasov , Elisabetta Sabini , Arnon Lavie , Michael Caffrey , 1,3 Wayne Anderson 1 1,3 1,3 1,3 2 2 Department of Molecular Pharmacology and Biochemistry, Feinberg School of Medicine, 2 Chicago, IL, United States, University of Illinois at Chicago, Department of Biochemistry and 3 Molecular Genetics, Chicago, IL, United States, Center for Structural Genomics of Infectious Diseases, Chicago, IL, United States The shikimate pathway links metabolism of carbohydrates to biosynthesis of aromatic compounds. In a sequence of seven metabolic steps phosphoenolpyruvate and erythrose 4phosphate are converted to chorismate, the precursor of the aromatic amino acids and many secondary aromatic metabolites. The third step in the pathway, consisting of the dehydration of dehydroquinate to dehydroshikimate, is performed by two distinct enzymes which catalyze the same reaction through disparate mechanisms. Crystal structures of the type I dehydroquinate dehydratase from Salmonella enterica typhimurium and Clostridum difficile are reported here. Structures of the un-liganded, substrate, and product bound enzyme illustrate protein movement over the course of the reaction. Upon substrate binding, a 7 residue flexible region hydrogen bonds with the substrate, closing over the active site. The substrate bound structure shows a 1.5 Å movement of histidine-143 to interact with the leaving hydroxyl. Following dehydration, the product bound structure shows histidine-143 returned to its original orientation. The direct interaction of histidine-143 with the leaving group positions the residue for proton shuttling between ring and leaving hydroxyl. The mechanism of histidine-143 proton transfer proposed here is consistent with previous observations of ciselimination in type I dehyroquinate dehydration (Harrison and Rose, 1963). The Center for Structural Genomics of Infectious Diseases has been funded by the National Institute of Allergy and Infectious Diseases under Contract No. HHSN272200700058C. S-068 Catalysis in the nitrilase superfamily amidases; implications from active site structure Serah Kimani , Brandon Weber , Andrew Nel , Don Cowan , Trevor Sewell 1 1 2 3 3 2 Molecular and Cell Biology Department, University of Cape Town, Western Cape, South 2 Africa, Electron Microscope Unit, University of Cape Town, Western Cape, South Africa, 3 Department of Biotechnology, University of the Western Cape, Western Cape, South Africa The nitrilase superfamily amidases catalyze the conversion of various amides to their corresponding acid and ammonia using a highly conserved Glu, Lys, Cys catalytic triad in an acid-base catalysis mechanism. Some of these enzymes are potential biocatalysts in the fine + chemical industry, while others like the amidase domain of the NAD synthetase from Mycobacterium tuberculosis (MTB) are attractive drug targets. We have recently solved the crystal structure of the amidase from Geobacillus pallidus RAPc8. The most interesting observation in this structure arises from the size and the geometry of the active site pocket, which is arranged in such a way that the reaction intermediate restricts access to the glutamic acid (Glu59) previously thought to be the general base catalyst for the hydrolysis of the acyl intermediate. An alternative choice for a general base catalyst is another glutamic acid residue (Glu142), which has not been characterized before, and which we found to be highly conserved in other structures from the nitrilase superfamily. We have also very recently solved the structure of another amidase from Nesterenkonia sp. The position and coordination of the second glutamic acid residue (Glu139) is also conserved in this amidase. We have proposed a catalytic mechanism that postulates the involvement of this additional glutamic acid as a fourth catalytic residue in the amidases of the nitrilase superfamily. We are presently investigating the role of this residue using both biophysical and structural methods. Mass spectra from the Geobacillus and Nesterenkonia sp. amidase mutants where the proposed general base catalyst glutamic acid residue has been changed to a leucine and a glutamine respectively, indicate that tetrahedral intermediates of various substrates are being trapped in the active site. This confirms that this residue is indeed involved in catalysis. To further confirm these findings, crystals of the E139Q Nesterenkonia amidase mutant reacted with various substrates have been prepared. The progress on this work will be presented. S-071 Structural investigation of the biotin carboxylation reaction in the BC domain of Rhizobium etli pyruvate carboxylase Sudhanshu Kumar, Martin St. Maurice Marquette University, Milwaukee, WI, United States Pyruvate carboxylase (PC) is a biotin-dependent multifunctional enzyme which plays an important role in gluconeogenesis, lipogenesis, glucose mediated insulin secretion and neurotransmitter biosynthesis. PC contains three functional domains on a single polypeptide chain: an N-terminal biotin carboxylase (BC) domain, a central carboxyltransferase (CT) domain and a C-terminal biotin carboxyl carrier protein (BCCP) domain. BCCP-biotin “swings” between BC and CT active sites to affect catalysis. Recent X-ray crystal structures have revealed the complete domain architecture of PC, showing BCCP-biotin and pyruvate positioned in the CT domain and free biotin and ADP/ATP bound in the BC domain. However, the catalytically relevant position and orientation of BCCP-biotin in the BC domain remains unknown. To structurally investigate this unknown conformation, we rationalized crystallizing the kinetically incompetent mutant of Rhizobium etli PC, T882A, in the hopes of trapping the BCCP domain in the desired conformation. It has been proposed that the biotin carboxylation reaction is a stepwise process. First, bicarbonate is phosphorylated to form a carboxyphosphate intermediate which subsequently leads to carboxylation of biotin. So far, there has been no direct and conclusive evidence for the existence of this proposed intermediate. Thus, the R.etli PC T882A mutant was co-crystallized with the carboxyphosphate intermediate, phosphonoacetate, with the intent to mimic the binding of the proposed carboxyphosphate intermediate. The X-ray crystal structure at 2.6 Å resolution reveals BCCP-biotin bound in the BC domain of PC. Furthermore, the carboxyphosphate analogue, phosphonoacetate, is present in the active site. This structure offers new insight into the molecular basis of catalysis in biotin-dependent carboxylase enzymes and strengthens proposals invoking a carboxyphosphate intermediate in the catalytic mechanism for this reaction. S-074 Structural Studies on Mutants of HMG CoA Reductase from Pseudomonas mevalonii Moumita Sen, C Nicklaus Steussy, Chandra Duncan, Victor Rodwell, Cynthia Stauffacher Purdue University, West lafayette, United States HMG-CoA reductase catalyzes the four-electron reduction of HMG-CoA to free CoA and mevalonate. This is one of the few double oxidation/reduction reactions in intermediary metabolism that take place in a single active site. In addition to the unusual enzymology, this reaction is of interest because it is the committed step of the fundamental mevalonate isoprenoid pathway. In animals this pathway produces cholesterol, the steroid hormones and a variety of signaling molecules based on the isoprenoid building block (1). In bacteria the pathway is equally important, and has been shown to be essential to the virulence of Staphylococcal and Streptococcal bacteria (2). To better understand the nature of this reaction, our laboratory has undertaken a comprehensive structural study of the mechanism of HMG-CoA reductase in bacteria utilizing the enzyme from Pseudomonas mevalonii. HMG-CoA reductase is an obligate dimer, with each monomer consisting of a large domain, a small domain, and a flap domain (2, 3) that is disordered in the apoenzyme structure. The flap domain is ordered in the crystal structure only in the presence of ligand and co-factors, where it closes over the active site, positioned by a network of hydrogen bonds that include the ligand and co-factor. Two residues proposed to be important in flap domain movement have been mutated. Mutant proteins have been crystallized, soaked with various combinations of ligands and co-factors, and their structures have been solved at 1.95-2.40Å. These structures, reinforced with kinetic analysis of the mutants, demonstrate the essentialness of this closure in the reaction and reveal how these residues are involved in flap domain movement. 1. Goldstein JL, Brown MS, Nature, 343, 425-430 (1990) 2. Wilding, EI et al. J. Bacteriol., 182 5147-5152 (2000) 3. Lawrence CM et al. Science, 268, 1758-1762 (1995) 4. Tabernero L et al. Proc Natl Acad Sci USA, 96, 7167-7171 (1999) S-077 Structural studies of thrombin-PAR1 interaction Prafull S. Gandhi1,2, Zhiwei Chen1, Enrico Di Cera1 Saint Louis University Medical Center, Saint Louis, MO, United States, 2Washington University School of Medicine, Saint Louis, MO, United States Abundant structural information exists on how thrombin recognizes ligands at the active site or at exosaites separate from the active site region, but remarkably little is known on how thrombin recognizes substrates that bridge both the active site and exosite I. Likewise, abundant data exist on how binding of ligands to exosite I alters the activity of thrombin, but the molecular basis of this long-range allosteric effects has remained elusive. The protease activated receptor PAR1 is particularly relevant in view of the plethora of biological effects associated with its activation by thrombin. Here we present the 1.8 Å resolution crystal structure of thrombin S195A in complex with a 30-residue long, uncleaved extracellular fragment of PAR1 that documents for the first time a productive binding mode bridging the active site and exosite I. We also show how binding of the cleaved extracellular fragment of PAR1 to exosite I causes a massive conformational change of thrombin from the inactive E* to the active E form, as well the details of the long-range allosteric communication between exosite I and the active site. These results fill a significant gap in our understanding of the mechanisms of substrate recognition by thrombin and the molecular basis of its allosteric function. 1 S-080 Structural and Functional Studies of Staphylococcus aureus Pyruvate Carboxylase Linda Yu, Song Xiang, Liang Tong Columbia University, New York, United States Pyruvate carboxylase (PC) catalyzes the ATP-dependent transformation of pyruvate to oxaloacetate, which marks the first step in gluconeogenesis. Oxaloacetate is also an important intermediate in the tricarboxylic acid cycle. Therefore, PC is considered an essential enzyme in intermediary metabolism. The structural architecture of PC consists of four domains, the biotin carboxylase (BC) domain, the carboxyltransferase (CT) domain, the biotin carboxyl carrier protein (BCCP) domain, and a novel PC tetramerization (PT) domain. It belongs to a group of biotin dependent enzymes where the biotin is covalently bound to BCCP, which swings between the distinct active sites on the BC and CT domains to carry out the catalysis. The newly discovered PT domain is essential for keeping the tetramer intact and mutations in this domain disrupt the tetramer in both human and Staphylococcus aureus PC. In terms of regulation, it is known that PCs from vertebrate sources are highly activated by acetyl-CoA, while PCs from prokaryotes have varying degrees of dependency on acetylCoA. We have recently reported the crystal structure of the S. aureus PC, alone and in the presence of acetyl-CoA. The acetyl-CoA binding site can be found at the interface where the BC dimer meets one PT domain. Upon CoA binding, the enzyme becomes more symmetrical, which is supported by cryo-EM data. Activator binding also seems to stabilize the BC dimer, which is hypothesized to be a possible mechanism by which acetyl-CoA enhances enzyme activity. The latest results from our structural and biochemical studies on this important enzyme will be presented. S-083 Residue 143 is the Key Residue for the Thrombin Activation by Na + Weiling Niu, Zhiwei Chen, Leslie Bush-Pelc, Alaji Bah, Prafull Gandhi, Enrico Di Cera Saint Louis University, Saint Louis, MO, United States Thrombin is a Na -activated trypsin-like protease for which the binding of Na enhances allosterically activity toward synthetic and physiological substrates. Although this effect has been known for over three decades, no information has been available on its molecular basis. We extended the classical Botts-Morales theory for the action of a modifier on an enzyme to + account for the contribution of the E*, E and E:Na forms of thrombin and established that + analysis of kcat unequivocally identifies allosteric transduction of Na binding into enhanced catalytic activity. Next, we constructed and purfied to homogeneity the thrombin mutant N143P to selectively destabilize the environment of the oxyanion hole of the enzyme. The + + mutant binds Na with an affinity comparable to that of wild-type but features no Na + dependent enhancement of kcat. The crystal structure of N143P in the absence of Na is in the + + inactive E* form. Binding of Na converts the enzyme to the active E:Na form. The N143P mutation abrogates the important H-bond between the backbone N atom of residue 143 and the carbonyl O atom of E192, which in turn controls the orientation of the E192-G193 peptide + bond and the correct architecture of the oxyanion hole. We conclude that Na activates thrombin by securing the correct orientation of the E192-G193 peptide bond. Conservation of the 143-192 H-bond in trypsin-like proteases and the importance of the oxyanion hole in + + proteases function suggest that this mechanism of Na activation is present in all Na activated trypsin-like proteases. + + S-086 Role of His 265, the most conserved residue for a family of C-C bond hydrolases, in the catalytic mechanism of BphD from Burkholderia xenovorans LB400 Subhangi Ghosh , Shiva Bhowmik , Geoff Horsman , Lindsay Eltis , Jeffrey Bolin 1 2 1 1,3 2,4 2 1 Purdue University, West lafayette, IN, United States, University of British Columbia, 3 Vancouver, BC, Canada, The Scripps Research Institute, La Jolla, CA, United States, 4 University of Wisconsin, Madison, WI, United States BphDLB400, a C-C bond hydrolase from the biphenyl degradation pathway of Burkholderia xenovorans LB400, is a key determinant in the degradation of biphenyl and PCBs. Homologs play a similar role in the degradation of dioxins and other xenobiotic pollutants as well as steroids. BphDLB400 catalyzes the cleavage of the C5-C6 bond of 2-hydroxy-6-oxo-6phenylhexa-2,4-diene (HOPDA). The reaction is believed to proceed via two steps and is known to depend on residues S112 and H265. In the first step HOPDA undergoes tautomerization to yield a keto intermediate, which facilitates the second step, hydrolysis of the C5-C6 bond. The present study will further explore the role of H265 in the first step. For the wild type enzyme, stopped flow spectrophotometry demonstrated the rapid formation of an intermediate species with a spectrum red shifted (λ max=492nm) from that of the substrate (λ max=434nm). The intermediate decays concurrently with the formation of spectral features corresponding to the product. In the BphDLB400 S112A mutant, this intermediate decays extremely slowly and is effectively trapped. In BphDLB400 variants carrying the mutation H265A, the intermediate species is not observed. Crystal structures of enzyme:HOPDA complexes revealed remarkably different conformations of HOPDA for the S112A and S112A/H265A variants. In the S112A:HOPDA complex, the dienoate portion HOPDA adopts a non-planar conformation with the 2-hydroxy/oxo oxygen near H265. In the S112A/H265A:complex, HOPDA is in a planar conformation with the 2-hydroxy/oxo oxygen distant from H265. The difference in conformation could be driven by the ability of H265 to act as a base or its hydrogen bonding capacity of H265. To resolve this issue, the present study investigates the interaction of HOPDA with BphDLB400 in the H265Q mutant, a variant that preserves hydrogen bonding while ablating the ability of the residue to function as a base. Data from stopped-flow spectrophotometry and crystal structures of mutants BphDLB400 H265Q, S112A/H265Q and their complexes with HOPDA will be presented. Microspectrophotometry on single crystals of the complex of BphDLB400 S112A with HOPDA, before and after X-ray diffraction data collection, are being performed to correlate the crystal structures of enzyme:HOPDA complexes with the transient or trapped species observed in solution. S-089 Structural basis for substrate recognition by mouse N-acetylglucosaminyltransferase II (GnT II) Zhijie Li , Malathy Satkunarajah , Jenny Tan , Jayaraman Seetharaman , James Rini 1 2 1 1,2 1,3 4 1,2 Department of Biochemistry, University of Toronto, Toronto, ON, Canada, Department of 3 Molecular Genetics, University of Toronto, Toronto, ON, Canada, Division of Structural 4 Biology and Biochemistry, The Hospital for Sick Children, Toronto, ON, Canada, Biology Department, Brookhaven National Laboratory, Upton, NY, United States GnT II is a Golgi-resident glycosyltransferase in the N-glycan biosynthetic pathway of multicelluar organisms. It recognizes the GnT I and mannosidase II processed N-glycan and catalyzes an essential step in the biosynthesis of complex N-glycans. Here we report the Xray crystal structure of the soluble catalytic domain of mouse GnT II in both its apo form and in complexes with its substrates/products: i) UDP-Mg2+; ii) UDP-GlcNAc-Mg2+; iii) UDPMg2+ and GlcNAc-Man3-octyl and iv) GlcNAc-Man3-octyl alone. The enzyme is metal ion dependent and as shown by the structure a member of the GT-A fold family. The tetrasaccharide acceptor substrate, GlcNAc-Man3-octyl, is well-ordered and extensive interactions with the β 1,2-linked GlcNAc moiety reveals the structural basis for GnT II's requirement for the prior action of GnT I. Comparison with other GnT I dependent enzymes shows that recognition of the β 1,2-linked GlcNAc does not stem from evolutionarily conserved structural features. A flexible loop covering the nucleotide sugar binding site was ordered in the ternary complex of GnT II with UDP and the acceptor sugar. Unlike some other glycosyltransferases, this loop does not form a binding surface for the acceptor sugar. However, a partial stacking of the donor and acceptor substrates, suggested by the superimposition of the donor and acceptor complexes, may contribute to the observed Bi Bi ordered sequential kinetics shown by GnT II. S-092 Novel chemistry in the ureide catabolism pathway as revealed by structural and functional analysis of enzymes from the Klebsiella pneumoniae Hpx gene cluster Jarrod French, Katherine Hicks, Steven Ealick Cornell University, Ithaca, New York, United States The degradation of ureides into more soluble compounds occurs in many organisms as diverse as mammals, plants and bacteria. In plants, some fungi and several bacteria the catabolism of these molecules provide a source of nitrogen, carbon and energy. While much of the biochemistry of this degradation has been worked out, there are still many questions to be answered. Recent genetic studies on Klebsiella pneumoniae have uncovered a gene cluster believed to contain all of the enzymes required for the breakdown of uric acid to allantoin and those responsible for the further catabolism of allantoin to carbon dioxide and ammonia. In this work, insights into the novel chemistry that occurs along this pathway are provided by several crystal structures and supporting biochemical studies. Recent debate over the mechanism of the decarboxylation of 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline is discussed in the context of the 1.6 – 1.8 Å crystal structures of the enzyme, HpxQ, in unliganded and product-bound forms. The 2.3 Å structure of HpxA, the first reported structure of an allantoin racemase, sheds light on the mode of ligand binding and the two-base mechanism of catalysis within the active site. Finally, the biochemical and structural characterization of HpxJ, an enzyme that catalyzes a novel aminotransfer reaction, is presented. S-095 Human UDP-glucose Dehydrogenase and the Pin in Fischer’ s Lock Renuka Kadirvelraj , Stephen Weitzel , Nicholas Sennett , Samuel Polizzi , Zachary Wood 1 2 1 2 1 1 1 University of Georgia, Athens, GA, United States, University of Oregon, Eugene, OR, United States Human UDP-glucose dehydrogenase (hUGDH) catalyzes the NAD -dependent oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronic acid, a key building block of the extra cellular matrix. hUGDH is believed to be allosterically regulated by the feedback inhibitor UDP-xylose (UDP-Xyl). The substrate UDP-Glc and inhibitor UDP-Xyl are isosteric except at the C5’ position; UDP-Glc has a hydroxymethyl substituent at C5’, while UDP-Xyl has a hydrogen. We report two crystal structures of the hUGDH:UDP-Xyl complex and compare them to an existing hUGDH:UDP-Glc structure. We show that both substrate and inhibitor UDP-sugars bind in the hUGDH active site. When UDP-Glc binds, a buried, mobile loop in the active site adopts a conformation that promotes three hUGDH dimers to oligomerize into a torus-shaped + hexamer. When UDP-Xyl binds, the buried loop repacks, blocking the NAD binding site and altering the hUGDH oligomer interface. This results in an inactive, horseshoe-shaped hexamer. Sedimentation velocity studies confirm that the binding of either UDP-Glc or UDPXyl favors hexamer formation. Thus, the hUGDH active site is bifunctional in that it carries out both catalysis and regulation. This is in contrast to classic, heterotropic allostery, where the functional and regulatory sites are separate. We present evidence showing how this bifunctional active site evolved from a non-allosteric, ancestral UGDH. + S-098 The Role of Structural Change in UGDH Regulation Nicholas Sennett, Renuka Kadirvelraj, Zachary Wood University of Georgia, Athens, GA, United States Human UDP-glucose dehydrogenase (UGDH) catalyses the NAD -dependent oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronic acid. We show UGDH activity is linked to the oligomeric state of the enzyme. In solution apo-UGDH favors a dimeric state but can also oligomerize to form tetramers and hexamers. Binding of the substrate UDP-Glc causes a buried loop to adopt a conformation that induces UGDH to form a torus-shaped hexamer. In contrast, the feedback inhibitor, UDP-xylose (UDP-Xyl), shifts the buried loop to favor a stable horseshoe-shaped hexamer. To see how the buried loop moves we designed a mutant in the protein core. The crystal structure of the mutant shows that the domain containing the loop opens with a clamshell-like motion, allowing the loop to repack. Crystal structure and sedimentation velocity results confirm that the loop mutant forms the torus-shaped hexamer, suggesting that domain opening and substrate exchange can occur without dissociation of the oligomer. To determine if the hexamer state is required for activity we designed a mutation to stabilize the dimer state. Sedimentation velocity results confirmed that the mutant is a dimer and kinetics showed the mutant to be inactive. We propose that the loop of dimeric UGDH freely moves between its two conformations and that substrate binding drives the dimer to the active torus-shaped hexamer while inhibitor binding drives the dimer to the inactive horseshoe-shaped hexamer. + S-220 Apo Form of Human FABP5 Solved at High Resolution in the Inactive Conformation Eric Armstrong, Eric Ortlund Emory University, Atlanta, GA, United States The fatty acid binding protein (FABP) family includes nine known members, each ~14-15kDa in size and found throughout the animal kingdom. Though they share relatively little sequence homology, all form a twisted β -barrel, composed of 10 anti-parallel β -strands arranged into two orthogonal β -sheets, with a helix-turn-helix lid near the N-terminus. Belonging to the superfamily of intracellular lipid binding proteins (iLBP), they have traditionally been thought to be mainly involved in the solubilization/protection of their various hydrophobic cargos, facilitating ligand transport via passive diffusion across the various compartments of the cell. However, research within the past decade has increasingly bestowed a newfound importance upon the iLBPs as specific mediators of vital signaling pathways. FABP5, like its family members, displays a rather promiscuous ligand binding profile, and has been shown to form a complex with numerous long chain fatty acids as well as several synthetic small molecules. Interestingly, only a subset of these have been demonstrated to serve as "activators," i.e., to result in the protein's nuclear translocation from the cytoplasm in cellular assays. We hypothesize that this differential response upon binding can be explained structurally via an activator-unique conformational change in FABP5, leading to the formation of a tertiary nuclear localization sequence. Surprisingly, incubation of the protein with non-activating ligands facilitated the crystallization of a new apo form that has been solved at 1.67Å. To our knowledge this is the first high resolution structure of an empty iLBP, which we believe will provide a basis for understanding the molecular switch that triggers FABP5 nuclear import. S-101 Structural Studies of Wolinella Succinogenes O-acetylhomoserine Sulfhydrylase Timothy Tran , Andrew Torrelli , Kalyanaraman Krishnamoorthy , Tadhg Begley , Steve 1 Ealick 1 1 1 1,2 1,2 Cornell University, Ithaca, NY, United States, Texas A&M University, College Station, TX, United States O-acetylhomoserine sulfhydrylase (OAHS, EC 4.2.99.10) is a pyridoxal-5’-phosphate (PLP) dependent sulfide-utilizing enzyme in L-cysteine and L-methionine biosynthetic pathways of various enteric bacteria and fungi. As the name implies, OAHS catalyzes the conversion of Oacetylhomoserine to homocysteine using hydrogen sulfide in a process known as direct sulfhydration. However, the source of sulfur has not been identified and no structures of OAHS have been reported. Using molecular replacement, the crystal structure of Wolinella Succinogenes OAHS (WsMetY) has been determined at 2.2 Å resolution. WsMetY crystallized in space group C2 with two monomers in the asymmetric unit. Size exclusion chromatography showed that the biological unit exists as a tetramer. This observation is supported using crystallographic twofold symmetry to generate the tetramer, and confirmed by the free energy calculation from the PISA server. The structure has been refined to a current R/Rfree of 23.0/25.7. Superposition of homologous structures revealed that WsMetY has the same fold as cystathionine gamma lyase and methionine gamma lyase. Their active sites, containing PLP, are also very similar to one another. The structure of WsMetY, together with biochemical data, provide useful insight to the mechanism of sulfur transfer. 2 S-104 Crystallographic studies on two nickel-alkyl species in methyl-coenzyme M reductase. Peder E. Cedervall , Xianghui Li , Mishtu Dey , Stephen W. Ragsdale , Carrie M. Wilmot 1 2 1 2 2 2 1 University of Minnesota, Minneapolis, MN, United States, University of Michigan, Ann Arbor, MI, United States In methanogenic archaea, methyl-coenzyme M reductase (MCR) catalyzes the final and ratelimiting step in methane biogenesis; the reduction of methyl-coenzyme M (methyl-SCoM) by coenzyme B (CoBSH) to methane and a heterodisulfide (CoBS-SCoM). MCR is a 300 kDa protein with six subunits arranged in a α 2β 2γ 2 oligomer. Crystallographic studies have shown that the two active sites of MCR each contain a highly reduced nickel-tetrapyrrole, coenzyme F430, that sits at the base of a 30 Å long substrate channel. No true catalytic intermediate for MCR has ever been observed so the reaction mechanism remains illusive. Based on mechanistic studies in solution, DFT calculations and previous X-ray crystal structures three different mechanisms have been proposed. One of the proposed mechanisms involves a high valent Ni(III)-alkyl intermediate. This species can artificially be produced by treating the enzyme with either methyl iodide or bromopropanesulfonate, generating Ni(III)-methyl and Ni(III)-propylsulfonate, respectively. Here we present the crystal structures of MCR in complex with these two alkyl species. The resulting structures show a mixture of the expected alkyl species and the substrate analogue HSCoM (demethylated methyl-SCoM), which co-purifies with MCR and cannot be fully removed by extensive buffer exchange. By using multiwavelength X-ray diffraction studies we were able to differentiate the components via anomalous electron density maps and allow structural analysis of the Ni-alkyl species. S-107 Structural Insights into Thiopurine S-Methyltransferase Pharmacogenetics Yi Peng , Qiping Feng , Dennis Wilk , Araba Adjei , Oreste Salavaggione , Richard 2 1 Weinshilboum , Vivien Yee 1 1 2 1 2 2 Case Western Reserve University, Cleveland, OH, United States, Mayo Clinic College of Medicine-Mayo Clinic, Rochester, MN, United States Thiopurine S-methyltransferase (TPMT) modulates the cytotoxic effects of thiopurine prodrugs such as 6-mercaptopurine (6MP) by methylating them in a reaction using Sadenosyl-L-methionine (AdoMet) as the donor. Patients with TPMT variant allozymes exhibit diminished levels of protein and/or enzyme activity and are at risk for thiopurine drug-induced toxicity. We have compared the catalytic activity of wild-type and two variant TPMTs, and found that enzyme activities were decreased for both variants while expression levels were substantially decreased for only one, compared to the wild-type enzyme. We have also determined an ensemble of murine TPMT (mTPMT) crystals structures, as binary and ternary complexes containing either wild-type or a variant TPMT. Comparison of the wild-type structures reveals that an active site loop becomes ordered upon acceptor substrate binding. The positions of the two ligands are consistent with the expected SN2 reaction mechanism. Crystal structures and molecular dynamics (MD) simulation calculations for one variant reveal conformational differences in the active site loop that could explain compromised acceptor substrate binding and reduced enzyme activity. Computational modeling and MD simulation calculations for the second variant are consistent with the observed decrease in both enzyme expression and activity. Together, the crystallographic and computational structural studies provide a detailed view of the molecular contributions in a classic example of pharmacogenetics. 2 S-111 Structural insight into the Corynebacterium glutamicum allosteric inhibition of aspartate kinase from Ayako Yoshida, Takeo Tomita, Tomohisa Kuzuyama, Makoto Nishiyama Biotechnology Research Center, The University of Tokyo, Tokyo, Japan Aspartate kinase (AK) is the enzyme that catalyzes the first committed step of the biosynthesis of aspartate family amino acids; lysine, threonine, and methionine. AK is known to be regulated by the end products via feedback inhibition as seen in other enzymes at the first step in amino acid biosynthetic pathway. AK from Corynebacterium glutamicum, a bacterium used for industrial fermentation of amino acids including glutamate and lysine, is inhibited by lysine and threonine in a concerted manner. AK from C. glutamicum (CgAK) also has a characteristic 2 2-type heterooligomeric quaternary structure. The 2 2-type structure is composed of two subunits and two subunits, which are encoded by in-frame overlapping gene. To elucidate the unique regulatory mechanism and quaternary structure, we determined the crystal structures of CgAK in several forms; an inhibitory T-state form complex with both lysine and threonine, an active R-state form complex with only threonine, and feedback inhibition-resistant mutant complex with both lysine and threonine. We previously showed that threonine binding stabilizes an interaction between subunit and the regulatory domain of the subunit, which is essential for catalytic regulation, by the crystal structure determination of regulatory domain ( subunit) dimer with threonine and some mutational experiments. In T-state, we showed the allosteric binding sites of both inhibitors, and comparison of the crystal structures between T and R-state revealed that lysine binding causes a conformational change to a closed inhibitory form, and the interaction between the catalytic domain in subunit and subunit (regulatory subunit) is a key event for stabilizing the inhibitory form. We propose that the regulatory mechanism of CgAK is composed of two steps, i) the interaction between regulatory domain (subunit) triggered by threonine-binding, ii) the conformational change at the C-terminus of subunit to interact between catalytic and regulatory domain provoked by lysine-binding. This study shows not only the first crystal structures of 2 2-type AK but also the mechanism of concerted inhibition. Moreover, since AK is a candidate of antibacterial drug because of its absence in humans, this study will lead to the development of novel antibacterial drugs. ¢ S-114 Introduction of a disulfide bond leads to stabilization and crystallization of a ricin immunogen Jaimee R. Compton , Patricia M. Legler , Mark A. Olson , Benjamin V. Clingan , Charles B. 2 Millard 1 1 1 3 1 Walter Reed Army Institute of Research, Silver Spring, MD, United States, U.S. Army 3 Medical Research & Materiel Command, Frederick, MD, United States, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD, United States 2 RTA1-33/44-198 is a catalytically inactive, single-domain derivative of the ricin toxin A-chain (RTA) engineered to serve as a stable protein scaffold for presentation of native immunogenic epitopes. To improve the stability and solubility of RTA1-33/44-198, we have undertaken the design challenge of introducing a disulfide (SS) bond. Nine residue-pairs for placement of the SS-bond were selected based on molecular dynamics simulation studies of the modeled single-domain chain. Disulfide formation at either of two positions involving a specific surface loop (44-55) increased the protein melting temperature by ~5° compared with RTA1-33/44C 198 and by ~13° compared with RTA. Prolonged stabil ity studies of the R48C/T77C variant C confirmed a >40% reduction in self-aggregation compared with RTA1-33/44-198 lacking the SS-bond. Introduction of the disulfide bonds promoted crystallization, and the X-ray structures of the variants were solved. The structures confirm formation of a SS-bond and a single-domain structure that is globally compacted in volume compared with RTA. Loop 4455 is partly disordered as predicted by simulations and is positioned to form self-self interactions between symmetry-related molecules. We discuss the importance of loop 34-55 as a nucleus for unfolding and aggregation and draw conclusions for ongoing structure-based minimalist design of the RTA immunogen. This work was funded by the U.S. Defense Threat Reduction Agency JSTO award S.S.0003_06_WR_B (CBM) and National Institutes of Health U01 A1082120-01 (CBM). The opinions or assertions contained herein belong to the authors and are not necessarily the official views of the U.S. Army or the U.S. Department of Defense. S-117 Mechanism for allosteric regulation of glutamate dehydrogenase from Thermus thermophilus Takeo Tomita, Tomohisa Kuzuyama, Makoto Nishiyama Biotechnology Research Center, The University of Tokyo, Tokyo, Japan Glutamate dehydrogenase (GDH) catalyzes the reversible conversion between glutamate and 2-oxoglutarate using NAD(P)(H) as coenzymes. Due to the important role in balancing nitrogen metabolism in cells, GDH is widely distributed among living organisms. An extremely thermophilic bacterium, Thermus thermophilus, possesses two glutamate dehydrogenase genes, gdhA and gdhB, in a tandem manner on its genome. To elucidate the functions of these genes, the gene products were expressed, purified, and characterized. GdhA showed no GDH activity, while GdhB showed GDH activity for reductive amination activity 1.3-fold higher than for oxidative deamination. When GdhA was expressed with his-tag fused GdhB, GdhA was co-purified with the his-tagged GdhB. The co-purified GdhA/GdhB had decreased reductive amination activity and increasing oxidative deamination activity, resulting in 3.1-fold preference of oxidative deamination to reductive amination. These results demonstrate that GdhB undergoes conformational change through hetero-complex formation with GdhA. Addition of leucine elevated the GDH activity of co-purified GdhA/GdhB hetero-complex by 974 and 245% for reductive amination and oxidative deamination, respectively, while GdhB alone did not show such a strong activation by leucine. These results suggest that the allosteric activation by leucine occurs through formation of hetero-complex, where GdhA and GdhB act as a regulatory subunit and as a catalytic subunit, respectively. To elucidate the allosteric mechanism of GdhA/GdhB, we determined the crystal structures of GdhA and GdhB complexed with glutamate at 2.2 and 2.1 Å resolution, respectively. GdhA consists of catalytic domain and nucleotide binding domain (NBD) and takes a homotetrameric structure with novel subunit interfaces between its NBDs, while small type GDHs including GdhA and GdhB are known to have homohexameric structure. GdhB takes a homohexameric structure and glutamate are bound in the active sites and the subunit interface position far from active sites. Glu in the active sites are recognized by several electrostatic interactions and hydrogen bonds similar with the other GDH/Glu complexes. Glu in the subunit interface are recognized by the residues from three distinct subunits. This observation raises the hypothesis that the second Glu site may function allosteric effector binding site for recognition of leucine. S-120 Protein-protein complexes of the E. coli phenylacetic acid utilization pathway Andrey Grishin , Eunice Ajamian , Limei Tao , Linhua Zhang , Allan Matte , Miroslaw 1,2 Cygler 1 1 1 2 2 2 Department of Biochemistry, McGill University, Montreal QC, Canada, Research Institute, National Research Council, Montreal QC, Canada 2 Biotechnology The metabolic pathway of phenylacetic acid utilization in E. coli K12 is poorly characterized both biochemically and structurally. This pathway is important as it represents an aerobic route for the utilization of phenylacetic acid as a coenzyme A derivative. Of the five consecutive reactions utilized to metabolize phenylacetic acid, three are presumed to involve enzyme complexes. The oxygenase reaction of this pathway is catalyzed by PaaA-E. We performed a search for stable protein-protein complexes involved in the oxygenase reaction of phenylacetic acid metabolism by co-expression of various combinations of pathway enzymes (PaaA-B-C-D-E) followed by co-purification experiments. These studies revealed that PaaA-B-C and PaaA-C could be purified as complexes, although no strong interaction was found between either PaaE or PaaD with PaaA-B-C. The presence of functional protein complexes was verified by detecting reaction products using LC-MS. Our studies show that PaaA, PaaB, PaaC and PaaE, but not PaaD, are indispensable for activity in vitro. The crystal structure of PaaA-C was determined as well as its complexes with coenzyme A, 3hydroxybutyrl-coenzyme A, benzoyl-coenzyme A and the true substrate, phenylacetylcoenzyme A. Despite low sequence identity, PaaA and PaaC are structurally similar to methane monooxygenase and to other di-Fe monoxygenases. This represents the first structure of a multi-component monoxygenase that utilizes a CoA-derivative of an aromatic compound as substrate. The search for additional protein-protein interactions for other components of this pathway, as well as their biochemical and structural characterization, is in progress. Supported by CIHR. S-123 Structural analysis of Bacillus phytase in complex with phytate and metal ions Yi-Fang Zeng, Rey-Ting Guo, Je-Ruei Liu Institute Of Biotechnology, National Taiwan University, Taipei, Taiwan The hydrolysis of phosphomonoesters in biological systems is an important process. Phytase, which hydrolyzes phytate to less phosphorylated myo-inositol derivatives and inorganic phosphate, is widespread in nature. Bacillus phytases, which exhibit their desirable activity profile under neutral pH, higher thermal stability, and strict substrate specificity for the calcium–phytate complex, have considerable potential for commercial and environmental applications. Thus, we are interested to know how the substrate binding domain of Bacillus subtilis phytase PhyC attracts the substrate and metal ion involvement. Here, we have determined the high resolution X-ray structures of the Bacillus subtilis phytase PhyC in complex with phytate analog in the presence of 5 mM CaCl2. In this structure, the phytate analog was bound the catalytic site of beta-propeller phytase. In addition, we 2+ 2+ determined the enzyme activity in both Ca and Cd loaded state from the structure, and 2+ 2+ found the Cd competed against Ca in the metal ion binding position. Thus, the phytase 2+ activity of PhyC was greatly inhibited by metal ion Cd . These findings provided the evidence 2+ for the binding interaction between the enzyme and substrate in the present of Ca and/or 2+ Cd metal ions. S-126 The crystal structure of Thermotoga maritima CelA Ya-San Cheng, Je-Ruei Liu, Rey-Ting Guo Institute of Biotechnology, National Taiwan University, Taipei, Taiwan Plant matter is the most abundant renewable biomass on earth and cellulose is the major component of plant cell wall. Cellulose is a kind of polysaccharide consisting of glucose linked together via -1,4-glycosidic bond. Thermotoga maritima is an anaerobic hyperthermophilic bacterium and it can produce some thermostable carbohydrate-degrading enzymes which have potential industrial applications. CelA from T. maritima is a thermostable -1,4endoglucanase which is classified into glycoside hydrolase family 12. The three-dimensional structure of TmCelA has not been solved yet. Therefore, we are interested in knowing the TmCelA protein structure and the catalytic mechanism by solving the X-ray structure. We already purified the TmCelA protein and obtained the well diffracted protein crystals. The phase problem was solved by using some heavy atom derivatives recently and the structure refinement is ongoing now. Once we obtained the TmCelA protein structure, we can have more understanding of the catalytic mechanism. We may also have more idea of knowing CelA protein properties such as hyperthermostability by analyzing TmCelA protein structure. £ £ S-129 Structural analysis of Piromyces rhizinflata 2301 CelAcd Chih-Wen Tseng, Rey-Ting Guo, Je-Ruei Liu Institute of Biotechnology, National Taiwan University, Taipei, Taiwan The enzymatic degradation of the plant cell wall is environmentally friendly routes to biomass conversion, including the production of biofuels. Cellulase program is the most widely used scheme in green energy developments. The Piromyces rhizinflata CelA2, a bifunctional endoand exoglucanase, belong to the glycosyl hydrolase family 5 and showed hydrolysis activity toward barley β -glucan、 lichenin、 oat spelt xylan, and carboxymethyl cellulase（ CMC） . The three dimensional structure of the catalytic domain of CelA has not been solved yet. Here we conduct a study to determine the structure of CelA by solving its X-ray structure. So far, the recombinant CelA was purified to homogeneity by immobilized metal ion-affinity chromatography and DEAE-Sepharose ion exchange column. The molecular weight of the purified CelA was estimated 49 kDa on SDS-polyacrylamide gel electrophoresis. Afterward, the catalytic mechanism and detail binding interactions between enzyme and substrates will be studied by solving the protein structure. S-132 Structure of RedJ: A Thioesterase from the Prodiginine Biosynthetic Pathway Jonathan R. Whicher , Galina Florova , Kevin A. Reynolds , Janet L. Smith 1 2 University of Michigan, Ann Arbor, MI, United States, Portland State University, Portland, OR, United States Microbial polyketide synthase (PKS) and non-ribosomal peptide synthase (NRPS) pathways synthesize small-molecule natural products from acylCoA and amino acid precursors, respectively. Modular PKS and NRPS are arranged in an assembly line fashion, with each enzyme catalyzing a specific elongation or modification of an intermediate in the biosynthetic pathway to the natural product. The natural products are secreted by the microbes and have multiple biological activities, which are thought to provide the producing organism with a competitive advantage in its environment. Most natural products are cytotoxic, and as a result, they have been investigated as possible therapies for human diseases. Thus the study of the enzymes involved in the PKS and NRPS systems has become an important area of research in hopes of engineering these pathways to produce novel therapeutics. Prodiginines are a class of tripyrrole small- molecule natural products produced by hybrid PKS/NRPS systems. Analogues of prodiginines have immunosuppressant and anticancer activity. As a result, the prodiginine biosynthetic pathway in Streptomyces coelicolor has been extensively studied and most enzymes within this pathway have been assigned functions. However, one enzyme, RedJ, has an unknown function. Biochemical, bioinformatic and genetic evidence indicate that RedJ is a thioesterase with a novel role in facilitating transfer of a 12-carbon intermediate from one enzyme to another. The 2.12-Å crystal structure of RedJ presented here suggests a structural basis for RedJ specificity for long-chain aliphatic substrates. In addition, distinct conformations of an active-site “lid” region in different crystal forms provide insights into the mechanism by which RedJ regulates access of substrates to its active site. 1 2 2 1 JRW is supported by an NIH Chemistry Biology Interface Training Grant. Research supported by NIH grant DK42303 to JLS. S-135 Crystal structure of Plasmepsin I (PM-I) from Plasmodium falciparum Prasenjit Bhaumik , Yasumi Horimoto , Huogen Xiao , Alexander Wlodawer , Yoshiaki Kiso , 1 Gustchina Alla Protein Structure Section, Macromolecular Crystallography Laboratory, National Cancer 2 Institute at Frederick, Frederick, Maryland, United States, University of Guelph, Guelph, 3 Ontario, Canada, Kyoto Pharmaceutical University, Kyoto, Japan Malaria is contributing to death of nearly two million people every year, most of them children. Although several antimalarial drugs are available, rapid development of resistance to the currently available treatments makes it necessary to discover and develop a new generation of therapeutics. Plasmodium falciparum, the parasite that causes the deadliest form of malaria, consumes large amounts of hemoglobin from the blood cells of the human host to generate amino acids for its growth and maturation. Hemoglobin is degraded by several aspartic proteases (plasmepsins) present in the acidic digestive food vacuole of the parasite. Plasmepsin I (PM-I) is one of the enzymes directly involved in hemoglobin degradation, thus it is considered a promising target for new antimalarial drugs. We have crystallized the recombinant PM-I complexed with a potent inhibitor of several plasmepsins, KNI-10006. Crystal structure of the complex was determined with data extending to the resolution of 3.1 Å, with R-factor and R-free of 21.1% and 29.9%, respectively. The PM-I-KNI-10006 complex crystallized in the tetragonal space group P43 with four molecules in the asymmetric unit, related by non-crystallographic symmetry. The structure elucidates the unique binding mode of KNI-10006 in the PM-I active site, with the central hydroxyl group of the inhibitor positioned between two catalytic aspartates, Asp32 and Asp215. Analysis of the PM-I-KNI-10006 complex and its comparison with the structures of other plasmepsins will help to elucidate the inhibition mechanism of KNI-10006, and also should guide future design of specific inhibitors that could be developed into antimalarial drugs. 1 1 2 2 1 3 S-138 An X-ray Crystallographic Investigation of Nitric Oxide Binding to Beef Liver catalase. Namrta Purwar , Jennifer McGarry , A. Andrew Pacheco , Marius Schmidt 1 1 2 2 1 2 UW milwaukee, Department of Physics, Milwaukee, WI, United States, UW-Milwaukee, Departmen of chemistry and Biochemistry, Milwaukee, WI, United States The function of catalase in the elimination of H2O2 from living aerobic organisms has drawn the interest of scientists for a long time. The enormous enzymatic activity makes this enzyme very efficient. Catalase catalyzes the heterolytic decomposition of H2O2 into non-toxic water and oxygen, thus avoiding the formation of highly reactive and toxic radicals from homolytic H2O2 decomposition. Herein we report on the interaction of NO with Beef liver Catalase (BLC). NO mimics H2O2 binding at the active site, but does not undergo further reaction to compound I. Using X-ray crystallography on BLC crystals, we show how NO binds to the heme iron of the catalase. X-ray data of three BLC species were collected at BioCARS 14BMC, Advanced Photon Source, Argonne IL. Initially, single crystals of BLC were grown in th the presence of trace NH4OH, and had an NH3 ligand bound at the 6 coordination site of the heme-iron (occupancy 100%). After soaking the crystals in NH3 free buffer, a second species th was characterized, which had no electron density at the 6 coordination site. To investigate NO binding, crystals were soaked in 100mM of 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEANO), which releases controlled amounts of NO when dissolved in neutral or acidic solutions. Crystals soaked with DEANO showed about 60% NO occupancy at the iron binding site. An NO molecule fit to the electron density displays a Fe-N-O angle that is significantly 3+ bent away from the normal of the heme plane. Typically, NO bound to (ferric) Fe displays 2+ linear or nearly linear Fe-N-O angles, whereas NO bound to (ferrous) Fe shows substantial bending. In catalase the iron is in the ferric Fe(III) state. Therefore the large bending angle is a remarkable result. Two possible explanations are: (i) the proximal ligand of the heme in catalase is Tyr 357, whose deprotonated hydroxyl group might donate substantial electron density to the iron, so that it resembles more an Fe(II); (ii) by exposing the crystals to X-rays during data collection, photo-electrons are generated that might reduce the Fe(III) to Fe(II). S-141 Crystal Design Based on Charge-assisted Hydrogen Bonds Yuzhou Liu, Michael Ward New York University, New York, United States Inclusion compounds comprise host frameworks, assembled through non-covalent interactions, and “guest” molecules. A particularly attractive feature of inclusion compounds is the ability to incorporate functional guests within a host framework with controllable architecture. Whereas most crystal engineering strategies rely on structural control and function combined in the same molecular building block, inclusion compounds allow separation of structure and function, permitting improved control of material properties. One challenge that frequently appears is the ability to manipulate and control the host architecture while varying guest molecules. We have reported inclusion compounds based on hydrogen bonded frameworks comprising + + the guanidinium cation (G =C(NH2)3 ) and organosulfonate anions including organomonosulfonate (MS=R-SO3 )and organodisulfonate (DS= O3S-R-SO3 ; R=alkyl, arene). The three-fold symmetry of the ions enable self-assembly, through charge-assisted (N-H· · · O) hydrogen bonds, into infinite 2-D quasi-hexagonal guanidinium sulfonate (GS) sheet decorated with organic groups that project from the surface of the sheet. This GS sheet can be described as an assembly of GS ribbons connected by hydrogen bonds acting as “hinges”. This feature enables the GS network to pucker like an accordion so that its inclusion compound can achieve dense packing. The distance between adjacent sheets is primarily dictated by the dimension of the R groups. Most of our work has the similarity that different sulfonate nodes in one GS sheet belongs to different sulfonate molecules, and the inclusion cavities are formed between the organic parts of different sulfonate molecules. Intra-connecting sulfonate nodes in the GS sheet, which can be achieved by using multi-sulfonates with suitable sulfonate spacing for GS sheet formation, will introduce the capsule structures. This will lead to a way to systematically construct capsule structures from interchangeable molecular modules with predicated crystal structures. The impulse to construct capsule structures comes from their interesting application as molecular containers. Therefore constructing molecular capsules using GS system will not only advance the ability in controlling crystal packing motifs three dimensionally, but also benefit the development of functional materials which always suffers from the poor control over the solid state structures. S-144 The Crystal Structures of 4,4’ -(methylenediimino)bis-1,2,5-oxadiazole-3-carboxylc acid i and carboxamide Mark Frisch , Jeffrey Deschamps , Rodney Willard 1 1 1 2 The Laboratory for the Structure of Matter, Naval Research Laboratory, Washington, DC, 2 United States, School of Polymers and High Performance Materials, University of Southern Mississippi, Hattiesburg, MS, United States The needs of the US defense for advanced energetics have been evolving over the past several years. Energetic materials, compounds which under certain stimuli will release large amounts of energy, are essential ingredients in explosives and rocket propellants. An important property to take into account when designing organic compounds for use as energetic materials is the density; density is directly related to performance. This program to produce densely packed organic compounds suitable for use as energetic materials led to the synthesis of two compounds derived from amino-1,2,5-oxadiazole. The bis-carboxamide and bis-carboxylic acid analogues were characterized by single crystal X-ray diffraction using MoK . Both of these compounds crystallize in a monoclinic space group however the bis3 carboxamide is calculated to have a higher density (1.800 vs. 1.623 Mg/m ). Presented herein will be a comparison of the two compounds along with a detailed crystallographic description. ¤ S-147 Characterization of a Novel Pathway Essential For Survival In Macrophages Across Distally Related Pathogens Rodrigo Torres, Benson Lan, Celia Goulding Unviversity of California Irvine, Irvine, CA, United States The ability of pathogens to evade our immune defenses has been well documented and is the cause of many diseases today. One subset has the ability survive in macrophages and includes Yersinia pestis, the causative agent of plague, and Salmonella enterica, the cause of food poisoning. These pathogens are currently treated with various antibiotics, but further developments are needed to identify new targets for treatment, due to the advent of drug resistant strains and the possibility of reemergence of potent pathogens such as Y. pestis, as a possible source of bioterrorism. Of note, a novel rip (required for intracellular proliferation) operon has been specifically shown to be involved in Y. pestis and S. enterica survival when endocytosed into activated macrophages. This rip operon is conserved among a distallyrelated subset of macrophage-residing pathogens, including Burkholderia mallei, suggesting that this uncharacterized pathway involving the Rip proteins (RipA, RipB, RipC) is required for their survival. Thus, we purpose building a structural understanding of these three proteins, followed by testing a functional hypotheses about their substrates, products and interaction partners, in order to shed light on their involvement in pathogenicity. To that end, preliminary structures have been obtained for RipA to 1.9A and RipC to 2.6 A, with RipB currently under crystallization trials. In addition, initial activity assays for RipA suggest CoA transferase activity, the proposed function based on structural homology searches. S-150 X-ray powder diffraction residues characterization to avoid water contamination in oil fields. Maria Lara , Samantha Rendon , Monty Rendon , Luis Rendon marudecori consultants, cuernavaca, Mexico, jiutepec, Mexico 1 2 1 1 1 2 Mexican Institute of Water Technology, When petrochemical equipment is subject to maintenance it is common to obtain residues from inside natural gas containers, these residues most of the cases are non hazardous materials, nonetheless it is always a good practice to make a complete characterization of them. X-rays powder diffraction characterization is a excellent method to avoid water contamination in oil fields facilities; water is a subject of environmental interest. The use of X-rays powder diffraction characterization as a tool to avoid its contamination is growing continually, as an alternative to a regular and tedious chemical analysis. Petrochemical industry is a dormant risk due to the formation of chlorinated organic compounds or any other kind of hazardous compound which are considered to be toxic or carcinogenic. Prevention appears to be a more convenient method in comparison with any water treatment of industrial residual water containing waste from processes sources such as oil industry, textiles, agriculture, paper and pulp; that leave residues that are fairly easy to remove. We are presently experimenting a great deal of success with preventing the contamination process and avoiding the removal of organic residues in water. HS-004 HFE: An Iron Uptake Regulation Molecule Justin Fu, Yuhan Chen, Bryan Dongre, Shariq Moore, Nick Nabar, Vick Nabar, Nikil Prasad, Joshua Speagle, Sai Vangala, Louise Thompson 1 Brookfield Central High School, Brookfield, WI, United States, Blood Center of Wisconsin, Milwaukee, WI, United States Accumulation of excess iron results in a common hereditary disease, Hereditary Haemochromatosis (HH). There are various genetic mutations that lead to different forms of the disease. HH-I is a form of this disease in which iron accumulates in hepatocytes and intestinal epithelial cells and is associated with a mutation in the HFE (high iron protein) gene. The Brookfield Central SMART Team (Students Modeling A Research Topic) developed a model of HFE using 3D printing technology. The HFE gene encodes for a non-classical MHC class I protein. In physiological conditions, HFE is expressed and translocated to the cell surface where it may interact with a transferrin receptor (Tfr). The binding of the 1/2 domains of HFE to the Tfr allows for controlled release of iron bound to transferrin-transferrin receptor complex. A mutation (845G>A) causes the replacement of a cysteine with a tyrosine (C282Y). This replacement prevents the 3 subunit of HFE from folding properly and from interacting with 2 microglobulin, abrogating the translocation of the HFE-microglobulin complex to the cell membrane and promoting its rapid degradation. This defect hinders the regulatory capability of HFE. Current treatments include phlebotomy to prevent organ damage from accumulated iron. Further study to increase understanding of the regulatory mechanism may lead to improved treatment design. Supported by a grant from NIH-NCRR-SEPA. 2 HS-003 SMART Teams: Using 3D Rapid Prototyping Technology to Model DNA Replication Mechanisms and Engage High School Students in the Scientific Process. Susan Huang , Aimee Marceau , Nicholas P. George , Muhammad Cheema , Zoe Havlena , 2 1 2 2 2 Amy Hua , Basudeb Bhattachryya , Dylan Meacham , Jade Moon , Connie Wang , David L. 1 1 Nelson , James L. Keck Madison West Senior High School, Madison, WI, United States, Madison, WI, United States 1 2 1 1 1 2 2 Univ. of Wisconsin, DNA replication is a vital process in all organisms and understanding the fundamental biochemical interactions that drive replication is essential. Single-stranded DNA-binding (SSB) proteins form an important component of the replication machinery that facilitates the transfer of RNA primers from the enzyme primase to the replicative polymerase. This activity occurs throughout lagging-strand DNA replication. The crystal structure of the E. coli has modeled this interaction using 3D Rapid Prototyping Technology to gain insights into the physical interactions that drive DNA replication. The SMART team program allows students to experience the scientific process beyond the textbook by investigating the experimental methodology of structural biology and takes students out of the classroom and into the laboratory. Supported by grants from the Howard Hughes Medical Institute and NIH-NCRRSEPA. HS002 Lighting Up Science: Firefly Luciferase. Elana Baltrusaitis, Allyson Bigelow, Rachel Brielmaier, Pamela Burbach, Jake Dowler, Johnny Fuller, Caroline Hildebrand, Teagan Jessup, Molly Jordan, Josh Kramer, Jenna Lieungh, Alex Mikhailov, Pat O’Grady, Andrew Pelto, Quin Rowen, Rachelle Schmude, Bobby Schultz, Katherine Seubert, Alex Sherman, Parker Sniatynski, Alex Venuti, Erin Verdeyen, Molly Wetzel, Donna LaFlamme. St. Dominic School, Brookfield, WI, Medical College of Wisconsin, Milwaukee, WI. Luciferase is the generic name for an enzyme responsible for bioluminescence reactions and is commonly associated with fireflies. It is also found in many other organisms including bacteria, fungi, anemones, and dinoflagellates. Since the gene for the North American firefly (Photinus pyralis) luciferase was cloned in 1985, scientists have been genetically engineering the gene into living cells. The luciferase reaction is now widely used in scientific research to study protein production in cells, to analyze gene promoter activity, to study stem cell function in vivo, and in cancer studies, to trace the metastasis of cancer cells in living test animals. The scientific study of the luciferase enzymes themselves is also continuing. In recent research, single amino acid mutations to the active site cause the emission of different colored light in a predictable way. The uses of and improvements in bioluminescent imaging are increasing exponentially in cell biology, molecular biology, and in medical research. The St. Dominic SMART Team (Students Modeling A Research Topic) developed a model of luciferase using 3D printing technology. S-163 Isovaleryl-CoA Dehydrogenase: Dehydrogenate This! Nick Grabon, Beth Bougie, Matt Cira, Colin Erovick, Anne Fahey, Kelsey Jeletz, Elanore Kukla, Matt Murphhy, Tim Rohman, Alyssa Sass, Laura Tiffany, Sam Wolff, Karen Tiffany, Jung-Ja Kim. Cedarburg High School, Cedarburg, WI, Medical College of Wisconain, Milwaukee, WI. Although rare, isovaleric acidemia (IVA) is a potentially fatal metabolic disorder that affects one in every 250,000 people in the US. IVA results from lack of an enzyme, isovaleryl-CoA dehydrogenase (IVD), involved in the breakdown of leucine. Without this enzyme, leucine catabolism stops and organic acids accumulate within the body, causing symptoms of IVA, including vomiting, diarrhea, and fatigue. IVD belongs to a family of related enzymes called acyl-CoA dehydrogenases. IVD catalyzes the dehydrogenation, or removal of a pair of hydrogen atoms, of a small, branched-chain substrate, isovaleryl-CoA, during the third step of leucine catabolism. Glutamate 254 of IVD removes one hydrogen as a proton from the substrate, and flavin adenine dinucleotide, FAD, a cofactor of the enzyme, takes away the other hydrogen from the substrate. The three-dimensional structure of IVD, as determined through X-ray diffraction, illustrates how a small-branched chain substrate is able to fit into the active site of this enzyme and enables further investigation of how mutation of the IVD gene could affect IVD function, thus resulting in IVA. To further understand the structural impact on substrate specificity, a physical model of IVD has been designed and built by the Cedarburg High School SMART (Students Modeling a Research Topic) Team using 3D printing technology. Supported by a grant from NIH-NCRR-SEPA. S-166 Crystal Structures of Flt3 and cFMS in Complex with Inhibitors Hu Pan, May Lin, Kari Callaway, Terence Hui, Weimei Xing, Shendong Yuan, Jeanne Baker, John Anderson, Ying-Zi Xu, Hing Sham, Frederique Bard, Brian Wipke, Rick Artis, Nanhua Yao Elan Pharmaceuticals, S. San Francisco, CA, United States FLT3 and cFMS are type III receptor tyrosine kinases and play important roles in innate immunity, cancer, and inflammatory diseases. As part of a structure-based drug discovery project, we have determined a number of crystal structures of the kinase domain of Flt3 and cFMS in complex with inhibitors of different chemo types, respectively. These structures revealed the inhibitory mechanism of the JM domain. The different sequences and conformations of JM domains adopted by Flt3 and cFMS in crystal structures imply conformational flexibility in this region, which could be exploited for developing more selective inhibitors. Detailed structural analysis of these co-crystal structures provides great insights into the binding modes and selectivity of the inhibitors among the members of type III RTK family and guides the design of novel inhibitors targeting autoimmune diseases. S-170 Combining 3D Structures and Sequences to Assign Substrates and Functions of 16,000 Enzymes. Robert Huether , Jimmitti Teysir , David Dziak , Courtney McEachon , Dana 1 1,2 Hogan , William L. Duax 1 2 1 1 1 1 Hauptman Woodward Medical Research Inst., Buffalo, NY, United States, State Univ. of New York at Buffalo, Buffalo, NY, United States We have been able to align 16,000 short chain oxidoreductase enzymes that have the Rossman fold recognition element TGxxxGxG and the catalytic hexad (N)SYKP(T) (acronym TGYK). The alignment is sufficiently accurate that we can separate gram-positive from gram-negative bacteria and isolates all members of most bacterial classes, orders, families and genus. We can correlate variation in 2 positions that determine cofactor recognition with 5 residues that define at least 100 known or potential substrates with additional residues that determine the details of specific oligomeric aggregation. On the basis of amino acids in three positions in the sequence, we can separate the two largest TGYK subfamilies, the 1800 member β -keto acyl carrier protein reductase family present in all bacteria and the acetoacetyl CoA reductase family that is present only in α , β and γ proteobacteria. We achieve accurate alignment by locating a few residues (primarily Gly, Pro, Ala and Arg residues, GARP) that are fully conserved in all 16,000 members of the family and by determining precisely the location and minimum size of indels required to align all members of family. The GARP residues are critical to the alignment because of their stereochemical properties. Glycines, having positive phi values that were embedded early in folded proteins, are conserved throughout the evolution of those proteins families. These results support conclusions based upon analysis of multiple open reading frames and codon bias in actinobacteria and proteobacteria that some species in these phylums evolved at a time when the defined genetic code was composed of only triples that end in G and C. Support in part by: Mr Roy Carver, Stafford Graduate Fellowship, Caerus Forum Fund and The East Hill Foundation. S-172 Disrupting Quorum Sensing: Two Mechanisms Underlying Antagonist Function Guozhou Chen , Lee Swem , Danielle Swem , Philip Jeffrey , Bonnie Bassler , Fred 1 Hughson 1 1 1 1 1 1,2 Princeton Univ, Princeton, NJ, United States, HHMI, Chevy Chase, MD, United States 2 Quorum sensing bacteria communicate via small molecules called autoinducers to coordinate collective behaviors. Gram-negative bacteria employ acylated homoserine lactones (AHLs) as autoinducers. AHLs enter cells and bind dimeric LuxR-type transcription factors, which subsequently regulate quorum-sensing target genes. Membrane-permeable quorum-sensing antagonists that prevent population-wide expression of virulence genes offer a potential route to novel antibacterial therapeutics. Here, we report structure-function analyses of a LuxRtype protein called CviR from Chromobacterium violacein. We find that two CviR antagonists function by distinct mechanisms: one by blocking RNA polymerase engagement, the other by preventing operator DNA binding. In the former case, RNA polymerase binding is blocked by the relocation of only two non-hydrogen atoms in the LuxR-type receptor. In the latter case, the bound antagonist acts by stabilizing a domain-swapped configuration in which the receptor’s DNA-binding helices are held apart by ~60 Å, double the ~30 Å separation required for operator binding. S-174 Crystal Structure of RHCC Interacting with the Anti-cancer drug (Cis-platin) and its Potential as Novel Chemotherapeutic Delivery System in cancer Efehi Ogbomo , Suat Ozbek , Sabine Hombach-Klonisch , Thatchawan Thanasupawat , 1 1 1 Jerry Krcek , Thomas Klonisch , Joerg Stetefeld University of Manitoba, Winnipeg, Manitoba, Canada, Heidelberg Institute of Zoology, Heidelberg, Germany Right handed coiled‐ coil (RHCC) is a 24 kDa tetrameric protein that originates from the archaebacterium Staphylothermus marinus. S. marinus is an extremophile capable of surviving wide ranges of temperature, salt, pressure and pH. The crystal structure of RHCC reveals a new structural motif with four large cavities inside the tetrameric channel. The 3 cavities vary in size (320 - 360 Å ) and can be loaded with metallic compounds. Based on our new Cis‐ platin‐ RHCC crystal structure, we hypothesize that the binding properties of the cavities make RHCC a potential storage and delivery system for one of the most efficient anti‐ cancer drugs. Here we present the crystal structure of the chemotherapeutic drug Cis‐ platin bound to RHCC at 3.2 Å resolutions. RHCC was crystallized in space group P3121 with unit cell dimensions of a, b=112.8 Å, c=71.6 Å and α , β =90°, γ =120° Employing . fluorescence microscopy we show that Alexafluor labelled RHCC molecules are internalized by the human hypopharyngeal squamous carcinoma cell line FaDu, human glioblastoma cell line T98G, and primary glioblastoma cells from patients. RHCC may provide a novel mode for the delivery of chemotherapeutic drugs into tumour cells and represent a unique and novel approach in the treatment of cancer patients. 1 2 1 2 1 1 S-175 Molecular characterization of a Class I P450 electron transfer system from Novosphingobium aromaticivorans DSM12444 Wen Yang , Stephen Bell , Hui Wang , Weihong Zhou , Mark Bartlam , Luet-Lok Wong , Zihe 1,3 Rao 1 1 2 3 1 1 2 Nankai University, Tianjin, China, University of Oxford, Oxford, United Kingdom, Tsinghua University, Beijing, China 2 3 Cytochrome P450 enzymes of the CYP101 and CYP111 families from the oligotrophic bacterium Novosphingobium aromaticivorans DSM12444 are heme monooxygenases that receive electrons from NADH via ArR, a ferredoxin reductase, and Arx, a [2Fe-2S] ferredoxin. These systems show fast NADH turnovers that are efficiently coupled to product formation. The three-dimensional structures of ArR, Arx and CYP101D1, which form a physiological class I P450 electron transfer chain, have been solved by X-ray crystallography. The general structural features of these proteins are similar to their counterparts in other Class I systems such as putidaredoxin reductase (PdR), putidaredoxin (Pdx) and CYP101A1 of the camphor hydroxylase system from Pseudomonas putida, and adrenodoxin (Adx) of the mitochondrial steroidogenic CYP11 and CYP24A1 systems. However significant differences in the proposed protein-protein interaction regions of the ferredoxin reductase, ferredoxin and P450 enzyme are found. There are regions of positive charge on the likely interaction face of ArR and CYP101D1 and a corresponding negatively charged area on the surface of Arx. The [2Fe-2S] cluster binding loop in Arx also has a neutral, hydrophobic patch on the surface. These surface characteristics are more in common with those of Adx than Pdx. The observed structural features are consistent with the ionic strength dependence of the activity. S-177 “Structure-based identification of ceruloplasmin as a hypothetical human p53-binding protein and its possible role in carcinogenesis” Chris Pacheco Rivera , Birgit Eisenhaber , Chandra Verma 1 1 2 2 2 Universidad Peruana Cayetano Heredia (UPCH), Lima, Peru, Bioinformatics Institute (BII), A-STAR, Singapore, Singapore The p53 tumor suppressor protein is the most commonly mutated protein identified in cancer. p53 activation promotes the upregulation of various target genes responsible for cell cycle arrest or apoptotic cell death depending on the cellular environment, a critical role in cellular defense against cancer. Molecular interactions between the p53 protein and azurin, a redox Pseudomonas aeruginosa protein, were demonstrated to trigger apoptosis in human cancer cells. Since the protein-protein interaction between azurin and p53 allows the stabilization of the latter and cancer regression, it was of great importance to determine the domains involved in their physical association. Models based on crystal structures of p53 and azurin suggest that their interaction take place at the DNA-binding core domain of p53, where the 95% of cancer-associated mutations take place. Thus, p53 DNA-binding domain, might potentially represent a new target for tumour cell death induction or growth arrest in cancer treatment. Here, by means of tertiary structure alignment methods, we report a hypothetical human p53 binding protein, ceruloplasmin, a multicopper oxidase protein comprised of multiples domains each of which has the typical fold of a single domain of cupredoxin proteins which shows a significantly tertiary structural similarity to azurin. Protein docking approaches allowed us to identify a potential p53-ceruloplasmin binding interface by the extrapolation of residues involved in the p53-azurin complex formation; experimental data suggest the involvement of ceruloplasmin in cancer development. S-179 Local caching to improve efficiency of CIF DDLm function import references in SBEVSL Elena Zlateva, Herbert Bernstein Dowling College, Oakdale, NY, United States The introduction of the DDLm import attributes into CIF [1] has allowed for the development of specific domain dictionaries without the overhead of redundant common definitions. These modularized dictionaries can be located anywhere on the web, and importation facilitates the access and sharing of specialized definitions. In an attempt to increase efficiency of DDLm dictionary expansion through importation, we have created an expansion utility, CIFGET (available for download at http://sourceforge.net/projects/cifget/), that follows all importation tags, much like HTML links, and fetches a local copy files tree of referenced dictionaries. The advantages of having a local copy of the tree are similar to the benefit of running local database queries and transactions versus querying a remote database. Local queries provide virtually no delays. Thus, running a dictionary expansion utility on a local dictionary tree is faster and more reliable than fetching dictionary definitions only when needed. Local database queries are an efficient way to create look-ups of any dictionary definition or value. The Structural Biology Extensible Visualization Scripting Language Project has proposed an extension to the current DDLm specification standards to allow for function definitions anywhere within a CIF data file. This would allow user-defined functions to manipulate the data from the data file to provide scripting, data conversion, or animation capabilities. These functions could be exported into modularized dictionaries, thereby creating a library of functions, which would then be accessible through importation. Function calls from a CIF data file to a SBEVSL functions dictionary, fetched on the local files tree, will be very fast as they are analogous to querying a local database with key-value pairs of function names and definitions. Work supported in part by grants from IUCr, NIH and DOE. [1] Hall, SR, Spadaccini, N., Westbrook, J., “Dictionary Definition Language DDLm”, IUCr, 2007. http://www.iucr.org/__data/assets/pdf_file/0020/16382/DDLm_spec_aug08.pdf S-181 Host Cell Pre-Selection, Towards the Development of a Generally Applicable “Tough” Protein Over-Expression in Escherichia coli Jiapeng Ruan, Wayne Anderson Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, United States Overproduction of proteins from heterologous genes has limitations in the production of functionally folded soluble proteins. For the Escherichia coli expression system, although numerous efforts have published, the benefits from current strategies have been casespecific. Establishing generally applicable soluble expression methods for protein overexpression remains a significantly challenging task. We have determined that soluble expression in E. coli requires the coupling of tightly controlled target gene expression and target protein specific folding environments. To alleviate solubility problems, we chose E. coli host cells containing vectors for target protein expression and the co-expression of transcription factors, followed by pre-selection of host cells under protein folding and overexpression stress. Selected host strains were then used for the induction of overexpression. We show here that host pre-selection optimizes the overexpression of some previously totally insoluble “tough” proteins with dramatically improved solubility. Approaches to generate expression regulation and host pre-selection are discussed and evaluated. S-184 In-silico docking study of Mitogen-activated Protein Kinase Kinase 4 (MAP2K4/MEK4) with Genistein and its Analogs Sankar Narayan Krishna, Li Xu, Rebecca Farmer, Xiaoke Huang, Antoinette Nibbs, Karl Scheidt, Wayne Anderson, Raymond Bergan Northwestern University, Chicago, IL, United States Metastasis or spread of Prostate Cancer (PCa) to other parts of the body is the second highest cause of death due to cancer among men in the United States. Our lab has shown that 4,5,7-trihydroxyisoflavone (genistein), inhibits the initiating step of cell invasion, as well as the downstream formation of metastasis associated with PCa, by inhibiting Mitogenactivated protein kinase kinase 4 (MAP2K4/MEK4) activity. In particular, it has been shown that MEK4, a 399 amino acid protein, activates the established pro-invasion protein, p38 MAPK. MEK4 increases cell invasion and induces matrix metalloproteinase type 2 (MMP2). Genistein inhibits MEK4 kinase activity in vitro, and MEK4-mediated signaling in intact PCa cells. In related studies, a series of genistein analogs have been synthesized and tested for their ability to inhibit prostate cell invasion, using a Boyden chamber assay system. In order to investigate the structure-activity relationship of MEK4 with genistein and synthetic analogs, an in silico model of MEK4 was constructed. Using this model, while blinded to the Boyden chamber assay results, activity prediction after virtual docking (Autodock 4.0) was done. In particular, the analogs and genistein were first segregated based on their optimal scoring binding sites on the protein target and then the Autodock estimated Inhibition constant (Ki) was determined. In silico results were then compared to Boyden chamber assay results. The similarities and differences between in silico and experimental findings and thus the potential of using this in silico modeling technique for predicting analog activity will be discussed. As a direct extension of this study, efforts are now being directed at crystallizing MEK4 and deriving it both with and without genistein and relevant analogs. The biochemical mechanism by which genistein and selected analogs inhibit MEK4 kinase activity will be determined by measuring their e ect upon MEK4 enzyme kinetics in vitro. These results combined with in silico will be crucial in guiding the synthesis of new lead compounds with higher specificity and lower cytotoxicity. S-187 Structural studies of topoisomerase V, a novel type IC topoisomerase Rakhi Rajan, Alexandra Gast, Bhupesh Taneja, Alfonso Mondragon Northwestern University, Evanston, United States Topoisomerases are ubiquitous enzymes that regulate the topology of DNA inside the cell and thus facilitate several fundamental cellular processes. They function by creating a transient DNA break and passing another DNA through this break before resealing the break. Topoisomerase V (Topo-V) is a novel type IC topoisomerase, and is unique because it contains both topoisomerase and DNA repair activities within the same protein. The topoisomerase domain is located at the N- terminus of the protein and this is followed by twelve helix-hairpin-helix (HhH)2 domains. Previous studies of an N-terminal 61 kDa fragment of Topo-V (Topo-61) revealed that the topoisomerase domain of Topo-V has a new fold entirely different from other topoisomerases. In the present study, different fragments of TopoV containing either the topoisomerase domain (Topo-44) or both topoisomerase and repair domains (Topo-78) have been constructed and analyzed by structural and biochemical methods. Crystal structures of Topo-44 determined under three different conditions show significant conformational changes in the (HhH)2 domain near the topoisomerase active site compared to Topo-61 crystal structure. These conformational changes are required for exposing the topoisomerase active site so that DNA can gain access to the active site. Five phosphate ions bound in the topoisomerase active site helped to model a DNA molecule, and the model gives initial information on how the protein and DNA interacts. Biochemical studies are in progress to understand the cleavage/religation mechanism and DNA and metal binding characteristics of Topo-V. Crystallization studies of Topo-78 gave a model for the th topoisomerase domain and the first seven (HhH)2 domains. The last (8 ) (HhH)2 domain, which has the DNA repair activity, is highly disordered and hence studies are in progress with a deletion mutant of Topo-78 to understand the structure of the repair domain. In addition, biochemical experiments are being carried out to identify the residues involved in DNA repair activity of Topo-78. Successful structure determination of Topo-78 will give structural details of the topoisomerase and repair active sites for the first time and show whether these two domains interact each other. S-190 The Crystal Structure of CARDS Toxin from Mycoplasma pneumoniae Argentina Becker , Alexander B. Taylor , Ahmad Galaleldeen , Olga N. Pakhomova , 1,2 3 3 1,2 Stephen P. Holloway , T.R. Kannan , Joel B. Baseman , P. John Hart Department of Biochemistry, UTHSCSA, San Antonio, Texas, United States, The X-ray Crystallography Core Laboratory, UTHSCSA, San Antonio, Texas, United States, 3 Department of Microbiology and Immunology, UTHSCSA, San Antonio, Texas, United States Mycoplasma pneumoniae is a bacterial pathogen that colonizes the lung and is known to cause asthma, pneumonia and other infections in humans. M. pneumoniae produces a toxin known as CARDS (Community-Acquired Respiratory Distress Syndrome) toxin that has been shown to display cytotoxic effects on mammalian cells similar to those observed during M. pneumoniae infection, suggesting that the toxin plays a key role in M. pneumoniae pathogenesis. The 591 amino acid protein has ADP-ribosylating and vacuolization activities and biochemical evidence suggests that it gains entry into host cells through binding to surfactant protein A (SP-A), an abundant glycoprotein in the lung. Here, we present the crystal structure of CARDS toxin determined to 2.6 Å resolution. The crystals grow in space group R3 with one molecule in the asymmetric unit. The catalytic N-terminal CARDS domain shares structural similarity with the S1 subunit of the toxin from Bordetella pertussis (Pertussis toxin) while the receptor-binding C-terminal domain, with no detectable sequence homology to any known proteins, folds into two topologically similar subdomains. The new structural data provide insight into various aspects of CARDS toxin trafficking and function. 1 2 1 1,2 1,2 1,2 S-193 Evaluating the Bruker SMART X2S bench-top system: A means to bringing x-ray crystallography into the undergraduate curriculum Uzma Zakai, Ilia Guzei, Nicholas Hill University of Wisconsin-Madison, Madison, United States X-ray crystallography is a powerful and increasingly common tool for routine structural characterization yet remains poorly represented at the undergraduate level. Several strategies have been adopted to bring X-ray crystallography into the mainstream undergraduate experience, including forming alliances between institutions to economize equipment and establish joint teaching and research projects. Herein we report our evaluation of Bruker SMART X2S, a single crystal X-ray diffractometer designed for institutions lacking any crystallographic infrastructure. Bruker SMART X2S is a portable benchtop diffractometer that requires only a 110 V outlet to operate. The instrument operation is intuitive and facile with an automation layer governing the workflow from behind the scenes. Based on our examination of 19 samples, the Bruker SMART X2S yields publishable quality data. Although data quality is a function of sample quality, this instrument is a bold advance towards bringing chemical crystallography in the undergraduate curriculum. S-196 Assessing the stability of theophylline cocrystals in the presence of competing coformers in the solid state Jennifer Urban, Heba Abourahma The College of New Jersey, Ewing, NJ, United States Cocrystals have become an increasingly popular subject of study in the chemistry community. It is generally known that physical properties of a compound, including solubility, hygroscopicity, and stability, can be altered when cocrystallized with other molecules, termed "coformers". Our goal is to assess the stability of a cocrystal in the solid state in the presence of competing coformers and determine the possibility of displacing one coformer in a cocrystal with another. The results could be important in the pharmaceutical industry if a cocrystal were to make it to the formulation stage as it would have to be ground into a tablet with excipients, binding materials and others. We have focused our attention on theophylline, an active pharmaceutical ingredient (API) that is used in asthma medication. Theophylline makes a good model system for this study since a number of theophylline cocrystals with a variety of coformers have been reported to date. To achieve our goal, two general types of experiments were conducted: selectivity and competition experiments. In the selectivity experiment the theophylline is wet-ground with equimolar amounts of two coformers and the relative affinity of the theophylline to the coformers is determined. In the competition experiment, a prepared cocrystal of theophylline is wet-ground with a competing coformer that is known to form a cocrystal with theophylline. With each experiment, grinding time is varied between 20, 40, and 60 minutes to determine its effect on the experimental outcome. Cocrystals of theophylline that were studied in this project include those with 4-nitrophenol, 4-hydroxybenzoic acid, salicylic acid, 2hydroxynaphthoic acid, melamine and acetamide. The products from each experiment were analyzed by powder x-ray diffraction (PXRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and infrared spectrometry (IR). This poster will present the results from the selectivity and competition experiments of theophylline cocrystals with the coformers mentioned above. S-199 Cocrystals as a Means to Control Polymorphism in Pyrazinamide Devon Cocuzza, Heba Abourahma The College of New Jersey, Ewing, New Jersey, United States Polymorphism is the ability of a molecule to exist in more than one possible form in the solid state. Each polymorphic form has its own unique crystal structure which is responsible for determining the physical properties such as stability, solubility, hygroscopicity and dissolution rate among other things. The objective of our research is to examine cocrystals as a method to control polymorphism. Cocrystals are crystalline materials that are comprised of at least two different components that are solid at room temperature and are held together by non-covalent interactions. The target polymorphic compound in our study is pyrazinamide (PZA). PZA is an active pharmaceutical ingredient (API) used for the treatment of tuberculosis and is known to exist in four different forms: alpha, beta, gamma, and delta. Since amides are known to have high affinity to carboxylic acids and amides, we considered cocrystal formers (coformers) that contain these functionalities such as benzoic acid, anthranilic acid, 3,5-dintrosalicylic acid, isophthalic acid, and nicotinamide. Our experiments consisted of reacting equimolar amounts of PZA with each coformer via liquid-assisted solid state grinding and in solution. The products were characterized using Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis (TGA), Nuclear Magnetic Resonance (NMR), Infrared Spectroscopy (IR) and Powder X-Ray Diffraction (PXRD). We further examined the persistence of a particular cocrystal formation in the presence of different solvents during liquid-assisted solid state grinding including methanol, ethanol, THF, DMSO, DMF, acetone, acetonitrile, and water. Herein we will present the synthesis and characterization of PZA cocrystals obtained to date from the above mentioned experiments. S-202 Why is P21/n a Standard Non-Standard Space Group? James Haestier1, Amber L. Thompson1, David J. Watkin1, George C. Feast2, Jeremy Robertson2, Lee W. Page3 Chemical Crystallography, Oxford, United Kingdom, 2Chemistry Research Laboratory, Oxford, United Kingdom, 3GlaxoSmithKline, Harlow, United Kingdom The structure of an unusual methylene aziridine was initially determined in the monoclinic space group P21/n, with a cell of a = 13.8593(3) Å, b = 10.5242(2) Å, c = 14.8044(4) Å and β = 92.0014 (7)° and two molecules in the asymmetric unit. , Prior to publication [1], the CIF was verified with checkCIF, which gave the following alert: PLAT128_ALERT_4_G Non-standard setting of Space-group P21/c…P21/n The hyperlink explains further: ‘The reported monoclinic space-group is in a nonstandard setting. Transformation to the conventional setting is indicated unless there is a good (scientific) reason not to do so.’ The unit-cell obtained from the initial indexing was then transformed, and reprocessed (including integration, scaling and cell final refinement) to give the data in the space group P21/c, with a cell of a = 13.8594(2) Å, b = 10.5243(2) Å, c = 19.9230(3) Å and β = 132.0439(7)° The atomic coordinates from the original . structure were transformed, and the structure re-refined. It has long been known that refinements in oblique cells have increased correlation between selected parameters, potentially making refinements less stable [2]. A comparison of these results in P21/n and P21/c clearly displayed an increase in the correlation between coordinates in the ac plane for the oblique cell. The increase in the corresponding covariances makes a significant contribution to the standard uncertainties of derived parameters, e.g. bond lengths. Thus, there are clear, scientific advantages to reporting this (or any structure) in the “more orthogonal” space-group setting. 1. Feast G. C. et al. (2009). Acta Cryst. C65, o635–o638. 2. Dunitz, J. D. (1979). X-ray Analysis and the Structure of Organic Molecules, p 205–206. Cornell University Press. 1 S-205 Structural Determination and Identification of Receptor Binding Site of Adeno Associated Virus Serotype 6 using X-ray Crystallography Robert Ng, Govindasamy Lakshmanan, Hyun-Joo Nam, Brittney Gurda, Jude Samulski, Robert McKenna, Mavis Agbandje-McKenna 1 University of Florida, Gainesville, FL, United States, University of North Carolina, Chapel Hill, NC, United States Adeno-associated viruses (AAVs) are nonpathogenic single-stranded DNA viruses which belong to the Parvoviridae family and the genus Dependovirus. Due to their nonpathogenicity, attention has been directed towards their development as gene therapy vectors. Representative members of the AAV antigenic clades/ clonal isolates display capsid sequence-associated receptor attachment, tissue tropism, transduction efficiency, and antigenic reactivity properties. To identify the structural features that dictate these different properties, we have initiated the structure determination of these viruses. Unlike most of the AAVs that either bind sialic acid or heparin sulfate as a primary cell surface receptor, AAV6 is a unique serotype that binds both carbohydrate on cell surfaces. To identify the AAV6 capsid regions involved in these interactions we aim to determine the structure of this serotype alone and complexed with carbohydrate ligands. The structure of AAV6 determined to 9.7Å resolution by cryo-electron microscopy and image reconstruction shows the capsid features already described for the AAV. An X-ray diffraction data has been collected for AAV6 alone and AAV6 complexed with heparin sulfate or sialic acid, and structure determination using our available crystal structure of AAV1 as a phasing model is underway. These structures will be used to identify variable surface loop regions on the AAV6 capsid that dictate its unique dual carbohyrdrate binding phenotype. In addition, a comparison of the AAV1 and AAV6 structures will provide information on the role of the capsid in dictating the differential liver and lung tropism observed between these two viruses which differ by only 6/736 amino acids in their capsid viral protein. This data will impact our understanding of the capsid determinants of tissue tropism and transduction efficiency for the AAVs and will be applicable for the engineering of recombinant AAV vectors for improved tissue targeting specificity. 2 S-208 Structural Studies of MG289, an Extracytoplasmic Thiamine Binding Lipoprotein from the Sexually Transmitted Infection Mycoplasma genitalium Katherine Sippel, Balasubramanian Venekatakrishnan, Susan Boehlein, Govindasamy Lakshamanan, Yoshihisa Sakai, Jeanne Quirit, Mavis Agbandje-McKenna, Charles Rosser, Robert McKenna University of Florida, Gainesville, Florida, United States Mycoplasma genitalium (Mg) is a sexually transmitted infection that causes non-gonococcal urethritis in men and endocervicitis in women. This pathogen is resistant to tetracyclines and azithromycin indicating the need for novel drug targets to treat this disease. Studies have identified MG289 as a minimal gene necessary for Mg survival. It is homologous to Cypl, an extracytoplasmic thiamine pyrophosphate (TPP) binding lipoprotein from Mycoplasma hyorhinis. The structure of Cypl was solved previously using magic triangle providing a molecular replacement model for structure solution. The crystal structure of MG289, has been solved and refined to 2.1 Å resolution. The current model has an Rcryst of 18.6% and Rfree of 23.1%. MG289, like Cypl, is a mixed α /β protein with two well-defined domains, which are separated by a deep cleft. As predicted from sequence alignments and homology modeling, the structure shows thiamine bound to MG289, rather than the TPP seen in Cypl. Analysis of the thiamine in the binding cleft shows numerous interactions that can be translated into an in vitro fluorescence-based kinetic assay for drug binding. The insights derived from the structure solution and characterizations of MG289 are being used as a starting point for structure-based drug design of a novel antibiotic to fight mycoplasmal infection. S-211 Structural Investigations of TopBP1 in DNA Replication Stress Charles Leung, Mark Glover University of Alberta, Edmonton, AB, Canada Replication stress often compromises the replication machinery and can lead to DNA damage at replication forks. This induces a complex repair mechanism, which is characterized by the early accumulation of Replication protein A (RPA) onto chromatin. Studies have shown that independent loading of RPA onto exposed ssDNA and the trimeric ring complex Rad9-Hus1Rad1 (9-1-1) at DNA junctions facilitates activation of the crucial signalling kinase, Ataxia telangiectasia and Rad3 related (ATR), via the ATR activating function of Topoisomerase II binding protein 1 (TopBP1). The ability of TopBP1 to activate ATR results from its ability to form protein-protein interactions. We are currently investigating the structural biology of TopBP1 involved in these specific processes. ¥ S-214 Structural Investigation of trans-4-hydroxynonenal-derived Adduct in protein-DNA complex 1,N -deoxyguanosine 2 Surajit Banerjee, Plamen P. Christov, Albena Kozekova, Carmelo J. Rizzo, Michael P. Stone Vanderbilt University, Nashville, TN, United States trans-4-Hydroxynonenal (HNE) is produced by peroxidation of -6 polyunsaturated fatty acids. HNE exhibits a range of biological effects, from alteration in gene expression and cell signaling to cell proliferation and apoptosis. The Michael addition of deoxyguanosine to HNE 2 yields four diastereomeric exocyclic 1,N -dG adducts. Site specific mutagenesis reveals that these induce primarily G to T mutations. Here we present the replication bypass studies and structures of the Sulfolobus solfataricus DNA polymerase Dpo4 with the template:primer 5'TCAYXGAATCCTTCCCCC-3' 5'-GGGGGAAGGATTC-3', where X is the site of adduction with the (6S,8R,11S) HNE-dG adduct and Y is either C or T for two sequences respectively. In-vitro bypass studies with Dpo4 shows misincorporation of dATP at the adduct site for both 2 sequences. The 1,N -HNE-dGua adduct maintains the exocyclic structure in case of -1 primer and intercalates between the neighbouring template bases. 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MinE is a topological specificity factor that counters the activity of MinCD division inhibitor at the mid-cell division site. Its structure consists of an anti-MinCD domain and a topology specificity domain (TSD). Previous NMR analysis of truncated Escherichia coli MinE showed that the TSD domain contains a long α helix and two antiparallel β -strands, which mediate formation of a homodimeric α /β structure. Here we report the crystal structure of full-length Helicobacter pylori MinE and redefine its TSD based on that structure. The N-terminal region of the TSD (residues 19-26), previously defined as part of the anti-MinCD domain, forms a β -strand (β A) and participates in TSD folding. In addition, H. pylori MinE forms a dimer through the interaction of anti-parallel β Astrands. Moreover, we observed serial dimer-dimer interactions within the crystal packing, resulting in the formation of a filamentous structure. We therefore redefine the functional domain of MinE and propose that a multimeric filamentous structure is formed through antiparallel β -strand interactions. S-226 ER α -glucosidase I in the N-glycosylation pathway Megan Barker , David Rose 1 1 2 2 University of Toronto, Toronto, Ontario, Canada, University of Waterloo, Waterloo, Ontario, Canada Many proteins on the eukaryotic cell surface are covalently linked to complex carbohydrates, leading to a heterogeneously sugar-coated cell. The process of protein N-glycosylation begins in the endoplasmic reticulum (ER) with the transfer of a standard N-glycan, Glc3Man9GlcNAc2, to an asparagine residue of a nascent protein. The terminal glucose residue is subsequently cleaved by the transmembrane enzyme ER α -glucosidase I (GluI), followed by further processing in the ER and Golgi. Recent studies on the S. cerevisiae homolog of GluI have determined several key residues within the catalytic domain, located in the ER-lumenal C-terminal region (Faridmoayer et al, 2007). However, the structure of GluI is presently unknown. I have achieved secreted expression of a transmembrane-deletion construct of S. cerevisiae GluI using the methyltropic yeast Pichia pastoris, giving purified protein at a yield of three milligrams of protein per litre of growth culture. Optimizing crystal growth by microseeding from an initial hit, I obtained rod-like crystals 0.7mm in length. Using a high-pressure cooling system (Kim et al, 2005) developed at the Cornell High Energy Synchotron Source (CHESS), and paratone oil as a cryoprotectant, 2.1Å datasets of the native protein and with an inhibitor soak have been collected. Work towards solving the phase problem is currently in progress. This poster will describe the ongoing efforts to determine the structure and understand the catalytic mechanism of this key member of the N-glycosylation pathyway. S-229 E2 Interaction and Dimerization in the Crystal Structure of TRAF6 and Structural Basis for the Lack of E2 Interaction in the RING Domain of TRAF2 Qian Yin , Su-Chang Lin , Betty Lamothe , Miao Lu , Yu-Chih Lo , Gregory Hura , Lixin 1 3 2 5 4 Zheng , Rebecca L. Rich , Alejandro D. Campos , David G. Myszka , Michael J. Lenardo , 2 1 Bryant G. Darnay , Hao Wu Weill Medical College, New York, NY, United States, Univ . of Texas, Houston, TX, United 3 4 States, ALS Lawrence Berkely National Laboratory, Berkely, CA, United States, NIAID, NIH, 5 Bethesda, MD, United States, Univ. of Utah, Salt Lake City, UT, United States Tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins are intracellular signal transducers for a number of immune receptor superfamilies. TRAF2 interacts with members of the TNF receptor superfamily and connects the receptors to downstream signaling proteins; whereas TRAF6 mediates signalling emanated from both TNF receptors and interleukin-1 receptor/Toll-like receptors. Both TRAF2 and TRAF6 are proposed to function as E3 ubiquitin ligase, eliciting NF-κ B activation via Lys63-linked polyubiquitination. E3 ligase activity has been mapped to their N-terminal RING and zinc finger domains. Here we report the crystal structures of N-terminal TRAF6 and its complex with the ubiquitinconjugating enzyme (E2) Ubc13, and the structure of the RING and the first zinc finger domains of TRAF2. The RING and zinc fingers of TRAF6 assume a rigid, elongated structure. Interaction of TRAF6 with Ubc13 involves direct contacts of the RING and the preceding residues, and the first zinc finger has a structural role. Unexpectedly, this region of TRAF6 is dimeric both in the crystal and in solution, different from the trimeric C-terminal TRAF domain. Structure-based mutagenesis reveals that TRAF6 dimerization is crucial for polyubiquitin synthesis and autoubiquitination. Fluorescence resonance energy transfer analysis shows that TRAF6 dimerization induces higher-order oligomerization of full-length TRAF6. The mismatch of dimeric and trimeric symmetry may provide a mode of infinite oligomerization that facilitates ligand-dependent signal transduction of many immune receptors. On the other hand, although TRAF2 adopts similar linear arrangement of RING and zinc finger domains and same dimeric status, its RING structure displays multifaceted differences from that of TRAF6. These structural differences prevent TRAF2 from interacting with Ubc13 and other related E2s due to steric clash and unfavorable interfaces. Our structural observation should prompt a re-evaluation of the role of TRAF2 in TNFα signaling and may indicate that TRAF2associated proteins such as cIAPs may be the ubiquitin ligases for NF-κ B signaling. 1 2 1 1 1 1 1 3 S-232 Functional Structures: Repair 1 XLF and XRCC4 in Mammalian DNA Double-strand Break 2 2 3 1 Sara Andres , Sunetra Roy , Katheryn Meek , Mauro Modesti , Murray Junop 1 2 McMaster Univ., Hamilton, ONT, Canada, Michigan State Univ., Madison, WI, United 3 States, Aix-Marseille Univ. CNRS, Marseille, France DNA double-strand breaks are one of the most lethal forms of DNA damage that can occur in a mammalian cell. However, some double-strand breaks are part of programmed genomic rearrangements, such as V(D)J recombination. Non-homologous end-joining is the predominant repair pathway to fix these breaks and requires a core set of proteins to do so. Two of these proteins, XLF and XRCC4, interact with one another and are essential for nonhomologous end-joining, yet have no enzymatic function. They carry out their roles strictly through their architecture. To elucidate the mechanism by which these two proteins function in complex, we solved the structure of human XLF (1-224) to 2.5 Å using SAD. This structure bears similar resemblance to human XRCC4 (PDB 1FU1), except for the striking difference in the elongated tail of XRCC4, compared to the tail of XLF, which winds back up and around towards the head domains of the protein. Using information from both protein structures and conserved regions, we identified amino acids that were key to the interaction of both proteins. This interaction is necessary for repair, as XRCC4 mutants that were unable to bind to XLF caused a decrease in frequencies of coding joint formation during V(D)J recombination. How this physical interaction actually occurs, though, is still unknown. Therefore, we attempted crystallization of an XLF-XRCC4 complex. Crystals were obtained for multiple truncations of both proteins, but overall diffracted to >20 Å. Application of microseeding, however, and extreme dehydration led to crystal diffraction at 4.6 Å. Herein we describe our work towards solving and refining the low-resolution structure of an XLF-XRCC4 complex, and the insights it provides on the XLF-XRCC4 complex function in DNA double-strand break repair. S-235 Small changes on the substitution pattern of pyridines and pyridine-N-oxides: Their influence on the molecule aggregation Vera Vasylyeva, Klaus Merz Ruhr-University Bochum, Bochum, Germany One challenge in chemical engineering is the lack of correlation between crystal packing and the molecular structure. The nature of self-organisation in the solid state is complicated and depends on different parameters such as symmetry, secondary interactions and supramolecular synthons. Our strategy for analysing weak dipole-dipole-interactions is to reduce the complexity of parameters and investigate small molecules, such as fluoro- and deutero-substituted pyridines and pyridine-N-oxides. The in situ crystallisation with an IR-laser and a low temperature device allows a crystallisation of the compounds with a low melting point under the direct control of the crystal growth via X-ray analysis. Fluorine is known to influence the electronic structure of aromatic backbone and therefore the entire molecules but the nature of the C-F…H hydrogen bond is discussed controversially. On the other hand, fluorine forms only weak intermolecular interactions and seems to have no influence on the crystal packing. Pauling’s definition of the hydrogen bond would imply that fluorine, as the most electronegative atom, should be a stronger hydrogen-bond acceptor then oxygen and nitrogen. But the C-F group, the so-called “organic fluorine”, does not form hydrogen bonds commensurate with electronegativity considerations in contrast to the C-O and C-N groups. Hydrogen/deuterium exchange is without doubt the smallest possible alteration of the molecular structure. Very few examples are known that show a remarkable influence of deuterium substitution on the aggregation of molecules. Recently pentadeuteropyridine was found to crystallise completely different in comparison to a notdeuterated pyridine [1]. We pose two questions: How can the influence of fluorine/deuterium on molecule structure be useful for crystal engineering? A comparison of fluoro-substituted pyridines shows different intermolecular interactions depending on the substitution pattern of the fluorine atoms at the pyridine backbone [2]. Furthermore already a partial deuteration of pyridine-N-oxide leads to great changes in the crystallisation behaviour [3]. The second question is: Can the weak influence of secondary interactions of fluorine/deuterium substituents on the crystal packing be amplified through the increase of the number of F/Datoms? [1] R. Boese et. al, Angew. Chem. Int. Ed., 2009, 48, 755-757 [2] V. Vasylyeva, K. Merz, J. Fluor. Chem., 2010, 131(3), 446-449 [3] V. Vasylyeva, T. Kedziorski, C. Schauerte, K. Merz, Angew. Chem., 2010, submitted S-238 Fragment-Based Lead Discovery for Urokinase-Type Plasminogen Activator Inhibitors Li Qiu , Longguang Jiang , Mingdong Huang , Edward J. Meehan , Liqing Chen 1 2 1 2 2 1 1 University of Alabama in Huntsville, Huntsville,AL, United States, State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou,Fujian, China The Urokinase-Type Plasminogen Activator (uPA) is a trypsin-like serine protease that activates plasminogen to plasmin. uPA also plays crucial roles in regulating arterial remodeling, angiogenesis, cell migration and proliferation. uPA has been widely recognized as a target against tumor metastasis in various animal models. Upregulation of uPA expression has been shown to correlate with cellular proliferation and invasiveness. Various strategies have been proposed to intervene with either uPA proteolytic activity or the zymogen activation. High affinity inhibitors have been identified that intervene the uPA proteolytic activity. However, these uPA inhibitors are typically quite basic and thus have poor bioavailability. Fragment-based screening using high throughput X-ray crystallography is an established method to identify low molecular weight fragments that can specifically bind to the target active site. Those fragments may be evolved to larger lead compounds, either by linking or merging fragments together or by growing the fragments to pick up additional interactions. In this investigation, high quality uPA crystals were soaked with a library of 384 low molecular weight fragments and screened by X-ray diffraction for compounds binding. Data were collected at the SER-CAT synchrotron beamlines 22ID/22BM at the Argonne National Laboratory. Small molecule fragment specifically binds to uPA as determined by structural analysis was biochemically tested on its binding/inhibition profile. The results of this study will be implemented towards the development of novel drug compounds. S-247 Supramolecular Structural Variation in a Series of Ni(X)(NO)(PPh3)2 Complexes. Nongnaphat Khosavithitkul, Kenneth J. Haller Science, Muang, Nakhon Ratchasima, Thailand Concerted weak interactions are important building blocks of extended solid state structures. One such building block for molecular compounds is the multiple phenyl-phenyl edgeto-face (ef) C−H∙ ∙ ∙ π attractive noncovalent interactions of the concerted sextuple phenyl embrace (6PE). The resulting sum of interaction energy is sufficient to make it a dominant supramolecular motif for crystals of complexes containing triphenylphosphine or similar ligands. The supramolecular structures of a series of three four-coordinate Ni(X)(NO)(P(C6H5)3)2 − − − complexes, where X = NCS , N3 , and Cl are examined. Although the molecular complexes are quite closely related, their supramolecular structures are different. The primary extended interactions in the isothiocycanato complex are zig-zag chains of the expected 6PE between adjacent triphenylphosphine ligands. Chains are connected together into a three dimensional network by additional weaker concerted interactions. The chloro complex also contains onedimensional chains of 6PE. However, it crystallizes with a benzene molecule of solvation, which accommodates considerably more C−H∙ ∙ ∙ π interactions than a phenyl ring, thus becoming the major link interconnecting three of the 6PE chains via ten C−H∙ ∙ ∙ π interactions centered about the benzene of crystallization. While the azido complex does not contain 6PE, it does contain one-dimensional chains of highly concerted noncovalent interactions involving the azido group as well as phenyl rings. Thus, as the strongest hydrogen bond acceptor, the azido ligand becomes perhaps the most important determiner of the supramolecular structure. Additional phenyl-phenyl interactions are abundant and important in determining finer details of the crystal structures. S-250 The structure of 2-(naphthyl)-3-pyridinyl-1,3-thiazolidin-4-one Jose Luis Pinto , Jose Antonio Henao , Vladimir Kouznetsov , Diego Fernando Amado , 2 2 Teresa González , Alexander Briceño Universidad Industrial de Santander, Bucaramanga, Santander, Colombia, Venezolano de Investigaciones Científicas, Caracas, Distrito Capital, Venezuela 1 2 1 1 1 1 Instituto Heterocyclic compounds are largely studied due to the showed biological activities of most of the heterocycles. Thiazolidinones are important heterocyclic compounds, which exhibit a broad range of biological activities, including interesting profile as fungicidal, pesticide, antibacterial, anticonvulsant, antihistaminic, antioxidant, anti-inflammatory and antinociceptive agents, etc. As a consequence many different protocols allowing the synthesis of 4thiazolidinone skeletons have been developed The compound 2-(naphthyl)-3-pyridinyl-1,3-thiazolidin-4-one was synthesized by means of a multicomponent reaction with a Dean-Stark trap promoted by glacial acetic acid, in which stoichiometric amounts were used of the reagents: α -aminopyridine, α -naphthylaldehide, and thioglycolic acid, in anhydrous toluene. The molecular characterization was carried out by means of the techniques IR, MNR and GC-MS. The X-ray diffraction data were collected on an AFC7S single crystal diffractometer using MoK radiation ( = 0.71070Å). The structural solution and refinement were made with Shelxs-97 software package. The 2-(naphthyl)-3-pyridinyl-1,3-thiazolidin-4-one (C18H14N2OS), M = 306 g/mol, crystallizes in the monoclinic system, space group P21/c [No. 14] with unit cell parameters a = 11.958 (3) Å, 3 b = 9.656 (2) Å, c = 12.662 (2) Å, β = 97.010° (5), Vol = 1451.09 (2) Å and Z = 4. Key Words: Crystal structure; thiazolidin-4-ones. S-253 Crystal Structure and Molecular Modeling of Analogue of Chalcone C20H22NO3. William B. Fernandes, Caridad Noda Perez, Hamilton B. Napolitano Department of Chemistry, UnUCET, State University of Goias, Anapolis/Goias, Brazil Chalcones are considered precursors of the biosynthesis of flavonoids and are obtained by condensation reaction of Claisen-Schmidt between a ketone and an aromatic aldehyde in the presence of basic catalysts. The importance of chalcones is based upon the wide variety of chemical and biological properties presented by them. Furthermore, chalcones have been the subject of several theoretical and experimental studies, that aimed at determining their molecular structures, chemical reactivity, antimicrobial activity, among other applications in [1,2] the therapy field . In order to develop new drugs the analogue of chalcone of retinoid type (1E,4E)-1-(4-nitrophenyl)-5-(2,6,6-trimetilciclohex-1-enyl)-penta-1,4-dien-3-one was obtained from the equimolar coupling of the β -ionona with p-nitrobenzaldeide by classical ClaisenSchimidt condensation using lithium hydroxide as catalyst.The single crystal growth was obtained by indirect diffusion technique using a system of hexane and methanol. The structure was solved by Direct Methods and refined by full matrix Least Square methods on 2 [3] F using WINGX package . Non H atoms were refined anisotropically and all H atoms were placed geometrically. The compound crystallizes in the P21/c monoclinic space group and the cell dimensions are: a = 11.593(2) Å, b = 11.715(2) Å, c = 14.202(2) Å, α = γ = 90° and β = 110.147(5)°; Z = 4 and V = 1810.8(4) ų. 17572 measured reflections with 3211 unique and 2166 observed [I > 4σ (I)]. The final residual factor R1 is 0.0525 for 226 refined parameters. ... One non-classical intra-molecular hydrogen bonds C–H O [2.856(3)Å] stabilizes the molecule in partially planar arrangement. The nature of the observed disorder was investigated theoretically within Density Functional Theory using pseudopotentials and planewave basis set by Nudged Elastic Band Method. The transition state structure was obtained and the calculated potential barrier energy is 9.22 eV. The effect of crystal packing was also examined. This work was financed by CAPES (process 2036-09-6) and PrP/UEG. [1] Dominguez, J. N., Charris, J. E. (2001). European J. of Med. Chem., 36, 555 - 560. [2] Valla, A., Cartier, D. (2006). European Journal of Medicinal Chemistry, 41, 142 - 146. [3] Farrugia, L. J. (1999). Journal Appl. Cryst., 32, 837 - 838. S-256 Experimental Electron Density Determinations of CB1 Receptor Agonists Steven Fournet, Edwin D. Stevens, Mark L. Trudell Department of Chemistry, University of New Orleans, New Orleans, LA 70148, United States Of the two cannabinoid receptors that have been identified, CB1 and CB2, the CB1 receptor is found primarily in the central nervous system. Some agonists of the CB1 receptor, including the drug Rimonabant, have been shown to be effective in the treatment of obesity in clinical trials. Recently, a new series of compounds have been synthesized that have been shown to be agonists with a wide range of binding affinities for the CB1 receptor in rat brains. We have undertaken the high-resolution measurement of the electron density distributions of several of these compounds in order to correlate features of the electron structure with -1 biological activity. Highly redundant, high-resolution (typically sin max/ 1.1 Å ) x-ray diffraction data sets have been collected at 120 K using MoK radiation, and the data refined using the Hansen-Coppens aspherical atom multipole model with the XD2006 program. Analysis of the resulting electron density distributions includes deformation density maps and topological parameters obtained using the Atoms in Molecules theory. Of particular interest are plots of the electrostatic potential calculated on the surface of the molecular electron density distribution. The molecular electrostatic potential provides information on chemical reactivity, identifying for example sites of protonation and electrophilic attack, and is a major component of intermolecular interaction energies. Funded in part by the National Institute on S-259 Experimental Electron Density Distribution of Dimethoxygossypol, a Derivative of the Disesquiterpene Gossypol Isolated from Cotton Plants Carlos Zelaya , Michael K. Dowd , Edwin D. Stevens 1 1 2 1 Department of Chemistry, University of New Orleans, New Orleans, LA 70148, United 2 States, Southern Regional Research Center, USDA, New Orleans, LA 70124, United States Gossypol is a natural product isolated from the cotton plant that is of interest because of its wide sphere of bioactivity. We have isolated and synthesized a number of derivatives of gossypol to explore their anticancer and antifungal activity. Crystals of the 6,6’-dimethoxy derivative were found to be suitable for a high-resolution study of the electron density distribution. A highly redundant set of x-ray diffraction intensity measurements was -1 collected to (sin / )max of 1.19 Å at 120 K. The experimental electron density distribution was obtained by least-squares refinement of the x-ray data using the Hansen-Coppens aspherical atom multipole model. §¦ In addition to maps of the molecular deformation density, the topology of the electron distribution of dimethoxygossypol has been analyzed using the Atoms in Molecules approach. The locations of the critical points of the electron distribution, and the values of the 2 density, (rb), Laplacian (rb), and bond ellipticity, , at the bond critical points have been determined for the wide variety of different covalent bonds and hydrogen bonds present in the structure. S-262 N-alkyl-DABCOnium trihalozincates: Privileged scaffolds for the preparation of noncentrosymmetric solids Aaron Finke, Danielle Gray, Jeffrey Moore University of Illinois, Urbana-Champaign, Urbana, IL, United States A major requirement for the development of materials with nonlinear optical susceptibilities is the absence of inversion centers in the solid state, i.e. crystallization in noncentrosymmetric (NCS) space groups. Nonetheless, a priori determination of a compound's preference for acentricity remains a challenge. Few achiral, organic moieties are known to act as privileged NCS scaffolds, due in part to the difficulty of systematic investigations of such moieties on solid-state morphology. We present studies of a new, achiral, metal-organic scaffold, which exhibits an unusual preference for polar, NCS space groups. Compounds containing the scaffold are easily prepared from DABCO (1,4-diazabicyclo[2.2.1]octane), ZnBr2, and an alkyl bromide; all compounds are highly crystalline solids, which are stable under ambient conditions. A library of compounds containing the scaffold was analyzed by single-crystal X-ray diffraction. Systematic modification of the alkyl functionality was crucial to understanding the role of the scaffold toward preferential crystallization in NCS space groups. S-264 Rapid temperature switching for time resolved measurements Kevin Beyer , Peter Chupas , Karena Chapman , Mark Newton 1 1 1 1 2 Argonne National Laboratory, Argonne, Illinois, United States, ESRF, Grenoble, France 2 The ability to probe materials and reactions in real time under real operating conditions is pivotal to understanding of their structure and functional behavior. Towards this goal it is important to develop appropriate sample environments generating non-ambient operating conditions. In particular, probing the kinetics and mechanism for a reaction rely on the ability to initiate the process on a time scale that is fast relative to the reaction itself. Here we present apparatus that enables the rapid switching of temperature or reactive gas streams to initiate and characterize solid state reactions. S-265 Structural Analisys of the Phenyl Sulfonylamide Acetophenone as an intermediary of a Sulfonamide Chalcone Lorraine Malaspina , Carlito Lariucci , William Fernandes , Caridad Perez 1 2 1 1 2 2 Federal University of Goias, Goiania, Goias, Brazil, State University of Goias, Anapolis, Goias, Brazil Chemically, the chalcones are flavonoids with open-chain, in which the two aromatic rings are connected by a system of three carbons, forming ketones α , and β unsaturated, where both the carbonyl and the olefinic portion are linked to aromatic groups. They are found in nature, in undergrowth plants, in different plants organs, especially at the flowers. They constitute a class of antifungal and anticancer agents that, according to some authors, has shown promising therapeutic efficacy against a wide variety of tumor cells both in vivo and in vitro, especially in the treatment of stomach cancer. The importance of chalcones is due to the wide variety of chemical and biological properties that they present. For this reason, chalcones have been the subject of several theoretical and experimental studies, mainly aimed at determining their molecular structures, their chemical reactivity, its antimicrobial activity, its capacity of inhibition and enzyme induction, among other applications in the therapy field. The compound object of this work was obtained by Prof. Caridad Noda Perez from the State University of Goiás and her student William Borges Fernandes. Crystal Data: Data collection in a KappaCCD diffractometer, MoKα radiation. The solution, anisotropic refinement, geometrical calculations, molecular packing and drawings were done with the program package WINGX. Molecular formula: C14H13NO3S. Structure: a = 12.5179(6) Å, b = 8.3615(4) Å, c = 13.0007(5) Å, α = γ = 90º, β = 98.118(3)º, monoclinic, space group P21/c, Z = 4, V = 1347.13(6) ų. 9797 measured reflections with 3009 unique and 6061 observed. Final indices R1 = 0.0456 for 177 refined parameters. There is an intra-molecular hydrogen bond with N−H…O angle equal to 137.58º and distance equal to 2.182 Å, and symmetry [ x, y+1, z]. Acknowledgements: This work was partially financed by CNPq, CAPES and FUNAPE/UFG. The Data Collection were done by Prof. Carlos Alberto Simone at the Institute of Physics of USP-São Carlos (IFSC). S-268 Common Recognition Motifs used by Prokaryotic LysR-type Transcriptional Regulators are Evident from the Structures of BenM DNA Binding Domain with its Operator-promoter DNA Amer Alanazi, Ellen Neidle, Cory Momany University of Georgia, Athens, GA, United States The LysR-type transcriptional regulator (LTTR) BenM is involved in controlling benzoate degradation in the soil bacterium Acinetobacter baylyi strain ADP1. The 1.8 Å resolution crystal structure of the unbound BenM DNA binding domain subunit (BenM DBD) confirmed that the BenM DBD forms a compact globular domain composed of three helices with a winged helix-turn-helix motif (a2-a3) and longer linker-helix resembling that of the DBD of winged helix proteins. BenM DBD was crystallized with its cognate benA and catB DNA promoter sites in two different crystal-packing arrangements. In these nucleic acid complexes, BenM DBD dimers span a large region of bent DNA where the DNA recognition helices (a2) of one dimer bind into two consecutive DNA major grooves in a sequence-dependent manner. The specific DNA major groove interactions that define the LTTR conserved recognition motif (T-N11-A) include van der Waals interaction of two proline residues at the N-terminal end of the recognition helices with the methyl group of the thymine base of the recognition motif. Also involved in sequence specific interactions are the side chain of Gln 29 with the imino and amino groups of the recognition motif adenine base respectively (5'-ATAC-3') and the side chain of Arg 34 with the carbonyl oxygen of guanine (5` -GTAT-3` ) in the complementary strand. The wing of the winged HTH motif interacts mainly with the phosphate backbone of the DNA minor groove and assists in the proper positioning of the N-terminal end of the recognition helix. S-271 Understanding blue-to-red conversion in monomeric fluorescent timers and hydrolytic degradation of their chromophores Sergei Pletnev , Fedor Subach , Zbigniew Dauter , Alexander Wlodawer , Vladislav 3 Verkhusha SAIC-Frederick, Frederick, IL, United States, National Cancer Institute, Frederick, IL, United 3 States, Albert Einstein College of Medicine, New York, NY, United States Crystal structures of a fast fluorescent timer (Fast-FT) and its precursor with blocked blue-tored conversion (Blue102) have been determined at the resolution of 1.15 Å and 1.81 Å, respectively. Structural data suggest that blue-to-red conversion, taking place in Fast-FT and in related fluorescent timers (FTs) of the same family, is associated with the oxidation of Cα 2Cβ 2 bond of the chromophore. Site directed mutagenesis revealed a crucial role of Arg70 and Tyr83 in the delayed oxidation of Cα 2-Cβ 2 bond, introducing the timing factor in maturation of the fluorescent timer. Substitutions Ser217Ala and Ser217Cys in Fast-FT substantially slow down formation of an intermediate blue chromophore but do not affect much blue-to-red conversion, whereas mutation Arg70Lys, having little effect on the blue chromophore formation rate, markedly accelerates formation of the red chromophore. The chromophore of FTs adopts a cis-conformation stabilized by a hydrogen bond between the phenolate oxygen of the chromophore and the side chain hydroxyl of Ser146. In case of Blue102, a bulky side chain of Ile146 precludes the chromophore from adopting a “cis-like” conformation, blocking its blue-to-red conversion. Both Fast-FT and Blue102 structures revealed hydrolytic degradation of the chromophores. In Fast-FT, chromophore-forming Met66 residue is eliminated from the polypeptide chain, whereas Leu66 in Blue102 is cleaved out from the chromophore, and although decarboxylated, remains attached to the preceding Phe65. Hydrolysis of the chromophore competes with chromophore maturation starting from ether the keto or enolate intermediates and is driven by the same residues that participate in chromophore maturation. 1 2 1,2 3 2 2 S-274 Structural and functional analysis of LR11 Vps10p domain Zenzaburo Nakata, Masamichi Nagae, Norihisa Yasui, Terukazu Nogi, Junichi Takagi Institute for Protein Research, Suita, Osaka, Japan LDLR relative with 11 binding repeats (LR11) is a 250-kDa type-1 membrane protein highly expressed in cortex and cerebellum. This protein contains a domain that is structurally similar to the Vacuolar protein sorting 10 protein (Vps10p), a sorting protein in yeast. LR11 is known as a major risk factor of Alzheimer disease and is hypothesized to be involved in the intracellular trafficking of the amyloid presursor protein, regulating the production of amyloidgenic- peptide. Here we have analyzed the structure and function of LR11 Vps10p domain to gain insights into the mechanism of intracellular protein sorting mediated by this domain. The binding affinity of LR11 Vps10p domain to a ligand, its own propeptide, was measured by fluorescence polarization assay at various pH. The binding was markedly reduced at acidic pH. This suggests that LR11 Vps10p domain may be involved in the intracellular sorting in a pH dependent manner. The crystal structure of LR11 Vps10p domain under the acidic condition was solved at 2.3 Å resolution. Vps10p domain assumes a tenbladed -propeller fold followed by two small domains, designated 10CC-a and 10CC-b domains. Superposition of Vps10p domain of LR11 with that of sortilin reveals that the putative ligand-binding site is masked by a loop connecting blade 6 and 7 of LR11. This result suggests that the ligand recognition property of LR11 Vps10p domain is completely different from that of sortilin. ¨ ¨ S-277 Characterizing heme uptake and iron storage from pathogenic and non-pathogenic Mycobacteria. Lisa Marie McMath , Michael Tullius , Lana Cong , Nicholas Chim , Cedric Owens , Christine 1 2 1 Harmston , Marcus Horwitz , Celia Goulding University of California- Irvine, Irvine, CA, United States, University of California- Los Angeles, Los Angeles, CA, United States Iron is essential for virtually all forms of life. Similar to most pathogens, Mycobacterium tuberculosis (Mtb) must import iron from its host. We aim to understand novel mechanisms of mycobacterial iron metabolism to potentially provide new avenues for anti-Mtb therapeutic development. We have shown that Mtb has a newly discovered heme uptake system, and have identified the genomic region responsible. Found encoded within this genomic region is a secreted protein that binds heme tightly, which we propose to be a hemophore. We have solved its structure, and are currently attempting to solve its structure in complex with heme. Additionally, we observed that non-pathogenic Mycobacterium bovis BCG (BCG) has an attenuated heme uptake system in comparison with Mtb. By sequence alignment, the homologous hemophore in BCG is identical to that of the Mtb hemophore, except for lysine 87 substituted with threonine (K87T). We hypothesize this substitution may contribute to the attenuation seen in the BCG pathway. To address structural consequences, we have crystallized the BCG apo-hemophore, and are currently attempting to crystallize it in complex with heme. Furthermore, preliminary heme transfer experiments suggest an inefficient transfer of heme from the BCG hemophore to one of the potential heme transporters. Bacteria usually have cytosolic iron storage proteins. The mycobacterial ferritin (BfrB), a structurally conserved iron detoxification and storage protein, has been identified and crystallized. However, our preliminary crystallographic data show that the C-terminus of each subunit in the 24-mer complex is disordered. By sequence alignment, we observed that the Mtb BfrB polypeptide is longer than most ferritins, and its secondary structure prediction is coiled. Thus, we engineered a truncated BfrB without the last 15 residues at the C-terminus. This truncated BfrB still assembles into a 24-subunit oligomer, readily crystallizes, and we hope to solve its structure by X-ray crystallography. 1 2 1 2 1 1 1 S-280 NorthEastern CAT Beam Lines at the Advanced Photon Source Kay Perry, Steven Ealick, Malcolm Capel, Anthony Lynch, Frank Murphy, Igor Kourinov, David Neau, Kanagalaghatta Rajashankar, Cynthia Salbego, Jonathan Schuermann, Narayanasami Sukumar, James Withrow Cornell University, Argonne, IL, United States The NorthEastern Collaborative Access Team (NE-CAT) focuses on the design, construction, and operational support of synchrotron X-ray beamlines for the solution of technically challenging structural biology problems and provides an important resource for the international research community. Currently there are two operational undulator beamlines: 24ID-C - fully tunable in the energy range from 6 to 22keV and 24ID-E - fixed energy at ~12.66keV. These operational beamlines are currently open to institutional members and general APS users. Both beamlines are equipped with MD2 microdiffractometers, Q315 detectors and robotic sample automounters. NE-CAT provides stable, well-collimated beam from 5 to 100 microns in diameter, tools for on-axis visualization of micron-sized crystals, focused and de-focused beam, detector two-theta rotation, a mini-kappa goniometer for optimal alignment of crystals, automatic data collection strategy prediction and automatic data processing. NE-CAT maintains a website at http://necat.chem.cornell.edu/. Funding for NE-CAT is provided through a P41 grant from the National Center for Research Resources and from the NE-CAT member institutions. S-281 Structural studies of the interaction between MD-1 and lipid IVa. Sung-il Yoon, Minsun Hong, Gye-Won Han, Ian Wilson The Scripps Research Institute, La Jolla, CA, United States MD-1 is a secretory protein that can form a stable complex with RP105 on the cell surface. The MD-1/RP105 complex can regulate the biological function of its evolutionarily related complex, MD-2/TLR4, which recognizes bacterial lipopolysaccharide (LPS) and initiates innate immune responses. Here, we report structural and biophysical data to demonstrate a previously unidentified LPS binding activity for MD-1. The crystal structure of chicken MD-1 (cMD-1) was determined at 2.0 Å resolution by SIRAS method. MD-1 exhibits a β -cup like fold containing a large hydrophobic cavity between two β -sheets, similar to that seen in MD-2. Based on the structural similarities between MD-1 and MD-2, we hypothesized that MD-1 directly interacts with LPS. Indeed, LPS was identified as an MD-1 ligand by electrophoresis and gel filtration analyses. Moreover, the interaction was supported by the 2.4 Å resolution crystal structure of cMD-1 complexed with an LPS precursor, lipid IVa. The complex structure reveals that the MD-1 cavity embeds one lipid IVa molecule in a mode that differs from that used by MD-2. These results imply an important biological role for soluble MD-1 as a regulator of the host LPS response. S-283 The SER-CAT / UGA Crystal Shipping Kit for Data Collection John P. Rose, James Tucker Swindell II, John Chrzas, John Gonczy, Bi-Cheng Wang SER-CAT, Department of Biochemistry & Molecular Biology University of Georgia, Athens, GA, United States s›? ¡‹ ·‒¡? ⁄\ ? ? «¡« ¡‒ ? ⁄\ ¡? ⁄¡? ›› ? ‹¡¦¡ \‒„? ›? ¦\‒‒„›· ? ¡¢¢ ¦ ¡‹ ? \ \? ¦› ¡¦ ›‹? \‹ ? ¦‒„ \ ¦‒¡¡‹ ‹£? ›‹? ? ¡\« ‹¡ K? ⁄¡? r›· ⁄¡\ ? q¡£ ›‹\ ? b› \ ›‒\ ¡? `¦¦¡ ? s¡\«? GrdqLb`sH? ⁄\ ¡ ¡ ›fi¡ ? ?›•‹?¦‒„ \ ? ⁄ fifi ‹£? „ ¡«? \ ¡ ?›‹? ⁄¡?a¡‒¤¡ ¡„N`kr?fi·¦¤M??s⁄¡?rdqLb`s?N?tf` ¦‒„ \ ? ⁄ fifi ‹£? ¤ ? ? «\‹·¢\¦ ·‒¡ ? \‹ ? › ? „? ⁄¡? t‹ ¡‒ „? ›¢? f¡›‒£ \? h‹ ‒·«¡‹ ? r⁄›fi? \‹ ¦›‹ \ ‹ ? \ ? ⁄¡? ¡fl· fi«¡‹ ? ‹¡¦¡ \‒„? ›? ›\ K? ⁄ fi? \‹ ? ‒¡¦› ¡‒? US? ¦‒„ \ ? ›? ⁄¡? ¡\« ‹¡? · ‹£? \ \‹ \‒ ?s\„ ›‒?v⁄\‒ ›‹?bwqLPOO? ‒„? ⁄ fifi¡‒?G‹› ? ‹¦ · ¡ HM???r⁄ fifi ‹£?¤ ?¦›«fi›‹¡‹ ? ‹¦ · ¡Y S?`kr? „ ¡?fi·¦¤ P?o·¦¤? ⁄ fifi ‹£?¦‒\ ¡?GS?fi·¦¤ H P?l\£‹¡ ¦?fi·¦¤?⁄› ¡‒ P?o·¦¤? ¡fi\‒\ ›‒ P?h‹ ‒·¦ ›‹\ ?bc rdqLb`s?⁄\ ?\ ›? ¡ ¡ ›fi¡ ?\?‹› ¡ ? ¡ ¦¡?¢›‒? ›\ ‹£?\‹ ?·‹ ›\ ‹£?fi·¦¤ ?¢‒›«? \«fi ¡ ?¦›‹ \ ‹¡ ‹?¦‒„› \ K? ⁄¡?rdqLb`s?o·¦¤?k›\ ¡‒M The Puck Loader is a simple "hands-free" tool, which eliminates common errors associated with manually loading (and unloading) ALS style pucks from cryovials. The Puck Loader has no moving parts and allows the experimenter to load a single puck within a fraction the time required for traditional manual loading / unloading methods. Importantly the Puck Loader allows the user to load pins directly into the puck base while still in the cryovial thus preventing loss or damage to crystal due to operator error. The Puck Loader also allows the experimenter to safely recover pins from the puck directly into the cryovial for storage. Stop by the SER-CAT booth to see the SER-CAT Shipping Kit and Puck Loader and to get a price list and order form. Work supported by the SER-CAT Member Institutions, University of Georgia Research Foundation and the Georgia Research AllianceM P?e›\«?c¡•\‒ P?a¡‹ ?⁄¡«› \ P?a¡‹ ?¦‒„› \ ?⁄› ¡‒ P?g\«fi ›‹?¦‒„ \ ?•\‹ S-284 Regulation of the essential M. tuberculosis peptidoglycan-precursor flippase Christine Gee , Kadamba Papavinasasundaram , Sloane Blair , Christina Baer , Arnold 2 2 3 1 Falick , David King , Christopher Sassetti , Tom Alber Department of Molecular and Cell Biology, QB3 Institute, University of California, Berkeley, 2 Berkeley, Ca, United States, Howard Hughes Medical Institute, University of California, 3 Berkeley, Berkeley, Ca, United States, Department of Molecular Genetics and Microbiology, 4 University of Massachusetts Medical School, Worcester, MA, United States, Australian Synchrotron, Clayton, Vic, Australia Receptor Ser/Thr kinases control broad aspects of physiology in bacteria, but little is known about how the kinases regulate cellular pathways. Among the Mycobacterium tuberculosis (Mtb) proteins essential in an animal model is the flippase that delivers the peptidoglycan (PG) precursor, lipid II, to the cell surface for incorporation into the cell wall. In mycobacteria and several other actinomycetes, the flippase has accessory domains, which presumably add functionality or regulate the activity. We found that the Ser/Thr protein kinase, PknB, efficiently phosphorylates the intracellular pseudokinase domain of the Mtb flippase, and this single modification creates a binding site for the forkhead associated (FHA) domain protein FhaA in vitro and in vivo. To define the mechanisms of recognition, we determined the crystal structures of the flippase extracellular domain and the intracellular pseudokinase alone and in combination with FhaA. The extracellular accessory domain is homologous to a galactose binding domain, while the intracellular domain has a highly diverged, inactive protein-kinase fold. These results suggest support a model in which extracellular PG regulates PG synthesis by controlling assembly of a protein complex containing the lipid II flippase. 1 1,4 3 1 1 S-288 GfcC shows similarities to Wza and is important for group 4 capsule polysaccharide expression. Karthik Sathiyamoorthy , Ilan Rosenshine , Mark Saper 1 1 2 1 2 University of Michigan, Ann Arbor, MI, United States, The Hebrew University of Jerusalem, Jerusalem, Israel Many bacteria produce a polysaccharide capsule necessary for resisting host defenses and biofilm formation. The group 4 capsule operon (gfc) in enteropathogenic E. coli contains seven genes (gfcABCDE, etp and etk), each important for polysaccharide synthesis and export [1]. Homologs of gfcE, etp and etk are present in many other capsule systems including a group 1 capsule system (wza, wzb and wzc respectively) also present in E. coli. The four other gfc genes encode secreted proteins of unknown function. GfcB and GfcD are putative outer membrane lipoproteins, while GfcC is a periplasmic protein. We have determined the crystal structure of GfcC at 1.8-Å resolution by the single wavelength anomalous diffraction method. GfcC has two β -grasp domains similar to domains 2 and 3 of the periplasmic region of Wza, but with little sequence identity. The Wza structure has a Cterminal amphipathic helix that forms a novel transmembrane helical pore (~17Å) in the observed octamer [2]. This was proposed to be the exit hole for the growing polysaccharide chain [2]. GfcC also has a C-terminal amphipathic helix, but in contrast to Wza, it packs against the β -sheet of one β -grasp domain and is partially occluded by a helical hairpin insert from the other β -grasp domain, a structure that is clearly absent in Wza. As a result, GfcC behaves as a soluble monomer in vitro. Although the Wza homolog GfcE (95% identity) is also encoded in the gfc operon, the unique presence of gfcABCD suggests that the mechanism for polysaccharide translocation may be more complex. Structural predictions of the GfcD sequence suggest that it forms a transmembrane β -barrel structure, a structure known to be important in other polysaccharide export complexes. Interestingly, homologs of gfcC and gfcD are fused in some Burkholderia genomes suggesting that GfcC may make important interactions with GfcD. Experiments to understand the function of GfcC in polysaccharide export are in progress. [1] Peleg, A., et al. (2005). Identification of an Escherichia coli operon required for formation of the O-antigen capsule. J Bacteriol. 187(15): 5259–5266. [2] Collins, R.F., Beis, et al. (2007). The 3D structure of periplasm-spanning platform required for assembly of group 1 capsular polysaccharides in Escherichia coli. Proc Natl Acad Sci USA 104(7): 2360–2365. S-290 Structural basis of phosphatidylinositol 4-phosphate recognition by Fapp1 PH domain Ju He , Robert Stahelin , Joanna Gajewiak , Glenn Prestwich , Tatiana Kutateladze 1 1 2 3 3 1 2 University of Colorado Denver School of Medicine, Aurora, CO, United States, Indiana 3 University School of Medicine-South Bend, South Bend, IN, United States, University of Utah, Salt Lake City, UT, United States The four-phosphate-adapter protein (Fapp1) regulates formation and fission of post-Golgi vesicles and directs the endocytic transport from the trans-Golgi network (TGN) to the plasma membrane. Upon activation, cytosolic Fapp1 is recruited to the TGN membranes through the interaction of its N-terminal Pleckstrin Homology (PH) domain with phosphatidylinositol 4phosphate [PtdIns(4)P] and the small GTPase ADP-ribosylation factor 1 (ARF1). Despite the important role of Fapp1 in secretory membrane transport, the structural and biochemical mechanisms by which Fapp1 exerts its functions have not been established. We applied high field Nuclear Magnetic Resonance (NMR), Surface Plasma Surface (SPR), X-ray crystallography and Mutagenesis experiments to investigate the dual recognition of Arf1 and PtdIns(4)P by Fapp1 PH domain. Our data reveal that Fapp1 interacts with two anchoring components using distinct binding interfaces. Mutations in these interfaces can specifically block the Fapp1 binding to each component. Based on the structure model, we have developed a metabolically-stabilized PtdIns(4)P analogue that selectively targets Fapp1 PH. Our work shows great promise for the future development of therapeutics for Fapp1 related disorders such as Legionnaires' disease. S-292 Structural Interactions of Pilocarpine with Human Cytochrome P450 Enzymes Aaron Bart, Natasha DeVore, Emily Scott University of Kansas, Lawrence, KS, United States Cytochrome P450 (P450) enzymes are a large family of heme thiolate proteins involved in the metabolism of both endogenous compounds and xenobiotic compounds, including drugs. Xenobiotic-metabolizing P450 enzymes can each bind and metabolize a diverse set of substrates and often produce a variety of metabolites. Structures of the P450 enzyme family reveal a highly canonical global protein fold, but with large variations in the active site size, topology, and conformational flexibility. Though in vivo and in vitro metabolism data demonstrate both overlapping substrate selectivity and substrate specificity, the structural basis for this is often difficult to surmise. The goal of the current work is to determine how a related set of human cytochrome P450 enzymes bind and interact with the common inhibitor and clinical muscarinic receptor agonist pilocarpine. Pilocarpine inhibition of CYP2A6, CYP2A13, and CYP2E1-mediated metabolism of the substrate p-nitrophenol revealed significant differential inhibition, with pilocarpine inhibiting CYP2A13 much more efficiently than CYP2E1. In order to elucidate key amino acids that are involved in pilocarpine binding, a 2.8 Å X-ray structure of CYP2A13 has been determined with pilocarpine in the active site. Data collected at SSRL indicated a P1 space group with 12 molecules in the asymmetric unit. The CYP2A13/pilocarpine co-crystal structure was solved by molecular replacement. Several previously determined CYP2A13 structures were used as search models to locate molecules in the asymmetric unit that have varying conformations. Pilocarpine binds in the CYP2A13 active site, forming a coordinate covalent bond to the heme iron. Comparison of this structure with other structures currently being generated of pilocarpine bound to CYP2A6 and CYP2E1 is providing an understanding of how these closely related enzymes each interact with the inhibitor pilocarpine. S-293 Alternating access in E. Coli maltose transporter mediated by rigid-body rotations Michael Oldham, Shanshuang Chen, Cedric Orelle, Amy Davidson, Jue Chen Purdue University, West Lafayette, IN, United States ATP binding cassette (ABC) transporters couple ATP binding and hydrolysis to the translocation of substrates across the membrane bilayer. ABC transporters are composed of two transmembrane domains (TMDs) coupled to two cytoplasmic nucleotide-binding domains (NBDs). Bacterial importers, including the well-studied E. coli maltose transporter, also employ a periplasmic substrate-binding protein for delivery of the substrate to the transporter. We have previously reported structures of the maltose transporter in two independent conformations: an inward-facing, nucleotide-free, resting state in which the transmembrane translocation cavity is closed to the periplasm while the cytoplasmic NBDs are held open; and an outward-facing conformation in which the TMDs outline a substrate-binding pocket open toward the periplasm while ATP is poised for hydrolysis along the closed dimer interface of the NBDs. We report here the structure of an intervening, nucleotide-bound, substrate pretranslocation state in which a closed substrate-loaded binding protein is docked atop the closed periplasmic gate of the inward-facing TMDs. Comparison of all three structures reveals that alternating access of the substrate involves rigid-body rotations of the TMDs that are coupled to the closure of the NBDs around the ATP to be hydrolyzed. Prior to docking of the binding protein, key residues that position the ATP gamma-phosphate for hydrolysis are sequestered away from the nucleotide-binding pocket. Docking of the binding protein brings the coupled NBDs closer together to sense the nucleotides bound at their pre-formed dimer interface. S-294 Comparison of the SH3-guanylate kinase (GUK) module of the tight junction protein ZO-1 with various MAGUK core modules reveals interdomain flexibility between the SH3 and GUK domains Ming Lye , Alan Fanning , Ying Su , James Anderson , Arnon Lavie 1 2 1 2 1 2 1 University of Illinois at Chicago, Chicago, Illinois, United States, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States Zonula Occludens-1 (ZO-1) is a membrane-associated guanylate kinase-like (MAGUK) protein critical for the formation of tight junctions. This property of ZO-1 and its ability to localize to the membrane has been attributed to the core module, which consists of the two consecutive protein-protein interaction domains Src Homology 3 and guanylate kinase, and an insert in the SH3 domain called the U5 region (SH3-U5-GUK). We have solved the structure of the ZO-1 core module to 2.6 Å and observed the conservation of interdomain beta ( strand interactions as predicted by the previously solved core module structure of the homologous MAGUK protein post-synaptic density-95 (PSD-95). This is mediated by main chain interactions between the two beta ( strands flanking either side of the GUK domain to form a unit that completes the SH3 fold. Interestingly however, comparison of the core module of ZO-1 with that of PSD-95 and ZO-3 revealed significant differences in the conformations of the core module of all three MAGUKs due to interdomainangle differences between the SH3 and GUK domains. The ZO-1 core module adopts a more open and more closed conformation compared to PSD-95 and ZO-3, respectively. These conformational differences between the different MAGUKs reflect general variations within MAGUK core modules that could have functional and regulatory implications. We have also made the novel discovery that the unique 6 (U6) region of ZO-1, composed of a stretch of acidic residues immediately C-terminal to the GUK domain, binds to the core module in a bivalent-cation dependent manner through electrostatic interactions. Using pull-down assays, we have shown that the U6 region and the calcium sensor protein calmodulin both compete for similar binding sites on the core module. This direct binding interaction between the U6 region and the core module may be one general mechanism by which U6 regulates core module function. © © S-297 Crystal Structure of the MyD88:IRAK4:IRAK2 Complex Reveals A Hierarchical Helical Oligomer in TLR/IL1-R Signaling. Su-Chang Lin, Yu-Chih Lo, Hao Wu Weill Cornel Med College, New York NY, United States MyD88, IRAK4 and IRAK2 are critical signaling mediators of the TLR/IL1-R superfamily. Here we report the crystal structure of the MyD88: IRAK4: IRAK2 death domain (DD) complex, which surprisingly reveals a left-handed helical oligomer that consists of 6 MyD88, 4 IRAK4 and 4 IRAK2 DDs. The assembly of this helical signaling tower is hierarchical, in which MyD88 recruits IRAK4 and the MyD88: IRAK4 complex recruits the IRAK4 substrates IRAK2 or the related IRAK1. Formation of these complexes brings the kinase domains of IRAKs into proximity for phosphorylation and activation. Composite binding sites are required for recruitment of each of the individual DDs in the complex, which are confirmed by mutagenesis and previously identified signaling mutations. Specificities in the MyD88: IRAK4 interaction and in the recruitment of IRAK2 are dictated by both detailed molecular complementarity and correspondence of surface electrostatics. The MyD88: IRAK4: IRAK2 complex provides a template for Toll signaling in Drosophila and an elegant mechanism for versatile assembly and regulation of DD complexes in signal transduction. S-298 SrRietveld: A Next Generation Rietveld Refinement Program Jiwu Liu , Peng Tian , Wenduo Zhou , Yingrui Shang , Chris Farrow , Pavol Juhas , Simon 1,2 Billinge Columbia University, New York, NY, United States, Brookhaven National Laboratory, 3 Brookhaven, NY, United States, Michigan State University, East Lansing, MI, United States SrRietveld is a highly automated software tool kit for Rietveld refinements. Compared to traditional refinement programs, it is more efficient and easier to use. It is designed for modern high throughput diffractometers and is capable of processing large volume of data. SrRietveld currently makes use of conventional Rietveld refinement engines, such as GSAS and FullProf. It is built to automate and extend the functions of those engines in a flexible and uniform way so that new refinement engines can be incorporated easily as they become available. SrRietveld is an open source software. 1 2 1 1,3 1 1 1,3 1 S-300 Effect of ILE274 Mutation on the Structure and Function of Catalase HPII of Escherichia coli Vikash Jha, Peter Loewen University of Manitoba, Winnipeg, Canada Catalase or hydroperoxidase HPII of Escherichia coli is the largest known catalase, a class of enzyme that degrades hydrogen peroxide (H2O2) with a high turnover rate, attributed to the presence of more than one channel leading from the molecular surface to the active site heme. The channel approaching the active site perpendicular to the plane of heme has been considered as the main channel for the ingress of substrate H2O2 and has been the focus of several studies. The second channel, which approaches the heme laterally, has not been investigated in as much detail. Ile274 is located at the entrance to the lateral channel in close proximity to the vinyl group of ring I of heme. This study investigates the effect of Ile mutations on the structure and activity of catalase HPII. Site directed mutagenesis of the katE gene of E.coli was used to change Ile274 to Gly, Ala, Val, Phe, Ser, and Cys. The Ile274Gly and Ile274Ala variants exhibit 80% and 60% reduction in activity, respectively, whereas the Ile274Val variant retained 70% of wild type activity. The Ile274Phe mutation and most surprisingly the Ile274Ser mutation interfered with the folding of the protein such that no variant protein accumulated. The results indicated that the size and the hydrophobicity of the residue at this location are important determinants of enzyme activity and protein folding. The Ile274Cys variant folded correctly but retained only 40% activity as compared to wild type. The heme of the Ile274Cys variant could not be extracted by acetone-HCl suggesting covalent cross-linking to the heme and this was confirmed by mass spectrometry and its unreactivity with the thiol reactive reagent DTNB. Crystal of the four variants including Ile274Gly, Ile274Ala, Ile274Val, and Ile274Cys were obtained by hanging drop vapour diffusion method and crystal structures have been determined at ~1.6Å using X-ray technique. Two significant changes in the structures of the variants compared to the native enzyme include the heme being present in two orientations and the presence of an oxoferryl species that was sensitive to X-irradiation. S-301 Fine Tuning the Activity of Liver Receptor Homologue -1, an Orphan Nuclear Receptor Manish Pathak, Eric Ortlund Emory University School of Medicine, Atlanta, Ga, United States Liver Receptor Homologue-1 (LRH-1), an orphan nuclear receptor, center to the breast and colon cancer development has also been implicated in Diabetes, Obesity, Bileacid homeostasis and Steroidogenesis. Being at the center of multiple pathways raises the pharmaceutical importance of the ligand on the consequent impact. Unlike human Lrh-1 Ligand Binding Domain (hLrh-1), mouse LRH-1 (mLrh-1) and Drosophila ortholog Ftz-F1 use distinct strategy to abolish ligand binding to stay constitutive. While, Ftz-f1 redirects helix H6 into its own pocket, mLRH-1 closes the pocket mouth. Borrowing six residues from mLrh-1 mouth into hLrh-1 converted it into an unliganded & unrecruited form akin to mLRH-1 shown by x-ray crystal structure, which retained the ability to recruit coactivator in vitro. The change induces a series of conformational changes in the Lrh-1 mouth that accommodates the phosphate head group. Here we show the ligand modulated coactivator recruitment by Lrh-1. Apparently constitutive, phospholipid stripped hLRH-1 recruits co-activator 13.4 times weaker whereas choline enhances the recruitment suggesting a hierarchy of ligands involved. Reduced activity with liver extract prompts us to envisage a theory of fine tuning of the ligand mediated coactivator recruitment instead of binary active-inactive forms. The putative hypothesis refers that lower activity is a good activity. Identification of few ligands using mass-spectroscopy will also be explained. Moreover, change in the secondary structure content of apo LRH-1 seen by Circular Dichroism adds into the fine-tune theory from molecular dynamics point of view signifying controlled execution highly imperative. A diverse comparative study with other nuclear receptors highlight the versatility associated with LRH-1 and its multifaceted role in cellular system at different stages of development. S-302 Toward multi-sample data collection for macromolecular crystals: frozen crystal nonisomorphism Rita Giordano, Sean McSweeney, Alexander Popov ESRF, Grenoble, France The amount of diffraction data that can be obtained from a single protein crystal, is limited by the radiation damage to the sample and this effect cannot be avoided. In those cases where several crystals of the same type are available, the result of a structural study may, potentially, be improved by using all crystals. This experimental method is especially applicable to a set of micro crystals. In general less data can be obtained from a small crystal, before significant radiation damage occurs because the diffracting volume is lower. In order to gain from the use of multiple-crystal data collection strategies, the experiment has to be properly constructed. There are no well developed protocols for organizing this kind of measurement. The challenging task is the development of a new crystal ranking method that will be based on the determination of isomorphism between crystals. The experiments have been carried out on the beam line ID 23 EH-1 at ESRF, where 46 data set of cubic (space group I213) Zn-free bovine pancreatic insulin were collected to 1.5 Å. The data sets were processed using XDS (Version 30 January 2009) and the diffraction intensities were put on a common scale using the program XSCALE (Version 30 January 2009). One arbitrary data set was used as a reference for XDS; this reference data set has a completeness 99.4%, R factor 3.1% and I/sigmaI 41.75 (value calculated using XDS). Using the statistical computing program R (http://www.r-project.org/) a multivariate statistical analysis was executed. A hierarchical cluster analysis of the matrix of the correlation coefficients of the scaled intensities was performed in order to select the best data sets collected. This analysis revealed two principal clusters of insulin datasets. Additionally, the principal component analysis (PCA) was used to identify patterns in the data, to highlight similarity and differences between data sets. Other statistical methods were applied, for example, distance matrix analysis to differentiate the two clusters of insulin datasets. S-304 X-ray-induced deterioration of disulfide bridges at atomic resolution Tatiana Petrova , Stephan L. Ginell , Andre Mitschler , Youngchang Kim , Vladimir Y. 2 1 3 3 Lunin , Grazyna Joachimiak , Alexandra Cousido-Siah , Isabelle Hazemann , Alberto 3 1 1 Podjarny , Krzysztof Lazarski , Andrzej Joachimiak 1 1,2 1 3 1 Argonne National Laboratory, Argonne, IL, United States, Institute of Mathematical Problems of Biology, Russian Academy of Sciences, Pushchino, Russian Federation, 3 Département de Biologie Structurale et Génomique, IGBMC, CNRS, ULP,INSERM, Illkirch, France Overall and site-specific X-ray-induced damage to porcine pancreatic elastase was studied at atomic resolution at temperatures 100K and 15K. The experiments confirmed that the irradiation causes the small movement of protein domains and bound water molecules in protein crystals. These structural changes occur not only at 100K but also at as low as 15K. An investigation of the deterioration of disulfide bridges demonstrated that: (i) a decrease in γ the occupancy of S atoms and the appearance of new cysteine rotamers occur γ simultaneously; (ii) the occupancy decrease is observed for all S atoms, while new rotamers arise for some of cysteine residues; the appearance of new conformations correlates with the accessibility to solvent (iii) the sum of the occupancies of the initial and new conformations of a cysteine residue is approximately equal to the occupancy of the second cysteine residue in 7 the bridge; (iv) the most pronounced changes occur at absorbed doses below 1.4*10 Gy; with only small changes occurring at higher doses. The comparison of the radiation-induced changes in an elastase crystal at 100 and 15K suggested that the dose needed to induce the deterioration of disulfide bonds and atomic displacements at 15K as those seen at 100K is two times higher. 2 S-306 Structural Study on Metal Aluminium Amides M[Al(NH2)4]x (M = Li, Na, K, Mg, Ca; x = 1, 2) Masami Tsubota , Taisuke Ono , Keiji Shimoda , Takayuki Ichikawa , Yoshitsugu Kojima IAMR, Hiroshima University, Higashi-Hiroshima, Japan, Higashi-Hiroshima, Japan 1 2 1 2 1 1,2 1,2 ADSM, Hiroshima University, The search for alternative fuel is an urgent problem to be solved. One of the forerunners is hydrogen. In these days, much interest has been focused on the hydrogen storage materials composed of light elements, so-called chemical hydride. A composite technique is quite powerful to improve gas desorption properties for chemical hydrides. Recently, Janot et al. focused on lithium aluminium amide LiAl(NH2)4 and reported that the composite of LiH and LiAl(NH2)4 released more than 5 mass% H2 below 130 ° Thermal decomposition pathway of C. the composite was also proposed. However, the composite become amorphous during decomposition and the detailed reaction products are still unclear. Therefore, it is needed to clarify the NH3 desorption mechanism of pristine LiAl(NH2)4 for better understanding the complex thermal reaction of the composite from the structural point of view. In this study, we have investigated the detailed structural properties of the decomposition products by using in situ synchrotron X-ray diffraction and X-ray total scattering techniques as well as the thermal gas desorption properties by thermogravimetry-mass spectroscopy. Especially, the thermal decomposition pathway of M = Li had been re-examined. M[Al(NH2)4]x was synthesized by milling the raw materials in liquid NH3. From the results of synchrotron radiation X-ray diffraction, it was found that LiAl(NH2)4, NaAl(NH2)4, KAl(NH2)4, Mg[Al(NH2)4]2, and Ca[Al(NH2)4]2 could be indexed with single phases with monoclinic (a = 9.50 Å, b = 7.37 Å, c = 7.42 Å, = 90.1 ), monoclinic (a = 13.24 Å, b = 6.05 Å, c = 7.34 Å, and = 94.0 ), orthorhombic (a = 11.36 Å, b = 8.85 Å, c = 6.15 Å), hexagonal (a = 12.10 Å, c = 7.95 Å), and orthorhombic (a = 12.29 Å, b = 6.45 Å, c = 6.44 Å) unit cells, respectively. For M = Li, the results of high temperature in situ X-ray diffraction measurements showed that LiAl(NH2)4 became amorphous phase with NH3 desorption above 135 ° and the results of C PDF showed that a tetrahedral AlN4 unit was kept during decomposition. The decomposition mechanism will be described. Acknowledgement This work was partially supported by NEDO under “Advanced Fundamental Research Project on Hydrogen Storage Materials”. S-308 Structural Determination of Synthetic Intermediates of Disubstituted Amino Acids – Critical Building Blocks in the Design of Inhibitors of Amyloid Beta Aggregation Gregory McCandless, Frank Fronczek Department of Chemistry, Louisiana State University, Baton Rouge, LA, United States Peptide aggregation is a common theme among major human disorders, such as Alzheimer’s disease and Type II diabetes. The aromatic amino acids (phenylalanine and tyrosine) within the peptide sequence of amyloid beta (linked to Alzheimer’s disease) and islet amyloid polypeptide (associated with Type II diabetes) are believed to be the key aggregating initiators via π -stacking. This aggregation process can be inhibited by using short peptide mimics that will bind via self-recognition and block subsequent bonding using sterically bulky side chains. Peptide inhibitors containing the disubtituted amino acid, dibenzyl glycine, have been very effective (even in sub-stoichiometric amounts) in previously published in vitro results involving aggregation studies with amyloid beta. However, the low yielding dibenzylation step with benzyl bromide in the synthesis of this unnatural amino acid has been disheartening – it is the first step of a series of four reaction steps. Nonetheless, a new disubstitution strategy was established with moderate increases in product yield using halogen exchange (similar to the Finkelstein Reaction) and new analogues were developed using para electron donating substituents to help stabilize a positive charge on the carbon undergoing nucleophilic attack. Also, a different synthetic mechanism has been discovered that undergoes a radical disubtitution reaction using poor halide leaving groups and para electron withdrawing substituents. Single crystal X-ray diffraction results confirmed the desired synthetic intermediates for the low yielding dibenzylation step as well as the competing substitution reaction pathways (SN2 or SN1 vs. SRN1) and types of benzylation (C-benzylation vs. O- benzylation). S-310 Structural Biology of Rift Valley Fever Virus Nucleoprotein Donald Raymond, Mary Piper, Sonja Gerrard, Janet Smith University of Michigan, Ann Arbor, MI, United States Rift Valley fever virus (genus Phlebovirus, family Bunyaviridae) is an arthropod-borne pathogen endemic to sub-Saharan Africa that has spread to North Africa and the Arabian peninsula. The virus infects livestock and humans and is primarily transmitted by infected mosquitoes. The enveloped virus has a segmented negative-sense RNA genome that is completely encapsidated by a nucleoprotein (N). We developed a procedure to separate the RNA from the recombinant N (recN) by denaturation, resulting in predominantly monomeric refolded N with ~10% existing as a dimer. The approximate number of RNA nucleotides in the nucleocapsid complex was determined by extracting the RNA from the natively purified N-RNA complex. Binding affinity and stoichiometry were determined using a reconstituted recombinant N-RNA complex. Native crystals of refolded N diffracted to 1.9 Å and the structure was solved using a 2.4-Å twowavelength MAD dataset from a crystal of SeMet N. N crystallized as a symmetric dimer in which the N-terminus and an internal loop-helix are engaged in the subunit interface. The compact subunit consists of two helical lobes, each with a novel fold. Potential RNA-binding sites on opposite sides of the dimer were identified on surfaces that are uniformly electropositive among the highly conserved phlebovirus N. The fold, dimeric organization, lack of an electropositive cleft and EM micrographs of viral and reconstituted RNPs differ substantially from N of other negative-sense RNA virus families, indicative of a novel RNA encapsidation strategy. Supported by NIH grant P01-AI055672 to JLS and an NIH Biophysics Training Grant to DDR. S-312 Syntheses and Crystal Structures of Some Solid-State Uranium Compounds George N. Oh Northwestern Univ., Evanston, IL, United States In our continuing efforts to discover new solid-state uranium compounds and characterize their physical properties we have recently examined compounds of the type A/U/M/Q, where A is an alkali metal, M is Pd or Pt, and Q is S or Se. No previous work appears to have been done in this area. From high-temperature reactions we have obtained two distinct compositions, namely A2UM3Q6 and A2U6M4Q17, as deduced from single-crystal X-ray diffraction determinations. A single reaction can yield both compositions.. The A2UM3Q6 compounds crystallize in the NaBa2Cu3O6 structure type. In this structure the M atoms are coordinated in a square-planar manner by four Q atoms and these MQ4 units edge share to form hexagons. The A2U6M4Q17 compounds crystallize in a new structure type that consists of a network of MQ4 square-planar units, UQ7 and UQ8 units, as well as ordered AQ9 units. Rb2U6Pt4Se17, as opposed to Rb2U6Pd4Se17, differs structurally in that the Rb atom is disordered over two sites. S-314 Growth of large Scandium substituted Barium hexaferrite single crystals BaScxFe12xO19 with small structure defects Juergen Kraeusslich , Carsten Dubs , Ortrud Wehrhan , Peter Goernert 1 1 2 1 2 University, Jena, Germany, INNOVENT, Jena, Germany 2 Due to its outstanding magnetic properties, M-type hexagonal ferrites such as Barium hexaferrite single crystals (BaFe12O19) or with still improved characteristics Scandium substituted Barium hexaferrite (BaScxFe12-xO19) are a very suitable basic material for highfrequency filter components used in the 40 to 100 GHz range of modern microwave measurement techniques. The growth of large Scandium substituted Barium hexaferrite single crystals up to 2 cm edge length had been succeeded using the Top Seeded Solution Growth (TSSG) technique (Fig. 1). Away from the seed growth sectors emerge and form larger regions y c with nearly perfect crystal structure. The high resolution X-ray transmission topography (Fig. 2), carried out at the synchrotron facility in Grenoble (ESRF), had only revealed a few dislocations and growth striations due to the marginal fluctuation of stoichiometric composition. 10 mm a1 Fig. 1: Large Ba(ScFe)12O19 single crystal grown by Top Seeded Solution Growth (TSSG) technique Fig. 2: High resolution X-ray transmission topograph of a ccut Sc substituted Ba hexaferrite single crystal slice. The shown growth sector is almost perfectly, only few dislocations and growth striations are seen. g - X-ray diffraction vector ┴ (1-10 0) g growth sector 2 mm S-316 Structure-Properties Relationship experimental and Theoretical study 1,2 2 along the (Co1-xNix)Al2O4 1 Spinel 2 Series: An Delphine Gout , Gregory MacDougall , Thomas Brueckel , David Mandrus 1 2 JCNS, Oak Ridge, TN, United States, ORNL, Oak Ridge, TN, United States The Spinel structure is one of the most common structural arrangements and mineral phases on earth. We have recently reinvestigated the (Co1-xNix)Al2O4 Spinel series using both neutron and X-ray powder diffraction. We have refined the crystal structures, atomic distributions of five different Co1-xNixAl2O4 spinel compounds for x=0, 0.25, 0.5, 0.75 and 1 and the refinements indicate specific site distributions between Ni and Co in the octahedral and tetrahedral sites. Our goal was to understand the chemical influence on the magnetic and optical properties. Theoretical calculations as well as optical measurements have been performed to understand the structure-properties relationship in the spinel series. S-318 “Unpublishable” Data: Does My R-factor Look Big in This? Amber L. Thompson, David J. Watkin Chemical Crystallography, Oxford, United Kingdom One of the most common questions asked by synthetic chemists is to crystallographers is, “Is the R-factor less than 5%?” Although often a good indicator of the quality of the data and refinement, a bad structure may have a low R-factor and a good structure may have a high Rfactor. Thus this (and other indicators) can condemn structures to languish unpublished, while adding conviction to incorrect results (see 70 publications in Acta Crystallographica Section E by Zhong and Liu from Jinggangshan University, China for examples). There is no question that in an ideal world every structure would be perfect, but we don’ t all work with perfect crystals all of the time. Recollecting data to improve these statistics consumes instrument time, samples and manpower as well as potentially delaying publication of results. In cases where the structure determination is part of a package of analytical techniques, a cost-benefit analysis may show that this is a poor use of resources if it makes no difference to the conclusions drawn. So, what governs whether a structure is correct and publishable? We believe that this depends on the context, i.e. the required chemical information coupled with the crystallographic “problems”. For example, if the starting materials are known, a chemically sensible structure that agrees with *ALL* the available data (including bulk analysis techniques like NMR) should be reported in the literature, even if there are crystallographic difficulties. Based on a clearly stated "fitness for purpose", very few structures become “unpublishable”; indeed, even where there is an unidentified problem giving a poor refinement, a partial solution may be useful corroborative evidence when taken with other results. A selection of examples will be presented including good structures with high R-indices; bad structures with low R-indices; interesting structures published with an unknown spacegroup; published structures with exceptionally poor data, and structures with other, sometimes unidentified difficulties. S-320 The X-ray Beam Crystallography. and Biological Crystal Visualization for Macromolecular K. J. Gofron, M. Molitsky, R. W. Alkire, N. E. C. Duke, A. Joachimiak Argonne National Laboratory, Argonne, IL, United States The SBC on-axis visualization system allows viewing of X-ray beam and biological crystal from X-ray beam direction, and without parallax distortion. The system was constructed using non-dispersive optics: a long working distance Maksutov–Cassagrain reflective microscope, and right angle (45º) mirror. This on-axis geometry allows crystal visualization during diffraction data collection with full Kappa geometry. An x-ray beam and biological crystal imaging system during data collection has been developed. The direct X-ray beam uses X-ray excited ultraviolet (UV) fluorescence. The highenergy radiation such as X-ray and Middle UV (MUV) radiation excite “visible” light luminescence from biological materials, which can be imaged with CCD cameras. The fluorescence from biological crystals is primarily emitted as near UV (NUV) wavelengths between 300-360 nm depending on a biological material and surrounding environment. We demonstrate detection of biological crystal location using X-ray excited UV fluorescence. We discuss techniques for biological crystal location using intrinsic X-ray excited and MUV excited, UV fluorescence from biological crystals. The X-ray beam can be characterized using a scintillator (phosphor or a single crystal) that converts X-ray photons into visible light photons, which can be imaged using SBC on-axis optics. The X-ray penetration is dependent on the composition of the scintillator (especially effective Z) and X-ray energy. Several scintillators have been used to visualize X-ray beams. Here we compare CdWO4, PbWO4, Bi4Ge3O12, Y3Al5O12:Ce (YAG), and Gd2O2S:Tb (phosphor). The synchrotron X-ray beam profile studies were done using on-axis and off onaxis imaging. We determined that scintillators made of CdWO4 and similar high-Z single crystal materials are best suited for the energy range (7-20 keV) and are most suitable for beam visualization for macromolecular crystallography applications. These scintillators show excellent absorption, optical, and mechanical properties. This work was supported by the U.S. Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357. S-322 THE STRUCTURE OF THE MUSCLE GROWTH INHIBITOR MYOSTATIN BOUND TO FOLLISTATIN 288: INSIGHTS INTO RECEPTOR UTILIZATION AND HEPARIN BINDING JENNIFER CASH , CARLIS REJON , ALEXANDRA MCPHERRON , DANIEL BERNARD , 1 THOMAS THOMPSON UNIVERSITY OF CINCINNATI, CINCINNATI, OH, UNITED STATES, MCGILL 3 UNIVERSITY, MONTREAL, QC, CANADA, NATIONAL INSTITUTES OF HEALTH, BETHESDA, MD, UNITED STATES Myostatin is a member of the transforming growth factor- (TGF- ) family and a strong negative regulator of muscle growth. Here, we present the crystal structure of myostatin in complex with the antagonist follistatin 288 (Fst288). We find that the prehelix region of myostatin very closely resembles that of TGF- class members and that this region alone can confer signaling through the non-canonical type I receptor Alk5. Furthermore, the N-terminal domain of Fst288 undergoes conformational rearrangements to bind myostatin. Additionally, a unique continuous electropositive surface is created when myostatin binds Fst288, which significantly increases the affinity for heparin. This translates into stronger interactions with the cell surface and enhanced myostatin degradation in the presence of either Fst288 or Fst315. Overall, we have identified several characteristics unique to myostatin that will be paramount to the rational design of myostatin inhibitors that could be used in the treatment of muscle-wasting disorders. 1 2 1 2 3 2 S-324 Structure of a leucine-rich repeat domain in the NLR family. Minsun Hong, Sung-Il Yoon, Ian Wilson The scripps research institutue, La Jolla, CA 92037, United States Host pattern recognition molecules (PRMs) activate the innate immune system by recognizing conserved microbial associated molecular patterns (MAMPs) and dangerassociated molecular patterns (DAMPs). TLRs (Toll-like receptors) represent a class of membrane-spanning PRMs that have been extensively studied in the past decade. NOD-like receptors (NLRs, Nucleotide-binging domain, Leucine-Rich repeat containing protein) have recently emerged as a second family of PRMs that are located intracellularly and detect various MAMPs and DAMPs inside the host cells. Approximately 20 NLR proteins have been found in the mammalian genome and all are characterized by three distinct domains: an Nterminal protein-protein interaction domain, central NOD domain and C-terminal LRR. Mutations in NLR genes have been associated with complex chronic inflammatory barrier diseases (e.g. Crohn's disease, bronchial asthma). However, understanding of NLRs in a molecular level has been limited due to lack of biophysical and structural knowledge about NLR proteins and their ligands since the various expression systems have failed to provide enough materials of recombinant proteins for biochemical characterization. To answer questions regarding how NLRs interact with their ligands and initiate signaling, we have been pursuing x-ray crystallographic studies on NLR members. We have successfully expressed and determined the crystal structure of an NLRLRR protein which encodes a signature LRR domain in NLRs. The three-dimensional structure of the NLR-LRR and its novel insights into other NLR, as well as other LRR protein, families will be discussed. S-326 Recombinant techniques for the production of protein-protein complexes for structural characterization Gyorgy Babnigg, Robert Jedrzejczak, Boguslav Nocek, Adam Stein, William Eschenfeld, Lance Bigelow, Changsoo Chang, Gekleng Chhor, Marianne Cuff, Yao Fan, Grazyna Joachimiak, Youngchang Kim, Hui Li, Jurek Osipiuk, Ella Rakowski, Kemin Tan, Christine Tesar, Alicia Weger, Ruiying Wu, Andrzej Joachimiak MCSG, Argonne National Laboratory, Argonne, IL, United States X-ray crystallography provides an unparalleled insight into the functions of proteins and became a method of choice for the understanding biology at the atomic level. While proteinprotein complexes perform most functions in cells, far fewer structures are available of them than those of monomers or homooligomers. Since a large fraction of cellular heterooligomeric complexes are stable, they can be directly purified from their native host for structure determination. However, this approach is feasible for abundant protein-protein complexes. Less abundant complexes can be expressed by recombinant techniques and reconstituting complexes, or by co-expressing the interacting partners using a bicistronic vector. These techniques however are neither compatible with high-throughput (HTP) operations, nor are they economical. We have recently developed a technique amenable to HTP operation using a standard Ligation Independent Protocol (LIC). Target selection strategies are based on existing interaction data generated by experimental methods and on bioinformatics analyses. A bioinformatics pipeline was developed to identify a set of 384 protein-protein complexes from the MCSG reagent genomes utilizing the IrefIndex protein interaction database and the MicrobesOnline resource. We employed two HTP cloning strategies: the co-expression of the interacting partners as an operon (‘Operon-strategy’), and as a salvage pathway, as a bicistronic cassette (‘Eps-RBS-fusion strategy’). We are able to co-express proteins from up to three genes using this approach. We have processed more than 50 complexes in large-scale purification and obtained crystals for more than 20 of them. Structures of 3-oxoadipate coAtransferase and molybdopterin converting factor from Helicobacter pylori have been determined. We demonstrate that our target selection strategy combined with the experimental methods can lead to the successful HTP and low cost structure determination of proteinprotein complexes. This work was supported by National Institutes of Health Grant GM074942 and by the U.S. Department of Energy, Office of Biological and Environmental Research, under contract DEAC02-06CH11357 S-328 Determining the 2.2 Å Structure of Human Notch NRR1 Bound to the Fab Fragment of an Antagonistic Antibody Gladys de Leon, Chris Seibel, Sarah Hymowitz Genentech Inc, South San Francisco, United States The Notch signaling pathway is a key component in mammalian cell fate and growth. Aberrant signaling through each receptor has been linked to numerous diseases, particularly cancer, making the Notch family a compelling target for new drugs. To better elucidate the discrete functions of the individual Notch receptors, phage display technology was used to generate anti-Notch1 and anti-Notch2 inhibitory antibodies. To understand the molecular basis of the specificity and inhibitory mechanism of these antibodies, we determined the 2.2 Å crystal structure of the Fab fragment of the anti-Notch1 antibody bound to the Notch1 Negative Regulatory Repeat (hereafter referred to as NRR1). Initial non-single blade-like crystals were readily obtained with commercial screens. Using synchrotron radiation sources we collected a data set with a nominal resolution of 3.5 Å, but with significant anisotropy and high Rsym values. Successive rounds of microseeding led to crystals with improved 3dimensionality and more uniform composition. A 2.2 Å data set was collected using sychrotron radiation. Using Phaser, we found a solution with two complexes forming the asymmetric unit with parallel orientation of the two complexes. The refined structure revealed that NRR1 forms a compact structure very similar to that of human Notch2 or NRR1 with 2+ three Ca ion-binding LNR (Lin-Notch Repeat) modules wrapped around the core HD domain. The structure reveals that the apparent effect of Fab binding is to stabilize the LNRHD interactions such that a critical cleavage site is not accessible, thus keeping the Notch1 signaling cascade quiescent. This hypothesis is supported by the observation that the Fab does not directly occlude the processing site, but instead binds at the interface between the HD module, LNR1 and LNR2. S-330 Structural Basis for Recognition of Diubiquitins by NEMO Yu-Chih Lo , Su-Chang Lin , Carla C. Rospigliosi , Dietrich B. Conze , Chuan-Jin Wu , 2 1 1 Jonathan D. Ashwell , David Eliezer , Hao Wu Weil Cornell Medical College, New York, NY, United States, National Cancer Inst. NIH, Bethesda, MD, United States NEMO is the regulatory subunit of the IkappaB kinase (IKK) in NF-kappaB activation, and its CC2-LZ region interacts with Lys63 (K63)-linked polyubiquitin to recruit IKK to receptor signaling complexes. In vitro, CC2-LZ also interacts with tandem diubiquitin. Here we report the crystal structure of CC2-LZ with two dimeric coiled coils representing CC2 and LZ, respectively. Surprisingly, mutagenesis and nuclear magnetic resonance experiments reveal that the binding sites for diubiquitins at LZ are composites of both chains and that each ubiquitin in diubiquitins interacts with symmetrical NEMO asymmetrically. For tandem diubiquitin, the first ubiquitin uses the conserved hydrophobic patch and the C-terminal tail, while the second ubiquitin uses an adjacent surface patch. For K63-linked diubiquitin, the proximal ubiquitin uses its conserved hydrophobic patch, while the distal ubiquitin mostly employs the C-terminal arm including the K63 linkage residue. These studies uncover the energetics and geometry for mutual recognition of NEMO and diubiquitins. 1 2 1 1 1 2 2 S-332 X-ray Structural Studies of Four Symmetrical and Unsymmetrical Nonlinear Optical Organic Dyes Paul Tongwa, Andrii Gerasov, Artem Masunov, Olga Przhonska, Eric Van Stryland, Tatiana Timofeeva New Mexico Highlands University, Las vegas, NM, United States We discuss the structural characterization of four linear and nonlinear dyes, including the effects of conjugation length and terminal groups. Understanding the relations between molecular structure and nonlinear optical (NLO) properties in organic materials has been of interest for many years. We report the single crystal X-ray diffraction studies of some anionic symmetrical A - π -A, (I, II, IV), and neutral asymmetrical D - π - A, (II) cyanine dyes. The extended π -electron delocalizations/excitations that occur in these and other highly conjugated and polycyclic organic molecules is the basic origin of their observed spectral properties. Bond length alternation values confirm π - conjugations in these materials. Spectral studies leading to large two-photon absorption (2PA) and excited state absorption (ESA) cross-sections are also reported. SP.02 Uranium and Neptunium Solid-State Crystallography, and Characterization. James Ibers Northwestern Unvi., Evanston, IL, United States The structural chemistry of the lighter actinides, though to a reasonable extent accessible, has been neglected. This talk with discuss some aspects of our recent work on solid-state uranium and neptunium chalcogenides and pnictides. Safety issues, problems with syntheses, crystallographic problems, and uncertainties in complete characterization will be illustrated for systems ranging from the cubic A5Cu12U2S15 compounds to the AAn2Q6 compounds, where A = alkali metal; An = U or Np; Q = S or Se. Some structural relationships will be illustrated for systems ranging from the AnCuOP compounds to neptunium thiophosphates. Compounds: Adventures in Syntheses, *Supported by U. S. Department of Energy, Basic Energy Sciences, Chemical Sciences, Biosciences, and Geosciences Division and Division of Materials Sciences and Engineering Grant ER-15522. TR.01.1 The USC-BNL Collaboration: 25 Years of Metal Hydride Structures Thomas F. Koetzle Brookhaven National Laboratory, Upton, New York, United States Beginning in 1973, and continuing for 25 years, a collaboration spearheaded by Robert Bau resulted in a series of pioneering neutron diffraction studies of transition-metal hydrides. In work carried out at the Brookhaven High Flux Beam Reactor, the USC-BNL collaboration produced in total more than 30 hydride structures, featuring among them terminal, edge- and face-bridging, and interstitial hydride ligands. A wealth of information was obtained on the bonding of hydrogen to metals. Important trends emerged including, for example, estimates of the variation of M-H distance with H coordination number. This talk will present highlights from the 25 years of metal hydride structures. A detailed review is included in Bau, R.; Drabnis, M. H. Inorg. Chim. Acta 1997, 259, 27–50. Work at BNL was supported by the United States Department of Energy Office of Basic Energy Sciences. TR.01.2 Commissioning of the Time-Resolved Laue Single-Crystal Neutron Diffractometer (TOPAZ) at the Spallation Neutron Source Xiaoping Wang, Christina M. Hoffmann, Matthew J. Frost Oak Ridge National Laboratory, Oak Ridge, United States The TOPAZ single-crystal neutron diffractometer entered the commissioning phase in September 2009. It uses multiple area detectors (48 when all installed) that are highly efficient and capable of resolving time-of-flight pulsed neutrons in microsecond time scale. This efficient use of neutron beam flux allows data collection times on the order of minutes rather than hours for many samples. After successful completion of the commissioning phase, we expect that TOPAZ will be well positioned to collect data on sub-millimeter size single crystal samples. The choices of sample environment now range from a three-circle goniometer with fixed chi-axis for data collection at ambient temperature, a cryogenic goniometer for low temperature experiments, to an option to mount a 5 Tesla cryogenic magnet for magnetic structure measurements. The instrument team is working with the SNS Sample Environment Group to expand the sample handling capabilities to accommodate the requests from the science community. During commissioning TOPAZ will be continuously tested and improved to accept friendly user proposals in Fall 2010. This research is supported by UT Battelle, LLC under Contract No. DE-AC05-00OR22725 for the U.S. Department of Energy, Office of Science. TR.01.3 Big Metals, Small Ligands: Characterization of the 15-Coordinate Complex Thorium Aminodiboranate [Th(H3BNMe2BH3)4] by Single Crystal Neutron Diffraction Paula Piccoli , Scott Daly , Arthur Schultz , Tanya Todorova , Laura Gagliardi , Gregory 2 Girolami 1 1 2 1 3 4 Argonne National Laboratory, Argonne, IL 60439, United States, University of Illinois at 3 Urbana-Champaign, Urbana, IL 61801, United States, University of Geneva, 1211 Geneva 4, 4 Switzerland, University of Minnesota, Minneapolis, MN 55455, United States The anionic borohydride ligand, [BH4] , is known to have monodentate, bidentate or tridentate binding modes, making it a useful ligand for the achievement of high coordination numbers in inorganic and organometallic complexes. Employing a metal center with a large atomic radius, such as an actinide, with smaller chelating ligands like borohydrides, increases the chance of obtaining complexes with coordination numbers higher than 12. While 14coordinate actinide complexes such as [U(BH4)4(thf)2] have been previously reported, no complex with higher coordination numbers has been able to be unambiguously structurally characterized. Due to the low sensitivity of X-rays for the detection of hydrogen atoms and the domination of the heavy metal in the scattered X-ray data, neutron diffraction data is invaluable for accurately characterizing such complexes. Reaction of ThCl4 with Na(H3BNMe2BH3) in tetrahydrofuran produces the monomeric complex [Th(H3BNMe2BH3)4] (1), a molecule for which DFT calculations predict a theoretical maximum 1 coordination number of 16 in the gas phase. Neutron diffraction data collected at the IPNS at low temperature reveal that all but one of the hydrogen atoms on the chelating aminodiboranate ligands coordinate to the thorium center, bringing its Werner coordination number to 15 in the solid state, the highest for any structurally characterized complex to date. Upon heating, complex 1 undergoes a thermal reaction to produce the sterically less crowded 14-coordinate [Th(H3BNMe2BH3)2(BH4)2]. - 2 Herein we describe the synthesis and structural characterization of complex 1, its characterization with complementary spectroscopic methods, DFT calculations and comparisons to analogous high coordination actinide complexes containing hydride or borohydride ligands. 1 S. R. Daly et al, Angew. Chem.. Int. Ed., DOI: 10.1002/anie.200905797 TR.01.4 Making the Molecular Movie: First Frames R. J. Dwayne Miller 1 1,2 2 University of Toronto, Toronto, Ontario, Canada, Center for Free Electron Laser Science and University of Hamburg, Hamburg, Germany Femtosecond Electron Diffraction has enabled atomic resolution to structural changes as they occur, essentially watching atoms move in real time -- directly observe transition states. This experiment has been referred to as "making the molecular movie" and has been previously discussed in the context of a gedanken experiment. With the recent development of femtosecond electron pulses with sufficient number density to execute single shot structure determinations, this experiment has been finally realized. A new concept in electron pulse generation was developed based on a solution to the N-body electron propagation problem involving up to 10,000 interacting electrons that has led to a new generation of extremely bright electron pulsed sources that minimizes space charge broadening effects. Previously thought intractable problems of determining t=0 and fully characterizing electron pulses on the femtosecond time scale have now been solved through the use of the laser pondermotive potential to provide a time dependent scattering source. Synchronization of electron probe and laser excitation pulses is now possible with an accuracy of 10 femtoseconds to follow even the fastest nuclear motions. The camera for the “molecular movie” is now in hand. Atomic level views of the simplest possible structural transition, melting, have been obtained for a number of systems involving both thermal and purely electronically driven atomic displacements. Optical manipulation of charge distributions and effects on interatomic forces/bonding can be directly observed. New phenomena involving cooperative phase transitions in strongly correlated electron systems have also been observed. The primitive origins of molecular cooperativity has also been discovered. These new developments will be discussed in the context of developing the necessary technology to directly observe the structure-function correlation in biomolecules -- the fundamental molecular basis of biological systems. TR.01.5 Neutron Protein Crystallography. --------Hydrogen- and Structural Biology. Nobuo Niimura Ibaraki University, Tokai-mura,Ibaraki-ken, Japan The three dimensional structure determinations of biological macromolecules such as proteins and nucleic acids by X-ray crystallography have improved our understanding of many important life processes. In many cases, these results have clearly suggested that hydrogen atoms and water molecules around proteins and nucleic acids could play a crucial role in many physiological functions. However, since it is very hard to determine the positions of hydrogen atoms in protein molecules using Xrays alone, a detailed discussion of protonation and hydration sites often involves some guesswork. In contrast, it is well known that neutron diffraction provides an experimental method of directly locating hydrogen atoms, but unfortunately, to date, there are relatively few examples of biological systems studied by single-crystal neutron crystallography since the collection of a sufficient number of Bragg reflections is a time-consuming process. And, perhaps more importantly, the requirement of large single crystals (with volumes in the range of 1 - 10 mm3) has been a serious limitation. Breakthrough technical events in the neutron macromolecular field have been the development of the neutron imaging plate (NIP), the adoption of Laue methods at reactor sources, and most recently the LANSCE time-of-flight electronic detector for neutron protein crystallography. Thus these three technical developments have allowed exploration of the main frontiers of the capability of neutron protein crystallography. The current development of "next generation" spallation neutron sources, such as the J-PARC (Japanese Proton Accelerator Research Complex) in Japan and the SNS (Spallation Neutron Source) in the USA, will enable several more powerful protein crystallographic instruments to be installed. In these new spallation sources, a gain in neutron intensity of almost two orders of magnitude is expected. At that point, the use of neutron diffraction is expected to greatly expand the field of structural biology, that is, hydrogen- and hydration-sensitive structural biology which are beyond the folding structure. The recent results of our group will be introduced in the Symposium. Hydration- Sensitive TR.01.6 Polarised-neutron Laue diffraction on a crystal containing spin-polarised hydrogen Maths Karlsson , Florian Piegsa , Ben van den Brandt , Patrick Hautle , Ton Konter , Colin 1 4 2 2 Carlile , Ted Forgan , Oliver Zimmer , Garry McIntyre European Spallation Source, Lund University, Lund, Sweden, Institut Laue-Langevin, 3 4 Grenoble, France, Paul Scherrer Institute, Villigen, Switzerland, School of Physics & Astronomy, University of Birmingham, Birmingham, United Kingdom Neutron diffraction on samples with large hydrogen content, e.g. on organometallic and protein samples, generally suffers from a strong featureless background due to strong incoherent scattering from the protons. This is particularly evident in the two-dimensional projection of Laue diffraction, a technique which is otherwise undergoing a renaissance thanks to the development of large-solid-angle image-plate detectors, as illustrated by numerous recent studies from VIVALDI [1] at the ILL by Bob Bau and colleagues [2]. Despite the strong incoherent background, the larger coherent neutron scattering length of hydrogen relative to other elements compared with the situation in X-ray diffraction more readily yields answers to specific questions on, e.g. protonation states, hydrogen positions, and dynamic disorder of hydrogen. Deuteration can be used to reduce the incoherent background, but sample growth may be difficult or even impossible, and there may be an isotopic difference in the structure. An intriguing alternative is parallel polarisation of the incident neutron beam and the hydrogen spins [3]. We have performed a proof-of-principle polarised-neutron Laue diffraction experiment on a spin-polarised single crystal of Nd-doped lanthanum magnesium nitrate hydrate (LMN), La2Mg3(NO3)12.24H2O. The experiment was carried out on the FUNSPIN beam line [4] at the continuous spallation neutron source SINQ (PSI) with the proton spins aligned by dynamic nuclear polarisation. It demonstrates that not only is the incoherent background indeed reduced but also that the intensity of the Laue reflections can be enhanced or diminished significantly to give a form of contrast variation. In the longer term, we foresee that the technique can be employed to improve substantially the poor signal-to-noise ratio in neutron Laue diffraction experiments on biological crystals. 1 2 1 2 3 3 3 [1] G.J. McIntyre et al., Physica B 385-386 (2006) 1055-1058. [2] R. Bau et al., Inorg. Chem. 43 (2004) 555-558; T. Stewart et al., Inorg. Chim. Acta 363 (2010) 562-566; and five papers in between. [3] J.B. Hayter, G.T. Jenkin & J.W. White, Phys. Rev. Lett. 33 (1974) 696-699. [4] J. Zejma et al., Nucl. Instr. and Meth. A 539 (2005) 622-639. TR.01.7 Hydrogen, Neutrons and Energy Ian Anderson Oak Ridge National Laboratory, Oak Ridge, TN, United States Hydrogen, the first element, plays a major role in a sustainable energy economy through multiple mechanisms. Neutron scattering is a well established and ideal probe for determining the structure and dynamics of hydrogen in materials and therefore remains a critical tool in support of basic energy research. This presentation will provide an overview of the range of studies being carried out using neutron scattering from hydrogen, with an emphasis on the applications to energy technology. TR.01.8 Structure and properties of (hydroxy)alkylammonium salts of flurbiprofen Carl Schwalbe , Miren Ramirez , Barbara Conway , Chris Bache , Simon Coles , Peter 3 Timmins Aston University, Birmingham, United Kingdom, University of Southampton, Southampton, 3 United Kingdom, Bristol-Myers Squibb, Moreton, Cheshire, United Kingdom Starting with the t-butylammonium salt of the anti-inflammatory drug flurbiprofen (FTbut), we have systematically increased the hydrophilicity of the cation by successively changing methyl to hydroxymethyl groups. Hydrogen bonding by the added OH groups is expected to influence the solubility and compactability of these salts. 1 2 1 1 1 1 2 Me COO F + H3N CH 3 CH 3 CH 3 + to H3N CH 2 OH CH 2 OH CH 2 OH In FTbut donation of hydrogen bonds from NH3 to COO forms ladders built out of R4 (10) rings. Substitution with one OH group to make FAmp does not change this pattern: the OH group lacks a credible hydrogen bond acceptor and is disordered. However, when a second OH group is introduced (FAmp2), the pattern changes fundamentally. Cations deploy one NH and one OH hydrogen atom in hydrogen bonds to one anion. The other OH group links intermolecularly to the first one while NH finds OCO . The third amino H atom pairs with OH 2 2 2 as acceptor to form a centrosymmetric dimer. Thereby R2 (9), R3 (9), and R2 (10) rings are formed. The Tris salt (FTris) exists in two polymorphs. Crystals of form II are well ordered with similar hydrogen bonds to those in FAmp2. Crystals of FTris form I are triclinic with Z’ = 2, the independent ions being related by pseudosymmetry and also showing disorder. Each carboxylate O atom accepts only one hydrogen bond. Whereas the twist angle between rings in the biphenyl moiety of F is 44-46° in the other structures, it is 55° and 61° here. These factors should imply increased energy for this form, yet FTrisI melts at a higher temperature than FTrisII. Despite the presence of a reasonable slip plane, FtrisI displays poor mechanical properties, producing weak compacts with troublesome elastic recovery. FTrisII has a slightly wider slip plane, and indeed strong tablets with shiny faces and excellent mechanical properties are formed. We are grateful for use of the Diamond synchrotron to collect data on FAmp and FTrisI. + - 3 TR.01.9 Structural Coordination Chemistry beyond Routine Diffraction: Single Crystal Transformations, Shape Changes and Ambiguities, and Neutron Scattering Larry R. Falvello University of Zaragoza - C.S.I.C., Department of Inorganic Chemistry and I.C.M.A., Zaragoza, Spain Diffraction analyses outside the realm of routine structure determination have been used to explore chemical and physical phenomena that would be difficult to observe either in the synthetic laboratory or in rapid, albeit high quality, x-ray structural studies. Chemical reactions within single crystals have been observed to yield products that would not be expected at the benchtop. The 1-D coordination polymer in {Cs2[Co7(citr)4(H2O)13.5]}2∙ 15H2O -(4-) [citr = citrate, C6H4O7 ] in the crystalline state undergoes a concerted reaction to give a 2-D polymer in Cs2[Co(H2O)6]{[Co6.5(citr)4(H2O)9]}2∙ 3H2O, cross-linked by an unusual Co(II) center surrounded by seven O-coordinated ligands. The experimentation needed to discover and characterize this process is described, along with potential efficiencies for future studies of this type. A different Co(II) citrate complex which forms a 2-D net in molecular crystals (4n-) (formula { -[Co(C2H6O2)(H2O)2]2[Co4(C6H4O7]4}n ), is seen to have magnetic properties that are tunable through small structural changes outside the magnetic mesh, and which would benefit from studies by "higher-throughput" neutron diffraction. Seemingly more routine structural phenomena such as the planar aqua ligand in transition metal complexes are subject to new insights through the application of high-throughput neutron diffraction and inelastic neutron scattering, as an example system shows. A final study involves trans[Ni(cyan)2(NH3)4] (cyan = cyanurate, C3H2N3O3 ), a simple paramagnet that forms molecular crystals with a second-order phase transformation that leads to a continuous molecular shape change as either pressure or temperature is varied. This system was studied by neutron diffraction to give a cogent conclusion regarding the cause of the transformation. TR.01.10 The rotational dynamics of the dihydrogen ligands in RuH 2(η -H2)2(PCyp3) as seen by neutron diffraction analysis. Alberto Albinati , Silvia Capelli , Sax Mason 1 2 1 2 2 2 University of Milan, Milan, Italy, Institut Laue-Langevin, Grenoble, France The study of the interaction of an intact H2 molecule with a transition metal center has been a very active area of research since the discovery of this type of complex by G. Kubas in 1984. The nature, the stability, and the fluxionality of many metal-dihydrogen complexes have been studied by diffraction methods, NMR, inelastic neutron scattering (INS) and theoretical calculations. Single crystal neutron diffraction has been of paramount importance in providing accurate structural parameters; however information on the dynamics of the dihydrogen ligands have been obtained mainly by INS and NMR. The analysis of the temperature dependence of the Anisotropic Displacement Parameters allows dynamic information to be extracted from diffraction data, that are useful to distinguish between molecular flexibility and disorder or between kinematic and dynamic interpretation of structural features. We have collected very accurate single crystal neutron diffraction data on the complex 2 RuH2(η -H2)2(PCyp3) (1) at 20, 60 and 100K on the ILL D19 diffractometer and analyzed the data sets using a physical model (2) that explicitly accounts for the effects of temperature and describes the structure of the complex (at the three temperatures) with a unique set of parameters: the normal mode frequencies and associated wave vectors that describe the rigid-body librations and translations of the complex core, and two additional rotations of the dihydrogen moieties. This analysis yielded values for the rotational frequencies of the H2 -1 -1 groups of 104(17) cm and 170(40) cm , in good agreement with previously determined INS values and theoretical calculations. (1) M. Grellier, L. Vendier, B. Chaudret, A. Albinati, S. Rizzato, S. Mason, S. Sabo-Etienne J. Am. Chem. Soc. (2005) 127, 17592. (2) H.B. Bürgi, S. C. Capelli Acta Cryst. (2000) A56, 403. TR.01.11 Neutron Diffraction Studies of Metal-Hydrides Muhammed Yousufuddin University of Texas at Arlington, Center for Nanostructured Materials, Arlington, TX, United States Neutron diffraction is the method of choice when locating chemically interesting hydrogen 1 atoms, particularly in systems containing a heavy metal. In this talk, two fascinating complexes containing hydrogen atoms that were unambiguously located with neutron diffraction will be highlighted. The first complex, OsH3Cl(PPh3)3, is a Kubas-type complex 2 containing a rare elongated dihydrogen ligand. Results from the neutron diffraction study showed that the dihydrogen ligand demonstrated an H…H distance of 1.48(2) Å. In the second study, a four-coordinate hydrogen atom was located and measured for the first time 3 using neutron diffraction. Results from this study showed that the hydrogen atom occupied the interstitial space of a tetrahedral Yttrium (Y4) cluster. This work was performed in the laboratory of Professor Robert Bau at the University of Southern California for completion of a doctoral dissertation and will be presented at the transaction symposium being held in Prof. Bau’ s memory and honor. 1. M. Yousufuddin and R. Bau “Neutron Diffraction” in Applications of Physical Methods to Inorganic and Bioinorganic Chemistry, edited by Robert A. Scott and Charles M. Lukehart, Chichester, UK; John Wiley & Sons Ltd. ISBN 978-0470-032176, 2007, 271. 2. M. Yousufuddin, T. Wen, S. A. Mason, G. J. McIntyre, G. Jia, R. Bau Angew. Chem. Int. Ed. 2005, 44, 7227. 3. M. Yousufuddin, M. Gutmann, J. Baldamus, O. Tardif, Z. Hou, S. A. Mason, G. J. McIntytre, R. Bau J. Amer. Chem. Soc., 2008, 130, 3888. TR.01.12 Molecule-based Magnets: New Materials, Chemistry, Physics, and Crystals for this Millennium Joel Miller University of Utah, Salt Lake City/UT, United States Molecule-based materials exhibiting the technologically important property of bulk magnetism have been prepared and studied in collaboration with many research groups worldwide frequently exhibit supramolecular extended 3-D structures. These magnets are prepared via conventional organic synthetic chemistry methodologies, but unlike classical inorganic-based magnets do not require high-temperature metallurgical processing. Furthermore, these magnets are frequently soluble in conventional solvents (e. g., toluene, dichloromethane, acetonitrile, THF) and have saturation magnetizations more than twice that of iron metal on a mole basis, as well as in some cases coercive fields exceeding that of all commercial magnets (e.g., Co5Sm). Also several magnets with critical temperatures (Tc) exceeding room temperature have been prepared. In addition to an overview of magnetic behavior, numerous examples of structurally characterized magnets made from molecules will be presented. Our groups has discovered 7 families of molecule-based magnets, mostly organic-based, and have significantly contributed to an eight family based upon the Prussian blue structure. Four o examples magnetically order above room temperature and as high at 127 C. These will III III include [M (C5Me5)2][A], [Mn (porphyrin)][A] (A = cyanocarbon etc. electron acceptors) as well as M[TCNE]x, which for M = V is a room temperature magnet that can be fabricated as a thin film magnet via Chemical Vapor Deposition (CVD) techniques. A newer class of magnets of [Ru2(O2CR)4]3[M(CN)6] (M = Cr, Fe; R = Me, t-Bu) composition will also discussed. For R = Me an interpenetrating, cubic (3-D) lattice forms and the magnet exhibits anomalous hysteresis, saturation magnetization, out-of-phase, "(T), AC susceptibility, and zero field cooled-field cooled temperature-dependent magnetization data. This is in contrast to R = tBu, which forms a layered (2-D) lattice. Additionally, new magnets possessing the nominal Prussian blue composition, M'[M(CN)6]x and (Cation)yM'[M(CN)6], but not the common fcc cubic Prussian blue structure will be described. Also discussed will be Prussian blue- like magnets that have a different composition, namely, (Cation)2Mn3(CN)8 and (Cation)Mn3(CN)7 compositions, whose structures and magnetic behaviors will be reported. TR.01.13 High Throughput Neutron Diffraction – New Opportunities in Hydrogen Location in Molecular and Materials Structure Chick Wilson , Paul Henry , Marc Schmidtmann , Lynne Thomas , Valeska Ting , Mark 2 Weller 1 1 3 1 1 2 University of Glasgow, Glasgow, United Kingdom, University of Southampton, 3 Southampton, United Kingdom, Helmholtz Centre Berlin (HZB), Berlin, Germany Many of the areas recently advanced in structural studies of small molecule materials rely heavily on the ability to study structures on a shorter timescale, either to examine a series of samples or to study a single sample under a range of conditions. This “high throughput” approach has only recently become feasible for neutron diffraction studies of molecular materials as a result of a substantial revolution in the provision of instrumentation with massive detector arrays for single crystal diffraction, and with very high count rates for powder diffraction. This has allowed neutron chemical crystallography to respond in a very successful fashion to modern trends in structural molecular science, extending the applications of neutron diffraction in the area of chemical crystallography and molecular materials; many of these exploit the power of neutron diffraction in determining accurately the hyrogen atom parameters in materials. This high throughput capability allows the powerful information available from neutron diffraction to be harnessed, with complementary X-ray and computational input, to tackle more of the problems at which neutron diffraction excels, particularly those involving hydrogen atom location. These include prominent examples in hydrogen bonding including both strong and weaker hydrogen bonding interactions, the location of hydrogen in inorganic systems, in complementing charge density studies and in studying materials under conditions of variable temperature and variable pressure. The potential of this new capability will be illustrated by a range of recent studies on proton migration, hydrogen atom disorder and transfer, polymorphism in molecular complexes, water location in materials, thermochromic materials, hydrogen-bonding interactions and in the location and full description of hydrogen in inorganic materials. The powerful complementary use of both X-ray and computational methods with neutron powder and single crystal diffraction in these studies will also be highlighted. 2 TR.01.14 Hydrogen and Hydration: The 15K neutron structure of W3Y single mutant rubredoxin from Pyrococcus furiosus Dean Myles , Robert Bau , I. Tsyba , Matthew Blakeley 1 1 2 2 3 2 Oak Ridge National Laboratory, Oak Ridge, TN, United States, University of Southern 3 California, Los Angeles, CA, United States, Institute Laue-Langevin, Grenoble, France Neutron crystal structures provide insights into hydrogen bonding interactions, protonation states and details of hydration states in protein and nucleic acid crystal structures that are not available from x-ray analysis alone. The iron-sulfur redox protein rubredoxin from Pyrococcus furiosus (PfRd) is stable for days in boiling water, whereas most other bacterial rubredoxins are readily denatured within minutes at 373K. Using the LADI instrument at the Insititute Laue Langevin, we collected high-resolution (1.7 Å) neutron diffraction data at 15K on a single mutant form of PfRd (W3Y-PfRd) in order to probe its temperature stability. By collecting the 3 data at 15K we illustrated the feasibility of cryo-cooling large (>1 mm ) protein crystals and hence collecting high-resolution neutron diffraction data at cryo-temperatures. Comparison of the single mutant solvent structure at 15K against the wild-type and triple mutant forms indicates that by lowering the data collection temperature we have observed a more complete picture of the hydration shells of the protein. Detailed analysis of the hydrogen bonding interactions may help explain why the triple and single mutant forms of PfRd are found to be less stable at low pH than the wild-type form. TR.01.15 The New Microporous materials: MOFs, COFs and ZIFs Michael O'Keeffe , Omar Yaghi 1 1 2 2 Arizona State University, Tempe, Arizona, United States, Angeles, California, United States University of california, Los In the last decade there has been explosive growth in the synthesis and characterization of new microporous materials particularly those known as metal-organic frameworks (MOFs), covalent organic frameworks (COFs) and zeolitic imidazolate frameworks (ZIFs). MOFs are typically neutral frameworks in which inorganic clusters (secondary building units - SBUs) are joined by organic linkers into periodic frameworks. Ideally the inorganic part has a structure which can be abstracted as a simple polygon or polyhedron. The organic component may be ditopic in which event the structure topology is that of the SBU shapes joined by links. The organic component may be polytopic and again abstracted as a polygon or polyhedron shape and the MOF topology then corresponds to two shapes joined by linkers. COFs are similar except for replacement of the inorganic component by an organic SBU. Recognition of the fact that there are just a small number of known default topologies (ideally those in which there is just one kind of link) leads to the possibility of synthesis of materials with targeted structure, including pore size and functionality - so-called reticular chemistry. ZIFs are rather different. They are metal imidazolates that generally adopt the structures of simple zeolites and other zeolite-like frameworks. Here the fuctionalization of the basic imidazole ring (e.g. as methyl- or nitro-imdazole) determines the framework topology. The new materials show unprecedented capacity for adsorption of gases such as dihydrogen, methane and carbon dioxide and clearly have great promise in emerging clean-energy technologies. TR.01.16 Beyond Single Crystal Structure Determination – Interpretation of 3D Disorder Diffuse Scattering Hans Beat Buergi , Michal Chodkiewicz , Vickie Lynch 1 2 1,2 2 3 3 Univ. of Zurich, Zurich, Switzerland, Univ. of Bern, Bern, Switzerland, Oak Ridge National Laboratory, Oak Ridge, TN, United States Single crystal structure determination from Bragg diffraction has become a largely routine operation: the process is well understood theoretically, experiments and data interpretation are automated to a large extent, as is the validation of results. The information obtained is limited, however: it is the content of the crystallographic unit cell averaged over the time of the experiment and the volume of the crystal. If the type and distribution of atoms differ between unit cells, i.e. if a crystal structure shows occupational and displacive disorder, some of the scattered intensity is lost from the Bragg peaks and distributed throughout reciprocal space as diffuse scattering. Some examples from material science will illustrate some prototypical diffuse scattering: 1D streaks, 2D planes and 3D continuous diffuse signals. The interpretation of such scattering is far from routine. Sometimes the important information can be obtained from qualitative, ad hoc arguments and some simple modeling calculations, sometimes significant computational resources are required to perform elaborate Monte Carlo modeling. Both types of interpretation will be illustrated with examples. The collaborative effort between groups at Oak Ridge National Laboratory and the University of Zürich to construct a more general tool for the interpretation of diffuse scattering will be sketched. 07.07.1 The Use of in Situ GISAXS and GIXAS Techniques at the Design of New Classes of Bond-Selective Catalytic Materials in the Sub-Nanometer and Nanometer Size Regime Stefan Vajda , Sungsik Lee , Byeongdu Lee , Soenke Seifert , Randall Winans , Yu Lei , 1 1 1 1 5 Faisal Mehmood , Larry Curtiss , Jeffrey Greeley , Michael Pellin , Luis Molina , Alessandro 4 3 3 3 Fortunelli , Kristian Sell , Viola von Oeynhausen , Karl-Heinz Meiwes-Broer , Maria Flytzani7 2 2 8 8 Stephanopoulos , Lisa Pfefferle , Gary Haller , Sonja Wyrzgol , Johannes Lercher 1 1,2 1 1 1 1 1,6 Argonne National Laboratory, Argonne, Illinois, United States, Yale University, New Haven, 3 4 Connecticut, United States, Universitaet Rostock, Rostock, Germany, IPCF-CNR, Pisa, 5 6 Italy, Universidad Valladolid, Valladolid, Spain, University of Illinois, Chicago, Illinois, United 7 8 States, Tufts University, Medford, Massachusetts, United States, Technische Universitaet Muenchen, Muenchen, Germany The elucidation of the size/composition/shape/structure and function correlation, the effect of support along with the determination of the nature of the catalytic particles under realistic reaction conditions are instrumental on the way to the design of new classes of catalytic materials. Uniform particles on technologically relevant supports are prerequisites for such studies, making size-selected clusters of few atoms to several nm in size as ideal model systems. 2 The experimental studies are based on 1) size-selected cluster deposition on technologically relevant support, 2) electron microscopy of nanoclusters, and 3) in situ synchrotron X-ray characterization of clusters under working conditions, combined with mass spectroscopy analysis of reaction products. Density functional theory calculations are used to understand the activity of clusters and the underlying reaction mechanisms. Using metal and metal-oxide based catalytic systems, in this presentation examples will be given on bridging the size gap between the sub-nm and nm size regime as well as on bridging studies of model and “real” catalysts. Select reactions will include e.g. C-H, C=C bond activation, where new highly selective and active catalyst systems performing at low temperatures were identified for dehydrogenation, epoxidation and other reactions. The applied in situ X-ray techniques proved to be instrumental at gaining fundamental insights about the properties of (sub)nanoscale matter. 07.07.2 Atomic-scale X-ray studies of redox-induced cation dynamics for oxide supported monolayer catalysts Michael Bedzyk1, Zhenxing Feng1, Chang-Yong Kim2, Jeffrey Elam3, Qing Ma1, Zhan Zhang3, Martin McBriarty1, Donald Ellis1, Peter Stair1 1 Northwestern University, Evanston, IL, United States, 2Canadian Light Source, Saskatoon, SK, Canada, 3Argonne National Laboratory, Argonne, IL, United States Metal oxides anchored to oxide supports often exhibit greater catalytic activity as monolayers than as thicker films. Understanding this phenomenon requires a chemically sensitive, atomicscale view of the interfacial processes. We use in situ X-ray standing wave (XSW) 3D atomic imaging combined with ex situ X-ray photoelectron spectroscopy (XPS) and X-ray absorption fine structure measurements to follow the redox-induced surface site exchange of cations on a single crystal oxide support as well as the concurrent changes in the oxidation states of the supported cations. This is then compared to density functional theory. As an example, we 1 follow the reversible changes during the redox cycle of a /3 ML WOX / -Fe2O3 (0001) interface grown by atomic layer deposition. The XSW measured W atomic maps and XP spectra show dramatic changes for the as-deposited, oxidized and reduced interfaces, which are explained by models that account for W incorporation at Fe sites with various coordination schemes.[1] The 3D W atomic map for each condition is measured by the summation of the XSW measured hkl Fourier components for the XRF selected W distribution.[2] This strategy was then also applied to redox-induced structural and chemical changes for the sub-ML and 2 ML VOX / -TiO2 (110) interfaces. [1] Z. Feng, C.-Y. Kim, J.W. Elam, Q. Ma, Z. Zhang, M.J. Bedzyk, J. Am. Chem. Soc. 131, 18200 (2009). [2] L. Cheng, P. Fenter, M. J. Bedzyk, N. C. Sturchio, Phys. Rev. Lett. 90, 255503 (2003). 07.07.3 Ordering and kinetics of two-dimensional self-assembly of nanoparticles at surfaces – a study by in situ and time-resolved grazing-incidence small-angle x-ray scattering Zhang Jiang , Xiao-min Lin , Michael Sprung , Suresh Narayanan , Jin Wang , Carlee 2 2 2 Ashley , Darren Dunphy , Jeffrey Brinker 1 1 1 1 1 1 Argonne National Laboratory, Argonne, IL, United States, Albuquerque, NM, United States 2 University of New Mexico, The characterization of ordering and kinetics at nanostructured surfaces and interfaces has become increasingly critical to understand and optimize the synthesis of ordered nanomaterials on nanometer length scales. This requires high-resolution in situ and timeresolved surface probes that can also be applied to various experimental environments. Grazing-incidence x-ray scattering is an ideal tool for the studies. At the Advanced Photon Source, using high-resolution small-angle x-ray scattering in grazing-incidence geometry (GISAXS), we successfully captured the growth kinetics of two-dimensional metal nanocrystal superlattices at the liquid/air interface and identified their ordering, phases and phase transitions in a quantitative manner. GISAXS was also applied to study the convective transport and the effects of non-volatile solvent during the convective assembly of twodimensional superlattices of virus-like nanoparticles. 07.07.4 Role of Solvent Content, Thickness, and Composition on Surface Morphology in Diblock Copolymer Thin Films Yan Sun , Kevin Henderson , Zhang Jiang , Joseph Strzalka , Jin Wang , Kenneth Shull 1 1,2 1 2 2 2 1 Northwestern University, Evanston, IL, United States, Argonne, IL, United States 2 Argonne National Laboratory, The phase behavior of diblock copolymer melts and solutions offers a novel means for templating nanoscale patterns and particles for a variety of applications. In a solution containing a block-selective solvent, enthalpic interactions between the non-soluble block and the solvent govern the phase behavior. In the case of poly(methyl methacrylate) (PMMA), this interaction is temperature dependent, with the disordered state attainable around 70-80C for solvent fractions > 0.7. However, in solventless conditions, phase behavior is entirely governed by the XN interaction between copolymer blocks. While the phase behavior of bulk polymer solutions and melts have been well characterized both experimentally and with selfconsistent mean field simulations, the role of confinement and surface effects in thin film equivalents have not been as extensively studied. Though recent work has focused on the effects of film thickness, controlling the solvent content in these films has received much less attention. Here, we employ a poly(tert-butyl methacrylate)-poly(methyl methacrylate) (PtBMAPMMA) diblock to study the role of solvent content, film thickness, and block copolymer composition. In bulk solutions, this diblock exhibits thermoreversible micellation in alcohols, so we have chosen butanol as our solvent of interest. Using grazing incidence small-angle xray scattering (GISAXS) and atomic force microscopy (AFM), we have investigated the phase behavior in three different diblocks under various solvent conditions up to 160C. Comparisons to bulk phase behavior and self-consistent mean field calculations are discussed. 07.07.5 Complex Epitope Engineering: A Rational Approach to Improve Protein Crystallization Victor Naumov , Nicholson W. Price , Samuel K. Handelman , John F. Hunt 1 2 2 2 2,1 2 Northeast Structural Genomics Consortium, New York, New York, United States, Columbia University, New York, New York, United States In previously published work, we have shown that the prevalence of well-ordered surface epitopes is a dominant factor controlling protein crystallization behavior (Price et al., 2008, Nature Biotech. 27:51-57). However, straightforward efforts to exploit this insight to improve protein crystallization have been frustrated by the fact that most single residue mutations lowering surface entropy compromise protein solubility. In contrast, many naturally occurring proteins have excellent solubility characteristics and nonetheless yield high quality crystals. We infer that these proteins possess more complex surface epitopes that maintain high solubility in dilute aqueous buffers while having a high propensity to mediate stable interprotein packing interactions under the low-water-activity conditions used for protein crystallization. In order to identify such epitopes, we have developed a set of new computational methods that have been applied to all crystal structures in the Protein Data Bank (PDB). The resulting analyses demonstrate that, on average, ~50% of surface residues participate in packing interactions, explaining why surface mutations generally alter crystallization behavior. They also demonstrate that protein crystals are inherently polymorphic, with the details of intermolecular packing typically varying significantly even in crystal structures with equivalent lattice symmetry and unit cell parameters. Most importantly, these analyses demonstrate that most packing epitopes are small and local in the polypeptide chain and that some sub-epitopes are highly overrepresented in crystal packing contacts compared to their frequency of occurrence on protein surfaces in the PDB. These overrepresented sub-epitopes generally interact with a wide variety of partner epitopes, suggesting that they are promising candidates to use in mutational engineering of proteins to improve their crystallization behavior. We have written software that takes a multiple sequence alignment and determines all ways in which these overrepresented sub-epitopes can be introduced into a protein in a manner consistent with the sequence profile of the corresponding protein family. These computational results and tools form the foundation of a novel approach to rational engineering of improved protein crystallization. 07.08.1 The Canadian Macromolecular Crystallography Facility at the Canadian Light Source James Gorin , Pawel Grochulski , Michel Fodje , Shaun Labiuk , Russ Berg , Peter Thorpe , 1 2 3 Shawn Carriere , Natalie Strynadka , Mirek Cygler Canadian Light Source Inc., Saskatoon, SK, Canada, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, BC, 3 Canada, National Research Council Canada Biotechnology Research Institute, Montreal, QC, Canada The Canadian Macromolecular Crystallography Facility (CMCF), which serves more than 60 protein crystallographers located across Canada, currently consists of two beamlines. The first, an insertion device beamline (08ID-1) illuminated by a small-gap in-vacuum hybrid undulator (SGU) located in the upstream half of the straight section, is capable of satisfying the requirements of the most challenging and diverse crystallographic experiments (i.e., physically small crystals with large unit cell dimensions). It has been in operation since 2006 and is producing a growing number of pdb deposits and scientific publications. More than a year ago, Mail-in Crystallography was introduced at the CMCF. It is run by a dedicated member of our team and has proven to be very successful. Up to 25% of available beamtime is reserved for commercial users. We typically observe increased activity of commercial users while the US synchrotrons are closed for maintenance. The second, a bending magnet beamline (08B1-1), has recently begun accepting users and is designed for high-throughput data collection with the capability of being remotely accessed. Together, the complementary beamlines will constitute a single facility, allowing for the screening and collection of data from a variety of projects. To date, there are in excess of 40 publications and 70 pdb depositions from 30 laboratories from Canada, the United States and the United Kingdom using CMCF beamlines. To facilitate remote access, SSRL Stanford-type automated mounting (SAM) robots have been installed for both beamlines and are undergoing final commissioning. The robot is used in combination with Uni-Pucks or cassettes which provides sufficient capacity for a shift of screening and data collection. Data collection software has been developed in-house and has similar functionality as BluIce. Features of the final software will include the automatic alignment and configuration of the beamline hardware, automatic crystal mounting and centering of crystals in the X-ray beam, automatic measurement of fluorescence spectra for MAD experiments, automatic screening and analysis of crystals in order to assess crystal quality and determine optimum parameters and strategies for data collection, automatic data collection and data processing. 1 2 1 1 1 1 1 1 07.08.2 Technological advances in synchrotron data collection from the perspective of a young crystallographer. Gina Ranieri, Richard Walter Shamrock Structures LLC, Woodridge, IL, United States Synchrotron X-ray data collection is in the midst of a technological revolution. From new software to detectors to protocols there is an unprecedented ability to collect more, higher quality data in a shorter amount of time than there ever has been in the history of the science. From hardware improvements like fast, reliable sample automounters, new detector technologies, all-in-one “microdiffractometers”, and auto-optimized focusing optics to software breakthroughs like user-friendly GUI controls, diffraction centering, and automated spiral data collection all elements are coming into place to apparently transform the discipline into a fully automated, remotely operated, push button service. The primary drivers behind this generation of technology are cost and capacity. On the plus side, the capacity added by these new capabilities has increased the access of these methods to more researchers. On the downside, we wonder what might get lost, potentially in data quality, probably in basic learning and understanding, and whether some of these new efficiencies are truly efficient. As a young professional who has traveled throughout the world for the last two years “learning on the job” how to quickly and efficiently collect high-quality X-ray data, I will discuss some of the technologies and protocols that have been developed at various facilities to attempt to deliver faster, more efficient, higher quality X-ray data. I will cite statistics from my data collection experiences at various facilities and discuss what I feel works and what does not work in the name of efficiency and quality. 07.08.3 On the front lines: Using high throughput synchrotron data collection to increase productivity downstream Virginia L. Rath Reciprocal Space Consulting. L.L.C., Oakland, CA, United States Reciprocal Space Consulting, LLC, located in Oakland, California is a contract research organization providing expertise in all aspects of structure based drug design from protein purification through crystallization experiments to data collection and structure solution. Utilizing both the Advanced Light Source in Berkeley and the Stanford Synchrotron Laboratory in Menlo Park, we logged over 2000 hours of synchrotron beam time in 2009 providing synchrotron support for academic institutions as well as Biotech and Fortune 500 pharmaceutical companies. Both ALS and SSRL continue to develop robotic and software systems to support efficient screening and data collection and these advancements have helped drive crystallography to a new level. Protein structure information is now an essential component in drug discovery from lead identification through lead optimization. With our support, customers can focus on high value downstream areas such as structural analysis of drug-protein complexes and compound design. 07.08.4 Automated Macromolecular Crystallography Facilities for Proprietary Research Elspeth Gordon European Synchrotron radiation facility, Grenoble, France The ESRF Structural biology group runs a six macromolecular crystallography (MX) beamlines and a facility for bioSAXs. This portfolio of beamlines includes three of fixed wavelength, one of which has microfocus capability. The three variable wavelength beamlines include two facilities with a relatively small focal spot size (50 x 30 m2) where long wavelength ( ~2.3 A) data collections can be performed. This suite of beamlines meets the needs of the even the most demanding experiments. To optimise use of the beamlines and to simplify support issues we have embarked upon a process of automation and standardisation. All beamlines are equipped with ESRF/EMBL developed sample changer robots and are controlled via a common, intuitive graphical user interface, mxCuBE. Within mxCuBE options including: remote access, quick realignment of the beamline; crystal visualisation and centring; sample characterisation and experimental strategy calculations; data collection; energy scans and the collection and analysis of X-ray Fluorescence (XRF) spectra. A beamline database (ISpyB) allows for real-time at-a-distance monitoring of experiments via a web interface. Up to 30% of the beamtime on the ESRF's MX beamlines is available for those users wishing to carry out proprietary research. Industrial users can either come to the ESRF and carry out their experiments themselves or, once they are familiar with the technology, control their experiment remotely from their home laboratory. The ESRF also offers a data collection service where frozen samples are shipped to the ESRF and inhouse staff carry out the data collection. The increased number of samples brought to the beamlines and the high demands on throughput and efficiency of our industrial clients have been a major driving force for technical developments on the ESRF's MX resources. This presentation will outline key features of the developments put in place at the ESRF and describe our plans for the future. 07.08.5 High throughput fragment screening- new reasons and new software Thomas Peat , Janet Newman , Vincent Fazio , Tom Caradoc-Davies , Kim Branson 1 2 1 1 1 2 3 CSIRO, Parkville, VIC, Australia, Australian Synchrotron, Clayton, VIC, Australia, Stanford University, Palo Alto, CA, United States To provide an experimental basis for a comprehensive molecular modeling evaluation study, 500 fragments from the Maybridge fragment library were soaked into crystals of bovine pancreatic trypsin and the structures determined by X-ray crystallography. The soaking experiments were performed in both single and pooled aliquots to determine if the combination of fragments is an appropriate strategy. X-ray diffraction data were collected on over 1400 crystals at the Australian Synchrotron. These data were subsequently processed, and the preliminary analysis was performed with a custom software application (Jigsaw), which combines available software packages for structure solution and analysis. 3 The in silico modeling community lacks a public set of high-quality data against which predictive rather than retrospective studies can be performed. Previous attempts to perform such evaluations suffered from a lack of truly “blind” data. Recently, OpenEye Scientific Software has initiated the SAMPL (Statistical Assessment of the Modeling of Proteins and Ligands) meeting, which provides a forum for the prospective testing of concepts, algorithms, and approaches in computational chemistry and protein modeling. One core goal of modeling is rapid and reliable in silico screening, in which a compound library is evaluated against a protein target of interest to select a subset of compounds, enriched for potential activity against the target. The resulting subset can subsequently be assayed for activity using biochemical or biophysical methods. Recently, it has been shown that this approach may be feasible for enriching fragment libraries of low molecular weight (~200 Da) in addition to libraries of larger “lead-like” compounds (300-500 Da). I will discuss the strategies used to obtain this high throughput data set, the reason for developing Jigsaw and why it was important to develop a specific process for this project. 07.08.6 Integrating Single-Crystal Spectroscopy and X-ray Diffraction at Beamline X26-C of the National Synchrotron Light Source (NSLS) John Skinner , Deborah Stoner-Ma , Annie Heroux , Dieter Schneider , Robert Sweet , 2 1 Michael Skinner , Allen Orville Biology Department, Brookhaven National Laboratory, Upton, NY 11973, United States, High School Reasearch Program, Brookhaven National Laboratory, Upton, NY 11973, United States 2 1 1 1 1 1 1 Correlated optical absorption spectroscopy at beamline X26-C of the NSLS is routinely collected before, during, and after x-ray diffraction data used for crystal structure determination. A microspectrophotometer is mounted at the beamline such that it probes ~25µm diameter region of the crystal that intersects the x-ray beam. We have integrated all of the spectrophotometer controls with the x-ray diffraction data collection and beamline control software, CBASS. The Ocean Optics USB4000 spectrophotometer is controlled through an EPICS IOC developed at the Canadian Light Source, which CBASS communicates with over EPICS Channel Access. Users typically start by performing an automated spectroscopic screen consisting of 72 crystal images and absorption spectra collected at 5-degree increments throughout 360 degrees of rotation. A program developed at the European Synchrotron Radiation Facility for automatic crystal centering, C3D, is called from a CBASS macro. The macro determines the “optimal” orientation, i.e. the flat-face of the crystal/loop, for optical absorption spectroscopy. A Java Webstart application also allows the user to control a "movie" of the screening run in order to evaluate quickly the relationship between crystal orientation and the anisotropic spectroscopic data. The optimal phi/omega angle, or a userprovided best orientation, is then revisited and an optical spectrum is collected during the readout of the x-ray detector after each frame of the diffraction data set. Each optical absorption spectrum is overlaid on a plot to provide instant feedback about spectroscopic perturbations induced by x-ray dose. The entire family of spectra is documented and available to the user in several formats through the beamline’s experiment tracking database system, PXDB. The Protein Data Bank will link the resulting atomic coordinates and the family of spectra upon publication and release of the structure. We are also integrating Raman spectroscopy with two excitation lasers (785nm and 532nm) into the beamline controls. Our diffraction and spectroscopy facility is available full-time to the general user community at the NSLS. Supported by the NIH National Center for Research Resources and the DOE Office of Biological and Environmental Research. 07.08.7 SER-CAT Automation: Providing Users with A “Virtual Home Synchrotron” John Chrzas, Jim Fait, Zheng-Qing Fu, John Gonczy, Andy Howard, Zhongmin Jin, Gerd Rosenbaum, John Rose, B.C. Wang University of Georgia, Athens, Georgia, United States From its inception, SER-CAT has been working towards the concept of providing remote users with “Light When YOU Need It!” A web-based “virtual home synchrotron beamline” was envisioned that could be integrated into the user's research program, much like another piece of major research equipment in the X-ray lab down the hall. SER-CAT began exploring automation of its beamlines shortly after the signing of MOU with APS in March 1999. Working with Oceaneering Space Systems, a conceptual design for an automated data collection robot (ASTRO) was developed in 2000. In 2003, using funds from the Georgia Research Alliance, automation of the SER-CAT beamlines began with the installation of a highly modified Berkeley/ALS automounter on SER-CAT's 22BM. Key to the SER-CAT virtual synchrotron beamline (or remote access) is the integration of hardware with software. The SERGUI beam line control interface allows the remote user full control of the beamline from their home lab including beamline/goniometer optimization, wavelength selection, fluorescence scans, automated crystal screening and MAD/SAD data collection. Today over 80% of SER-CAT members routinely collect data remotely. Significantly, SER-CAT's robotics, system integration and added user support have allowed it to implement 12-hour shifts with 16-hour/day on-site user support. SER-CAT also provides its members access to automated data processing and structure solution pipelines. Command line scripts CMDDENZO, CMDDTREK and CMDXDS are available for automated data processing using HKL2000, d*TREK and XDS, respectively. A cluster-based version of the SGXPro Crystallographers Workbench for automated structure determination is available, which allows members to produce a fitted map from their SERCAT data in a matter of minutes. An overview of the SER-CAT’ s automation will be described and discussed. Work supported by the SER-CAT Member Institutions (www.ser-cat.org), University of Georgia Research Foundation and the Georgia Research Alliance. 07.08.8 Towards serial micro crystallography of acoustically mounted crystals Alexei Soares, Allen Orville, Marc Allaire, Matthew Engel, Ruchi Parekh, Annie Heroux, Robert Sweet, John Skinner, Joseph Olechno, Richard Ellson Brookhaven Naiotnal Laboratory, Upton, NY, United States Acoustic droplet ejection (ADE) was used to transfer 2.5µL droplets of crystal slurries from 396 well plates onto MiteGen© micro mesh pins. Multiple homogenous slurries were tested to verify that acoustic specimen preparation was effective and non-damaging for R3 insulin and P43212 lysozyme crystals of different sizes (5µ±2µm, 10µm±4µm, 20µm±8µm), different concentrations, and different overall purity and cleanliness. All tested conditions yielded accurately transferred droplets of precise volumes. All transferred droplets yielded undamaged and well diffracting crystals. Both the diffraction patterns and the resulting structures of the specimens prepared with ADE were comparable to crystals that were hand mounted from the same slurries prior to acoustic ejection. We conclude that ADE specimen preparation is a strong candidate to mount micro crystals automatically from the growth plates onto the data collection media. We discuss some difficulties unique to micro crystals; including surface tension induced clustering, preferential orientation, and inaccessible regions of reciprocal space. 07.09.1 Insights into Amyloid Structure from Short Assembling Peptides Kyle Morris , Karen Marshall , Joost Schymkowitz , Frederic Rousseau , Pawel Sikorski , 1 Louise Serpell 1 1 1 2 2 3 University of Sussex, Brighton, East Sussex, United Kingdom, Vrije Universiteit Brussel, 3 Brussels, Belgium, Norwegian University of Science and Technology, Trondheim, Norway A broad range of peptides and proteins are able to spontaneously self-assemble into fibrillar structures known as amyloid. This phenomenon is associated with a number of neurodegenerative diseases and remarkably also with functional materials in nature. The structural features of this unique conformation have historically been determined by X-ray fibre diffraction (XRFD) and may be generally represented by the cross-beta architecture where beta-strands run perpendicular to the fibre axis and are stabilised by hydrogen bonding parallel to the fibre axis. More recently advances in X-ray crystallography and ssNMR have consolidated features of the cross-beta architecture. By using a multi-technique approach, developing XRFD techniques and with increased access to new amyloid-like model systems new studies promise to reveal greater detail about the amyloid fold. Taken together our observations define whether overall sequence or position specific characteristics are key to amyloid formation and give insights into what stabilising interactions are common to these fibril-forming pathological peptides. 2 07.09.2 A first molecular view of amyloid-like fibrils bound to small molecules revealed by Xray microcrystallography Meytal Landau, Michael R. Sawaya, Kym F. Faull, Arthur Laganowsky, Jie Liu, Jorge Barrio, David Eisenberg University of California Los Angeles, Los Angeles,CA, United States Amyloid protein fibrils are associated with a group of devastating human diseases. Currently there is no approved therapeutic agent that regulates the formation of amyloid deposition and alleviates the symptoms. Several dozen small-molecule ligands have been suggested to affect fibrillation. In order to understand the nature of fibril-ligand interactions we sought to determine the crystal structures of their complexes by X-ray microcrystallography. Structure determination was impeded by the miniscule crystals, about one micrometer in width. The difficulties include fast decay, crystal polymorphism, indexing small unit cells and merging integrated intensities from several crystals. Co-crystallization of the fibril-segments with ligands was particularly challenging due to low affinity and specificity of binding. Frequently the ligands appear disordered in one dimension because their lengths span multiple unit cells of the fibril; that is, the dimensions of the small molecule and the fibril unit cell were incommensurate. Previous structures of fibril-forming segments from our lab have presented the first fully objective atomic model of the common β -spine structure of amyloids. Here we present a first molecular insight into the interactions of such segments with ligands. The structures indicate binding of the ligands along the fibril axis. Interestingly, in some cases the ligands induced a unique polymorph of the fibrillar form. These structures advance our understanding of fibrilligand interaction and offer a starting point for the design of highly specific, potent and nontoxic compounds that will inhibit fibrillation or will be used for diagnosis of fibrils. OVMOXMR wL‒\„?« ¦‒› ¢¢‒\¦ ›‹?›‹?‹¡·‒›¢ ‒ \‒„? \‹£ ¡ ?• ⁄ ‹? ‹ \¦ ?` ⁄¡ «¡‒☂ ? ¡\ ¡? ‒\ ‹? ¡¦ ›‹ n £\? `‹ fi› \PK? `‹£¡ \? f· › ¡ La›‹QK? k\·‒¡ ? u\‹\QK? d‒ ¦? k\‹ \⁄ RK? k¡ ¡‒? a ‹ ¡‒PK? r\‒\⁄? q ¦¡QK i› ¡fi⁄?n‒£¡ P P h ‹› ? h‹ · · ¡? ›¢? s¡¦⁄‹› ›£„K? b⁄ ¦\£›K? hkK? t‹ ¡ ? r \ ¡ K Qm›‒ ⁄•¡ ¡‒‹? t‹ ¡‒ „? e¡ ‹ ¡‒£ r¦⁄›› ?›¢?l¡ ¦ ‹¡K?b⁄ ¦\£›K?hkK?t‹ ¡ ?r \ ¡ K Rc¡o\· ?t‹ ¡‒ „K?b⁄ ¦\£›K?hkK?t‹ ¡ ?r \ ¡ m¡·‒›¢ ‒ \‒„? \‹£ ¡ ? Gmes H? \‒¡? ¢ \«¡‹ ›· ? fi› „«¡‒ ? ¢›‒«¡ ? ¢‒›«? ⁄¡? « ¦‒› · · ¡L\ ›¦ \ ¡ fi‒› ¡ ‹K? \·M? ? s⁄¡? fi‒¡ ¡‹¦¡? ›¢? mes ? ‹? ⁄¡? ‒\ ‹? ¦›‒‒¡ \ ¡ ? ‒¡¦ „? ›? ¦›£‹ ¡? «fi\ ‒«¡‹ ? ‹ ` ⁄¡ «¡‒☂ ? ¡\ ¡? 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G`orHM? ? t‹ ¤¡? „‹ ⁄¡ ¦? mes K? mes ? • ⁄ ‹? fi„‒\« \ ? ¦¡ ? ¢›‒«? fl·\ L ¦‒„ \ ‹¡? \‒‒\„ ? ›¢? ¢ \«¡‹ ? \ £‹¡ ? \ ›‹£? ⁄¡ ‒? ›‹£? \‚¡ M? v¡? · ¡ ? \‹? ‹L ‹¡? ¢ ·›‒¡ ¦¡‹¦¡ « ¦‒› ¦›fi¡? ¡ ·fi? ›? ¡‹ ¢„?¢ ·›‒¡ ¦¡‹ „? \ ¡ ¡ ?mes ?• ⁄ ‹?fi„‒\« \ ?¦¡ ?›¢?`c? ‒\ ‹? ¡¦ ›‹ K ⁄¡‹? fi¡‒¢›‒«¡ ? wL‒\„? ¢ ¡‒? « ¦‒› ¢¢‒\¦ ›‹? ¡‚fi¡‒ «¡‹ ? ›‹? ⁄¡? \«¡? ¦¡ M? s⁄¡? \‒‒\„¡ ? ‒·¦ ·‒¡? ›¢ mes ? • ⁄ ‹? ⁄¡ ¡? ‒›·£⁄ „? TO‚QOO? « ¦‒›‹? ¦¡ ? «\„? ¡‹⁄\‹¦¡? ⁄¡ ‒? wL‒\„? ¢¢‒\¦ ›‹? fi‒›fi¡‒ ¡ K ¡‹\ ‹£?⁄ £⁄L‒¡ › · ›‹? ‒·¦ ·‒\ ? ¡ ¡‒« ‹\ ›‹?›¢?mes ?• ⁄ ‹?`c? ‒\ ‹M 07.09.4 Structure of Point Mutants of β -Amyloid Associated with Familial Alzheimer’s Disease Robert Tycko , Kimberly Sciarretta , Adam Cloe , Joseph Orgel , Stephen Meredith 1 2 2 1 1 3 1 The University of Chicago, Chicago, IL, United States, National Institutes of Health, 3 Bethesda, MD, United States, Illinois Institute of Technology, Chicago, IL, United States We examined two mutant Aβ 40 peptides associated with familial Alzheimer's Disease, D23NAβ and Δ E22-Aβ (Iowa and Japanese, respectively). D23N-Aβ is also associated with cerebral amyloid angiopathy. We synthesized D23N-Aβ 40 and Δ E22-Aβ 39; both form fibrils very rapidly and with no lag phase. EM of D23N-Aβ 40 fibrils shows multiple morphologies with mean diameter = 6.90 nm, compared to 10.2 nm for wide-type Aβ 40. X-ray diffraction of D23N-Aβ 40 fibrils shows cross-β pattern, with reflections at 0.47 and 0.94 nm. In contrast, wild type Aβ 40 fibrils show reflections at 0.47 and 1.04 nm. For Δ E22-Aβ 39, fibril diameter 13 13 15 and x-ray diffraction are similar to those of wildtype Aβ 40. Solid state NMR ( C- C and N13 C dipole-dipole coupling) of D23N-Aβ 40 indicated molecular polymorphism of fibrils, with only a minority containing in-register, parallel β -sheets. The majority of fibrils had antiparallel β -sheets with 17+k ↔ 21-k registry. An intriguing possibility is that the aberrant structure of D23N-Aβ 40 fibrils is related to the unusual vasculotropic clinical picture in these patients. Δ E22-Aβ 39 formed fibrils instantaneously under many conditions, but without thioflavin (ThT) fluorescence. Direct ThT binding assay (by HPLC) indicates loss of one of four putative ThT binding sites in Δ E22-Aβ 39. Nevertheless, Δ E22-Aβ 39 fibrils have a β -sheet character. CD of wild-type Aβ 40 indicates “random coil”, developing β -sheet character after ~ 3 days. In contrast, Δ E22-Aβ 39 shows a β -sheet signature by CD immediately when put into aqueous media. Soluble oligomers of Δ E22-Aβ 39 are present at extremely low concentration and are highly transient species, which usually are not seen by size exclusion chromatographs. Critical concentration of Δ E22-Aβ 39 is ~ one-third that of the wild-type Aβ 40. Solid-state NMR studies of Δ E22-Aβ 39 fibrils are in progress, but preliminary studies indicate that these fibrils, like D23N-Aβ 40 fibrils, do not have the in-register, parallel β -sheet structure that is typical of wild-type Aβ 40 fibrils. 07.09.5 Structural Insights into Fungal Prion HET-s by X-Ray Fiber Diffraction William Wan , Wen Bian , Holger Wille , Michele McDonald , Gerald Stubbs 1 2 1 1 2 1 1 Vanderbilt University, Nashville, TN, United States, University of California, San Francisco, San Francisco, CA, United States Prions are infectious proteins thought to propagate through polymerization of a misfolded protein nucleus. The nucleus may act as a template to convert natively folded isoforms into the misfolded state, resulting in the formation of long unbranched amyloid fibrils. The nucleation event, and subsequent templating ability, is dependent on the environmental conditions such as pH, salt concentration and agitation. In the filamentous fungus Podospora anserina, the prion form of the HET-s protein is used in a self non-self recognition process known as heterokaryon incompatibility. In this study we have obtained fiber diffraction data from amyloids formed from the prion domain of the HET-s protein at pH 2.5 and pH 7. It has been shown in the literature that the pH 7 fibrils are infectious while the pH 2.5 fibrils are not. However, fiber diffraction has revealed that the two fibrils are very similar, despite infectivity and morphological differences. Our findings imply that the structural differences that cause infectivity are actually very subtle. Support for this research was provided by the National Institutes of Health, grants P01 AG002132 and T32 GM008320-21. 07.09.6 Structural studies of prions and other amyloids by X-ray diffraction Gerald Stubbs , Wen Bian , Michele McDonald , William Wan , Amy Kendall , Hayden Box , 1 2 Adrianne Eyman , Holger Wille 1 1 1 1 1 1 1 Vanderbilt University, Nashville, Tennessee, United States, University of California, San Francisco, California, United States 2 Amyloids are misfolded proteins forming unbranched filamentous assemblies (fibrils) that produce a characteristic apple-green birefringence when stained with Congo red. In fiber diffraction experiments, they exhibit characteristic meridional diffracted intensity at about 4.75 Å resolution, coming from cross- secondary structure ( -strands running approximately at right angles to the filament axis). For many years, all amyloids were assumed to have a common structure consisting of -sheets stacked together, with the sheets approximately parallel to the fibril axis and the strands at right-angles to the axis. In recent years, however, work from a number of laboratories has shown that amyloid structure is much more complex and diverse than this, although the cross- structure is always present. We have examined amyloids formed from short peptides, the 40-residue A peptide, the 37-residue IAPP peptide, the the prion domain of the HET-s fungal prion protein, and the mammalian prion protein PrP. Work with A , IAPP, and HET-s makes use of information from solid state NMR, while studies of PrP use data from electron microscopy. Fiber diffraction studies comparing brain-derived PrP amyloid with recombinant PrP amyloid clearly indicate significant differences in structure. Fiber diffraction data support the diversity of -structures in amyloids in general, distinguish among competing models, and provide insight into their protofilament packing. Supported by NIH grants P01 AG002132 and P01 AG010770. 07.10.1 Area Detector Calibration Hakon Hope University of California, Davis, Davis, CA, United States Apparently, most users of commercial, CCD-based area detectors assume, without asking too many questions, that all geometric correction factors will be automatically applied to their data. Let us do a check to see if this implicit trust is deserved. Here are a couple of lines copied from an otherwise innocuous shelx lst file: Resolution(A) .68 .72 .75 .78 .83 .87 .95 1.04 1.18 1.50 inf K 1.046 1.043 1.023 .992 .968 .965 .949 .959 .998 1.024 The scale factor K is not constant, but shows a systematic variation. These effects are not just local phenomena. Similar observations can be readily made with data deposited with Acta Cryst. G. Wu, B. L. Rodrigues and P. Coppens* have published a paper “The correction of reflection intensities for incomplete absorption of high-energy X-rays in the CCD phosphor,” J. Appl. Cryst. (2002). 35, 356-359. This paper may not have received sufficient attention. Some time later, a feature was quietly added to sadabs to take such effects into consideration, but to date, this author is not aware of a corresponding feature to be accessible in the Bruker Apex user interface. In order to simplify the assessment of detector response as a function of position on the detector surface, the following procedure is proposed: Perform a number of scans that cover the exact same range in crystal positions, but with the detector in different positions. For example, perform runs with detector settings at -30°, -25° … +30° with ω covering a 60° range. This will result in many identical reflections observed at different positions on the detector surface. The main result of several such experiments is that identical reflections show significant differences inmeasured intensities, varying systematically with angle of incidence on the detector face. Variations easily exceed 5%. Corrections for these effects lead to lower R index and smoother difference maps. 07.10.2 CrystalPlan, an experiment planning tool for crystallography Janik Zikovsky, Christina Hoffmann Oak Ridge National Lab., Oak Ridge, TN, United States Instrument time at large x-ray and neutron scattering facilities such as the Spallation Neutron Source at ORNL is always at a premium. To make the most of a user's short visit to one of these facilities, developing a plan for an efficient experimental run would be advantageous. The CrystalPlan program is a tool designed to take the guesswork out of acquiring large data sets. Written in Python, CrystalPlan can run on multiple platforms. The program features an attractive graphical user interface (GUI) including a 3D viewer. Using the instrument's detector positions and the sample orientation, a 3D map of the reciprocal space coverage is generated and displayed. Statistics such as the fraction of reciprocal space measured are calculated. As users build up a list of desired sample orientations, the reciprocal space coverage increases; the program also displays and calculates the redundancy – how many times a specific volume of q-space was measured. The limitations of the sample orientation goniometer, if any, are displayed to the user. Users of the software can enter or load the sample crystal’s lattice parameters and UB matrix; these are then used to calculate, for each H,K,L peak, whether or not it was measured, by which detector(s), at which sample orientations and what wavelengths. For particularly interesting reflections, users can ask the program to find the sample orientation that places the peak at an optimal position on a detector. Once an acceptable sequence of sample orientations has been found, data is acquired in automatic mode. We will show a comparison between the predicted peak positions and the actual data for a crystal measured at the TOPAZ beamline at SNS. This research is supported by UT Battelle, LLC under Contract No. DE-AC05-00OR22725 for the U.S. Department of Energy, Office of Science. 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Kanagalaghatta Rajashankar, Igor Kourinov NE-CAT, Cornell University, 9700 S. Cass Av., Argonne, IL-60439, United States MAD (multiple anomalous dispersion) method utilizes the anomalous scattering nature of specific heavy atoms at characteristic X-ray energies as well as the variation of atomic scattering factor as a function of energy near atomic absorption edges. To maximize the signature of anomalous scattering center, typically, diffraction data are collected at three wavelengths, namely, peak, inflection and high-energy remote. As result of evolution of synchrotron beamlines to deliver increasingly accurate intensity data and the development of more robust, and powerful software for macromolecular crystal structure analysis, has influenced SAD (single anomalous dispersion) method to take over MAD method. However, MAD remains in the main-stream as a method of choice for challenging projects. Several variants of MAD have been proposed that make best of both anomalous and dispersive differences. Even, a method for simultaneous collections of multiple MAD collections has been proposed, though no practical case been demonstrated till now. Here we propose an easy method for collection of two wavelength MAD data, but without changing energy. This method makes use of the inherent property of most of synchrotron beamlines, namely presence of multiple harmonic energies. A test case of this method will be presented and improvement over SAD phasing will be demonstrated. NE-CAT is supported by award RR-15301 from the NCRR. APS is supported by the U.S. Department of Energy 07.10.5 A Comprehensive Strategy - Is It Possible? Lee Daniels, Mark Pressprich Rigaku Americas Corp, The Woodlands, TX, United States A truly global data-collection strategy algorithm must consider a great number of variables. Ideally one should be able to consider any source, any goniometer and any detector. Then one must involve the relationships among these components, including the limits of motion and all possible collisions. A truly flexible routine also allows the user to add his own limits, such as additional restrictions for temperature, pressure, humidity (etc.) control devices. An even greater number of experimental variables, or concerns, ensue. The basic requirements are completeness and redundancy. Then there are the details: a good "distributed" redundancy (the target redundancy is distributed over most or all unique reflections), avoid including partial reflections due to factors such as high Lorentz factors or α 1-α 2 splitting, appropriate detector swing overlap when necessary, and support for ω and/or φ scans for any geometry, to name a few. The routine must work in automatic mode using reasonable defaults and also allow "experts" a great deal of flexibility. Then there is a crucial measure of a useful algorithm - it must be FAST. An algorithm that implements these ideas will be discussed and demonstrated. 07.10.6 What can a synchrotron do for Chemical Crystallography? Simon Teat Lawrence Berkeley National Laboratory, Berkeley, CA, United States Synchrotrons are not just the domain of protein crystallography. At the Advance Light Source station 11.3.1 is a dedicated chemical crystallography station. The high intensity of synchrotron radiation can yield high quality structures on crystals that are otherwise too small or weakly diffracting to produce anything on a laboratory source. The factors affecting a crystals ability to diffract will be outlined, along with how the synchrotron can aid and the data collection strategies that have been used to provide higher resolution data on weakly diffracting samples. Not all synchrotron data collections serve to determine an unknown structure, some are to see how a known structure is changed by the external environment. In many of these cases using a small crystal can make the difference between success and failure. 07.10.7 Data Collection Planning and Automation for Chemical Crystallography Joerg Kaercher , Michael Ruf , Rob Hooft 1 1 1 2 2 Bruker AXS Inc., Madison, WI, United States, Netherlands Bioinformatics Centre (NBIC), Nijmegen, Netherlands We report on the latest development of expert systems for automating data acquisition and structure solution for high end research applications. This development was made possible in part through advances in the analytical software, specifically new and improved algorithms and decision making expert systems. The technology automates many of the routine aspects of data collection and analysis but also still allows the expert user to interact or intervene in more challenging cases. By combining automation with the latest developments in area detector and X-ray source technologies the productivity of the instrument can be significantly enhanced. In this paper we present an overview of the decision tree implemented in a fully automated system that involves all the individual steps in producing publication quality structures from single-crystals without user intervention. The system is designed to make all decisions autonomously, while at the same time keeping the user informed about the progression of the experiment in an easily comprehensible way. Suitable remedies are suggested if the software encounters a problem it cannot tackle, such as radiation induced crystal decay or a temperature induced phase transition. The expert system proceeds through the following stages: quantify the crystal quality, determine the unit cell and the crystal symmetry, select a data collection strategy, acquire and reduce the diffraction data, scale the diffraction data, determine the space group, solve the phase problem, refine and validate the molecular structure, and finally generate a report. The results are provided as a Crystallographic Information File (CIF) and as a Hypertext Markup Language (HTML) report. 07.11.1 Reconstructing RNA folding intermediates from time-resolved SAXS data Lois Pollack Cornell University, Ithaca, NY, United States Small angle x-ray scattering (SAXS) provides information about the size and shape of macromolecules in solution. In a time-resolved mode, SAXS has the potential to reveal structure(s) of transient intermediates that occur as molecules function or fold. I will discuss successes and challenges we have encountered in reconstructing low resolution structures from time-resolved scattering data. The scientific focus of these studies is on RNA folding to biologically functional forms, such as ribozymes or riboswitches. 07.11.2 Extended structures in RNA folding intermediates are due to non-native interactions rather than electrostatic repulsion. Nathan Baird, Haipeng Gong, Syed Zaheer, Karl Freed, Tao Pan, Tobin Sosnick University of Chicago, Chicago, United States RNA folding occurs via a series of transitions between metastable intermediate states for 2+ Mg concentrations below those needed to fold the native structure. In general, these folding intermediates are considerably less compact than their respective native states. Our previous work demonstrates that the major equilibrium intermediate of the 154 residue specificity domain (S-domain) of the B. subtilis RNase P RNA is more extended than its native structure (1). We now investigate two models with falsifiable predictions regarding the origins of the extended intermediate structures in the S-domains of the B. subtilis and the E. coli RNase P RNA that belong to different classes P RNA and have distinct native structures (2). The first model explores the contribution of electrostatic repulsion, while the second model probes specific interactions in the core of the folding intermediate. Using small-angle X-ray scattering (SAXS) and Langevin Dynamics (LD) simulations, we show that electrostatics only plays a minor role, whereas specific interactions largely accounts for the extended nature of the intermediate. Structural contacts in the core, including a non-native base-pair, help to stabilize the intermediate conformation. We conclude that RNA folding intermediates adopt extended conformations due to short-range, non-native interactions rather than generic electrostatic repulsion of helical domains. These principles apply to other ribozymes and riboswitches that undergo functionally relevant conformational changes. Reference 1. Baird, N. J., Westhof, E., Qin, H., Pan, T. & Sosnick, T. R. (2005). Structure of a folding intermediate reveals the interplay between core and peripheral elements in RNA folding. J. Mol. Biol. 352, 712-22. Reference 2. Baird, N. J., Gong, H., Zaheer, S. S., Freed, K. F., Pan, T., Sosnick, T. R. (in press) Extended Structures in RNA Folding Intermediates Are Due to Nonnative Interactions Rather than Electrostatic Repulsion. J. Mol. Biol. 07.11.3 Kinetics of hepatitis B virus core assembly by time-resolved small angle x-ray scattering Hiro Tsuruta , Kelly Lee , Adam Zlotnick 1 1 2 3 2 Stanford University, Menlo Park, CA, United States, University of Washington, Seattle, WA, 3 United States, Indiana Unviersity, Bloomington, IN, United States Hepatitis B virus (HBV) is a major pathogen and one of the most prevalent causative agents of cancer in humans. HBV is one of the few systems for which interactions between virus and drugs targeted against its protective capsid have been well characterized both structurally and biochemically. It is thus an ideal system for exploring the relation of virus structure and function. We have applied synchrotron time-resolved small angle x-ray scattering (TR-SAXS) to elucidate the disassembly and assembly of HBV cores. Icosahedral T=4 core capsids dissociate into 120 dimers of subunits in vitro in the presence of chaotropes such as guanidine hydrochloride. We first studied the kinetics of chaotrope-induced disassembly process over a few minutes. We observed an oscillating dissociation/reassociation multiphasicity instead of a one-way disassembly process. This unusual disassembly process, however, turns out to be consistent with a theoretical prediction by a computational modeling study. We recently conducted TR-SAXS experiments on the assembly of the HBV core capsid. Our solution x-ray scattering studies in equilibrium indicated that a low concentration of urea keeps the capsid protein in an assembly-ready dimeric form at least for several hours. We conducted time-resolved studies of the assembly induced by a rapid salt concentration jump at a mildly alkaline pH value. The assembly reaction is surprisingly fast: the half-life of the order of a few seconds or shorter, depending on salt concentration. Our recent results suggest the presence of a transient assembly intermediate which is larger than T=3 or T=4 capsid and undergoes partial disassembly prior to incorporating the dimeric capsid protein to form the T=4 capsid. The assembly process also involves a slow annealing process after the formation of a capsid. 07.11.4 A Combined Crystallography, SAXS and Computational Approach to the structure of Gln4 – yeast glutaminyl tRNA synthetase Edward Snell , Thomas Grant , Joseph Luft , Jennifer Wolfley , Elizabeth Snell , Hiro 3 4 4 4,5 4,5 Tsuruta , Stephanie Corretore , Erin Quartley , Eric Phizicky , Elizabeth Grayhack Hauptman Woodward Medical Research Institute, Buffalo, NY, United States, Department of 3 Structural Biology, SUNY Buffalo, Buffalo, NY, United States, SSRL, Menlo Park, CA, United 4 States, Department of Pediatrics, University of Rochester Medical Center, Rochester, NY, 5 United States, Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, United States tRNA synthetases play an essential role in protein synthesis by covalently coupling the correct amino acid to the correct tRNA, prior to its use in translation. Unlike their prokaryotic homologs, eukaryotic tRNA synthetases often have additional domains appended to their N or C-terminus. In some cases, these domains are known to have non-specific RNA binding activity, but their function remains poorly defined in the context of a tRNA synthetase. In this report, we address the structure of yeast Gln4, the tRNA synthetase that covalently Gln couples glutamine to tRNA , and of its N-terminal domain. We have crystallographically solved the structure of full-length Gln4, but find that 216 residues comprising 95% of the Nterminal domain is not resolved, although biochemical analysis confirms its presence. The Nterminal domain appears to be ordered, based on low resolution refinement and comparison to diffraction data from a C-terminal truncated Gln4. To complement the crystallographic studies, we used Small Angle-X-ray Scattering (SAXS) to examine the structure of Gln4. Alignment of full-length Gln4, C-terminal domain and the N-terminal domain with the crystallographic results shows that the N-terminal domain is ordered and attached to the Cterminal body by a flexible linker. SAXS studies of a homologous structure from C. glabrata indicate a similar envelope, suggesting a common but as yet undefined function for this domain. In addition, we have solved the structure of the truncated N-terminal domain, which is currently undergoing refinement. We will describe these results, as well as our progress on the complementary use of crystallography and SAXS to develop and test hypotheses for structural and functional investigations. 1 2 1,2 1 1,2 1 1 07.11.5 MADMAX: Multi-wavelength anomalous diffraction using medium-angle x-ray solution scattering Lee Makowski , Diane Rodi , Dave Gore , Robert Fischetti 1 1 1 2 1 2 Argonne National Lab, Argonne, IL 60439, United States, Illinois Institute of Technology, Chicago, IL 60616, United States Anomalous diffraction from proteins in solution has the potential to provide detailed information about the relative positions of atoms. Its use is hampered by the extreme weakness of the signal. When x-ray energy is changed, small systematic changes in scattering occur even in the absence of anomalous diffraction. These are not informative of the sample structure and complicate observation of the anomalous signal. Knowledge of the structure of the absorption edge is used to inform the analysis of changes in scattering near the edge. We used a principal components analysis of patterns taken at x-ray energies near the absorption edge to isolate the anomalous signal from other effects and measure it to ~ 5 A spacing. Measured differences compare well to theoretical expectations calculated from the atomic coordinates of proteins of known structure. Application to Fe-containing proteins; seleno-met-labelled proteins and membrane proteins has been demonstrated. 07.11.6 Comprehensive structural analyses of proteins, nucleic acids and their complexes in solution. Robert Rambo , Greg Hura , Michal Hammel , Alexei Kazantsev , Susan Lees-Miller , Norm 3 3 1,2 Pace , Robert Batey , John Tainer Lawrence Berkeley National Lab, Berkeley, CA, United States, The Scripps Research 3 Institute, La Jolla, CA, United States, University of Colorado at Boulder, Boulder, CO, United 4 States, University of Calgary, Calgary, Alberta, Canada Synchrotron based small-angle X-ray scattering (SAXS) of biological macromolecules has developed into a powerful structural tool that complements X-ray crystallography (MX), Nuclear Magnetic Resonance, and Electron Microscopy. SAXS based measurements of proteins and nucleic acid complexes provides complementary data on small and large assemblies, extended conformations, and flexibly linked domains in solution at about 12 Å resolution. In practice, SAXS investigations of biological samples can show inconsistencies that suggest limitations to the subsequent computational analyses and modeling. Our results show that many of the problems with SAXS experiments derive from sample heterogeneity that can be effectively resolved to provide reliable SAXS data. Furthermore, we provide an analysis pipeline and suggest an appropriate statistical basis for data interpretation. Our results show that the combination high resolution information with accurate SAXS data can be used to effectively model the Ku/DNA-PKcs non-homologous end joining complex, the ligand free state of the SAM riboswitch and the complete architecture of RNase P. 1 2 1 1 1 1 4 07.12.1 The First Crystal Structure of an Intron Debranching Enzyme Eric Montemayor , Alexander Taylor , Joshua Combs , Scott Stevens , John Hart 1 1 1 2 2 1 Univ. of Texas Health Sci. Center at San Antonio, San Antonio, United States, University of Texas at Austin, Austin, United States The intron debranching enzyme (Dbr1) hydrolyses the unique 2’-5’ phosphodiester bond found within branched or lariat RNA species. Hydrolysis of this bond is necessary for the recycling of excised introns and the formation of several small nucleolar RNAs; hydrolysis of the 2’-5’ phosphodiester bond has also been shown to play a role in the movement of genetic elements by retrotransposition. Dbr1 enzymes contain a highly-conserved N-terminal domain that is homologous to the metallophosphatase superfamily of enzymes and a C-terminal domain with virtually no sequence similarity to any other protein of known structure. Here we present the structure of the 354 amino acid Dbr1 from the parasite Entamoeba histolytica. This structure reveals that the N-terminal domain of Dbr1 indeed adopts a metallophosphatase-like fold; however, a cysteine residue that is conserved among Dbr1 enzymes is in a position usually occupied by an aspartate in other metallophosphatases. The structure also provides a clear view of the C-terminal domain of Dbr1, a helical structure that forms an extensive interface with the metallophosphatase domain. This structural information is currently being used to design new experiments that will elucidate the mechanistic basis for recognition and hydrolysis of 2’-5’ phosphodiester bonds by Dbr1. 2 07.12.2 Structure and Regulation of Yeast Glycogen Synthase-2 Sulochanadevi Baskaran, Peter Roach, Anna DePaoli-Roach, Thomas Hurley Indiana University School of Medicine, Indianapolis, IN, United States Glycogen is a major energy reserve in most eukaryotes and its rate of synthesis is controlled by glycogen synthase. The activity of eukaryotic glycogen synthase is regulated by the opposing effects of glucose-6-phosphate and phosphorylation and a conserved arginine cluster is responsible for the regulatory control. We solved the crystal structure of yeast glycogen synthase-2 by multiple isomorphous replacement using two tantalum bromide cluster derivatives, combined with four-fold molecular averaging and partial model phase combination. The presence of a unique sequence insertion in the C-terminal Rossmanndomain forms the majority of the enzyme’s tetrameric interface. This interface is surprisingly small and underlies the extensive conformational flexibility critical for the regulation of enzymatic activity. The conserved cluster of arginine residues are localized within a single alpha-helix (termed the regulatory helix) positioned orthogonally to one of the molecular twofold axes. Site-directed mutants based on our initial structure demonstrate that arginines 583 and 587 are necessary and sufficient for activation by glucose-6-phosphate. A screen of new crystallization conditions compatible with the presence of glucose-6-phosphate was performed and a new crystal form that only grew in the presence of this activator was identified. The structure of the activated form was solved using the individual subunits of our previous structure for molecular replacement calculations. Binding of glucose-6-phosphate to the N-terminal ends of the regulatory helices induces a large conformational transition amongst the subunits, akin to the petals of a flower opening, which frees each of the subunits in the tetramer to open and close their inter-domain clefts in response to substrate binding and product release. Additional structures containing product UDP and substrate-analogs further define the catalytic cycle of this complex enzyme and provide detailed insight into the molecular bases for its activity states. 07.12.3 Crystal structure of the bacteriophage Mu transpososome Sherwin Montano, Ying Pigli, Phoebe Rice Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL, United States Bacteriophage Mu utilizes DNA-based transposition to integrate its genome into the genome of the host cell. It encodes a MuA transposase, a member of the DDE transposase/integrase family, that recognizes the ends of the phage, and brings them together to form a synaptic complex called the transpososome. Both the cleavage of the phage DNA and the strand transfer to the host DNA occur within the transpososome and are catalyzed by the MuA protein. In vitro, the simplest working system for Mu transposition comprises a tetramer of MuA bound to two right ends. We have crystallized this tetramer bound to two right ends in the presence of a target DNA, a complex that contains a total of ~240kDa of protein and 135bp of DNA. The crystals diffract anisotropically to 4.5 Å in one direction and 5.2 Å in the other two. Experimental phases were obtained using MIRAS with four derivatives which include Ta6Br12 clusters, mersalyl acid, SeMet modified protein, and brominated DNA where every T on one strand had been replaced with 5-Br-dU. Building the transpososome model at this resolution was facilitated by the fact that individual structures of the largest 3 of the 4 protein domains have been previously determined. Anomalous difference Fourier peaks from Se and Br served as markers in the placement of these domains as well as in the model building of the DNA. The resulting structure shows a highly intertwined complex that agrees with a vast array of biochemical data, but also reveals unexpected interactions. 07.12.4 Structural and biochemical studies of symplekin, a scaffold in pre-mRNA 3’ -end processing Kehui Xiang, Takashi Nagaike, Song Xiang, Turgay Kilic, Maia Beh, James Manley, Liang Tong Columbia University, New York, United States The maturation of most eukaryotic messenger RNAs requires extensive processing, including 3’-end cleavage and polyadenylation. The mammalian pre-mRNA 3’-end processing complex has an essential ~1160kD component symplekin, which was originally identified in tight junctions. Symplekin mediates interactions with many other 3’-end processing factors and, like its yeast homolog Pta1, is thought to be a scaffold in the 3’-end processing machinery. Here we report the crystal structure of the N-terminal domain of symplekin in a ternary complex with a RNA polymerase II C-terminal domain (CTD) Ser-5 phosphatase Ssu72 and a CTD phosphor-peptide. Our structure indicates the N-terminal domain of symplekin consists of 7 pairs of anti-parallel helical repeats in an overall shape of an arc. Ssu72 binds to the concave face of the arc, with its active site 25 Å away from the contacting surface. Unexpectedly, we found that the phosphatase activity of Ssu72 can be stimulated by symplekin, suggesting a potential regulatory role for symplekin rather than simply a passive scaffold in pre-mRNA 3’-end processing. More strikingly, the CTD phosphor-peptide in the active site of Ssu72 has a cis pSer5-Pro6 peptide bond configuration, which contrasts to other known CTD peptide conformations. Our studies provide molecular basis with which to understand symplekin as a scaffold and its new function in pre-mRNA 3’-end processing. 07.12.5 Crystal structure of an active Hsmar1 transposase in humans that has evolved into a novel DNA repair protein Millie Georgiadis, Kristie Goodwin, Tsuyoshi Imasaki, Suk-Hee Lee Indiana University School of Medicine, Indianapolis, IN, United States While transposase activity has played an important evolutionary role accounting for half of the present organization of the human genome, little is known about the role of transposases in humans today. The Hsmar1 transposon, a class II transposable element, is an ancient element within the human genome introduced at least 50 million years ago in ancestral primates. To date, only one example of an intact copy of the Hsmar1 transposase domain has been identified within the human genome. This “functional” Hsmar1 transposase domain exists within a chimeric fusion protein, Metnase (also known as SETMAR), which resulted from insertion of the Hsmar1 transposon downstream of a SET gene encoding a histone methyltransferase, ultimately fusing the SET and transposase domains. Metnase retains many of the transposase activities including terminal inverted repeat (TIR) specific DNAbinding activity, DNA cleavage activity, albeit uncoupled from TIR-specific binding, and the ability to form a synaptic complex. However, Metnase has evolved as a DNA repair protein involved in non-homologous end joining. In order to obtain crystals of the transposase domain, an homology structure-based protein engineering approach was used to introduce substitutions for several surface residues. Additional protein engineering efforts including substitution of a Leu residue within the predicted core of the enzyme with a Met in order to increase the expected anomalous signal for SeMet SAD phasing proved essential for the structure determination. The structures of two different crystal forms determined at 2.5 Å and 1.9 Å reveal a novel dimeric enzyme with unusual active site plasticity that may be involved in modulating metal binding. We show through characterization of a dimerization mutant that the dimeric form of the enzyme is required for its DNA cleavage, DNA-binding, and nonhomologous end joining activities. Our analysis suggests that the structure of the Metnase transposase has been remarkably conserved through evolution; however, there is a clustering of substitutions in the modern enzyme within the putative DNA-binding site that may have resulted in a loss of transposition specific DNA cleavage activity and the acquisition of DNA repair specific cleavage activity. 07.12.6 Mechanism of NADH/NAD Sensing by the Redox Sensing Repressor, Rex. Krystle J. McLaughlin, Claire M. Strain, Mark S. B. Paget, Clara L. Kielkopf Department of Biochemistry and Biophysics, University of Rochester School of Medicine and 2 Dentistry, Rochester, NY, United States, Department of Biochemistry, University of Sussex, Falmer, Brighton, United Kingdom The ratio of reduced to oxidized nicotinamide adenine dinucleotide (NADH/NAD ) has emerged as a sensitive indicator of the intracellular redox state. Yet, how the slight chemical + differences between oxidized NAD and reduced NADH are transmitted into signals for DNA + binding remains unknown. To elucidate the molecular basis for NADH/NAD -dependent gene regulation, we present three novel structures of a Rex family repressor (T-Rex), a key sensor of oxygen availability among Gram-positive bacteria. First, the 2.3 Å resolution structure of T+ Rex bound to NAD and a consensus Rex operator site is compared with our previous structure of the induced T-Rex/NADH complex. NADH releases the T-Rex dimer from DNA by a remarkable >40° rigid-body closure between the dimeric subunits. Second, structures of variants in the absence of ligand (at 2.4 Å and 2.5 Å resolution, respectively) reveal that T+ Rex is pre-configured for DNA binding in the absence of NAD . Complementary site-directed mutagenesis experiments establish the importance of highly conserved residues for + NADH/NAD sensing. The NAD(H)-sensing mechanism of Rex may serve as a prototype for a wide-range of redox-sensing proteins whose function is regulated by the intracellular + NADH/NAD ratio. + 1 + 07.12.7 Structural recognition and functional activation of Fc receptors by innate pentraxins Jinghua Lu , Kristopher D. Margon , Lorraine L. Marnell , Carolyn Mold , Terry W. Du Clos , 1 Peter D. Sun Structural Immunology Section, Laboratory of Immunogenetics,National Institute of Allergy 2 and Infectious Diseases, Rockville, MD, United States, Department of Internal Medicine and Department of Molecular Genetics and Microbiology, University of New Mexico, Albuquerque, 3 NM, United States, VA Medical Center, Albuquerque, NM, United States Pentraxins are a family of ancient innate immune mediators conserved throughout evolution. The classical pentraxins include serum amyloid P component (SAP) and C-reactive protein (CRP), which are two of the acute-phase proteins synthesized in response to infection. Both recognize microbial pathogens and activate the classical complement pathway though C1q. More recently, members of the pentraxin family were found to interact with cell-surface Fcγ receptors (Fcγ R) and activate leukocyte-mediated phagocytosis. Here we describe the structural mechanism for pentraxin’ s binding to Fc R and its functional activation of Fcγ Rγ mediated phagocytosis and cytokine secretion. The complex structure between human SAP and Fcγ IIa reveals a diagonally bound receptor on each SAP pentamer with both D1 and D2 domains of the receptor contacting the ridge helices from two SAP subunits. We further extend these findings in two aspects: 1) High-affinity IgG receptor, Fcγ RI, is also recognized by pentraxins. The crystal structure of human Fcγ RI reveals a unique architecture of the three-Ig domains of Fcγ RI, which is reminiscent of the head of a seahorse. Despite the additional third Ig domain (D3), the first two Ig domains (D1 and D2) of Fcγ RI adopt a similar structure to that of Fcγ RIIa and also confer the high affinity for the binding of IgG. Therefore, like Fcγ RIIa, Fcγ RI binds to two diagonally-spanned subunits of each CRP or SAP pentamer with both D1 and D2 domains of the receptor contacting the ridge helices from two SAP subunits. 2) We show that pentraxins also recognizes Fcα RI, both in solution and on cells. Fcα RI bound to the effector face of CRP and SAP in a region close to but not identical with that of Fcγ Rs. The binding of CRP to Fcα RI transfected RBL cells induced degranulation and the phosphorylation of Syk kinase. Mutational and binding studies show that pentraxins are diverse in their binding specificity for FcRs but conserved in their recognition structure. Taken together, these results establish antibody-like functions for pentraxins in the Fc receptor pathway and suggest a new crosstalk between the innate and adaptive immune systems. 1 1 2 2 2 2,3 07.12.8 Engineering a gp41 epitope-display scaffold protein Robyn L. Stanfield, Robert Pejchal, Johannes S. Gach, Michael B. Zwick, Ian A. Wilson The Scripps Research Institute, La Jolla, CA, United States Currently, only a handful of potent, broadly-neutralizing antibodies against the HIV-1 virus have been discovered. Structural studies of these antibodies in complex with their viral epitopes have led to a better understanding of how the antibodies achieve their effective neutralization, and many studies are underway to try to use this structural information to develop an effective vaccine. One epitope, the gp41 Membrane Proximal External Region (MPER) is located on the gp41 protein, adjacent to its’ membrane-spanning domain. Crystal structures of three different broadly-neutralizing antibodies against overlapping sections of the MPER region suggest that the MPER undergoes conformational change during viral fusion, and that the antibodies are recognizing different stages of that process. The epitope is probably only accessible to antibodies for a brief period of time during the pre-hairpin intermediate phase of viral fusion, adding to the difficulty of eliciting similar antibodies against this region. To try to stabilize the MPER epitope in a relevant conformation for use as an immunogen, we are using proteins to act as stabilizing scaffolds for the epitope. Based on the previously determined crystal structure of the anti-HIV-1 Fab Z13e1 in complex with a gp41 epitope peptide (1) we have engineered a scaffold protein that contains the Z13e1 epitope and binds Z13e1 with nanomolar affinity. The scaffold protein was developed by first searching the PDB for proteins with regions of main-chain structural homology to the Z13e1 epitope, and then introducing side chains critical for Fab recognition into that region. We have cloned and expressed the scaffold protein and determined its structure in complex with Fab Z13e1. The crystal structure shows almost perfect structural homology between the scaffold-constrained and peptide epitopes bound to the Fab. This Z13e1 epitope scaffold will be tested as an immunogen to elicit Z13e1-like antibodies against the HIV-1 virus, and will also be useful as a reagent to identify or select for antibodies against the same region. Pejchal, R. et al. A conformational switch in human immunodeficiency virus gp41 revealed by structures of overlapping epitopes recognized by neutralizing antibodies. J. Virol. 83: 8451, 2009. 07.12.9 Mapping of conformational epitopes in dust mite allergens Der f 1 and Der p 1 Maksymilian Chruszcz , Martin D. Chapman , Lisa D. Vailes , Jill Glesner , Anna Pomés , 1 Wladek Minor University of Virginia, Charlottesville, VA, United States, INDOOR Biotechnologies, Inc., Charlottesville, VA, United States The Group 1 mite allergens, Der f 1 and Der p 1, are potent allergens excreted by Dermatophagoides farinae and Dermatophagoides pteronyssinus, respectively. Monoclonal antibody-based epitope mapping studies identified multiple species-specific epitopes on the Group 1 mite allergens, and a unique cross-reactive epitope defined by mAb 4C1. Binding of 4C1 to this epitope inhibits human IgE binding to both allergens. In order to determine the molecular basis of the cross-reactivity, the crystal structures of Der f 1 and Der p 1, both in complex with 4C1, were elucidated. Structural data reveal the epitope that is common to both Der f 1 and Der p 1. In both allergens the epitope is not only formed by the same amino acids, but the conformations of the epitope forming residues are very similar. Moreover these amino acids have the same conformations whether the allergens are complexed with antibody or not, in the case of both Der f 1 and Der p 1. The crystal structure of 4C1 alone shows that the CDR regions of the antibody do not significantly change in conformation upon allergen binding. Identification of the key amino acids involved in the unique cross-reactive epitope on the Group 1 mite allergens in combination with site-directed mutagenesis will facilitate identification of IgE-binding epitopes. This approach will lead to design of modified allergen molecules that could be used in recombinant vaccines for the treatment of dust mite allergy. 1 2 1 2 2 2 2 07.12.10 In crystallo posttranslational dehydrogenase complex 1 1 modification 2 within a 3 MauG/pre-methylamine Carrie Wilmot , Lyndal Jensen , Ruslan Sanishvili , Victor Davidson 1 2 University of Minnesota, Minneapolis, MN, United States, GM/CA-CAT, Advanced Photon 3 Source, Argonne, IL, United States, University of Mississippi Medical Center, Jackson, MS, United States MauG is a highly unusual di-heme enzyme that completes the synthesis of the novel amino acid derived catalytic cofactor, tryptophan tryptophylquinone (TTQ) found in the enzyme methylamine dehydrogenase (MADH). TTQ is formed by post-translational modification of two Trp residues of the β polypeptide chain of MADH during which two atoms of oxygen are incorporated into the indole ring of β Trp57 and a covalent bond is formed between the indole rings of β Trp57 and β Trp108. The natural substrate for MauG (preMADH) is a 119kDa protein precursor of MADH with mono-hydroxylated β Trp57 and no cross-link (Fig 1). MauG catalyzes a six-electron oxidation to complete TTQ biosynthesis, and it can do this in a H2O2dependent or O2/reducing equivalents-dependent reaction. We have solved the X-ray crystal structure of MauG complexed with preMADH to 2.1 Å resolution (Rwork 13.5%; Rfree 18.9%). The c-type heme irons and the nascent TTQ site are separated by long distances over which electron transfer must occur to achieve catalysis. In addition one of the hemes has an atypical His-Tyr axial ligation. The crystalline protein complex is catalytically competent, as on addition of hydrogen peroxide MauG-dependent TTQ synthesis occurs. This structure, also to 2.1 Å resolution (Rwork 14.2%; Rfree 19.4%), identifies the heme to which H2O2 / O2 binds. Figure 1: TTQ synthesis M-002 Enhancing protein crystallization success: Exploring additives and crystal detection. Russell Judge, Sumiko Takahashi, Elizabeth Fry, Kenton Longenecker, Erin Fleck, Mark Chiu Abbott Laboratories, Abbott Park, IL, United States In exploring the use of additives to improve crystallization success, we have focused on the use of ionic liquids. First ionic liquids were tested in the crystallization of model proteins to determine the effectiveness of these solutions as precipitating agents and as additives. The ionic liquids produced changes in crystal morphology and mediated significant increases in crystal size in some cases. Based upon the experiments performed with model proteins, the ionic liquids were used as additives for the crystallization of the poorly diffracting monoclonal antibody 106.3 Fab in complex with the B-type natriuretic peptide (5-13). The ionic liquids improved the crystallization behaviour and provided improved diffraction resulting in the determination of the structure. Our second area of interest is in crystal detection, where the challenge in screening is to quickly identify and distinguish protein crystals from non-protein crystals, which often also form in crystallization experiments. Here we will discuss our experience with UV fluorescence imaging and fluorescent probe labelling for crystal detection. M-005 Crystal Structure of the Stress Response Regulator PerR from Streptococcus pyogenes Shu-Ying Wang, Shi-Yu Chao, Chih-Cheng Tsou, Jiunn-Jong Wu National Cheng Kung University, Tainan, Taiwan Streptococcus pyogenes is a significant human pathogen that causes life-threatening diseases such as necrotizing fasciitis and streptococcus toxic shock syndrome. Unlike other gram-positive bacteria, S. pyogenes does not produce catalase, an oxidoreductase that is essential in other bacteria for resistance to the damaging effects of growth in an aerobic environment. Therefore it would be interesting to understand how this organism copes with such stress. In S. pyogenes, PerR has been identified as a transcriptional repressor of Dpr, which is an iron-binding protein that confers resistance to multiple stresses. Studies have shown that Dpr expression is induced by high concentrations of iron, zinc, nickel, or hydrogen peroxide. In pursuit of understanding the molecular mechanism by which PerR regulates the expression of Dpr, we have solved the crystal structure of PerR to 1.7Å resolution. The structure shows that PerR is a homodimer with two zinc binding sites per monomer. It has been proposed that PerR dimerization is necessary for DNA-binding, and that metal coordination at the regulatory site causes a protein conformational change that affects the protein DNA-binding ability. The crystal structure of PerR provides a basis with which to test these hypotheses and to better understand this entire molecular mechanism. M-008 Crystal Structure and IgE-epitope Mapping of BG60 Tse-Hao Huang , Chun-Hsiang Huang , Song-Nan Su , Ho-Jen Peng , Shwu-Huey Liaw 1 2 1 1 2 2 1 National Yang-Ming University, Taipei, Taiwan, Taipei Veterans General Hospital, Taipei, Taiwan Up to 20% of the population in developed countries suffers from Type 1 allergic diseases such as rhinitis, bronchial asthma or conjunctivitis. The major cause of outdoor allergy is from airborne grass pollen such as Bermuda grass (Cynodon dactylon). A 60-kDa isoallergen mixture of Bermuda grass (BG60) have been isolated and characterized. Here we have reported a crystal structure of BG60 at 2.15 Å resolution, which is the first flavinylated allergen 6 177 113 with the C and the 8 -methyl group of the FAD cofactor cross-linking to Cys and His , respectively. The protein structure belongs to the vanilly-alcohol oxidase (VAO) superfamily, and possesses an open and large substrate-binding groove compared with other known o members. Large BG60 isoforms display a higher Tm value of ~20 C than the small isoforms. A short N-terminal segment is conserved in the superfamily and presence in the large but not the small isoforms, forms extensive interactions with surrounding residues hence greatly enhances the structural integrity. Putative IgE-binding epitopes were then predicted, and several peptide decamers were designed. A peptide representing residues 396-405 displayed strong IgE reactivity to four allergic sera and cross-reacted with BG60-binding IgE. M-011 Structural Basis for Unique Ganglioside Recognition by Botulinum Neurotoxin Type C Zhuji Fu, Abby Kroken, Andrew Karalewitz, Joseph Barbieri, Jung-Ja Kim Medical College of Wisconsin, Milwaukee, WI, United States Botulinum neurotoxins (BoNTs) are comprised of seven serotypes (A through G) and are the most toxic proteins known, causing rapid paralysis through inhibition of neurotransmitter release. BoNTs are synthesized as a single 150 kDa polypeptide. The N-terminal ~50 kDa domain (light chain) is a zinc protease and the C-terminal domain (heavy chain, HC) is composed of a translocation domain (HCT) and a receptor binding domain (HCR). The HCR domain of BoNT/A and BoNT/B bind motor neurons via a cell surface ganglioside and a protein receptor. While earlier studies by Kozaki and Binz and their collaborators showed that HCR/C binds gangliosides, there is limited information for the identity of the host receptor and the mode of neuron binding by BoNT/C. We have determined the crystal structure of HCR/C to 2.5 Å resolution and have studied HCR/C’ s affinitie to various gangliosides by s biochemical/cell-based assays. The overall structure of HCR/C is similar to the HCR domains of BoNT serotypes /A, /B, /E, and /F. However, there are several significant differences, most notably: 1) the relative orientation of the two sub domains is different, and 2) the “gangliosidebinding pocket” is different from that of HCR/A, HCR/B, or HCR/T, lacking the signature Tryptophan, indicating that HCR/C binds gangliosides via a unique mechanism different from other BoNT serotypes, consistent with our biochemical data. In a solid phase binding assay, HCR/C binding to gangliosides was dependent upon W1258 (which is located away from the homologous location of the ganglioside binding pocket of HCR/A), and HCR/C showed the highest affinity for gangliosides that contained two sialic acid moieties. In addition, unlike HCR/A and HCR/B, HCR/C bound primary neurons at 4 C, independent of synaptic activity, and mutation of W1258 drastically abolished the binding of HCR/C to the background level. The structure of the HCR/C, together with the biochemical results, reveals the structural basis for a unique ganglioside binding mode by BoNT/C and provides insight into the basis for BoNT/C entry into neurons. M-014 High-throughput pH Measurements: Seeing is Believing Roger Sayle , Vincent Fazio , Janet Newman 1 1 2 2 2 NextMoves Software, Santa Fe, NM, United States, CSIRO, Parkville, VIC, Australia Coaxing macromolecules into crystals suitable for X-ray diffraction analysis is a multivariate problem, dependent on the type, construct and purity of the macromolecule preparation, and additionally the chemical nature of the precipitation solution and the physical arrangement used to crystallise the protein sample. We are interested in characterizing the process of crystallization so as to understand the biophysical underpinnings, with the long term goal of making the production of suitable diffraction quality protein crystals a more robust and reproducible process. As component of this, we are developing high-throughput techniques to analyse the chemical properties of the precipitants used in crystallisation. We present here a novel method of determining the pH of a solution, which uses imaging technology rather than a physical probe. This allows the pH to be measured rapidly, and with sufficient accuracy, in 96 or higher density plates. We use this assay to return the pH values of 96 well crystallization screens, but perhaps more importantly, as a quality control check during the preparation and storage of crystallization screens. M-017 Center for Structural Genomics of Infectious Diseases (CSGID) Wayne Anderson , Elisabetta Sabini , Aled Edwards , Daved Fremont , Andrzej Joachimiak , 5 6 7 8 2 Wladek Minor , Christine Orengo , Zbyszek Otwinowski , Scott Peterson , Alexei Savchenko Northwestern University, Chicago, IL, United States, University of Toronto, Toronto, 3 4 Canada, Washington University, Saint Louis, MO, United States, University of Chicago, 5 Chicago, IL, United States, University of Virginia, Charlottesville, VA, United States, 6 7 University College London, London, United Kingdom, UTSW, Dallas, TX, United States, 8 JCVI, Rockville, MD, United States The Center for Structural Genomics of Infectious Diseases (CSGID, http://csgid.org/csgid/) has been funded by NIAID with the goal of applying structural genomics approaches to potential drug targets from NIAID category A, B, and C priority pathogens. The goal of CSGID is to determine approximately 400 protein structures during a five-year period. The targets that enter the high throughput pipeline of CSGID are proteins with biomedical relevance and potential therapeutic benefits and they include drug targets, essential enzymes, virulence factors, and vaccine candidates. All the steps, from target selection through structure deposition are available to the scientific community as a free service. Scientists can request structure determination by CSGID for targets of interest simply by filling an online submission form. Community requested targets that are considered to be feasible and biomedically relevant are submitted to NIAID for approval and then entered into the CSGID pipeline. As of April 2010, 21% of the target proteins selected at CSGID have been requested by the scientific community. A major focus of CSGID is to determine the structures of complexes of the target proteins with small molecule ligands such as natural substrates, cofactors and drug candidates, for drug discovery purposes. In order to identify small molecule ligands, target proteins are screened using a thermal denaturation shift assay and a smaller number of possible drug candidates are screened using different CSGID small molecules libraries. The Center also has access to the Life Sciences Collaborative Access Team (LS-CAT) beamlines at the Advanced Photon Source (APS) for screening of crystals using the robotic system for crystal sample changing and the state of the art equipment for crystallographic data collection. After 2½ years since the beginning of the contract, CSGID has deposited in the PDB more than 170 structures, with and without ligands bound, of which 24 belong to targets requested by the community. In addition to structure determination, CSGID also provides the scientific community with the protein expression systems (deposited in the Biodefense and Emerging Infections Research Resources Repository), and the results of ligand screens. CSGID is a consortium of institutions and investigators that have experience with high throughput approaches. Each institution contributes to different aspects of the project, working together to produce a complete, coordinated high throughput structure determination pipeline. CSGID has been funded with Federal funds from the National Institute of Allergy and Infectious Diseases under Contract No. HHSN272200700058C. 1 2 1 1 2 3 4 M-020 X-ray Crystallographic Studies on the Structure-Function Relationship of UDP-glucose4-epimerase from Aspergillus nidulans. Sean Dalrymple, Inder Sheoran, Amira El-Ganiny, Susan Kaminskyj, David Sanders University of Saskatchewan, Saskatoon, SK, Canada Morphologically simple eukaryotes, such as fungi, are becoming increasingly potent human pathogens because the resulting fungal diseases are quite often therapeutically intractable on account of their underlying metabolic similarities with animal systems. Even with aggressive treatment, fungal infections produce high mortality rates and many drugs are starting to lose effectiveness due to emerging fungal resistance. Typically, drugs are focused against fungal extracellular carbohydrates, the building blocks for fungal cell walls, because these components are not found in animal systems. To this effect, we are interested in Aspergillus nidulans UDP-glucose-4-epimerase, UgeA, which is responsible for the interconversion between UDP-glucose and UDP-galactose along the galactose metabolic pathway. As UDPgalactose is a precursor for lipopolysaccharide biosynthesis, UgeA is therefore an important target for antifungal drug development. Furthermore, UgeA exhibits interspecies variation and heterogeneity at both structural and functional levels. Subsequently, the structurefunction relationship differences between UgeA of the host and the pathogen can be exploited for targeted design of potential drugs. As such, detailed structural characterization of UgeA from Aspergillus nidulans is necessary for identifying potential inhibitors. In the present study, we report the preliminary structure of A. nidulans UgeA for which synchrotron diffraction data has been collected at the Canadian Light Source (CLS). The native UgeA data was processed in P1 with unit cell parameters a = 67.1 Å, b = 68.2 Å, c = 163.2 Å, α = 86.4 , β = 82.4 , and γ = 60.7 the unit cell contains six molecules packed as three sets of dimers. A discussion on the solution and refinement of the A. nidulans UgeA structure will be presented along with details of the co-factor binding site as well as the relevant structure-function relationship extracted from the experimental model. M-023 Structure of DUF1341 Representative – a Possible Aldose Norma Duke, Ruiying Wu, Brian Feldmann, Andrzej Joachimiak Argonne National Laboratory, Argonne, IL, United States The crystal structure of CAL13803, a protein found in Yersinia enterocolitica, a Gram-negative coccobacilli, belonging to the family Enterobacteriaceae, has been determined. Y. enterocolitica infections produce severe diarrhea and other symptoms in humans and a search for possible antibacterial targets is on going. The CAL13803 protein belongs to a large family of bacterial proteins of unknown function (DUF1341). Structure was determined to 1.90 Å, using SAD data collected at the 19-BM beamline of the Structural Biology Center. The CAL13803 structure is a classical barrel observed for Class I aldolases with the putative active site residues (K160, Thr190, Phe162) mapping to Escherichia coli 2-keto-3deoxy-6-phosphogluconate (KDPG) aldolase catalytic site template (1FQ0). However, in contrast with trimeric KDPG aldolase, CAL13803 appears to be a dimer. Sequence and structural searches have been conducted to further refine function prediction. BLAST searches find that the sequence is virtually identical to that of the protein ZP_04619510.1, from Yersinia aldovae, which is annotated as a 4-hydroxy-2-oxoglutarate aldolase/2-dehydro3-deoxyphosphogluconate aldolase. The closest structural homologue found, via DALI, is 2keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolase from E. coli (2V82). In addition, close structural homologies are found for pyridoxine 5’ ph osphate syntase from Yersinia pestes (3F4N), and thiamine phosphate pyrophosphorylase from Pyrococcus furiosus (1XI3). Structural comparisons and co-crystallization trials with various ligands are being initiated to help identify specific biochemical function of CAL13803 protein. Acknowledgement: This work was supported by the U.S. Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357, and by a grant from the National Institute of Health (GM074942). M-026 Percentile based spread: a better measure of structural difference Edwin Pozharski University of Maryland, Baltimore, United States Traditional measure of difference between two or more similar structures is the root mean square difference (r.m.s.d.). This estimate of the variation in atomic positions is sensitive to the presence of outliers, which contribute disproportionately to the overall r.m.s.d. With outliers present, the measure does not reflect the average atom shift since the assumption of normal distribution is violated. The percentile based spread is proposed as an alternative measure of structural difference. It is shown that interatomic distances generally follow the theoretically predicted distribution when isomorphous structures are compared. Multivariate approximation is described and possible sources of interatomic distance outliers are discussed, such as variation of model precision, conformational changes and modeling errors. Several examples are analyzed, including isomorphous crystal structures, proteins undergoing conformational changes and multiple model ensembles. M-029 Structures of the Interaction Protein KREPA6 of the Editosome in Complex with VHH Domains from Llama Antibodies Which Served as Crystallization Chaperones Young-jun Park , Meiting Wu , Els Pardon , Stewart Turley , Andrew Hayhurst , Junpeng 1 2 1 Deng , Jan Steyaert , Wim G. J. Hol Biomolecular Structure Center, Department of Biochemistry, School of Medicine, University 2 of Washington, Seattle, WA, United States, Structural Biology Brussels, Vrije Universiteit 3 Brussel, Brussels, Belgium, Department of Virology and Immunology, Southwest Foundation for Biomedical Research, San Antonio, Texas, United States Trypanosomes are protozoan parasites several of which are the causative agents of severe infectious diseases. In trypanosomes, a ~2 M Dalton multi-protein complex called the “editosome” plays a crucial role in mitochondrial gene expression by extensive Uinsertion/deletion editing of pre-mRNAs. Many key editing steps occur in three different types of editosomes, which share a core of 12 proteins. This common set of twelve proteins includes enzymes for uridylyl (U) removal and addition, two RNA ligases, two proteins with RNase III-like domains, and six proteins with predicted oligonucleotide binding (OB) folds. Biochemical results indicate that the OB-fold proteins form an extensive protein-protein interaction network that connects two trimeric subcomplexes that catalyze U removal or addition and RNA ligation. The key “interaction protein” KREPA6, plays a central role in interconnecting several editosome subcomplexes. Crystallization of KREPA6 appeared to be a tremendous challenge but was immediately successful once VHH domains from llama antibodies were employed as “crystallization chaperones”. We used both immune VHH libraries and a large semi-synthetic VHH libraries combined with phage-display techniques to generate and select for VHH domains. Three different crystal structures of KREPA6-VHH domain complexes were solved. In each case a KREPA6 dimer occurred in the center of the heterotetramer. Interestingly, biochemical solution studies showed that the VHH domains dissociate the KREPA6 homotetramers and form heterotetramers as seen in the crystal structures. Solution studies also indicated that the C-terminal tail of KREPA6 is involved in the dimerization of KREPA6 dimers to form tetramers. The crystal structures of two of the VHH domains showed a novel parallel arrangement of β -strands from antibody and protein antigen. The three structures of KREPA6 in complex with VHH domains show how the antibodies facilitate protein crystallization by taking care of the majority or all of the crystal contacts. These studies show that llama VHH domains are versatile tools to crystallize recalcitrant proteins. 1 1 1 2 1 3 M-032 IMCA-CAT Insertion Device Beamline Upgraded for High-Throughput, Shutterless, Continuous-Rotation Data Collection Anne M. Mulichak, Kevin P. Battaile, Joe Digilio, Rong Huang, J. Lewis Muir, Eric Zoellner, Ann Bertling, Lisa J. Keefe IMCA-CAT, Argonne National Laboratory, Argonne, IL, United States The IMCA-CAT insertion device beamline, 17-ID, has been upgraded to a micro-focused high-flux diffraction beamline for automated high-throughput macromolecular crystallography. The energy range is 6–20 keV, allowing MAD/SAD experiments at energies for commonly used derivatives. The full beam is focused to 65 m x 30 m at the sample position and the GM/CA-CAT mini-quad collimators provide the user with selectable beam sizes of 300, 20, 10, and 5 m. Beam stability is achieved with custom software that automatically positions the beam with ±2 m positional accuracy. Automated sample mounting is performed with the Rigaku ACTOR robot, and samples are viewed with the Maatel on-axis viewing system. The new ALIO goniometer has a small (1.2 m) sphere of confusion, thus maintaining accurate sample positioning. A new detector, the PILATUS 6M pixel-array from DECTRIS, permits shutterless, continuous-rotation data collection. The high volume of data is managed with a 64 TB storage system that consists of a highly-available Lustre distributed parallel file system with Fibre Channel and InfiniBand interconnects. Custom software provides an intuitive interface for controlling the beamline. Rigaku JDirector software for data collection enables queuing of data collection jobs for automated data acquisition. Both unattended and remote data collection modes are supported. The automated and rapid data collection capabilities of beamline 17-ID are ideally suited for high-throughput crystallography projects such as pharmaceutical industry drug discovery programs. ! ! ! ! ! M-036 Crystal structure of PAN C-termini bound to 20S proteasome reveals AAA+ ATPase open the 20S gated channel with a mechanism different from that of 11S activator. Yadong Yu , David Smith , Alfred Goldberg , Yifan Cheng 1 2 1 2 2 1 UCSF, San Francisco, CA, United States, Harvard Medical School, Boston, MA, United States The primary site for protein degradation in eukaryotic cell is the 26S proteasome, which is composed of the 20S proteolytic core and two 19S regulatory particles that each contains a hexameric ATPase ring. The ATPases activate proteolysis by docking their C-termini with a conserved hydrophobic-tyrosine-X (HbYX) motif into pockets in the 20S to stimulate the opening of a gated substrate entry channel into the 20S. In contrast, 11S activators use Ctermini for binding while the additional structural element, activation-loops open 20S gate. To understand how PAN relies only on C-termini to activate 20S, we inactivated PA26 by mutating its activation-loops and replaced its C-termini with those from PAN. The resulting fusion protein formed tight complex with 20S. Cryo-EM reconstruction and crystal structure of this complex were solved at 8 and 4Å respectively. Both structures show a rotation in 20S ring compared to that in 20S structure itself. The crystal structure defined the detailed interactions between the critical C-terminal HbYX motif and the 20S -subunits. In particular the H-bond, cation-π and hydrophobic interactions contributed by the highly conserved tyrosine residue largely trigger the radial movement of -subunit N-terminal fragments which lead to gate opening. Upon binding of HbYX motif, 20S pocket undergoes an induced-fit conformational change and becomes much tigher than those with 11S activitor. Altogether these 7 tightened pockets transform into a rotation of -ring. The findings imply that AAA+ ATPases use C-termini of higher specificity to activate 20S. It may further help understand how hexameric AAA+ ATPase can work out the symmetry mismatch with heptameric 20S proteasome. " " " " M-039 Crystallographic and Cryo-EM Studies of the HK97-like Bacteriophage DNA Packaging Portal Bogi Nocek , Dong-Hua Chen , Adam Stein , Rory Mulligan , James Abdullah , Robert 1 2 1 Jedrzejczak , Wah Chiu , Andrzej Joachimiak Midwest Center for Structural Genomics, Argonne, Il, United States, National Center for Macromolecular Imaging, Houston,Tx, United States During large dsDNA virus assembly, viral DNA synthesized using bacterial resources is transferred into preformed empty prohead. The assembly and packaging of viral DNA is driven by a translocation motor system composed of several proteins and is powered by hydrolysis of ATP. A key component of the packaging machine is a portal protein. This protein assembles into a large ~500 kDa ring-shaped portal with a central channel. The motor connects the head of the phage to its tail and promotes translocation of the dsDNA into the prohead during packaging. At present, the structural information of phage portals is limited to two representatives, SPP1 and phi29. Here we present the 2.9 Å crystal structure and low resolution cryo-EM results of the dsDNA bacteriophage HK97 family portal. HK97 bacteriophages are widespread and they infect Gram-positive bacterial hosts. The portal is a 300-residue protein and shares very little sequence similarity to SPP1 and phi29 proteins. It assembles into a dodecamer with a characteristic funnel-like structure and a 40 Å wide central channel. The surface of the channel is mainly electronegative, but it includes three positively charged rings that may promote DNA transfer. Details of the HK97 structures will be presented. This work was supported by National Institutes of Health Grant GM074942 and by the U.S. Department of Energy, Office of Biological and Environmental Research, under contract DEAC02-06CH11357. 1 2 1 2 1 1 1 M-042 Capillary-top crystal mounting method for S-SAD phasing and semi-automated mounting device Nobuhisa Watanabe , Yu Kitago , Isao Tanaka 1 2 1,2 2 2 Nagoya University, Nagoya, Japan, Hokkaido University, Sapporo, Japan For the S-SAD phasing that utilizes single-wavelength anomalous diffraction from sulfur atoms, using longer-wavelength X-rays has advantages for the detection of small anomalous signals from them. However, the accuracy of the measured diffraction intensity decreases at longer wavelengths because of the greater X-ray absorption effect. We had improved the standard cryogenic crystal mounting method with a new tool, the capillary-top mounting method (formerly the loopless mounting method), by which we can remove the buffer around the protein crystal just before flash freezing of the crystals. This capillary-top mounting method makes it possible to eliminate amorphous ice around the protein crystal and reduces systematic errors in the evaluation of small anomalous differences. However, use of this method requires a large amount of skill. The processes of harvesting and flash freezing the crystal are performed using both hands, and the mouth is also used for cryo-solution aspiration. In order to reduce its laboriousness, we have developed a new device that can freeze the protein crystal semi-automatically using a micro-manipulator. Using this device, one can harvest the protein crystal from the crystallization drop, and further procedures, such as withdrawal of the solution around the crystal by suction and subsequent flash freezing of the protein crystal, are carried out automatically. The loop glued to the tip of the glass capillary of the custom cryo-pin for the capillary-top mounting method was also improved. The conventional nylon loop was replaced with a microlithography shaped polyimide film. We have recently designed a new pattern of the polyimide film and made a prototype of an implementation tool for the gluing process of the polyimide film at the tip of the glass capillary. These devices make it easy for structural biologists to use the capillary-top mounting method for S-SAD phasing using longer-wavelength X-rays. This work was supported by the Targeted Proteins Research Program (TPRP) of the Ministry of Education, Culture, Sports, Science and Technology, Japan. M-045 Function Assignment by Catalytic Site Alignment with ProMOL Paul Craig , Mario Rosa , Scott Mottarella , Greg Dodge , Sean Bourne , Luticha Doucette , 2 Herbert Bernstein 1 1 1 1 1 1 1 Rochester Institute of Technology, Rochester, NY, United States, Oakdale, NY, United States 2 Dowling College, The ProMOL plug-in for the PyMOL molecular graphics environment uses the geometry and measurement tools incorporated in PyMOL to identify and align the catalytic site motifs of enzymes. The motifs are described in the Catalytic Site Atlas (http://www.ebi.ac.uk/thorntonsrv/databases/CSA/). These motifs were used by Torrance et al., (J. Mol. Biol. 347:565-81, 2005) to create two sets of JESS templates that were distributed broadly across the six classes of the Enzyme Classification system. The first template set was based on two atoms (JESS CaCb: C-alpha and C-beta of each catalytic site residue) while the second template set was based on three atoms (JESS FA: C-alpha, C-beta, and one side chain atom) to explore enzyme family homology. We have used a subset of the same structures (20 total PDB entries) to prepare motifs in ProMOL that are based on all side chain atoms of the catalytic site residues. The performance of the two JESS template sets and the ProMOL template set were compared for the template structures, their homologs and randomly chosen structures from the Protein Data Bank. The ProMOL motifs returned an exact identification of templates and known homologs 74% of the time, while the JESS CaCb templates returned exact identifications 8% of the time and the JESS FA templates returned an exact identification 18% of the time. These templates were then used to suggest functions for multiple PDB entries that are classified as "Structural Genomics, Unknown Function". Of 39 entries that were tested, ProMOL template alignment suggested 28 function assignments while the JESS FA template alignment suggested 4 function assignments. The library of ProMOL templates is being expanded and will be used to extensively test PDB structures of unknown function. The project is funded in part by NIGMS grants R15 GM078077-01 and 3 R15 GM07807701S1. M-048 How we got the chemical bond wrong (and how we can rescue it) I. David Brown McMaster University, Hamilton, Ontario, Canada How do you define a bond? And why do we need both an ionic and a covalent model? Neither model provides a satisfactory definition of a bond, and the boundary between the two models is fuzzy. The concept of a localized bond, which is good for intuitive modeling, is only found in the covalent picture, but simulations of structures are only successfully performed using the ionic model. And quantum mechanics sees no clear difference between ionic and covalent bonds, so why do we still need two incompatible models to describe chemical structure? The mistake we have made is to assume that a chemical bond necessarily involves a pair of electrons. If we abandon this idea we can create a unified localized bond model using only two atomic properties: the valence (oxidation state) and the coordination number. The electrostatic field of the ionic model defines localized bonds that combine the intuition of the covalent model with the quantitative predictions of the ionic model. This results in a simpler, more revealing and powerful picture of how localized bonding can be used to predict chemical structure and properties for all types of bond. A key feature of the unified model is an electronegativity, defined as the valence of an atom divided by its coordination number. Atoms with electronegativity > 1 have more valence electrons than bonds. They tend to form saturated bonds with the remainder appearing as lone pairs. Atoms with electronegativity < 1 have more bonds than valence electrons and form extended structures. M-051 Conformational Polymorphism in Crystals of a Novel HCV Inhibitor Qi Gao , Antonio Ramirez , Fukang Yang , Soojin Kim , Baoqing Ma , Michael Galella 1 2 1 1 2 1 2 3 Bristol-Myers Squibb Company, New Brunswick, NJ 08903, United States, Bristol-Myers 3 Squibb Company, Wallingford, CT 06492, United States, Bristol-Myers Squibb Company, Princeton, NJ 08540, United States The subject of this paper is two neat polymorphic forms of bis-HCl salt of a potent Hepatitis C virus (HCV) inhibitor targeting the virus-encoded nonstructural protein 5A (NS5A). Crystallographic studies of the polymorphic N-1 and N-2 forms showed two dramatically different conformations that reflect the C2 symmetry in the chemical structure. In the crystal of N-1, the molecule sits on a crystallographic 2-fold axis, while in the crystal of N-2 it is found in general positions with a pseudo 2-fold axis that is perpendicular to the ideal 2-fold axis in the conformation of N-1. We have not been able to prepare N-1 since the first time when N-2 was crystallized. The extinct N-1 was reasoned to be probably a metastable kinetic form, although it was not experimentally confirmed due to the lack of material. X-ray crystal structures of N-1 and N-2 were analyzed to understand the relationship of intramolecular (conformer) and intermolecular (lattice) energy in the crystallization and stability of polymorphs. Computational methods were also applied to gain a semi-quantitative assessment of the relative stability between the two polymorphs. Semiempirical AM1 calculations suggested greater stability of the N-2 conformation and that this stability does not appear to be related to salt formation, although salt formation could be crucial for crystal packing and lattice energy. This is in agreement with our experimental observations that no crystallization has been achieved with the free base. MMFF coordinate scans for isolated dihedral angles indicated that central arylaryl angle bending in N-1 is an important source of destabilization and the -isopropylcarbamate side-chain conformations are unfavorable. # M-054 Crystal structure of phosphoglucosamine mutase from Bacillus anthracis, an enzyme in the peptidoglycan biosynthetic pathway. Ritcha Mehra-Chaudhary, Lesa Beamer University of Missouri, Columbia, MO, United States The enzyme phosphoglucosamine mutase (PNGM) participates in the biosynthetic pathway of peptidoglycan, a component of the bacterial cell wall. Enzymes in this pathway are considered excellent targets for antibacterial compounds. We present here the first structure of a PNGM, from the bioterrorism agent B. anthracis. Peptidoglycan is a major component of the protective coat of the bacterial spore, and plays a known role in the pathogenesis of anthrax. The structure of B. anthracis PNGM (447 residues) was determined by molecular replacement (MR) at a resolution of 2.7 Å. Limited sequence identity (~30%) between B. anthracis PNGM and the available models complicated the MR efforts. Conformational variation between the two molecules in the asymmetric unit (asu) was an additional hurdle to refinement of the structure. Anomalous difference Fourier maps from a 3.2 Å selenomethionine data set proved critical for identifying methionine residues and successful model building. The resulting structure reveals that PNGM shares the overall architecture common to the large a-D-phosphohexomutase enzyme superfamily, although domain 4 of the protein has topological differences from other structurally characterized members of the family. The structure also shows that while key catalytic residues are highly conserved, other residues within the active site vary, and may be responsible for the distinct substrate specificity of the PNGM enzymes. A novel observation from the crystal structure is a potential dimeric interface between the two molecules in the asu, which is also present in the crystal packing of several related enzymes, and suggests that PNGM enzymes may function as dimers in solution. The implications of the structure for understanding enzyme function and its utility for inhibitor design with applications to medicine and bioterrorism will be discussed. M-057 Structural Basis for Substrate Specificity of Inositol Pentakisphosphate 2-Kinase Varin Gosein, Gregory J. Miller McGill University, Montreal, Canada Inositol phosphate (IP) signaling pathways are critically involved in cell communication and IPs have been implicated in a number of diseases including cancer and diabetes. IP signaling pathways are maintained by the IP kinase family of enzymes that sequentially phosphorylate inositol triphosphate (IP3) to create an array of >30 signaling molecules with up to 8 phosphate groups on the inositol ring. One IP kinase, inositol pentakisphosphate 2-kinase (IPK1), catalyzes the conversion of inositol 1,3,4,5,6 pentakisphosphate (IP5) to inositol hexakisphosphate (IP6). Studies have shown that IP6 possesses anticancer activity in vitro and in vivo against numerous tumors. IP6 treatment of human leukemic cell lines in vitro significantly reduced the abnormal cell population while leaving normal leukocytes unaffected while IP6 given to animal models for colon, liver, lung, and skin cancers reveal the antitumor activity of IP6 in vivo. The precise mechanisms underlying the action of IP6 remain unresolved, as there are no selective inhibitors for IPK1 to modulate levels of IP6 and knockouts of IPK1 are embryonic lethal. A high-resolution molecular structure of IPK1 would facilitate the design of selective inhibitors for IPK1 and would provide the first insights into the catalytic cycle and mechanism of the enzyme. Studies in yeast have shown mutations in IPK1 can be rescued by IPK1 orthologs, which demonstrates functional conservation of IPK1 despite very low sequence similarity across species. Arabidopsis thaliana IPK1 (AtIPK1) shows highest conservation with human IPK1 (HsIPK1) within six short regions; however, the degree of similarity decreases significantly outside of these regions. It is anticipated that these regions assemble into commonly structured catalytic sites and, in fact, may adopt a novel fold. HsIPK1 accumulates in inclusion bodies when expressed in E.coli which has complicated structure determination of this IPK1, but bacterial expression of AtIPK1 has been proven to be soluble and active and amenable to crystallization. Our overall objective is (1) develop the first system for investigation of the catalytic cycle of IPK1 during IP6 synthesis, and (2) to generate a homology model of HsIPK1 using a crystal structure of AtIPK1. M-060 Resonant Multi-Wave Diffraction Study on Magnetite Shih-Chang Weng, Cheng-Gang Chen, Yen-Ru Lee, Chia-Hung Chu, Shih-Lin Chang Department of Physics, Natl. Tsing Hua Univ., Hsihchu, Taiwan Multi-wave diffraction under resonance conditions, relating to both atomic and electronic structures, provides site- and element-specific information of the crystal studied. This characteristic gives an opportunity to investigate the electronic configuration of interesting materials, such as transition-metal oxides. In this presentation, the resonant multi-wave diffraction is used to study the charge distribution of magnetite below the Verwey transition temperature. Due to the three-wave interference and resonant scattering, resonant multiwave diffraction could reveal the 3d-electron distribution in magnetite. Two three-wave diffractions, (002)/(-3-31) and (002)/(311), were measured in the vicinity of Fe K-edge. The self-normalized relative intensities were recorded for different photon energies and compared with the calculated results based on the dynamical diffraction theory with Born approximation. The variation of the diffracted intensity shows the evidence of charge-ordering on the octahedral iron sites of magnetite below the Verwey transition temperature. M-063 X-ray Diffraction Measurements of Substrate-Supported Crystals Using a Hexapod Lin Yang Brookhaven National Laboratory, Upton, NY, United States Crystals formed on a planar substrate usually have a common crystal plane parallel to the substrate while their in-plane orientation undefined. In diffraction measurements of these structures, it is often required to anchor the X-ray beam on a fixed spot on the sample, such as an optically visible crystallite or island, while rotating the sample to access different Bragg peaks, so that the lattice constants as well as the local orientation of the lattice can be defined. Here, a hexapod is used in place of a traditional multi-circle diffractometer to perform area-detector-based diffraction measurements on an organic transistor device that contains 6,13-bis(triisopropylsilyethynyl)-pentacene (TIPS-pentacene) crystals. The hexapod allows for sample rotations about any user-defined rotation center. Two types of complex sample motions have been programmed to characterize the structure of the TIPS-pentacene crystal: an in-plane powder average has been performed at a fixed grazing-incident angle to determine the lattice parameters of the crystal; then the in-plane component of the scattering vector was continuously rotated in transmission geometry to determine the local lattice orientation. M-066 Real space asymmetric units optimized for integer grids Marat Mustyakimov , Ralf Grosse-Kunstleve , Paul Langan 1 1 2 1 2 Los Alamos National Lab, Los Alamos, NM, United States, Berkeley, NM, United States Lawrence Berkeley Lab, Efficient handling of direct-space asymmetric unit information in crystallographic map calculations can significantly accelerate widely used algorithms, e.g. bulk-solvent correction in macro-molecular refinement. We have investigated various trade-offs in the choice of optimized asymmetric unit definitions. It has been found that a number of the established asymmetric unit definitions in the International Tables (IT, Vol. A) are not optimal for the handling of grids as used in map calculations. More suitable definitions are suggested. A major property of the alternative definitions is that the interior of each face or edge of the asymmetric unit polyhedron has a constant multiplicity. We observe that such an asymmetric unit exists for each space group type. The new definitions and related algorithms have been added to the cctbx library. M-069 Portable Temperature Controlled Microplate for Optimization of Protein Crystals Gabriela Juarez-Martinez, Philipp Steinmann Centeo Biosciences Ltd, Dumbarton, United Kingdom Using temperature as an additional crystallization variable can increase space search and help finding better crystallisation conditions. The correct management can aid in controlling crystal nucleation, growth and dissolution of defects on the surface of the crystal; can modify the solubility and super saturation of the sample in a reversible manner; can prevent denaturation of temperature sensitive proteins and in improving the reproducibility of results. Currently the crystallography community is restricted to use fridges/incubators and temperature controlled rooms, however these do not always offer the best solution. Problems arise when the samples are taken out of the fridge/incubator e.g. for inspection under the microscope. At that moment the temperature control is lost which in some cases can affect the systems in a negative manner (dissolution of the crystal, protein denaturation, destabilization of the system). Even if the unintended temperature fluctuation result in the formation of a crystal, without knowing anything about this temperature fluctuation, the results are often difficult to reproduce. In response to this problem, Centeo has designed an electronic portable temperature controlled microplate. The system consists of 5 rows with 8 wells per row, allowing screening up to 40 different experiments simultaneously. Each row can be independently controlled allowing to screen up to 5 different temperatures from 4° to 60° at once. When in use, the C C microplate resides in a docking station, from which it can be removed for portable, battery powered operation. This work will show the performance of the system including crystallisation results. M-072 High-throughput Crystallization: What have we Learned? Ievgeniia Dubrovska, Ludmilla Shuvalova Northwestern University, Chicago IL, United States Structural genomics has been around for about 10 years and produced numerous important new methods, automated procedures and inspired design of robotic instruments. These new methods and tools were rapidly incorporated into the structural genomics center’s pipelines and adopted by structural biology labs. One of the critical stages of protein structure determination, crystallization, was not an exception. With all that new technology do we have to change the strategy of the experiment? At Northwestern University group of Center for Structural Genomics of Infectious Diseases (CSGID) we have tried to analyze our observations of crystallization experiments accumulated in 2.5 years of our project. How does one decide when protein is ready for screening? Which screens and how many to use? How to best balance time and effort involved with sample requirements? Crystals growing “like mushrooms”…do we want them to? Does ligand screening help? For how long to keep the plate? These and some other questions addressed in our poster presentation. M-075 Synthesis, Structures and Characterization of Organotin complexes S. M. Lee, H. Mohd Ali, K. M. Lo University of Malaya, Kuala Lumpur, Malaysia Based on literature review, metal complexes are widely prepared and have been successfully applied in the treatment of numerous human diseases including cancer. Among the many metal complexes, organotin complexes have been widely studied for their biological activities such as anticancer, antihistamine, antifungal, biocides and anti-fouling. Schiff base derived from substituted salicylaldehyde has been widely used as polydentate ligands in the preparation of metal complexes. In the present studies, a series of Schiff base ligands were prepared by reacting 3-hydroxy-2-naphthoic hydrazide with substituted salicylaldehydes. The diorganotin complexes were subsequently prepared by adding the ligands with diorganotin dichloride or oxide in 1:1 molar ratio and were characterized by various spectroscopic methods including IR, NMR spectroscopies. The x-ray structures of some of the diorganotin complexes were reported. All the complexes were found to be isostructures and the tin atom in each of the complexes is in a distorted cis-C2NO2Sn trigonal-bypyrimidal coordination. The deprotonated ligand was coordinated as tridentate via the azomethine nitrogen and two phenoxo oxygens. The tridentate 5-bromosalicylideneaminato(3-hydroxy-2naphthohydrazidate) and 5-chlorosalicylideneaminato(3-hydroxy-2-naphthohydrazidate) dianions of each of the complexes were stabilized by an intramolecular hydrogen bonding OH—N. The distortion from the trigonal-bypyrimidal coordination was influenced by the presence of the R groups. M-078 A Structural Study of Second-order Jahn-Teller Distortions in CuX5 Complexes (X=Cl, Br). Bryan Reynolds, Marcus Bond Southeast Missouri State University, Cape Girardeau, MOI, United States While Jahn-Teller distortions in CuX6 and CuX4 complexes are well known and studied, the second-order Jahn-Teller distortions in CuX5 complexes have received less attention. Reinen and Astanasov (Chem. Phys. (1989) 136, 27) have mapped out the second-order Jahn-Teller 2+ active distortion modes for trigonal bipyramidal Cu following the discovery by Reinen and Friebel (Inorg. Chem. (1984) 23, 791) that [Co(NH3)6]CuCl5 contains disordered, distorted C2v3symmetry CuCl5 rather than a trigonal bipyramidal complex. Since that time many new pentacoordinate halocuprate(II) structures have been reported, some of which show distortions outside the direct Berry rotation pathway that interconverts square pyramidal and trigonal bipyramidal geometries. We have examined distortion modes, as defined by Reinen and Astanasov, of CuX5 complexes (both isolated and not) found in structures from the Cambridge Structural Database and from our own laboratory, and map out the frequency with which these modes are observed experimentally. In addition we examine the structural data to seek an empirically defined distinction between five-coordinate CuX5 complexes and 4+1 coordinate complexes in which one Cu-X bond is semicoordinate. M-081 Increasing accuracy in low-resolution structures Bradley Hintze, Christopher Williams, Vincent Chen, Dave Richardson, Jane Richardson Duke University, Durham, NC, United States Structural biology provides an unparalleled view of the molecular world, allowing researchers to uncover important mechanisms that give rise to a protein’s function, provided there is sufficient detail in the data (which is generally a function of the data’s resolution). Currently, much research in structural biology is devoted to structure determination of large proteins and complexes, which tend to yield low-resolution data and present many challenges to crystallographers. Despite a variety of computational techniques that can be employed to assist low-resolution structure determination, validation typically shows an order of magnitude more errors (such as atom-to-atom clashes and highly irregular secondary structure) in 3-4 Å models than in 1-1.5 Å models. The figure shows a squashed surface helix at 3.5 Å resolution with the sidechain density as small nubbins; fitting such density often leads to Ramachandran and rotamer outliers. The research presented here aims to identify systematic patterns of such errors and create techniques to diagnose and correct them. We will utilize more thorough restraints on the regularity of geometry, torsion-angle patterns, and secondary structure and will rely on the power of consistently satisfying both allatom contacts (with explicit hydrogens) and the diffraction data. Finding such solutions will require new calculation methods informed by analysis of how low-resolution systematically distorts electron data. Significantly better accuracy would lead to great improvements in the biological utility of low-resolution structures. M-084 Apex2 and Proteum2 Suites – Hints and Tips Garold L. Bryant, Jr., Bruce C. Noll, Michael Ruf, Charles F. Campana, Matthew M. Benning, Joerg Kaercher Bruker AXS Inc., Madison, WI, United States The Apex2 and Proteum2 suites represent at least 40 man-years of programming. Several online venues exist for finding help and even though every effort has been made to make the suites intuitive, new features and enhancements are sometimes overlooked. Many helpful features such as report and CIF file generation are now available. Helpful hints and tips from application scientists will cover a wide variety of questions usually encountered by customers. A helpful list of do’s and don’ts for Proteum2, Apex 2 and BIS will be presented. Getting the most from various configurations of Bruker instruments will be presented. M-087 Screening Based on Proximity to Crystallization Conditions, Not Crystals. Marc Pusey iXpressGenes, Huntsville, AL, United States Current crystallization screening protocols involve exposure of the macromolecule to a range of cocktail solutions, with the endpoint being the appearance of a crystal. Conditions resulting in clear or precipitated outcomes are dropped from subsequent consideration. We suggest that a more advantageous approach would be a method that indicates proximity to crystallization conditions, not one that relies on randomly getting lucky. We are developing such an approach based upon fluorescence anisotropy, a measure of the rotational rate (and thus volume or mass) of the protein in solution. The method involves measuring a series of dilutions of the protein in the crystallization solution. Lead conditions are optimized using the capillary counter diffusion method. Experiments with model proteins, where the plate-based outcomes were already known, indicated that ≥ 80% more crystallization conditions could be obtained on average. We are now using this as the sole screening method with test proteins and to date has found crystallization conditions for all of them. The data also indicates that the signal intensity as a function of concentration is also a significant lead indicator. Advantages are: Speed, screening results can be had within 6-30 hrs after having pure protein in hand; more leads, many conditions that would have been discarded are found to be leads; reduced protein consumption, fewer screens and assay drop volume reduction can reduce the protein required to ≤ 0.3 mg/96 condition screen. Finally, the data is obtained as numerical values, facilitating subsequent analysis. M-090 Crystallographic Studies of a Fluorescent Phytochrome Michele Auldridge, Kenneth Satyshur, David Anstrom, Katrina Forest University of Wisconsin, Madison, WI, United States The three dimensional structure of phytochrome from Deinococcus radiodurans provided an opportunity to design structure-based variants of this red light photoreceptor with altered photocycle properties. The most dramatic of these are a family of fluorophores with substitutions at the Asp207 position. Given that these novel proteins use a tetrapyrrole chromophore available within cells and have emission maxima above 700 nm, they are extremely promising biotechnology tools. We have solved the crystal structure of the Asp207His variant of the chromophore-binding domain (CBD) of D. radiodurans phytochrome, and at 1.24 Å this structure is the highest resolution DrBphP structure to date. An altered hydrogen bond network within the biliverdin-binding pocket is observed. His207 forms a direct hydrogen bond to the chromophore A ring oxygen, and an interaction between Tyr263 and the now-missing Asp207 is altered. Additionally, with the goal of decreasing the size of this fluorophore and facilitating its use as a fluorescent tag in vivo, we changed several residues in the native coiled-coil dimer interface. Most significantly, Leu311 and Leu314 were replaced with glutamate to introduce charge repulsion. Interestingly, recombinantly expressed Dr phytochrome with the Asp207His substitution surreptitously binds the biliverdin precursor, protoporphyrin IX. Structural studies of phytochrome with this cyclic tetrapyrrole are currently underway. M-093 The SMART X2S: Reports from the field. Experiences in Research and Teaching. Bruce C. Noll, Michael Ruf, Joerg Kaercher Bruker AXS Inc, Madison, Wisconsin, United States The Bruker SMART X2S automated benchtop diffractometer for single crystal diffraction has been installed in a number of facilities for teaching and for research. It is showing itself to be fully capable of accommodating both tasks. After some time in the field, the SMART X2S is producing publications and training students in the science of crystallography. The instrument has been used for undergraduate education, graduate research, and as a principal instrument at the ACA Summer School. At the 2009 Summer School, eleven structures were determined in nine days. A summary of these varied experiences will be presented. M-096 Progress on the Structure of the Essential Yersinia pseudotuberculosis Type III Secretion System Membrane Protein YscU Xiaoming Zhao , Innokentiy Maslennikov , Christos Tzitzilonis , Roland Riek , Partho Ghosh 1 2 1 2 2 2 1 University of California San Diego, La Jolla, United States, Salk Institute for Biological Studies, La Jolla, United States YscU is an essential inner membrane protein of the Yersinia pseudotuberculosis type III secretion system, which is responsible for translocating bacterial proteins from the bacterial cytosol into the cytosol of host mammalian cells. YscU is predicted to contain four transmembrane domains and a large, C terminal cytosolic domain. Intact YscU and two different truncation fragments of YscU, all containing the transmembrane regions, have been cloned as TEV protease-cleavable fusions to the protein MISTIC. Intact YscU and the truncation fragments of YscU were found to be expressed to levels required for crystallographic studies. These proteins were extracted from membranes with detergent and 2+ purified by Ni NTA chromatography, after which MISTIC was removed by cleavage with 2+ TEV protease. Cleaved proteins were reapplied to the Ni NTA column and lastly purified by size-exclusion chromatography. Detergent concentrations of purified protein samples were analyzed by NMR, and oligomerization states by static light scattering. Crystallization trials are underway. M-099 Enhancements in Motif Preparation and Identification in ProMOL Mario Rosa , Herbert Bernstein , Paul Craig 1 1 2 1 2 Rochester Institute of Technololgy, Rochester, NY, United States, Oakdale, NY, United States Dowling College, The ProMOL plug-in for PyMOL enables users to create motifs based on catalytic sites and then identify the function of protein structures by comparison to these motifs. Significant upgrades have been made in the functionality and coding of the plug-in. The Motif Maker function was rewritten to eliminate some earlier bugs and to expand its capabilities. It can now build a motif with up to 10 residues (rather than 5) and repeated residues in an active site (e.g., 3 histidines) are now accepted. Now it is possible to explore larger motifs such as those found in protein-protein interaction sites. Once testing is complete, the code written by Motif Maker will be readily incorporated directly into ProMOL, which will expand the capabilities of the Motif Finder. The code for the Motif Finder in ProMOL was expanded to include an expanded set of motifs (based on the JESS motif set) that more systematically covers the different enzyme classes. The results generated by Motif Finder can now be viewed on the screen directly or exported as a simple table with comma separated values in a format that can be read by most spreadsheet programs. In the future, we plan to change the motif finder to compare exact residue matches (rather than the atom counting currently in place) and to handle batch processing of structure submissions to the Motif Finder. As an open source plugin, ease of modification of ProMOL is important. The code used of ProMOL was originally stored in a single file, which hampered efforts at further development. Therefore, the file was split into several files. The original file retains the functions necessary to hook ProMOL to PyMOL and to initiate the user interface of ProMOL. The other files are stored within a directory 'ProMol_dir' and grouped according to their function in the overall program. For example, files that hold the graphic user interface are stored within the 'Tabs' folder, while files used in the methodology of the program are stored in the 'Methods' folder. The project is funded in part by NIGMS grants R15 GM078077-01 and 3 R15 GM07807701S1. M-102 Copper Binding and Dynamic Behavior in Doped Cadmium-Histidine Michael Colaneri , Kristin Kirschbaum , Jacqueline Vitali 1 1 2 3 2 SUNY at Old Westbury, Old Westbury, NY, United States, University of Toledo, Toledo, OH, 3 United States, Cleveland State University, Cleveland, OH, United States Electron Paramagnetic Resonance (EPR) spectroscopy and X-ray crystallography was employed to gain a better understanding of copper binding and dynamic behavior in doped histidine crystals. Recent EPR measurements on copper-doped Cd-histidine showed a significant temperature dependence that can be explained by copper site dynamics. At temperatures lower than 150K, the EPR spectra exhibit two copper patterns, each related by the crystalline two-fold axis that runs through the host cadmium ion and between the two coordinating histidines. At a temperature higher than 200K and up to room temperature, the EPR spectra consist of only a single copper species that is characterized by rhombic g and A hyperfine tensor values, which are close to the averaged values of the corresponding low temperature, symmetry site related tensors. Subsequent EPR experiments display a 1:1 conversion from the low to high temperature copper species within a relatively narrow temperature range near 175K. These observations differ from those found in a previous EPR and crystallographic study of the dynamic behavior of copper in doped Zn- histidine (Dalosto et al., J. Phys. Chem. 2001, A105, 1074). Here the authors propose that the copper undergoes a continuous 4-state dynamic Jahn-Teller distortion, having a transition temperature near 268K, and further suggest that the copper site dynamic behavior is correlated with a fluctuating disorder of a water found in the host structure. In order to investigate the possibility of a similar type of host structural explanation, the crystallographic analysis of Cd-histidine was performed at temperatures that straddle the transition temperature (at 130K and 200K). The results of this study as well as an interpretation of the 77K EPR spectral superhyperfine splittings will be discussed. M-108 A tale of two polymorphs: macroscopically observed phase changes and atypical hydrogen-bonding in four uranium-bearing supramolecular materials Nicholas P. Deifel, Christopher L. Cahill The George Washington University, Washington, DC, United States Synthetic efforts to explore coordination polymers of uranyl (UO2 ) systems have often focused on exploiting metal-ligand coordination to create new families of materials. Hydrolysis of the uranyl ion in these systems commonly results in the formation of polynuclear building units and thus accurate prediction of the coordination environment of inorganic building units remains difficult. It is now known that highly acidic aqueous reactions in high chloride and 2bromide media favours the formation of the uranyl tetrahalide anion ([UO2X4] ) over polynuclear building units. This structural unit has been shown to interact with protonated, linear dipyridyl amines and 1,4-diazabicyclo[2.2.2]octane (DABCO) in a predictable supramolecular synthon (NH· · · X2M). 2+ Presented will be a systematic overview of our success with the aforementioned system and two examples of polymorphism that contrast with the ‘rules’ determined thus far. The first example of these anomalous phases involves a pseduopolymorph of [UO2Cl4](H2DABCO) that results from hydration. This material, [UO2Cl4(H2O)](H2DABCO), does not form the predicted uranium-bearing building unit. Exposed to air, it undergoes a solid-state transformation to the dehydrated form which shows markedly different fluorescent properties. The second example is a true packing polymorph of [UO2Br4](C10H10N2). The first phase crystallizes in the triclinic P-1 space group with both the uranium and organic species sitting on an inversion center: the bifurcated NH· · · X2M synthon is observed. A second phase has recently been discovered that crystallizes in a monoclinic space group (P21/c) with two crystallographically distinct uranium and organic sites. The expected NH· · · X2M hydrogenbonding synthon is only observed in one of these pairings; in the other, atypical hydrogenbonding between the pyridyl group and the uranyl oxygen is observed. M-115 Ribosomal Protein Structures and Sequences Define the Prokaryotic Tree of Life William L. Duax , Robert Huether , David Dziak , Lukas Klein , Kevin Gibas , Brian Riefler , 1 1 Fiona Henning , Rasheen Powell Hauptman Woodward Medical Research Inst., Buffalo, NY, United States, State Univ. of New York at Buffalo, Buffalo, NY, United States Search vectors composed primary of Gly, Ala, Arg, and Pro residues (GARP) distributed across the entire protein sequence retrieve 98% of each of the ribosomal proteins in prokaryotic species with no false ³hits². Different combinations of G, A, R and P and insertions or deletions differentiate each ribosomal protein from all others. Specific combinations of amino acids in two sequence positions in perfectly aligned L1 ribosomal proteins from 1600 different prokaryotic species in the gene bank separate all Gram positive from Gram negative bacteria. We are able to identify site mutations that subdivide each ribosomal protein ensemble into the individual phylum of bacteria. Further subdivision into orders, families, genus, and species is trivial. For example, specific residues in three positions in the alignment of prokaryotic L1 ribosomal proteins isolate 44 L1 proteins from cyanobacteria and 17 L1 proteins from chloroplasts unequivocally supporting the postulated evolution of the latter from the former. While there are significant differences between the sequences of the ribosomal proteins in different classes and orders of prokaryotes, within each order the amino acid sequences have remained highly conserved since divergence and speciation. We have found that the total GARP content of the ribosomal proteins of each class and order is a marker of the order of evolution and that the last universal common ancestor (LUCA) appears to have been an Actinobacteria. Perfect alignment of thousands of members of a protein family is essential to determining the molecular level details of its evolution, the evolution of protein fold and function and the evolution of bacterial species. Three dimensional structural information played an essential role in developing a new GARP based technique to achieve perfect sequence alignment. In retrospect it is possible to understand why GARP residues are 100% conserved in specific positions in families of proteins present in all species. Support in part by: Mr Roy Carver, Stafford Graduate Fellowship, Caerus Forum Fund and The East Hill Foundation. 1 2 1,2 2 1 1 1 1 M-118 In Pursuit of Chemical Matter for Blood Coagulation Factor XIa by Fragment-Screening Technology Melissa Harris , Francis Rajamohan , Seungil Han , Tracy Brown-Phillips , John Bryant , 1 1 1 2 4 Dave Cunningham , Andrew Butler , Boris Chrunyk , Ronald Sarver , Joel Morris , Rick 5 1 6 2 1 1 Sciotti , Dave Hepworth , Ing-Kae Wang , Shengwu Wang , Jane Withka , Ravi Kurumbail 1 1 1 1 1 1 Pfizer Global R&D, Groton, CT, United States, Pfizer Global R&D, La Jolla, CA, United 3 4 States, Neogen Corp., Lansign, MI, United States, National Cancer Inst. NIH, Washington, 5 DC, United States, Walter Reed Army Inst. of Research, Washington, DC, United States, 6 Novartis Inst., Cambridge, MA, United States Cardiovascular diseases remain as the leading cause of death in most of the world. Despite technical advances, very few novel drugs have been approved for the treatment of acute coronary syndrome and atrial fibrillation, two of the more prevalent disabilities that result in high morbidity and mortality. The pursuit for novel medicines to alleviate these symptoms has targeted both the platelets and the coagulation system that together maintain hemostasis. Blood coagulation factor XIa (fXIa) functions upstream of factor Xa in the intrinsic coagulation cascade. Based on human genetics and animal studies, fXIa appears to be an attractive target for the development of safe anti-thrombotics. In an attempt to identify potential starting points for medicinal chemistry, we employed several fragment-screening techniques based on biochemical assays, isothermal denaturation, surface plasmon resonance, nuclear magnetic resonance spectroscopy (NMR) methods and crystallography. We also evaluated the druggability of the target by ‘de-construction’ of a known fXIa inhibitor. The poster highlights some of the key learnings and challenges we faced during our multi-disciplinary approach to identify suitable chemical matter for this challenging coagulation protease. 2 M-121 Automating Microseeding Protein Crystallography Set-Ups Using Mosquito Ben Schenker, Joby Jenkins, Rob Lewis, David Smith TTP LabTech, Cambridge, MA, United States Crystallising proteins, required for structure determination by X-ray diffraction, is a difficult and labour-intensive task. One of the many challenges facing the protein crystallographer is growing crystals of sufficient size and quality to successfully determine the protein’s structure (this typically requires crystals of around 100-300 µm). For structure-based drug design a further challenge is being able to generate a sustainable crystal system capable of producing liganded structures iteratively to support active chemistry. Microseeding, where small crystals are crushed and suspended in a slurry of crystallisation buffer to produce new nucleation sites, is a recognised technique to improve crystal quality as well as promote the growth of larger, single crystals. However, it requires experimentation with varying concentrations of solutions to achieve successful results and as a manual process this can be very consuming. ® One approach to increase the speed and efficiency of microseeding set-ups is through the automation of the seeding process. However, this is not a simple process because of the ® problems that crystallisation robots have with dispensing low volumes. The mosquito liquid handler (TTP LabTech) is ideally suited to automating the complex set-ups required for microseeding due to its ability to perform multiple aspirations and dispenses with each pipette and the precise handling of nanolitre volumes of solutions, regardless of their viscosity. Here we describe an automated approach to setting up microseeding protocols in 96-well plates using mosquito. M-124 BioCAT as a National Center for X-ray Solution Scattering of Biological Samples liang guo, thomas irving BioCAT Illinois Inst. of Technology, Chicago IL, United States The BioCAT beamline 18ID is a NIH Biotechnology Research Resource operated by the Illinois Institute of Technology at the Advanced Photon Source, Argonne National Laboratory. It is one of the premier x-ray facilities in the world offering researchers opportunities to perform solution scattering studies on samples of proteins and RNAs/DNAs, and their complexes. Both static and time-resolved small angle scattering (SAXS) and wide angle scattering (WAXS) experiments can be used to obtain high data quality suitable for structural modeling with currently available software packages. With high beam flux and efficient detectors at the beamline, typical sample consumption for static experiments is ~60 ul at a concentration of 2 mg/ml for small proteins or 0.5 mg/ml for RND/DNA samples. Timeresolved experiments can achieve 1 ms time resolution with a Pilatus 100K photon counting detector (Dectris) coupled with a Bio-Logic SFM-400 stopped-flow instrument, at a sample consumption around 8 mg per experiment for protein samples or 2mg for RND/DNA samples. Time-resolved experiments can be performed with a short dead time of 0.5 ms. Data quality is high enough to allow reliable shape and size determination, offering the capability to follow the structural change of biological molecules during their functioning process. Acknowledgments: Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Science, under contract No. W-31-109-ENG-38. BioCAT is a National Institutes of Health-supported Research Center RR-08630. M-127 A Nonlinear Optical Approach to Imaging for Crystal Centering David Kissick , Ellen Gualtieri , Kevin Bataille , Michael Becker , Robert Fischetti , Steve 2 2 2 3 1 Ginell , Lisa Keefe , Anne Mulichak , Vadim Cherezov , Garth Simpson 1 1 1 2 2 2 Purdue University, West Lafayette, IN, United States, Advanced Photon Source, Argonne, 3 IL, United States, The Scripps Research Institute, La Jolla, CA, United States 2 Second order non-linear optical imaging of chiral crystals (SONICC) was used to image protein crystals at cryogenic temperatures. The need for automated crystal centering has motivated the development of many techniques that determine the position of protein crystals frozen in loops (e.g. brightfield image analysis, birefringence, intrinsic UV fluorescence and Xray diffraction based centering). These techniques have been limited by a lack of contrast, minimum detectable crystal size, and sample damage respectively. SONICC provides the necessary contrast by effectively eliminating the background from centrosymmetric materials (e.g. amorphous water and cryoprotectants). Images of crystals grown in lipidic cubic phase (LCP) demonstrate the effectiveness of this approach in turbid environments. The locations of the crystals from LCP were confirmed with subsequent mini-beam, raster scanning diffraction experiments. In addition, preliminary diffraction studies showed no evidence of structural damage to crystals that were exposed for fifteen minutes with ~500mW laser power, where a typical image requires less than one minute of exposure at ~100mW. Further experiments are planned to assess the suitability of SONICC for automated crystal centering, especially with respect to crystal damage. M-130 CATS & G-ROB: 6-AXIS ROBOTIC-ARM-BASED CRYSTALLOGRAPHY 1 2 AUTOMATED SYSTEMS FOR 3 JEAN-LUC FERRER , NATHALIE FERRER , JEAN-LOUP RECHATIN , XAVIER 1 1 1 2 2 VERNEDE , JACQUES JOLY , FLORIAN BOUIS , PIERRE MAZEL , PIERRICK ROGUES , 3 MATTHIEU PRIVAS INSTITUT DE BIOLOGIE STRUCTURALE, GRENOBLE, 3 GRENOBLE, FRANCE, IRELEC, GRENOBLE, FRANCE 1 FRANCE, 2 NATX-RAY, CATS and G-Rob systems were developed on protein crystallography beamline FIPBM30A at the ESRF. CATS (Jacquamet et al., JSR 16, 2009, 14-21) is a sample changer currently now installed on various synchrotrons (SLS, BESSY, DLS, APS, ...). G-Rob, also a 6-axis robotic arm based system, is a fully integrated device for crystallography beamlines and laboratories. G-Rob is an “all in one” system, since it integrates the following functions: - sample changer, - goniometer for frozen samples, capillaries, … (Jacquamet et al., Acta Cryst. D60, 2004, 888-94), - crystallization plates/micro-chips screening for in situ analysis of diffraction condition and data collection (Jacquamet et al., Structure 12, 2004, 1219-25), - goniometer for non-classical sample environments (high pressure cells, …), - beam monitoring. G-Rob provides unique features. It is automated: thanks to its tool changer, it goes automatically from one application to another. CATS and G-Rob are also highly flexible: if a new application or a new sample format emerges in the community, a new tool can be designed to implement it. They are highly reliable systems, based on well-known, industrial quality equipments, with reduced maintenance. They are currently in use on beamline FIP-BM30A. It was made available to the research community in 2005 and up to now, users have expressed an unprecedented high degree of satisfaction. The crystallization plates screening capability for example appears to be a precious tool in several cases (crystals too small to be fished, or too fragile, of when there is no good cryoprotectant). Several results obtained on FIP-BM30A are presented, such as in situ screening of membrane proteins, ribosome, high pressure protein diffraction, etc. Recent experiments demonstrated also the possibility of the automated structural screening for the Fragment Based Drug Design strategy: the same crystal was reproduced in presence of a library of fragments. Systematic in situ data collection has shown some of the fragments present in the active site, without having to manipulate the crystals individually. Movies are available on www.natx-ray.com. M-133 SCrALS: Challenging Samples, Straightforward Solution. Allen Oliver , Jeanette Krause 1 1 2 2 University of Notre Dame, Notre Dame, IN, United States, Cincinnati, OH, United States Univeristy of Cincinnati, The Service Crystallography at the Advanced Light Source (SCrALS) project provides chemical crystallography synchrotron access for samples considered too small or poorly diffracting to collect on a regular laboratory-based system. Synchrotron sources generate a more brilliant beam with a significantly higher X-ray photon flux allowing such samples to have data collected. SCrALS is a mail-in service with data collected during regularly scheduled beam-time. This presentation will highlight selected projects that would otherwise not have been possible without access to the more intense beam provided by a synchrotron source. Contact the authors for more information on the SCrALS project:
[email protected] or
[email protected]. M-136 High-Throughput Automation and Remote Access at SSRL Protein Crystallography Beam Lines: Novel Tools for Training, Education and Collaboration Aina Cohen, Clyde Smith, Graeme Card, Tzanko Doukov, Thomas Eriksson, Ana Gonzalez, Scott McPhilips, Pete Dunten, Irimpan Mathews, Jinhu Song, Mike Soltis SSRL/Stanford University, Menlo Park, United States The ultimate goal of synchrotron data collection is to get the best possible data from the best available crystals and the combination of high-throughput automation and remote access at SSRL has revolutionized the way in which scientists interact with synchrotron beam lines to achieve this goal. This has also triggered a shift in the way crystallography students and novices are introduced to synchrotron data collection and trained in the best methods to collect high quality data. SSRL provides expert crystallographic and engineering staff, stateof-the-art crystallography beam lines, and a wide range of accessible tools to facilitate data collection and in-house “remote training”, and encourage the use of these facilities for education, training, outreach and collaborative research. Hands-on workshops are also offered by SSRL User Support, both at SSRL and at remote locations, to facilitate the education of the next generation of protein crystallographers. The SSRL Structural Molecular Biology group operates 7 crystallography beam lines on the SPEAR3 storage ring, BL1-5, BL7-1, BL9-1, BL9-2, BL11-1, BL12-2 and BL14-1. All of the beam lines are MAD-capable, with three of the stations (BL7-1, BL9-1 and BL11-1) using a single-crystal side-scattering monochromator with a limited energy range (typically 3000-4000 eV) and the other four using double crystal monochromators giving a much wider energy range capability (over 10000 eV). BL12-2 is a new undulator station optimized for data collection with microcrystals. This station has an in-house designed microdiffractometer and a newly-installed Pilatus 6M pixel array detector, which will result in data collection times on the order of only a few minutes. All of the beam lines are fully automated, with samples being mounted using the Stanford Automated Mounting system (SAM) and controlled with the BluIce/DCS software system. Images collected during sample screening are automatically analyzed and the results, including the number of spots, Bravais lattice, unit cell, estimated mosaicity and resolution, are visible almost immediately through Blu-Ice, and also via the internet through Web-Ice. The availability of this real-time analysis enables researchers to then make informed choices as to which samples and experimental parameters should be used to collect the best possible data. M-139 Automated data collection software and hardware for small angle scattering at the Cornell High Energy Synchrotron Source. Soren S. Nielsen, Richard E. Gillilan Cornell University, Ithaca, NY, United States Data collection software is becoming an increasingly important factor in high throughput protein structure analysis on modern SAXS beamlines. As acquisition time decreases and the number of processed samples increases, it becomes important to obtain instant feedback on measurements to avoid errors. Automation of as much of the data analysis as possible dramatically reduces the workload. For researches that want to push the boundaries of automated SAXS data analysis, the source code for the basic tools is often either difficult to obtain, too specialized for a particular beamline, or developed in widely diverse programming languages, which means algorithms will have to be reimplemented all over again. BioXTAS RAW is a free and open source software project that is aimed at developing tools for high throughput data reduction and analysis, not only intended to be used as an integrated part of the data collection software on a SAXS beamline, but also by users at home. This Pythonbased software, available on SourceForge, is being developed as an alternative to current closed source and commercial software for isotropic scattering data. BIOXTAS RAW is an outgrowth of the BioXTAS project, a collaboration between The Technical University of Denmark and The University of Copenhagen and has been used at the I711 beamline at the MAX-lab synchrotron in Sweden and other sources worldwide. It is currently being integrated as the primary data reduction and analysis software on the BioSAXS beamlines G1 and F2 at CHESS. In addition to software and algorithmic details, this presentation will also discuss the redesign of CHESS F2 beamline as a dedicated BioSAXS station with robotic sample loading, including plans for automated microfluidic mixing. M-140 Structure of the reassembled Venus Masami Isogai, Yoshihiro Kawamoto, Kazuto Inahata, Kenji Sugimoto, Toshiji Tada Osaka Prefecture University, Sakai, Osaka, Japan Bimolecular fluorescence complementation (BiFC) assay based on the association of a fluorescent protein fragments is widely employed to study protein-protein interactions in cells. Non-specific interaction is a significant problem, because it interferes with the BiFC assay. To understand the mechanisms of reconstitution, we have attempted to determine the structure of the fluorescent protein, Venus, complemented between the N-terminal and C-terminal fragments. The fragments were co-expressed in E. coli and purified by the affinity and ionexchange chromatographic techniques. The fluorescent fraction was crystallized by the sitting-drop vapor diffusion method. The diffraction data were collected to 2.5 Å resolution at 100 K using an ADSC CCD detector at the NW12 station of PF, Japan. The crystal belongs to P212121, with unit-cell parameters of a = 58.97, b = 116.02, c = 156.62 Å. Assuming three molecules of the reassembled Venus in the asymmetric unit, the VM value was calculated to 3 -1 be 2.9 Å Da . The crystal structure was solved by molecular replacement using the program MOLREP with the structure of Venus (PDB: 1MYW) as a search model. Completion of model building and structure refinement are underway. M-142 The wwPDB Common Annotation and Deposition Tool Development John Westbrook , Martha Quesada , Jasmine Young , Zukang Feng , Tom Oldfield , Sameer 2 2 3 4 4 Velankar , Jawahar Swaminathan , Takanori Matsuura , Eldon Ulrich , Steve Madding , 2 3 4 1 Gerard Kleywegt , Haruki Nakamura , John Markley , Helen Berman 1 1 1 1 1 2 RCSB PDB, Rutgers Chemistry, Piscataway, NJ, United States, PDBe, European 3 Bioinformatics Institute, Hinxton, United Kingdom, PDBj, Osaka University, Osaka, Japan, 4 BMRB, University of Wisconsin-Madison, Madison, WI, United States The Worldwide Protein Data Bank (wwPDB) is committed to using the highest standards of curation and processing for experimentally-determined 3D biomolecular structure data. The wwPDB Common Deposition and Annotation Tool project was initiated to produce a set of common deposition and annotation processes and tools across the wwPDB that will serve to support the goals of quality and dependability over the next 10 years. The new tools make use of interactive interfaces and best of breed visualization tools to ensure the quality of data curation and communication with the originating scientists. To date, the project has delivered a sequence processing workflow, supported by a graphical user interface and managed by a workflow engine. The annotation processes are described in XML and are accessed by the workflow engine through a Python API. The resulting architectural foundation will be used to complete the full deposition and annotation workflow. The new processes were defined using business process re-engineering methodologies resulting in process-driven requirements and the incremental design and delivery of products. The wwPDB Common Deposition and Annotation suite of tools are built on Python, the Boost.Python Library and C++, supporting work automation, as well as computationally intensive tasks. 2 wwPDB members are: RCSB PDB (supported by NSF, NIGMS, DOE, NLM, NCI, NINDS and NIDDK), PDBe (Wellcome Trust, EU, BBSRC, NIH and EMBL), PDBj (BIRD-JST) and BMRB (NLM). M-145 Rapid Automated Processing of Data (RAPD) Software Package Jonathan Schuermann, David Neau, Kanagalaghatta Rajashankar, Frank Murphy Cornell University (NE-CAT), Argonne, IL, United States RAPD (Rapid Automated Processing of Data) is a software package aimed to help users in collecting and processing meaningful crystallographic data from a synchrotron beamline. The package is coded in Python as independent but interacting modules which are designed to run with minimal user input. RAPD monitors the beamline for collection of a snapshot (or pair), automatically autoindexes the collected images and generates an optimal data collection strategy. RAPD runs Labelit for autoindexing, Mosflm for integration, Raddose for radiation damage calculation, and Best for data collection strategies. Error correction is incorporated automatically so programs are rerun adjusting certain parameters if errors are detected. RAPD takes advantage of multi-core CPU's by parallelizing certain tasks. Typical time from data collection to appearance of the strategy in the user interface is 25 seconds on an Intel Core i7. Collection of a sweep of data triggers automated processing using Xia2. All results are accessible on a secure AJAX-based website which displays results in variable levels of detail. Users may log in remotely and view, download or reprocess data from current or historic data collection trips. Current and future work involves several new features. First, implementing STAC to take full advantage of the MK3 mini-kappa, allowing users to continue data collection across different crystals. Second, near real-time processing of data from multiple sweeps. Third, modify the code to take further advantage of a computer cluster. Fourth, automated structure solution pipeline. M-148 Automated (and non-automated) data processing with autoPROC Clemens Vonrhein, Claus Flensburg, Peter Keller, Wlodzimierz Paciorek, Andrew Sharff, Thomas Womack, Gerard Bricogne Global Phasing Ltd., Cambridge, United Kingdom The processing of macromolecular diffraction data can often be quite challenging, especially to novice users. Extracting the best possible data from a large collection of different datasets, such as occur in binding studies or in complex protocols for MAD phasing (involving multi-axis goniometers, multiple and possibly interleaved wavelengths, inverse-beam strategies and combinations thereof), requires making systematic and thorough use of all available information. This includes prior knowledge about the sample (likely crystal forms, known or expected behaviour of crystals in the X-ray beam) as well as the relationships (e.g. goniometric) between the various datasets that should eventually contribute to making up the scaled (and optionally merged) dataset from which to carry out phasing or refinement. The autoPROC toolbox helps the unexperienced and the expert user alike to deal with a large variety of data collection scenarios, both by providing a collection of advanced tools for finetuning the processing steps and by producing easily understood feedback and diagnostics. It is centred around the processing programs XDS [1] and MOSFLM [2], and uses POINTLESS/SCALA [3] and other programs from the CCP4 [4] suite for the later stages. It has been in constant use and development for nearly 5 years and a public release is planned within 2010. [1] Kabsch, W. J. (1993). Appl. Cryst. 26, 795-800. [2] Leslie, A.G.W. (1992). Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography, No. 26. [3] Evans, P. R. (2005). Acta Cryst. D62, 72-82. [4] Collaborative Computational Project, Number 4 (1994). Acta Cryst. D50, 760-763 M-151 Automated in situ Diffraction Screening at Beamline X06DA at the Swiss Light Source Vincent Olieric, Rouven Bingel-Erlenmeyer, Meitian Wang, Roman Schneider, Claude Pradervand, Wayne Glettig, Takashi Tomizaki, Ezequiel Panepucci, Vincent Thominet, Joerg Schneider, Jose Gabadinho, Xiaoqiang Wang, Andreas Isenegger, Clemens Schulze-Briese Paul Scherrer Institut, Villigen PSI, Switzerland X06DA is the third macromolecular crystallography beamline at the Swiss Light Source. It has been designed to fulfill the requirements of both academic and industrial users. To achieve maximum efficiency, high degree of automation was implemented from the optics to the experimental environment. A Bartels dual channel cut monochromator (DCCM) ensures rapid energy changes with a true fixed exit. The obtained X-ray beam has a focal spot size of 80 x 45 microns at the sample position and a total photon flux of 5E11 photons/sec, which is comparable to an undulator beamline. The mini-hutch end-station allows both rapid manual mounting and robotic sample exchange. In addition, a crystallization facility, directly adjacent to the X06DA mini-hutch, has been implemented. Crystallization experiments are performed using nano-dispensing robots and drops inspection is done via an automated imaging system. The unique feature of this facility is the possibility to test the crystals for diffraction directly in the plates (in situ screening) by transfering them from the crystal hotel to the mini-hutch in an automated manner. Without any manipulation to the crystals, this gives users a rapid feedback on important parameters such as diffraction limit, anisotropy, cell parameters or mosaicity, and aids to prioritize subsequent optimization steps. Moreover, users are welcome to bring any kind of SBS standard TM crystallization containers, including microfluidic chips and the CrystalHarp which yield a particularly low background in the diffraction image. First results obtained at the crystallization facility and future improvements will be presented. Other methodological developments such as a new type of multi-axis goniometer and phasing with weak anomalous scatterers will be described as well. M-154 Minstrel™ HT UV: A Fully Automated and High Performance Imaging System for Protein Crystallization with UV Fluorescence Jian Xu, Michael Willis Rigaku Automation, 5999 Avenida Encinas, Suite 150, Carlsbad, CA, United States Rigaku introduces the Minstrel HT UV™, custom engineered to meet the increased demand for a high-throughput ultraviolet and visible crystal imaging and protein crystal monitoring system. We have focused our research and development efforts and the combined knowledge of experts in optics, photochemistry, illumination, and automation to develop a custom solution that provides the highest sensitivity, the highest optical resolution, and the least photo damage to a protein sample. The result of this effort provides a substantial leap forward in imaging technology in both UV and visible color spectrum. The optimal balance of high resolution and depth of field for the crystallographic application seamlessly images hanging drop, sitting drop, and microbatch experiments for all UV suitable plates. The Minstrel HT UV together with Gallery™ 700 Incubators provide around the clock unattended incubation with user specified temperature, retrieval, temperature controlled imaging, and analysis. Developed by crystallographers for high throughput labs, Rigaku’s automated highthroughput incubation and imaging solution delivers exceptional reliability with versatility and scalability into the future. In addition, the new Minstrel HT UV software provides scientists with an easy to use and intuitive flow. Validation studies with various proteins prove that the Minstrel HT UV provides researchers with a significant advantage in the protein crystallization process. M-157 BLUICE-EPICS: a modern control system with classical look for macromolecular crystallography at the Advanced Photon Source Sergey Stepanov , Oleg Makarov , Mark Hilgart , Sudhir Pothineni , Derek Yoder , Michael 1 1 1 1 2 Becker , Craig Ogata , Ruslan Sanishvili , Nagarajan Venugopalan , Janet Smith , Robert 1 Fischetti Argonne National Laboratory, Argonne, IL 60439, United States, University of Michigan, Ann Arbor, MI 48109, United States The trio of macromolecular crystallography beamlines constructed by General Medicine and Cancer (GM/CA) Institutes at the APS has been in growing demand due to their outstanding micro crystallography capabilities. To raise the efficiency of these beamlines a significant effort has been put into designing fast, convenient, intuitive and robust beamline controls that could easy accommodate new beamline developments and provide high level of automation. This resulted in a system combining the widely praised user interface of SSRL BLUICE as a frontend and the industrial power of EPICS as a backend. While the GM/CA controls have the look and feel of BLUICE, their software design is very different making them faster, simpler and more flexible than most similar systems. First, the software consists of only two layers: the BLUICE clients and the EPICS servers. The BLUICE clients are designed to operate in parallel with other beamline controls streamlining the tasks performed by staff such as beamline preparation, maintenance, auditing and user assistance. Then, BLUICE-EPICS deploys multiple plugins that can be written in any programming language thus involving more staff into the development. Further on, it makes use of unified motion controls allowing for onthe-fly scanning and optimization of any beamline component. Finally, even the graphical frontend is made language-flexible so that the old Tcl/Tk controls inherited from BLUICE are being gradually replaced by more modern Java controls and the two seamlessly coexist. From the users’ perspective BLUICE-EPICS provides: one-click change between 5, 10 and 20μ m beam sizes; one-click beamline energy change that may involve switching undulator harmonics, mirrors lanes and beam realignment; automated diffraction rastering for finding small crystals and ’sweet’ spots on poorly diffracting crystals with automated scoring of raster cells by the number of reflections; data collection along a vector; automated on-the-fly fluorescent rastering, a faster and lower-irradiation compliment to the diffraction rastering; fully automated fluorescence measurements for MAD that include signal optimization, fast onthe-fly energy scanning and automated adaption of scan range to chemical shifts; fly-scan minibeam realignment; automated loop and crystal centering, controls for sample automounter, automated screening, data collection auditing, remote access and a lot more. 1 2 1 1 1 1 1 M-160 Exploring the path towards autonomous protein crystal harvesting: First experiences with an operator-assisting crystal harvesting robot. Bernhard Rupp , Jace Walsh , Alex Melka , Sean Murphy , Robert Viola 1 2 1,3 1 1 2 1 Square One Systems Design, Jackson, WY 83002, United States, Johns Hopkins University 3 Applied Physics Lab, Laurel, MD 20723, United States, q.e.d. life science discoveries, Livermore, CA 94551, United States Robotic crystal harvesting has become reality with the installation of the first demonstration unit in mid-2010. The Universal Micromanipulation Robot (UMR) has advanced from an operator-assisted prototype to a complete operator-assisting crystal harvesting work cell. We discuss the challenges that had to be overcome and which still lie ahead for the design of a platform-integrated system capable of fully autonomous harvesting of protein microcrystals. During the transfer of manual harvesting protocols into robotic processes, a number of novel and generally applicable technologies have evolved. Tape cutting and re-sealing has been simplified and improved, and a universal and reliable cryo-protection procedure has been developed. Combining robotic low-viscosity oil cryo-protection with reliable hyper-quenching allows standardization of cryo-protocols. In addition, optional room-temperature diffraction characterization the same crystal prior to flash-cooling will allow consistent analysis and comparison of cryo-protocols, providing information about native versus flash-cooled diffraction properties. Algorithmic crystal localization and UMR control remain two significant hurdles in deploying a fully automated solution. While significant advancements have been made in the application of machine vision to protein crystal harvesting, real time image processing interfaced with mechanical control and feedback are at the cutting edge of technology, and the design of autonomous systems represents a research frontier in mechatronics. Advantages in overall harvesting success rate of a robotic platform will very likely lead to market acceptance of fully automated and integrated crystallization platforms. In addition to general throughput and reliability advantages, we believe that advanced micro-manipulation robotics will open the field to further new science and emerging crystallization technologies of potentially far reaching impact. Work sponsored by NIH STTR Phase II Grant No. 2 R42 GM073278-02A1. M-164 Remote access data collection at SPring-8 protein crystallography beamlines Kazuya Hasegawa , Go Ueno , Takaaki Hikima , Yukito Furukawa , Daisuke Maeda , 1 2 Takashi Kumasaka , Masaki Yamamoto 1 1 2 2 1 2 SPring-8/JASRI, 1-1-1, Kouto, Sayo, Sayo, Hyogo, Japan, RIKEN SPring-8 Center, 1-1-1, Kouto, Sayo, Sayo, Hyogo, Japan High intensity X-ray beams available at synchrotron beamlines are now indispensable for protein crystallography, because they enable not only rapid data collection but also data collection using micro size crystals. However, one inconvenience of using synchrotron beamlines is a time consuming and expensive trip to distant facilities. Remote access beamline control and data collection via the internet are solutions for this issue. At SPring-8, we have developed the remote access system and have started user operation at protein crystallography (PX) beamlines. 2 Our remote access system makes use of beamline automation system consisting of [1] [2] integrated beamline control software BSS and sample auto-changers SPACE . For the secure and stable operation from the distant places, three network sessions are provided, which correspond to a device-control command path, a streaming video image of samples, and a data management link, respectively. The first two sessions have been developed as a common platform of SPring-8 network, which can be used at all beamlines in the future. For data and sample management for PX users, the third session has been provided based on [3] database server D-Cha , which has been used originally for mail-in data collection at PX beamlines. The test use of the new system has started and is now open for users at RIKEN beamlines BL26B1 and BL26B2. Further implementation of this system at public beamlines, where anybody can apply a proposal, is also planned. The remote access data collection at SPring-8 will enhance the usability of beamlines and expand the opportunity of applications. [1] Ueno G. et al. (2005) J. Synchrotron Rad., 12, 380-384 [2] Ueno G. et al. (2004) J. Appl. Cryst., 37, 867-873 [3] Okazaki N. et al. (2008) J. Synchrotron Rad., 15, 288-291 M-169 Automated crystal centering implementation at GM/CA beamlines at the Advanced Photon Source using XREC Sudhir Babu Pothineni , David Watts , Sergey Stepanov , Mark Hilgart , Nagarajan 1 1 1 3 2 Venugopalan , Ruslan Sanishvili , Craig Ogata , Janet Smith , Victor Lamzin , Robert 1 Fischetti Argonne National Laboratory, Argonne IL 60439, United States, European Molecular 3 Biology Laboratory, Hamburg 22603, Germany, University of Michigan, Ann Arbor MI 48109, United States Automated crystal centering software XREC, developed at EMBL, Hamburg [S. Pothineni, et.al. Acta Cryst. D62, 1358 (2006)] has been made operating within BluIce-EPICS, the General Medicine / Cancer Institutes (GM/CA) beamlines control system. XREC processes a series of optical images recorded while a crystal is rotated on the goniometer, determines the crystal center and provides an estimate of solution reliability. Automated crystal centering is the main bottleneck of unattended screening. An additional challenge at the GM/CA beamlines is the small sizes of crystals and X-ray beams putting more stringent requirements for crystal centering. Automated 'loop' centering is regularly used at GM/CA beamlines while the automated 'crystal' centering is still under evaluation for crystals upto 20 microns. At the GM /CA beamlines the automated loop/crystal centering is a two step process. In the first step automated loop centering is based on the low resolution camera images, which puts the loop into the field of view of on-axis high resolution camera. In the second step the loop/crystal centering is based on high resolution camera. The process normally takes up to 30 sec. Occasionally the initial sample position may not be in the field of view of low resolution camera and in these cases the first step is automatically repeated after analyzing the XREC centering parameters and automatically moving the sample into field of view of low-res camera. The images used for loop centering are stored in a database for future comparison of crystal centering success rate against the crystal sizes. The condition for automated crystal centering is that the crystal must be visible for human eye. When this is not the case, the GM/CA control system offers automated X-ray diffraction and/or fluorescence rastering for centering tiny or otherwise invisible crystals. This process is planned to be optimized by defining a loop bounding box (region of interest) using the XREC algorithms. The GM/CA software also provides an option to collect the X-ray diffraction data from predefined, multiple segments rather than at a single point on the crystal. This is also planned to be improved by stretching the XREC algorithms to define the crystal as a whole (size and shape). The proof of principle of these applications will be presented. 1 2 1 2 1 1 M-180 Experimental Charge Density Study of 7-(4-trifluoromethyl)coumarin propargyl ether, a Potent P450 Inhibitor Cheryl Stevens , Naijue Zhu , Edwin Stevens 1 1 1 2 2 Xavier University of Louisiana, New Orleans, Louisiana, United States, University of New Orleans, New Orleans, Louisiana, United States 7-(4-trifluoromethyl)coumarin propargyl ether was synthesized as a potential therapeutic for treating nicotine addiction by inhibiting P450 metabolism. A total of 127,735 reflections were measured on a Bruker Kappa APEX II CCD X-ray diffractometer at 150(2) K to a 2θ max of 116.3° Integration of these intensities resulted in 11, 800 unique reflections with Rint = 0.0267. . Unit cell dimensions were determined to be a = 5.01500(9) Å, b = 10.2382(2) Å, c = 11.9391(2) Å, α = 114.8696(3)° β = 93.4358(4)° and γ = 94.0350(4)° for space group P1bar. , , A conventional refinement of 19 non-hydrogen and 7 hydrogen atoms resulted in R = 0.0466. A fit of the data to a radial model gave an R = 0.0449 while a full multipole refinement gave R = 0.0291. Deformation density maps of the propargyl ether group shows a buildup of density in the triple bond. Electrostatic potential maps have been calculated and show negative potential associated with regions of reactivity near the trifluoromethyl substituent and the ketone group. Critical points show the presence of C-H...F and C-H...O hydrogen bonds. NIH/MBRS-SCORE (SC1GM084722) support is gratefully acknowledged. M-183 The crystal structure of the C-terminal domain of Gcn2 reveals a novel 3-D domain swapped dimer Hongzhen He, Lakshmi Palam, Ronald Wek, Millie Georgiadis Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis,Indiana, United States In response to amino acid starvation, the protein kinase Gcn2 phosphorylates eukaryotic initiation factor 2 (eIF2). Phosphorylation of eIF2 then prevents the exchange of GTP for GDP on eIF2B thereby blocking initiation of protein translation. Gcn2 is itself regulated in part by its C-terminal domain, which facilitates interaction of Gcn2 with the ribosome and contributes to the activated and inhibitory conformations of Gcn2. The C-terminal domain is also involved in dimerization of Gcn2, which has been shown to be important for mediating its response to metabolic stress. Although the kinase domains are conserved in other eIF2 kinases, the Cterminal domain is found only in Gcn2. At the sequence level, the C-terminal domain of Gcn2 has no obvious homologues in other proteins. Limited proteolysis and mass spectrometric methods were used to identify domain boundaries within the C-terminal part of Gcn2. Following expression and purification in E. coli, diffraction quality crystals were then obtained of this domain. The crystal structure was determined at 1.9 Å by using single wavelength anomalous dispersion (SAD) phasing from a mercury derivative. The structure determination was facilitated by the use of HKL3000 at the GM/CA 23ID beam line at the APS. A 3-D search for related structures reveals no significant structural matches with our structure of the Cterminal domain of Gcn2 suggesting that it is a novel structure. An interesting feature of the structure is the extensive 3-D domain swapping within the dimer. Dimerization is mediated by formation of a beta sheet involving the polypeptide chains of each subunit (the 3-D swap) and associated hydrophobic interactions as well as a central interaction between the two subunits. In addition, a potential RNA/ribosome binding site has been identified based on our structural analysis. Delineating these structural features of Gcn2 will provide insight into the underlying mechanisms activating this eIF2 kinase in response to amino acid starvation. M-189 The Crystal Structure of the Secreted Chlamydial Protein Pgp3 Ahmad Galaleldeen, Alexander Taylor, Ding Chen, Guangming Zhong, P. John Hart UTHSCSA, San Antonio, United States Chlamydiae are gram negative intracellular pathogens that are known to cause various health problems in humans. Little is known about the molecular mechanism(s) through which Chlamydiae interact with the host cells and manipulate their cell signaling and immune response because they replicate, differentiate, and perform all biosynthetic processes within host cytoplasmic vacuoles, and this sequestration makes them very difficult to manipulate. It is generally accepted that pathogenesis is related to inflammatory responses caused by chlamydial infection. Several chlamydial species carry a 7.5 Kb cryptic plasmid that encodes eight putative open reading frames (pORFs). pORF5 encodes Pgp3, a 27 KDa protein that is first detected in the cytosol of Chlamydia-infected cells 12 hours post-infection. Pgp3 interacts with host cell signaling pathways, including the activation of host pro-inflammatory genes. Here we present the crystal structure of Pgp3, which reveals a trimeric protein comprised of 2 globular domains connected by a coiled-coil triple helix. The C-terminal domain is an all β structure with a jelly-roll fold that resembles members of the tumor necrosis factor (TNF) family of cytokines, a fold rarely observed in bacteria, while the N-terminal domain has a novel fold. M-192 Diphthamide Biosynthesis Requires a Novel SAM-dependent [4Fe-4S]-containing Enzyme. Andrew T. Torelli , Yang Zhang , Xuling Zhu , Michael Lee , Boris Dzikovski , Rachel M. 1 1 1 2,3 1 1 Koralewski , Eileen Wang , Jack Freed , Carsten Krebs , Hening Lin , Steven E. Ealick 1 1 1 1 2 1 Department of Chemistry & Chemical Biology, Cornell University, Ithaca, NY, United States, Department of Biochemistry & Molecular Biology, The Pennsylvania State University, 3 University Park, PA, United States, Department of Chemistry, The Pennsylvania State University, University Park, PA, United States 2 Diphthamide is a unique, posttranslationally modified histidine residue that is absolutely conserved in archaebacterial and eukaryotic translation elongation factor 2 (EF2). This modification is the target of irreversible ADP-ribosylation by lethal eubacterial factors, such as diphtheria toxin, exotoxin A and cholix toxin, which interferes with the ability of EF2 to catalyze tRNA translocation during peptide synthesis. Diphthamide represents one of the most complex posttranslational modifications and its biosynthesis occurs in three steps. The first step requires four gene products in eukaryotes and results in formation of a C-C bond between the poorly nucleophilic C-2 atom of the target histidine and the 3-amino-3carboxypropyl group of S-adenosylmethionine (SAM). Here we present the crystal structure of Dph2, the sole protein required for this reaction in the archaeon Pyrococcus horikoshii. Each monomer of the PhDph2 homodimer binds a [4Fe-4S] cluster using an unusual arrangement of three cysteines that are each contributed by structurally-conserved domains related by pseudo-three-fold symmetry. Biochemical evidence suggests the [4Fe-4S] cluster is used to homolytically cleave SAM to generate a 3-amino-3-carboxypropyl radical capable of modifying the target histidine in the first step of diphthamide biosynthesis. This is distinct from enzymes in the radical SAM superfamily, which use [4Fe-4S] clusters to cleave SAM molecules and produce 5`-deoxyadenosyl radicals. Thus, our findings suggest PhDph2 is a novel SAM-dependent [4Fe-4S]-containing enzyme that catalyzes unprecedented chemistry. M-195 Ultrasensitive protein crystal detection by second harmonic generation microscopy Garth Simpson, David Kissick, Ellen Gualtieri, Vadim Cherezov Purdue University, West Lafayette IN, United States, Scripps Research Institute, La Jolla CA, United States Second order non-linear optical imaging of chiral crystals (SONICC) is demonstrated as a sensitive and selective detection method for protein crystallization, with detection limits for the onset of crystallization corresponding to crystal dimensions well below the optical diffractionlimit. Validation of SONICC was performed in lipidic cubic phase crystallization trials of Gprotein coupled receptors through direct comparison with conventional image analysis methods currently used in high-throughput screening (e.g., bright-field, birefringence, uvexcited fluorescence, and trace fluorescent labeling). In cases scored highly for crystallization hits by manual expert image inspection of lipidic mesophase trials, a simple automated algorithm for analysis of the SONICC images typically also yielded positive hits. However, SONICC also identified almost twice as many possible hits than manual inspection of images obtained lipidic mesophoase trials using conventional approaches, suggesting a false negative rate as high as ~40% that is overcome by nonlinear optical imaging. 1 2 @ EB TUTSRGQG PDIGFBIHG FBCE DCBA@ &% '& 6(2 ')( 2 & 15 0 2& 12 871 (43 0 % 97 &15 3( &2 ) %$ ? ? False positives by conventional analysis. False negatives by conventional analysis. ? ? ? ? ? ? ? nmhbb? «\£¡ M-201 Characterization of novel LCP matrices for membrane protein crystallization by highthroughput SAXS Thomas Weiss , Ping Liu , Wei Liu , Vadim Cherezov , Hiro Tsuruta 1 2 1 1 2 2 1 Stanford University / SSRL, Menlo Park, CA, United States, Scripps Research Institute, La Jolla, CA, United States Membrane proteins are essential components of living cells. They perform a variety of functions including transport of ions and nutrients, energy transformations and transduction of signals across the cell membrane. Involvement of membrane proteins in many crucial cellular and physiological processes and their location at the cell surface makes them important pharmaceutical drug targets. High-resolution structural studies of membrane proteins depend on the availability of crystals diffracting to sufficiently high resolution. Crystallization in lipid mesophases (in meso), such as the lipid cubic phase (LCP), has proven to yield high quality crystals for an increasing number of membrane proteins. Broader application of this in meso crystallization approach requires identification of novel lipidic matrices with specific phase properties capable of stabilizing proteins with a wide range of sizes and architectures. We have developed an integrated method to assess the lipid phase behaviour at conditions closely mimicking crystallization trials in a high-throughput manner. In this method the lipid samples are prepared using an in meso crystallization robot in specially designed 96-well LCP sandwich plates, in which 50nL droplets of lipidic mesophase are overlaid with 1uL of screening solution and held between two x-ray transparent synthetic films with a thin spacer to form a 12x8 grid. After incubation at 20 ˚C the position of the lipid sample within each well on the LCP plate is recorded into a file using a crystal screening imager. The LCP plate is then transferred to a temperature controlled holder for the x-ray scattering measurements on the BioSAXS beamline BL4-2 at SSRL. A specifically developed software interface implemented as part of the data collection software Blu-Ice at the beam line reads in the sample position file, aligns each LCP sample in the beam and automatically collects SAXS data. The obtained two-dimensional scattering data is then fed into a newly developed software pipeline that automatically integrates the data radially, determines the peak positions and identifies the underlying lipid phase and its basic structural parameters. This highthroughput approach allows us to study effects of detergents, additive lipids, proteins as well as great variety of precipitants on the lipid mesophases in order to understand the phase and structural behaviour of novel lipidic matrices and their compatibility with in meso crystallization trials. M-204 Crystal Structure of Bacillus Subtilis Histidine Kinase KinD Sensor Domain in Complex with Pyruvate Ruiying Wu, MinYi Gu, Andrzej Joachimiak, Marianne Schiffer Argonne National lab, Argonne, IL, United States Sporulation in most Gram-positive bacilli is an example of adaptation of bacteria to starvation. In Bacillus subtilis, pyruvate is secreted into the extracellular medium; the external concentration of pyruvate could be acting as an important signal for the initiation of sporulation. In B. subtilis, the sporulation initiation is tightly controlled by the sporulation transcription factor, Spo0A through its phosphorylation by kinases. Here we provide the first crystal structure of kin-kinase family: KinD sensor domain complexed with pyruvate. The diffraction data for KinD (3fos) without the pyruvate was reprocessed and the electron density was improved. KinD sensor consists of two domains PAS-like first domain and a second domain that also has the characteristic beta-sheet of the PAS-like domains. Pyruvate was found bound to the first domain mainly through interactions with a pair of arginine residues (Arg95 and 116). Glycerol molecules, previously assigned as waters were also identified on the solvent accessible surfaces. The structural details, domains role and possible mechanism underlying signal reception will be discussed. This work was supported by National Institutes of Health Grant GM074942 and by the U.S. Department of Energy, Office of Biological and Environmental Research, under contract DEAC02-06CH11357. M-207 Proposed amino acid sequence and crystal structure determination of a chitinase at 1.49 Å from tamarind (Tamarindus indica) seeds Pravindra Kumar, Deepak Patil, Manali Datta, Shaily Tomar Indian Inst. of Technology, Roorkee, India Chitinases are hydrolases involved in plant defense against a variety of pathogens. A protein with chitinase (CHT) activity has been isolated and purified from tamarind (Tamarindus indica) seeds. N-terminal amino-acid sequence analysis of this protein confirmed it to be an endochitinase, which belongs to the acidic class III chitinase family. The protein was crystallized by the vapour-diffusion method using Polyethylene glycol (PEG). The crystal data was collected at home source (Bruker MicrostarH rotating anode and Mar345 dtb detector) at 1.49 Å resolution and structure was determined by molecular replacement method. Based on high resolution electron density map, all amino acids were identified and the complete primary structure of the CHT is proposed. The final refined model, consisting of 270 amino acid residues, 320 water molecules, and a single glycosylation site containing one Nacetylglucosamine unit, has a crystallographic R-factor of 17.8% and a free R-factor of 19.2% . M-210 Using Single Crystal X-ray Diffraction to Determine Equations of State: an Example of Ordered and Disordered Plagioclase Feldspars. Lindsay Sochalski-Kolbus , Ross Angel , Fabrizio Nestola , Emiliano Bruno , Piera Benna 1 2 3 1 1 2 3 3 Virginia Tech, Blacksburg, United States, University of Padua, Padua, Italy, Univeristy of Turin, Turin, Italy Advances in single crystal X-ray diffraction with lab sources allow very small differences in the equations of state (the pressure variation of volume) of crystalline materials to be determined. Our work focuses on a group of framework aluminosilicates which show unusual structural and thermodynamic properties at high pressures, and in which bulk moduli differences of 2% can be readily distinguished. The combination of a full 4-circle Eulerian diffractometer with unfiltered MoKα radiation (to provide a stable platform and stable profiles of rocking curves), point detector, full-profile fitting of the rocking curves, and 8-position centering was used to collect the cell parameters for the samples up to maximum pressures above 8.5 GPa. The samples were individually loaded into an ETH-designed diamond anvil cell with beryllium seats and 40 half-opening angle. Along with the samples, a quartz crystal and ruby chip were loaded as pressure calibrants. To ensure hydrostatic pressures, the cell was loaded with a 4:1 methanol/ethanol mixture. The unit-cell volumes of the samples and quartz crystals were measured at each pressure increment with errors of 1 part in 10,000. Given that quartz has a low bulk modulus, the pressure in the cell is determined through its equation of state with a typical precision and reproducibility of 0.01 GPa or better. This is a significant improvement over the use of ruby as a pressure marker, which has a typical reproducibility of the order of 0.03-0.05 GPa, in part as a result of the sensitivity of the frequency of the ruby fluorescence line to temperature fluctuations. Our data for the aluminosilicate framework mineral group plagioclase feldspars show unusual th compressional behaviour that requires fitting with a 4 order Burch-Murnaghan EoS. The introduction of Al/Si disorder in the Na end-member reduces the bulk modulus from 52.3(9) GPa (ordered) to 50.4(5) GPa (disordered). In a sample in which 20% of the Na is substituted by Ca, the bulk modulus changes from 61.2(5)GPa to 59.7(7)GPa on disordering, and for a sample with 77%Ca, there is no significant difference in the bulk moduli of ordered and disordered samples. V M-213 Single-crystal neutron diffraction study of hydrous wadsleyite: hydrogen positions for H2O incorporation into Earth’ s deep interior Steven D. Jacobsen , Joseph R. Smyth , Matthias Gutmann , Daniel J. Frost , Erik Hauri , 1 Craig R. Bina Department of Earth and Planetary Sciences, Northwestern University, Evanston, IL, United 2 States, Department of Geological Sciences, University of Colorado, Boulder, CO, United 3 States, Rutherford Appleton Laboratory, ISIS Facility, Chilton Didcot, United Kingdom, 4 5 Bayerisches Geoinstitut, University of Bayreuth, Bayreuth, Germany, Department of Terrestrial Magnetism, Carnegie Institution of Washington, Washington DC, United States Wadsleyite ( -Mg2SiO4) is a nominally anhydrous, high-pressure polymorph of olivine and considered one of the major mantle phases at 410-520 km depth. Wadsleyite (orthorhombmic, Imma) has a remarkable ability to include H at very high pressures and temperatures through hydroxyl defects associated with O1, an underbonded oxygen site that is coordinated to five octahedral-Mg sites and not to Si (e.g. Smyth 1987). At conditions of the upper mantle, wadsleyite can incorporate up to several weight percent of H2O into its structure, making it potentially one of the largest H2O reservoirs in the global water cycle. Xray crystallographic studies of H in wadsleyite have inferred proton positions from R(O…O) interatomic distances in conjunction with polarized infrared spectroscopy, however no direct refinement of hydrogen positions in wadsleyite has been possible because of limited hydrogen concentrations (2 wt% H2O in wadsleyite represents only about 2 H atoms per unit cell) and limited crystal sizes from high pressure synthesis above 12 GPa and 1200C. We report synthesis of a hydrous wadsleyite crystal about one mm in size using the large-volume, 5000 tonne multi-anvil press at Bayerisches Geoinstitut in Bayreuth, Germany. The sample contains 1.77 wt% H2O, as measured by secondary ion mass spectrometery. The structure was investigated on the single-crystal diffraction (SXD) beamline of the ISIS pulsed-spallation neutron source at Rutherford Appleton Laboratory, UK. We integrated over 3000 time-of-flight reflections from three 24-hour exposures and carried out GSAS and Maximum Entropy Method refinements of the structure. Two hydrogen positions in the structure have been refined from the data. Methods and hydrogen-bonding environments in wadsleyite will be presented in detail. 1 1 2 3 4 5 W M-216 Chem_BLAST – a rule-based method to develop advanced structural ontologies for chemical bioinformatics and the PDB, the PubCheM T.N. Bhat NIST, Gaithersburg MD, United States Today’s Chemical Bioinformatics community must interact with a variety of information standalone applications and ontologies. This limitation promotes the need to define and develop rule-based stringent ontologies for information processing and sharing. Chemical Block Layered Alignment of Substructure Technique (Chem-BLAST) first recursively dissects chemical structures into blocks of substructures using rules that operate on atomic connectivity and then aligns them one against another to develop first Chemical Resource Description Framework (RDF) and then chemical ontologies in the form of a ‘tree’ made up of ‘hub-and-spoke’. The technique was applied for (a) both 2-D and 3-D structural data for AIDS (http://bioinfo.nist.gov/SemanticWeb_pr2d/chemblast.do ); (b) to ~60000 structures from the PDB which is now available from the RCSB/PDB Web site (http://www.rcsb.org/pdb/explore/externalReferences.do?structureId=3GGT) and advanced features at http://xpdb.nist.gov/chemblast/pdb.html . Full description of the Chem_BLAST along with recent results and illustrations including those for approximately a million compounds from the PDB and PubChem will be presented. X M-222 Refined Crystallization Screens Hao An, Gyorgy Babnigg, Hui Li, Andrzej Joachimiak Argonne National Laboratory, Argonne, IL, United States A number of commercially available crystallization formulations and screens have been evaluated in the Midwest Center for Structural Genomics’ structure determination pipeline. These formulations were applied, using standard protocols, to thousands of unique proteins and domain constructs. The initial analysis of our results showed that many crystallization screens conditions are redundant, and some screens pointed to several less populated regions of crystallization space. To reduce the number of crystallization conditions and maximize their effectiveness, new refined crystallization screens (ANL-1 and ANL-2) were designed. These screens, ANL-1 and ANL-2, complement the Index very well. Although Index shows a slightly higher success rate (0.47), ANL-1 and 2 screens (both show combined success rate 0.74) contain a more diverse set of conditions and cover a larger crystallization space. Both of them are now available commercially. Recently, we analyzed data for more than 5000 successful crystallization trials stored in our LIMS and indentified the top 384 most successful crystallization conditions. Four new crystallization screens were designed (MCSG1-4) and are now available on the MCSG web site (http://www.mcsg.anl.gov). Recently, our LIMS also added the high throughput crystal data collection system. Using a rich client platform, the LIMS can display and update the whole data set from a 96-well screen plate. Users can report all the crystal results on one screen plate in a quick and easy way. This new on-line report system significantly increases the efficiency of data entry, which will lead to a faster and more precise selection of the best crystallization conditions. This work was supported by NIH Grant GM074942 and by the U.S. DOE, OBER contract DEAC02-06CH11357. M-228 autoBUSTER re-refinement reliably reveals interesting features in newly-released PDB structures Thomas Womack, Oliver Smart, Andrew Sharff, Claus Flensburg, Peter Keller, Wlodek Paciorek, Clemens Vonrhein, Gerard Bricogne Global Phasing, Cambridge, United Kingdom The BUSTER-TNT refinement program has long shown consistent capabilities to produce superior maps from a given model and a given X-ray dataset. Recent improvements have been made to improve its ability to refine models. In particular autoBUSTER now has a powerful optimizer, good molecular geometry restraint function, TLS and fully automated NCS setup. Refinement with autoBUSTER typically leads to lower Rfree, smaller Rfree-Rwork gaps, and improved Molprobity scores even without the explicit representation of hydrogen atoms. On every week of the last three years, Global Phasing has re-refined with autoBUSTER every PDB structure released that week, and analysed the results are with increasingly automated tools. Typically, every week there is a structure with a difference-density peak at +14 sigma or more, and it is rare for there to be fewer than ten structures with highest difference-density peak above 10 sigma. autoBUSTER routinely shows extra interpretable density at N- and C-termini, ranging from omitted OXT atoms to substantial extensions of the chain. About one deposition a week has a claimed ligand which just isn't there, and several will have a sugar or buffer molecule in a clearly wrong conformation. A gallery of typical errors will be presented, including ones where autoBUSTER density shows issues more clearly than EDS. This work was funded by Phases IV and V of the Global Phasing Consortium, and by the VIZIER FP6 Project under EC Contract LSHG-CT-2004-511960. M-231 SrReal - an open-source software library for local structure analysis Pavol Juhas , Christopher Farrow , Simon Billinge 1 1 1 1,2 2 Columbia University, New York, NY, United States, Brookhaven National Laboratory, Upton, NY, United States The atomic pair distribution function (PDF) and other total scattering techniques are essential tools for probing the local structure of nanomaterials, crystals with local distortions, or randomly oriented molecules. While there are several free programs available for PDF modeling (PDFgui/PDFfit2) and local structure determination (RMC suite, Liga algorithm), it is extremely hard for outside developers to customize them (e.g., use special particle shape damping) or combine with additional structure criteria. SrReal is an open-source library for Python and C++ that provides highly customizable calculators for various structure quantities such as PDF, Ewald sums, bond valence sums or overlap of empirical ionic radii. The SrReal library has been developed as part of DANSE, the distributed data analysis for neutron scattering experiments. The library can be used at both Python and C++ levels, where its Python interface was designed for ease of use and the C++ level for speed. The objects in the library were designed for maximum code reuse, clarity and flexibility. The PDF calculation can be easily modified by incorporating custom PDF scaling functions, peak width models or peak profile functions, at either Python or C++ levels. The library calculators can be used with arbitrary structure representations in Python or C++ by providing a simple adapter class. The SrReal calculators share a common recipe of iterating over all atom pairs and summing their pair-wise contributions. A new calculator, for example of pair potential, could be readily implemented by reusing the recipe and changing only the pair contribution function. The SrReal library will be demonstrated with several short python and C++ programs and on actual PDF simulation problems. M-234 MAIN 2010: finalizing the structure by validation driven structure improvement Dusan Turk, Martin Turk Jozef Stefan Inst., Ljubljana, Slovenia At the final stages of crystal structure determination conformations of side chains, peptide bond orientations considering electron density maps as well as hydrogen bonding networks and electrostatic stability and packing of conformations need to considered before the structure can be considered final and ready for deposition in PDB. In MAIN a procedure has been developed for automated improvements and completion of the structure. The procedure includes side chain and peptide bond density fitting combined with flipping in a combinatorial manner. At first the current state of the model, termed starting model, is validated towards density maps. Dead end elimination, exhausted, rotational search is used to fit atoms into electron density maps followed by the energy minimization. Next side chains of branched residues ILE, VAL, THR and LEU are flipped and adjusted to density by fragment and side chain fitting, each followed by minimization. Each state is validated and compared to the starting model. When local improvement is achieved, the geometry of the modified part replaces that of the starting model. A similar procedure considering peptide bond orientation follows. After an optimal fit to density maps is achieved combinatorial search considering packing of short range (below 4A) electrostatic and vdw interactions as well as hydrogen bond network is considered. To enable this explicit hydrogens are used. Side chain and residue flipping is at this stage applied to electrostatically asymmetric residues HIS, ASN, GLN, and solvent molecules in a combinatorial manner. Each of the states is saved together with their packing energy. The lowest energy state is at the end of procedure transfered to the working model, which is agaain energetically minimized - as always using real space refinement procedure. The structure can then be refined against crystallographic targets and the cycle repeated unless the structure is considered done. M-237 Multi-crystal Anomalous Diffraction for Low Resolution Macromolecular Phasing Qun Liu , Zhen Zhang , Wayne Hendrickson 1 1 2 2 2 New York Structural Biology Center, New York, United States, Columbia University, New York, United States Anomalous diffraction experiment from single crystal is one of most commonly used methods for macromolecular phasing. Here we show an alternative way for anomalous phasing with anomalous diffraction data extracted from multiple crystals. Our study demonstrates that multi-crystal anomalous data can significantly benefit low resolution phasing in both heavy atom substructure determination as well as electron density maps interpretation at low resolution. We propose that the multi-crystal strategy may help solve crystal structures of large macromolecular complexes and membrane proteins which are prone to be damaged by X-ray radiation. M-240 Structure of nanoparticles: frontiers in PDF modeling Christopher Farrow, Simon Billinge 1 Columbia University, New York, NY, United States, Brookhaven National Laboratory, Upton, NY, United States Nanoparticles are poised to become vital in the future of energy, medicine, computing and countless other fields. Despite their current and potential usefulness, there are no robust methods for determining the structure of nanoparticles with atomic resolution. Furthermore, the process of nanoparticle growth is still not well understood. These obstacles stand in the way of high precision design and fabrication of nanoparticles for industrial applications. The atomic pair distribution function (PDF) has proved to be a powerful tool for investigating the structure of nanoparticles. In this talk I will describe established and new methods for modeling nanoparticles with the PDF. I will discuss recent work where these methods have been applied to model noncrystalized nanoparticle precursor molecules. Finally, I will give an outlook for how we plan to combine structural information from various sources with the PDF in order to model complex strained, segregated and disordered nanoparticles. 2 M-243 A Structural View on Drug Design for an Acyl-Carrier Protein Synthase from Staphylococcus aureus, Bacillus anthracis and Vibrio cholerae Andrei Halavaty , Youngchang Kim , Ludmilla Shuvalova , George Minasov , Ievgeniia 1,5 1,5 2,5 3,5 4,5 Dubrovska , James Winsor , Min Zhou , Keehwan Kwon , Tatiana Skarina , Olena 4,5 3,5 2,5 3,5 Onopryenko , Leka Papazisi , Andrzej Joachimiak , Scott Peterson , Alexei 4,5 1,5 1,5 Savchenko , Wayne Anderson , Center for Structural Genomics of Infectious Diseases Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States, 3 Computational Institute, University of Chicago, Chicago, Illinois, United States, J. Craig 4 Venter Institute, Rockville, Maryland, United States, University of Toronto, Toronto, Ontrario, 5 Canada, Center for Structural Genomics of Infectious Diseases, Chicago, Illinois, United States 2 1 1,5 2,5 1,5 1,5 Viability of multi-drug-resistant strains of pathogenic microorganisms is determined by many complex and interconnected factors. The bacterial type II fatty acid synthesis (FAS) is a wellorganized and regulated pathway with individual soluble proteins that produce diverse array of vital intermediates, and hence, it represents a valuable source for drug design. While a number of enzymes of FAS II have been shown as important candidates for inhibition, a scientific and medical quest for new targets stimulates research in the field. In order for FAS II to function without a failure, a central component of the system, acyl carrier protein (ACP), has to constantly deliver the thioester intermediates to the discrete partners of the pathway. This cannot occur without an initial posttranslational modification by a holo-(acyl-carrierprotein) synthase (AcpS), which transfers the 4 -phosphopantetheine prosthetic group from coenzyme A (CoA) to a conserved serine residue of apo-ACP. In this respect, AcpS is important for keeping FAS II working, and therefore, finding inhibitors of AcpS activity can destabilize the system. Bacterial AcpSs function as trimers making them distinct from the host AcpSs, which can be a pseudo dimer or a single domain in the complex multidomain eukaryotic FAS I synthases. This substantial architectural difference suggests a strategy for inhibition. While there are a number of 3D structures of bacterial AcpSs available, the mechanism of the transferase activity and its inhibition are not entirely understood. The highresolution X-ray structures presented for apo-AcpS from Staphylococcus aureus, holo-AcpS from Bacillus anthracis and 3 , 5 -ADP-bound AcpS from Vibrio cholerae will contribute to understanding of the structural and mechanistic details of the AcpS-reaction also providing possible alternative approaches to its inhibition. Y Y Y M-246 Determination of Vault Symmetry by Strictly-Crystallographic Means Daniel Anderson , Michael Sawaya , Valerie Kickhoefer , Leonard Rome , David Eisenberg 1 2 1 1 2 2 1,2 HHMI at UCLA, Los Angeles, CA, United States, Dept. of Biol.Chem., UCLA, Los Angeles, CA, United States Can the symmetry of crystalline ribonucleoprotein vaults be determined with low resolution diffraction data, using only crystallographic methods and omitting external information? Previous crystallographic symmetry tests seemed to support the hypothesis that the vault shell contained 96 copies of major vault protein (48 NCS-rotated copies of 98kDa in the asymmetric unit). This report contains results and observations from Patterson-based and density modification-based analyses, expanded from the previous analyses, and using diffraction data from two crystal forms. This crystallographic re-examination suggests that the vault shell can form with at least two symmetries, and corrects our previous symmetry assessment. 48-fold (2QZV): Anderson, et al (2007) PLoS Biology 5, 2661-2670. 39-fold (2ZUO, 2ZV4, 2ZV5): Kato, et al (2008) Acta Crystallographica D64, 525-531; Tanaka, et al (2009) Science 323, 384-388. M-251 Cystal Structure of PCSK9 with Different Length Variants of the EGF-A Domain of LDL Receptor. Javed Khan , Joseph Myers , Gerald Duke , Steven Sheriff , Mark Witmer , Yaqun Zhang , 1 1 1 2 2 Claudio Mapelli , Brian Carpenter , Mian Gao , Doree Sitkoff , Michael Lawrence , Rex 2 Parker 1 1 1 1 1 1 1 Bristol-Myers Squibb, Princeton, NJ, United States, Bristol-Myers Squibb, Hopewell, NJ, United States Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates extracellular levels of lowdensity lipoprotein receptor (LDLR) by binding to the epidermal growth factor precursor homology domain A (EGF-A) of LDLR. This leads to degradation of cell surface LDLR in hepatocytes and elevation of plasma LDL cholesterol levels. To determine the residues of PCSK9 that interact with EGF-A, we have determined co-crystal structures of PCSK9 in complex with 2 different length variants of the EGF-A domain, a longer 40-mer and a shorter 27-mer form of the EGF-A domain peptide. These co-crystal structures reveal the binding mode and key interacting residues of PCSK9 and the EGF-A peptide, and can be used towards the discovery and optimization of therapeutic agents to disrupt the PCSK9-LDLR interaction. 2 M-254 TITLE MISSING Daniel Camac, Joseph Myers, Javed Khan, Yaqun Zhang, Vandana Hegde, Mian Gao, Yongmi An, James Tamura, Malcolm Davis, John Somerville, David Weinstein, John Sack Bristol-Myers Squibb, Princeton, NJ, United States ABSTRACT MISSING - abstract was not processed because the template was not used or was altered M-257 Lithium Ion Pathways in Lithium Phthalocyanine Electrolytes David Grossie , William Feld , John Kelley , Lawrence Scanlon 1 2 1 1 1 2 Wright State University, Dayton, OH, United States, AFRL, PRPS, WPAFB, Dayton, OH, United States The lithium phthalocyanine anion (LiPC ) has been found to show promise as part of the dielectric for lithium ion batteries. The structures of three structures have been determined in which the LiPC anion form similar channeled networks through the unit cell. All three compounds form small, flakey crystals from the few solvents in which they can be dissolved. Experimental evidence indicates that the lithium atoms within the solid structure migrate in the presence of an electric field. The determination of the crystal and molecular structures were initiated so as to determine the pathways through which the lithium ions move. N N N N N Li+ N N N Y+ - Where Y is Li(H2O)2(acetone)2 or bis-adamantylimidizolium cations M-260 A new mini-beam device for protein crystallography Randy Alkire, Michael Molitsky, Frank Rotella Argonne National Laboratory, Argonne, IL, United States In recent years there has been a steady increase in the use of smaller diameter beams (<30 m) for protein crystallography. This has been brought forth mainly thru the use of beam limiting collimators, like the one developed by GMCA CAT at the Advanced Photon Source and by the introduction of mini-beam collimators in commercial instruments like the BrukerASC MD2 diffractometer. These instruments have similar designs in that their small beams result from narrowly defined apertures inside long tubes with guard apertures. Alignment of these tubes requires several degrees of freedom, including yaw, pitch and vertical and horizontal translations. Unfortunately for the end user of these devices, yaw and pitch are manually controlled, making initial installation a very time consuming process. At the Structural Biology Center we have developed a new mini-beam apparatus that separates the beam defining aperture from the guard aperture, allowing independent positioning of each pinhole with simple translation stages, eliminating the need for the time consuming yaw or pitch adjustment. Modular design of the encased pinholes allows for quick change-out if different size apertures are required. This design currently has room for two sets of pinhole/guard pairs. Because these motions are fully motorized, movement from one pair to the other after setup requires only a single command. If desired, any pinhole/guard pair can be lowered completely out of the beam path. If a change in experimental setup requires the mini-beam hardware to be removed, this can be accomplished in less than 5 minutes, with a similar time required for re-installation. A description of the helium filled, motorized assembly will be presented, along with diagrams of the pinhole assemblies. ` M-263 Feasibility Study of Nano-scale Wide-Angle Incidence X-Ray Waveguides Using Surface Diffraction Hsin-Yi Chen, Jhih-Ren Huang, Li-Sin Cai, Chia-Cheng Lin, Yan-Zong Zheng, Yung-Shih Fang, Shih-Lin Chang Department of Physics, National Tsing Hua University, Hsinchu, Taiwan A wide-angle incident waveguide with nano-scale structures is designed using crystal asymmetric surface diffraction. The waveguides are composed of Au/Si/Au sandwiches with Si stripes as the guiding layers and Au thin films as the cladding layer. The photon energy 8.8785 keV for Si (113) as a surface diffraction, is determined from the crystal orientation and diffraction geometry. The effects of x-ray guiding in both lateral and vertical directions are observed if an angle between the surface diffracted beam and the Au layer inside a waveguide is smaller than the critical angle of total reflection for the Au/Si interface. The energy tenability is about 300 eV. M-266 X-Ray Back Reflection from Multi-Plate X-ray Crystal Cavity Ying-Yi Chang , Sung-Yu Chen , Mau-Tsu Tang , Yu. Stetsko , M. Yabashi , Shih-Chang 1 1 1 1 1 1 Weng , Chia-Hung Chu , Yi-Wei Tsai , Po-Yu Liao , Yu-Hsin Wu , Chien-Chung Chang , 1 Shin-Lin Chang Department of Physics, National Tsing Hua University, Hsinchu, Taiwan, National 3 Synchrotron Radiation Research Center, Hsinchu, Taiwan, REKEN/Spring-8, Hyogo, Japan High-resolution X-ray back reflection of (12 4 0) from multi-plate monolithic silicon crystal cavities has been carried out to investigate the possibility of enhancing the cavity resonance effect by increasing the reflectivity of the back reflection. According to the theoretical calculations based on the dynamical theory, the reflectivity can be raised by increasing the number of crystal plates. Hence, several 2-, 4-, 6-, and 8-plate crystal cavities of a gap of 100 μ m and the plate thickness of 20-70 μ m are designed and fabricated using the microelectronic lithographic technique. The diffraction experiments are then performed by using the synchrotron X-rays of 14.4388 keV with the energy resolution Δ E = 0.36 meV. Interference fringes due to the cavity resonance for various multi-plate cavities are observed and the corresponding finesse of each cavity is measured from the fringe spacing. It is found that the reflectivity, namely the finesse, increases as the number of crystal plates increases, as the calculations predicted. The best result is from the 8-plate cavity where the finesse is about 3.2, compared with 2.0 for a 2-plate cavity. This result is useful for designing better finesse crystal cavity for diffraction experiments. 1 2 1 1 2 2 3 M-269 Diagnostic Tools used for Calibration and Verification at Protein Crystallography Synchrotron Beam Lines F. J. Rotella, R. W. Alkire, N. E. C. Duke, M. J. Molitsky Argonne National Laboratory, Argonne, IL, United States A number of tools have been adapted and developed for use at the bending-magnet and insertion-device beam lines (19BM and 19ID) of the Structural Biology Center (SBC) at Argonne’s Advanced Photon Source (APS). These tools are used diagnostically in the calibration and operating verification of these synchrotron x-ray beam lines and constituent equipment. Examples of diagnostic tools used at the SBC are presented. Hen egg-white lysozyme crystals are a de facto standard that are widely used at protein crystallography synchrotron beam lines. Diffraction data from lysozyme single crystals are used to verify the operating fitness of the SBC beam lines at the beginning of every APS user beam run and during user beam runs to verify the beam lines after equipment is replaced. The calibration of the multi-module CCD area detectors used at SBC beam lines is verified using powder diffraction from frozen polycrystalline slurries of lysozyme. Powder diffraction from standard reference materials such as Si and LaB6 are used to calibrate the precise position – sample-to-detector distance and detector swing angle (2 ) – of the area detectors used at SBC beam lines. A bench top digital thermometer and type K thermocouple are employed to measure the temperature profiles and verify the operating fitness of the coldstreams that cool frozen samples at SBC beam lines. Diffraction from silicon single crystal 3 cubes, which are 0.8 mm in volume and mounted on sample loops, is a sensitive probe of sample motion induced by improper positioning of the cold-stream or other motion in the x-ray source or beam line apparatus. Sources of parasitic scattering from beam line components (e.g., beam collimators) observed in area detector x-ray images can be identified from knowledge of the materials comprising the components and the x-ray wavelength and sample-to-detector distance used to measure the diffraction images. This work was supported by the U.S. Department of Energy, Office of Biological and Environmental Research under contract DE-AC02-06CH11357. a M-272 The Cardiac Ryanodine Receptor Exon3 Deletion Is Rescued By Beta Strand Switching Filip Van Petegem, Paolo Lobo, Lynn Kimicka, Ching-Chieh Tung Univ. of British Columbia, Vancouver, Canada The contraction of heart muscle requires the rapid release of Ca from the sarcoplasmic reticulum into the cytoplasm. This event is mediated by opening of the cardiac Ryanodine 2+ Receptor (RyR2), a large Ca selective ion channel. Many mutations in RyR2 are known to lead to devastating genetic conditions, including catecholaminergic polymorphic ventricular tachycardia (CPVT). The latter is characterized by triggered cardiac arrhythmias and may lead to sudden cardiac death. 2+ The most extreme form of CPVT is caused by deletion of the entire third exon of RyR2. This small exon encodes an α helix and a β strand that is part of a β trefoil core in the N-terminal domain of the channel. Such a deletion would be predicted to cause misfolding of the protein, raising the question of why this disease deletion is not lethal. Surprisingly, thermal melt analysis shows that the Δ exon3 N-terminal domain is not misfolded, but instead has even gained thermal stability. We solved the crystal structures of both the wild type and Δ exon3 N-terminal domain at 2.5 Å and 2.2 Å, respectively. The deletion causes a very dramatic rearrangement, in which an otherwise flexible loop becomes a β strand and thus rescues the β trefoil domain. Other β strands partially rearrange to accommodate the rescue segment. These events underscore the unusual structural plasticity of the RyR2 Nterminal domain. Despite the rescue, the deletion still causes a very severe disease phenotype, which can be explained by the loss of key domain-domain interactions within the intact channel. The study provides rare detailed insights into a severe channelopathy. It raises the question for a functional role of the rescue segment in wild type RyR2. M-275 Synthesis of Nanoporous IsoReticular Metal Prophyrin Frameworks for Hydrogen Storage Claudia Rawn , Michelle Everett , Patrick Ward , Ruichang Xiong , David Keffer 1 2 1,2 1 1 1 1 University of Tennessee, Knoxville, TN, United States, Oak Ridge National Laboratory, Oak Ridge, TN, United States A hypothesized structure, analogous to IsoReticular Metal Organic Frameworks (IRMOFs) but with the organic component being replaced by a porphyrin, has been used to model the hydrogen adsorptive capacity. Various techniques have been employed for synthesizing an IsoReticular Metal-Porphyrin Framework (IRMPF) structure made up of a functionalized carboxylic tetraphenylporphyrin as the ligands and an oxygen coordinated zinc tetrahendron as the vertices. Early synthesis attempts in a sealed heated test tubes with the porphyrin and Zn(NO3)2 6(H2O) dissolved in various solvents resulted in a variety of low yield crystals. None of these crystals when examined by single crystal X-ray diffraction provided adequate data for a crystal structure solution. Later attempts used solvothermal synthesis with a variety o of solvents in various ratios. One in particular experiment was carried out at 100 C using DMF and CHCl3 resulted in suitable crystals for characterization and single crystal X-ray diffraction data revealed a Zn oxopolymer structure. The coordinated Zn compound was a promising result, however, there was no evidence of the porphyrin in the structure. The adsorption of H2 in five IRMPFs has been calculated using Path Integral Grand Canonical Monte Carlo (PI-GCMC) simulations using standard force fields. For comparison the adsorption isotherms of H2 in IRMOF-1 and IRMOF-10 have also been calculated. Liquid nitrogen temperature (77 K) and room temperature (300 K) were chosen for the temperature of the simulations and all calculations indicate that all but one of the IRMPFs adsorb a higher weight fraction of H2 than both IRMOF-1 and IRMOF-10, but are still well short of practical goals. We observe that IRMPFs provide additional adsorption sites for hydrogen due to the metal center of the porphyrin. Some, but not all, of the additional functional groups on the porphyrin enhance adsorption as well. This work is supported by the STAIR (Sustainable Technology through Advanced Interdisciplinary Research) program funded by the National Science Foundation through contract DGE-0801470. b M-278 Use of Complementary Methods to Determine the Quaternary Structure of Proline Utilization A (PutA) John Tanner University of Missouri-Columbia, Columbia, MO, United States Proline utilization A (PutA) proteins are large (1000-1300 residues) bifunctional enzymes that catalyze the two-step oxidation of proline to glutamate. We recently determined the first crystal structure of a PutA, but the quaternary structure was not obvious from the crystal structure. We therefore turned to the complementary techniques of small-angle X-ray scattering and analytical ultracentrifugation to study PutA in solution. Surprising, we discovered that the enzyme forms a donut-shaped, tetrameric assembly in solution. This work provides a good example of the power of combining complementary biophysical techniques with high resolution X-ray crystallography. Reference: Srivastava et al., PNAS, (2010) 107(7):2878-2883. M-287 Mystery Solved: X-ray Crystallography Explains the Cross-reactivity Between Structurally Diverse Immunodominant MART-1 epitopes Oleg Borbulevych, Brian Baker University of Notre Dame, Notre Dame, IN, United States Adaptive immunity mechanisms are based on antigen recognition by cytotoxic T cells. T-cell receptors (TCR) interact with certain peptide epitopes presented by class I MHC molecules, leading to an intracellular signaling cascade and a subsequent immune response. The MART-1 antigen is overexpressed by the majority of melanoma affected cells, and hence represents an attractive target for cancer immunotherapy using cytotoxic T cells. Immunodominant epitopes of MART-1 presented by the class I MHC HLA-A2 proteins comprise the decameric MART-1 26-35 epitope EAAGIGILTV and the nonameric MART-1 27-35 epitope AAGIGILTV. Our previous crystallographic study indicated that the conformations of those peptides in the HLA-A2 peptide binding groove differ significantly. Paradoxically, most MART-1 specific cytotoxic T cells can still cross-reactively recognize both MART-1 epitopes. In the present communication we report, for the first time, the crystallographic structures of the clinically relevant TCR DMF5 in the complex with MART-127-35/HLA-A2 and MART-12635/HLA-A2 to 2.3 Å and 2.7 Å resolution, respectively. These structures allow us to unravel the mechanism of cross-reactivity. Notably, recognition of the MART-127-35 nonamer peptide by DMF5 is accompanied by a significant rearrangement of the peptide backbone. In contrast, the conformation of the decameric peptide remains unchanged upon recognition, indicating that cross-reactivity occurs via a “induced molecular mimicry” mechanism. M-296 Mechanism of IL-10 Neturalization Elucidated by the Crystal Structure of an IL-10/anti-IL-10 Fab Complex Brandi Jones, Laura Baker Smith, Mark Walter University of Alamba at Birmingham, Birmingham, United States LPO? k PO ? ?\ ?サ ⁄¦\ ?¦„?fi‒› ·¦ ¡ ‹¡ ›‹?›¢?fi‒› ‹ ? ‹⁄›¤ \ ? ⁄¡ ?⁄\‹¢ « « \ ›‒„ «M¦ e ? k P ? s e サ \ ·?‹\ ¡ „?M?? k ? ‹ ?\¡ ›•‹?‒¡ › M£ · ¡ ? ¡¡ ‹?fi‒¡ £ ? £›‹? ¡ \ ‒¡⁄¡·‒¢\ fl ?¦¡fi ‹£ PO?\ ·‒¢\ ‹ ¦¡ ?¦¡ ? ?\· „?›¢?\ ? ¡ ?£‚?¦›‹ « ¦¡‹£k ‹??›¢? \¡ ?¦›« POqP? \ k? 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w s cs cr wg c PO?\\ ¦ ›‹ ‒¡¡ fi¦ ›‒ PO sc c M-303 THE STRUCTURE OF ADENO-ASSOCIATED VIRUS SEROTYPE 3B (AAV-3B), A GENE THERAPY VECTOR Thomas F. Lerch, Qing Xie, Michael S. Chapman Oregon Health & Sci Univ., Portland, OR, United States Adeno-associated viruses (AAVs) are leading candidate vectors for gene therapy applications. The most widely characterized AAV is serotype 2 (AAV-2), which enters cells through interactions with heparan sulfate proteoglycan (HSPG). The development of recombinant AAVs that target specific tissues has been accelerated since the determination of the AAV-2 atomic structure, which provided a roadmap for vector design. As much as 80% of the population has been exposed to AAV-2, and the presence of neutralizing antibodies to this serotype limits the efficacy of AAV-2 based vectors. The AAV-3b capsid is closely related to AAV-2 (87% identity), but sequence, and presumably structural differences lead to distinct properties in cell entry and immune recognition. Like AAV-2, AAV-3b requires heparan sulfate for cellular entry, yet key AAV-2 residues involved in heparan interactions are not conserved in AAV-3b. In an effort to understand these differences, and perhaps harness them, the structure of AAV-3b has been determined by X-ray crystallography. The crystals display varying levels of merohedral twinning that in earlier times would have rendered them unsuitable, but here is shown to be a tractable complication in structure determination. Structural comparisons of AAV-3b with AAV2 and other serotypes of known structure provide insights into how these viruses have naturally evolved to preserve receptor interactions while avoiding immune neutralization. M-305 The Structural Basis of 5 Triphosphate Double-stranded RNA Recognition by RIG-I CTD Pingwei Li, Cheng Lu Texas A&M Univ., College Station, TX, United States RIG-I is a cytosolic sensor of viral RNA that plays crucial roles in the induction of type I interferons. The C-terminal domain (CTD) of RIG-I is responsible for the recognition of viral RNA with 5 triphosphate (5 ppp). However, the mechanism of viral RNA recognition by RIG-I is still not fully understood. Here, we show that RIG-I CTD binds 5 ppp dsRNA or ssRNA, as well as blunt-ended dsRNA, and exhibits the highest affinity for 5 ppp dsRNA. Crystal structures of RIG-I CTD bound to 5 ppp dsRNA with GC- and AU- rich sequences revealed that RIG-I recognizes the termini of the dsRNA and interacts with the 5 triphosphate through extensive electrostatic interactions. Mutagenesis and RNA binding studies demonstrated that similar binding surfaces are involved in the recognition of different forms of RNA with or without 5 triphosphate. Mutations of key residues at the RNA binding surface afffected RIG-I signaling in cells. M-307 POWGEN: A New TOF Neutron Powder Diffractometer at the SNS Jason Hodges , Ashfia Huq , Olivier Gourdon , Luke Heroux 1 1 1 1,2 1 2 Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States, Forschungszentrum Juelich GmbH, Juelich, Germany POWGEN is a fundamental departure from previous designs for a time-of-flight powder diffractometer at a spallation neutron source and may be considered a third-generation design. The instrument is optimized for both parametric studies of materials under a wide range of conditions (T, P, H, flowing gases, etc) and ab-initio crystal structure determinations 3 of complex solid-state materials with asymmetric unit-cells of the order ~1500 Å . The geometric design of the instrument allows for all detected scattered neutrons to be focused onto a single diffraction profile yielding high count rate while preserving good resolution d/d = 0.0015 at d = 1 Å. The new time-event mode for data acquisition will permit stroboscopic experiments and filtering of the incoming data, neutron by neutron, allowing very high resolution diffraction profiles to be generated with d/d ~ 0.0007 at d = 1 Å. SNS is operated with the support from the Division of Scientific User Facilities, Office of Basic Energy Sciences, US Department of Energy, under contract DE-AC05-00OR22725 with UT-Battelle, LLC M-311 Synchrotron powder diffraction simplified: the high-resolution 11-BM diffractometer at the Advanced Photon Source Matthew Suchomel, Lynn Ribaud, Brian Toby, Robert Von Dreele Argonne National Laboratory, Argonne, IL, United States Synchrotrons have revolutionized powder diffraction. They enable the rapid collection of high quality powder diffraction patterns with tremendous resolution and superb signal to noise. In addition, the high penetration and exceptional data sensitivity possible at high-energy light sources like the Advanced Photon Source (APS) allows synchrotrons to explore trace containment levels, in-situ sample environments and crystallographic site occupancies. Despite all these advantages, relatively few scientists today consider using a synchrotron for routine powder diffraction studies. To help address this, the new high resolution synchrotron powder diffractometer beamline 11BM at the APS now offers rapid and easy mail-in access for routine structural analyses with truly world-class quality data. This instrument offers the highest resolution available in the Americas and is a free service for non-proprietary users. The instrument can collect a superb pattern suitable for Rietveld analysis in less than an hour, is equipped with a robotic arm for automated sample changes, and features variable temperature and in-situ sample environments. Users of the mail-in program typically receive their high-resolution data within two weeks of sample receipt. The diffractometer is also available for on-site user experiments requiring more specialized measurements. Our presentation will describe this instrument, highlight its capabilities, explain the types of measurements currently available, and discuss plans to improve access and available sample environments. We are particularly interested in seeking input from potential users within the crystallography community. More information about the 11-BM diffractometer and its associated mail-in program can be found at our website: http://11bm.xor.aps.anl.gov. M-315 SER-CAT’ s mission of "Light When YOU Need It" for Member Ins titutions and the APS General Zhongmin Jin, John Chrzas, Jim Fait, Zheng-Qing Fu, John Gonczy, Andrew Howard, Rod Salazar, Unmesh Chinte, Gerd Rosenbaum, John Rose, B.C. Wang University of Georgia, Athens, Georgia, United States SER-CAT’ s mission To provide "Light When YOU Need It" to its member institutions and : the APS General Users. 12-hour blocks and 16-hours/day on-site user support: Since summer of 2009, SER-CAT users can reserve time in 12-hour blocks if desired. In addition, SER-CAT is currently providing 16-hours/day on-site user support. Remote Access & Participation with Advanced Robotics: Advanced beamline control hardware, and software integration, makes it possible for SER-CAT users to have a “virtual synchrotron” at their home institutions. About 80% of SER-CAT users collect and process data remotely. The sample Dewars on 22ID and 22BM can hold 160 and 96 crystals, respectively. Micro Beam Capability: SER-CAT offers a set of highly machined pinhole collimator inserts ranging from 5 to 300 microns on both its beamlines. A new MD2 micro-diffractometer will be integrated into 22ID during the August - September shutdown. Sulfur SAD Phasing: Recent improvements to 22ID have opened up the possibility of protein structure determination using soft X-ray (6-8kev) based sulfur-SAS methods. MAD/SAD Experiments: The SER-CAT 22ID and 22BM lines were designed and optimized to provide tunable X-rays (6-15 keV) for MAD and optimized SAD experiments. Mail-in Crystallography Services: SER-CAT provides a mail-in data collection service to its members. Researchers can mail their crystals to SER-CAT and SER-CAT staff will collect data for them on a first come first serve basis. This is a special perk for SER-CAT institutional members. Work supported by the SER-CAT Member Institutions, the University of Georgia Research Foundation and the Georgia Research Alliance. M-319 IMAGINE, a Quasi-Laue Single Crystal Neutron Diffractometer At The High Flux Isotope Reactor Flora Meilleur , Tibor Koritsanszky , Robert Blessing , Bryan Chakoumakos , Dean Myles 1 2 1,2 3 4 2 2 Oak Ridge National Lab, Oak Ridge, TN, United States, North Carolina Univ, Raleigh, NC, 3 4 United States, Middle Tennessee State Univ., Murfreesboro, TN, United States, HauptmanWoodward Med Research Inst., Buffalo, NY, United States The acquisition, installation and operation of IMAGINE at he High Flux Isotope Reactor (HFIR) was proposed to the National Science Foundation by a group of researchers from Physics, Chemistry, Biology, Biochemistry and Geological and Earth Sciences at Middle Tennessee State University, North Carolina State University, Hauptman-Woodward Medical Research Institute and Oak Ridge National Laboratory, with 13 additional participants from US industry and academic facilities. The objective of the program, which received funding from NSF in July 2009, is to develop a state-of-the-art facility and user-access program for neutron-diffraction analysis of advanced, complex and functional materials at the HFIR. IMAGINE will have broad scientific impact and community use, providing new tools, capabilities and methods for the analysis of light atom positions in materials that will be of interest across the diverse fields of structural biology, pharmacology, chemistry, condensed matter physics, nano-structured materials, and in environmental, biomedical and geological sciences. The instrument will enable the neutron structure of supra and macro-molecules to 3 be determined at or near atomic resolutions (1.5 Å) from crystals with volume < 1mm and unit cell < 100 Å. IMAGINE will be commissioned in spring 2011. The IMAGINE team welcomes discussion and interaction with the community through the installation and commissioning phase of the instrument, and is excited to start working with the community to build an excellent education and science program. The poster will give an overview of the IMAGINE project at the HFIR. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. Department of Energy under Contract DE-AC05-00OR22725. M-321 Crystal structure of the novel PaiA N-acetyltransferase from Thermoplasma acidophilum involved in the negative control of sporulation and degradative enzyme production Ekaterina V. Filippova , George Minasov , Ludmila Shuvalova , Olga Kiryukhina , 2 2 1,2 Shonda Clancy , Andrzej Joachimiak , Wayne F. Anderson Department of Molecular Pharmacology and Biological Chemistry, Northwestern University, 2 Feinberg School of Medicine, Chicago, IL, United States, Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne, IL, United States The crystal structure of a putative Gcn5-related N-acetyltransferase (GNAT) Ta0374 from Thermoplasma acidophilum, a hyperacidophilic bacterium, has been determined in an apo-form, in complex with its natural ligand (acetyl coenzyme A) and in complex with a product of reaction (coenzyme A) obtained by co-crystallization with spermidine. Sequence and structure analysis reveals that N-acetyltransferase Ta0374 belongs to a novel protein family PaiA involved in the negative control of sporulation and production of degradative enzymes such as subtilisin, neutral protease, levansucrase, α -amylase, and alkaline phosphatases. The crystal structure of Ta0374 confirms that it binds acetyl coenzyme A in a way similar to other GNAT and is capable of acetylating spermidine. Based on structural and docking analysis we suggest that Glu53 and Tyr93 are the key residues for the spermidine recognition. In addition, we find that part of the purification His-Tag in the apo-form structure of Ta0374 blocks binding of acetyl coenzyme A in binary complex and affects a chain-flip rotation of “motif A” which is the most conserved sequence motif among canonical acetyltransferases. The details of the Ta0374 structure, conformational changes, ligand-binding site provide insights into the biological function of Pai regulation. 1 1,2 1,2 1,2 1,2 M-323 Impact of conserved 16S rRNA methylation by KsgA on the structure of the 30S ribosomal subunit Hasan Demirci , Frank Murphy IV , Riccardo Belardinelli , Ann C. Kelley , V Ramakrishnan , 1 1 1 Steven T. Gregory , Albert E. Dahlberg , Gerwald Jogl Brown University, Providence, RI, United States, NE-CAT/Cornell University, Argonne, IL, 3 4 United States, University of Camerino, Camerino, MC, Italy, MRC, Cambridge, United Kingdom The most highly conserved ribosome modification is the N6, N6-dimethylation of the universally conserved adenosines A1518 and A1519 in helix 45 of the small ribosomal subunit. Dimethylation of both adenines is catalyzed by the KsgA methyltransferase. The absence of these post-transcriptional modifications enhances initiation from non-AUG codons, increases decoding errors, and confers resistance to the antibiotic kasugamycin. To examine the function of A1518 and A1519 dimethylation, we determined the X-ray crystal structure of the unmodified 30S ribosomal subunit from Thermus thermophilus. We observe significant changes in helix 45 resulting from lack of methylation and conformational adjustments in neighboring helices 24a and 44. The unmodified tetraloop assumes a conformation similar to a canonical GNRA tetraloop fold, consistent with previous NMR studies predicting a function of adenine dimethylation in the stabilization of the extended tetraloop conformation observed in the wild-type ribosome. Conformational shifts in helices 24a and 44 explain the varied phenotypic properties of ksgA mutants and reveal the impact of loss of dimethylation on kasugamycin binding. Overall, our data show, for the first time, the significance of a post-transcriptional rRNA modification for ribosome structure and suggest a role for these modifications in the final stages of 30S subunit assembly. 1 2 1 2 3 4 4 M-325 Structural basis for p300 Taz2:C/EBP interactions. Maria Miller, Zbigniew Dauter Protein tructure Section, MCL, NCI-Frederick, Frederick, MD, United States, Synchrotron Radiation Research Section, MCL, NCI, Argonne, Illinois, United States CBP and its paralogue p300 are histone acetyltransferases that regulate transcription by interaction with multiple transcription factors. The crystal structure of the zinc finger Taz2 domain of the human p300 transcriptional coactivator was determined using an anomalous diffraction signal of the bound Zn ions. The structure comprises a helical bundle held by three Zn ions and is very similar to the solution structures determined for the shorter peptide corresponding to the evolutionarily conserved Taz2 domain from CBP [1] and p300 [2]. Residues 1813-1834 from the current construct form a helical extension of the C-terminal helix and make extensive crystal contact interactions with the peptide binding site of Taz2. Based on the analysis of these contacts, we previously proposed a hypothetical model of the Taz2:p53 binding [3]. In the current study we use the crystal contact interactions to investigate Taz2 binding to the transactivation domain of the C/EBP protein. 1 2 [1] De Guzman R.N., Liu H.Y., Martinez-Yamout M., Dyson H.J., Wright P.E. J. Mol. Biol, 2000, 303, 243. [2] Feng H., Miller Jenkins L.M., Durell S.R., Hayashi R., Mazur S.J., Cherry S., Tropea J.E., Miller M., Wlodawer A., Appella E., Bai Y. Structure 2009, 17, 2002. [3] Miller M., Dauter Z., Cherry S., Tropea J.E., and Wlodawer A., Acta Cryst. D65, 2009, 1301-1308. Key words: transcription regulation, zinc finger protein, anomalous diffraction M-327 Crystal Structure of a Dimeric Glycosylated IL-22/IL-22R1 Complex Ashlesha Desphpande, Brandi Jones, Mark Walter University of Alabama at Birmingham, Birmingham, AL, United States o xwtvffo ›? \ ‒¡ ›⁄„ ‒\ ¡ POq ‹ ? ? ⁄\ ? ¦‒„ ¡ ¡ \ \ ? ⁄¡ •¡ ¡ ⁄›•? k ·¡ ?‹ › ‒ ‹¢¦\ ? ‹¦¡ ? k ¦›« ‹¡· ·‒\? fi ‚›¡?•k ¡‹ ⁄?fi\ M??? ?? ‒·¦ ?›¢? ⁄¡ ? k ·‒¡ ‒·¦ ⁄¡ ? k qP?•⁄?P¦⁄ ⁄¡ \ ?サ P?¡› ?·‒fi‒ ?fi\ ¦›« ?« ›‹›« ¦ ¡?‹ ›« ‹£ ‒¦\⁄\ ‒„M?? ? ?¢ ‹ ‹£ ?›¢?›·‒ k ¦‒„ ¢›·‒?fiP‚¡ ⁄¡•⁄ \ ¦›‹ ? ‹?? \ ‒¦? ·‹ ·k ? „? ?‹? P?\¦›«„« ¦⁄ ¡ «? ¡ \ ‒¡k •›? k P?¦›« k ?? k P? ¦⁄ fi ¡‚¡ ›« ¡« ? M£ « ¡¦? ¡M› ‒„ M¡ ?‹ ›? ⁄¡ \¡? ¡ ¡ ? « ‹•¡ ‒« ››fi?‒¡ ¡ ? ‹· ? ? ›‹?›¢?¿ •›¢› ?‒¡\ ¡ ‒‹ ¡ ⁄¡ ?\ £ ‹ ›‹? ‒¢\ ¦¡ ??¢›‒« k ¦› ¡¡ ¡ ?« ¦?¦‒„ ?☜ ⁄\‒·¦¡ ·‒¡ ¦· ⁄ k ? fi¡« ? ⁄¡ «›fi ¡?\ M??? ? ‒\ ?☝?⁄\ ?‒¡ ¡ PO ? \‒¡\?¡¢›‒« ¦‒„ k ·‒¡›¢? ? ‹¡⁄\\ ?? ⁄›« ‒·¦ ? ‒·¦ ·‒¡›¢? ? k ? ¦„ ››¤ ⁄¡\ ¡ « ‒¡ ? ? M? k P? ‒fi ?¡ ?¡?·fi›‒· \¡ « › ?›‒?‒¡\ ¡?¦›«¡\⁄¡ ‚¡¡ ¦›? ¢?« ?fi‒› ?«\ ?\ › ‹¦¡ « ?›¢ ‒ k \ ?¦‒„ ⁄ «›¦„M???\›‒?¦⁄\ ¡ \?\ ⁄ ‹¦\ ·‒¡ ¦ ¡ ‒ ¦\›£ ¡‒·¦›£ ‒ \\ ›‹?¢›‒? ? ›‹?›¢? ? ¦›‹ ¡\ ¦?fi‒¡¡ ? ¡M ‹?• y qffo f ~ xwtvffo f ffo POq ‹‒¡· ‹£k ??fl ?›?„¦› £ ? \¡¡ „ \ ?¢›‒« o o f o z p{ f ~ o ffo o g n ml kjj i he ff g ff ed u qffo |xwtvffo p ~ rqffo |xwtvffo o ffo p } rqffo |xwtvffo o z wtvffo p pf o ffo L ? G L H? ? ? ? ¦‒ ‹? « ‹¦¡«¦fi‒› ¡ ¦\‒› ·?‹\ ? ·⁄\?? fi‒›£ ? ‹‹\ ‹£‒\ ? fi⁄¡ ? ¡¡ ? „? \\¢‒›« „? ‒ ¡ ¦¡ \ ›fi›‹‹ ?⁄‹„k ¦‹¢¡ ¦\›£·¡£¢¢⁄¡ ‒\?¡k ?‒¡ ⁄¡ ¦ ¡fl·?›¢?? ›‹M?? ?‒¡fl‹£⁄?\ ‒¡¡? ?‹ \ ? \ ¢¢k \ POq ? ‒¡¡ ›? ›¦‒‹\ ‹£ ‹ ‹„ ›•? ?? fi¦ ›‒? ¦\‒„?\ ⁄¡ ‹ ? ¦⁄\£ ? ¢›‒« ? ›£ „? ? ¡‹ ¡ ¦›« ?·¡ ?k fi ‚M? ?¡⁄\¡¡›fi ¡ o‒›‒? ‒¡ ? \\ « ‹ S? k M ? LPO? ¢\ ›¢? サ « „? ⁄¡¦„ ¦\ ? ‹¡ k M?›¤ ? fi\ \ ? „ yxwtvffo u to hs p prqff ffo z qffo |xwtvff p o z wtvffo v z z M-329 Mechanism of the UvrA•UvrB DNA Damage Sensor David Jeruzalmi, Danaya Pakotiprapha, Martin Samuels, Greg Verdine Harvard Univ., Cambridge, MA, United States There are several pathways in cells that monitor DNA and mount responses when damage is detected. One of these, the nucleotide excision repair (NER) pathway, processes bulky DNA lesions. We study the NER pathway in bacteria where the first steps are performed by three proteins: UvrA, UvrB, and UvrC. The UvrA•UvrB ensemble monitors DNA and recognizes damage. On encountering damage, UvrA exits the complex, leaving UvrB stably bound. Damage searching, formation of the DNA complex and dissociation of ‘A’ are regulated by ATP. ‘B’ then recruits the endonuclease UvrC, which catalyzes incisions on either side of the lesion. Additional processing reactions lead to restoration of the original DNA sequence. In order to better understand the initial steps of bacterial NER, we have determined two crystal structures, 1) full-length UvrA bound to ADP and 2) a complex between the two isolated interaction domains of UvrA and UvrB. The structure of isolated UvrA revealed its overall architecture and arrangement of its four-nucleotide binding sites. Structure-guided biochemical studies were used to identify surfaces that interact with UvrB and DNA. Our second structure focuses on the UvrA•UvrB complex. From the structure of the isolated interacting domains and the structures of the isolated components, we deduced a model for the complete damage-sensing complex. This work was supported by the National Institutes of Health (CA100742, GV) and the National Science Foundation (MCB 0918161, DJ). M-333 N-terminal Domain of Nop56/58 is Critical for Assembly and Methylation Activity of Archaeal Box C/D sRNP Complex Shyamasri Biswas , Keith Gagnon , Xinxin Zhang , Carla Mattos , Bernard Brown II , Stuart 1 Maxwell Department of Structural Biochemistry, North carolina state University, Raleigh, NC, United 2 States, Departments of Pharmacology and Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, United States Box C/D RNP complexes guide the 2’O-methylation of rRNA nucleotides which are critical for ribosome assembly and function. The archaeal sR8 box C/D RNP complex consists of L7Ae, Nop56/58 and fibrillarin core proteins and the sR8 sRNA. Although extensive structural studies have been carried out on the ‘kink turn' RNA-binding protein L7Ae and S-Adenosyl Methionine -binding methyltransferase protein fibrillarin, little is known about Nop56/58. Methylation of rRNA is initiated by L7Ae binding to kink-turn RNA motif followed by binding of the Nop56/58 and fibrillarin proteins. Here we describe the crystal structure of the N-terminal domain of the Methanocaldococcus jannaschii Nop56/58 core protein. This N-terminal domain interacts with fibrillarin and this complex exhibits exceptional stability resisting denaturation and melting temperatures above 100° Mutations in the N-terminal domain have identifi ed a C. single point mutant and a five amino acid deletion mutant that significantly reduce or abolish box C/D sRNP-guided nucleotide methylation in vitro, suggesting that these amino acids play a critical role in catalysis of 2’-O-methylation. Thus, the Nop56/58 core protein, either alone or in concert with fibrillarin, plays a functional role in nucleotide modification guided by the archaeal box C/D sRNP complexes. 1 1 1,2 1 1 1 07.14.1 Harnessing structural tuning parameters in a class of layer ordered CMR materials Omar Chmaissem , Bogdan Dabrowski , Stanislaw Kolesnik , Yang Ren , Dennis E 1 1 Brown , James Mais Northern Illinois University, DeKalb, IL, United States, Argonne, IL, United States 1 2 1,2 1,2 1 2 Argonne National Laboratory, In this talk, I will briefly review the main structural and physical properties of A-site layer ordered and disordered La1-xAxMnO3 (A = Ba, Sr, Ca; x~0.5) manganites and identify the characteristics they have in common and those that are distinctly different. Recent theoretical work successfully explained the colossal magnetoresistance (CMR) properties of the manganites in terms of parameters that include ferromagnetic (FM) double-exchange and antiferromagnetic (AFM) superexchange couplings, electron-phonon coupling, quenched disorder, etc. Here, I will discuss our observation of charge ordering (CO), orbital ordering (OO) and the presence of a multicritical point in the A-site layer ordered La1-xBa1+xMn2O6 (x~0.04) class of materials and the existence of a strong competition developing between three distinctly different magnetic ground states (FM, CO AFM and OO AFM). At the right composition and below some temperature, the three phases start to separate and eventually they freeze in their respective domains; thus giving rise to spin glass clusters. Neutron and xray data taken as a function of temperature and magnetic fields agree very well with the complex magnetic and resistive data. On the other hand, disordered La1-xBaxMnO3 (0.2 ≤ x ≤ 0.52) does not exhibit any such properties and the materials remain ferromagnetic at all temperatures. Furthermore, we find that the deliberate introduction of a small structural disorder on the otherwise perfectly ordered La or Ba layers results in modifying the system’s property to either move it closer or away from the multicritical point depending whether the disorder is on the La or the Ba sites. Our asymmetric results suggest that disorder is not the primary parameter controlling phase separation and the CMR properties. In fact, it may be used as a fine tuning tool to enhance/reduce the electron-phonon coupling near the multicritical point; thus, resulting in phase separation. Finally, in this class of materials, charge ordering and the observed phase separation (responsible for the CMR properties) can be suppressed under the application of low magnetic fields of 1-2 Tesla. This represents a huge improvement of an order of magnitude when compared with the large fields (10-60 T) required to suppress CO, structural transitions, and phase separation in the disordered 3D counterparts. 07.14.2 The Cosubstitution Reaction of In2O3 by ZnO and SnO2 as Characterized with X-ray Absorption Spectroscopy Cathleen Hoel, Jean-Francois Gaillard, Kenneth Poeppelmeier Northwestern University, Evanston, IL, United States Transparent conducting oxides (TCOs) are important materials to optoelectronics, such as flat-panel displays and photovoltaics, with tin-doped indium oxide (ITO) having the high electrical conductivity and optical transparency required for maximum device performance. The limited global supply of indium metal coupled with the increased demand has necessitated the need to develop indium-free TCOs. The cosubstitution of ZnO and SnO2 into In2O3 (ZITO) has demonstrated conductivities comparable to those of ITO, while 1,2 reducing the percentage of In by 40 mole %. The local structural changes that occur in In2O3 upon substitution with Zn and Sn have been investigated with X-ray absorption spectroscopy. Zn and Sn were observed to induce first shell oxide bond shortening owing to the smaller cationic radii compared to In. Zn was six-coordinate with oxygen and the Zn and Sn next nearest neighbor cation environment was consistent with the bixbyite framework. 1. G. Palmer, et al., Chem. Mater. 9 (1997) 3121 2. S. Harvey, et al., J. Am. Ceram. Soc. 91 (2008) 3683 07.14.3 Effect of lattice distortion via Phosporus doping in Fe-based Oxypnictides Clarina dela Cruz , W.Z. Hu , Shiliang Li , Qing Huang , Jeff Lynn , Mark Green , Miaoyin 2 2 5 3 1 6 2,1 Wang , Meng Wang , G.F. Chen , N.L. Wang , Herbert Mook , Qimiao Si , Pengcheng Dai 1 1 3 3 4 4 4 Neutron Scattering Science Division, Oak Ridge National Laboratory, Oak Ridge, TN, United 2 States, Department of Physics and Astronomy, University of Tennessee, Knoxville, TN, 3 4 United States, Institute of Physics, Chineses Academy of Sciences, Beijing, China, NIST 5 Center for Neutron Research, NIST, Gaithersburg, Maryland, United States, Department of 6 Physics, Renmin University, Beijing, China, Department of Physics and Astronomy, Rice University, Houston, TX, United States We use neutron diffraction to study the structural and magnetic phase diagram of P doped Fe-based oxypnictide superconductors and their corresponding parent compounds. First, we find that replacing the larger arsenic with smaller phosphorus in CeFeAs1-xPxO simultaneously suppresses the antiferromagnetic order and orthorhombic distortion near x=0.4, thus suggesting the presence of a magnetic quantum critical point. In this system, P doping drives the system through the quantum critical point to a paramagnetic state. Our detailed structural analysis reveals that the pnictogen height is an important controlling parameter for their electronic and magnetic properties, and may play an important role in electron pairing and superconductivity of these materials. Similar studies were done in the LaFeAs1-xPxO where P-doping drives the system into a superconducting state. Comparisons between the two systems were done to identify the correlation of lattice distortion on the resulting transport properties and magnetism. 07.14.4 Targeting New Materials Kenneth Poeppelmeier Northwestern University, Evanston, IL, United States An example of a new transition metal oxide fluoride, which was synthesized recently, is the high silver density material Ag4V2O6F2 (SVOF). Ag2V4O11, or silver vanadium oxide (SVO), is used commercially as the cathode material in primary lithium batteries for high rate applications, such as those used in implantable cardioverter defibrillators (ICDs). A long-term goal of the medical battery industry is to increase the capacity of the cathode above 3 V while maintaining electrode stability. Owing to the high mole fraction of silver and the replacement of oxide with fluoride, SVOF has a higher capacity above 3 V of 148 mAh/g in comparison to 100 mAh/g in SVO and the upper discharge plateau at 3.5 V is 300 mV higher than the silver reduction potential of SVO. The electrochemical behavior of SVOF and the significant impact new materials such as SVOF may have on the future generation of primary lithium batteries for ICDs will be highlighted. 07.14.5 Single Crystals of Functionalized Nanoparticles as a Medium for Structural and Spectroscopic Studies of Photosensitizer Dyes on Semiconductor Surfaces Jason Benedict, Philip Coppens University at Buffalo, Buffalo, NY, United States Single crystals of semiconductor nanoparticles functionalized with technologically relevant ligands offer the opportunity to obtain structural information that can be combined with theoretical calculations. The latter are based on the experimental geometry and used to interpret spectroscopic and other information. A series of Ti/O clusters with diameters up to approximately 2.0 nm have been synthesized and functionalized with a variety of small molecule ligands which exhibit distinct binding modes. DFT calculations complement spectroscopic measurements which examine the band gap of the nanoparticles pre- and postfunctionalization and elucidate the influence of adsorbate geometry as well as size quantization effects on the band structure.The detailed structure of the nanoparticles deviates significantly from that of anatase, which is commonly used as a structural model for the substrate used in photovoltaic cells. The structures for the first time unambiguously reveal the † different binding modes of the adsorbant molecules. l Work was funded by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy through Grant DE-FC0202ER15372 † J. B. Benedict & P. Coppens, JACS 2010, 192, 2938-2944. 07.14.6 Local structure and its relationship to the properties of “ReO 3” type frameworks showing positive, low and negative thermal expansion Angus Wilkinson , Benjamin Greve , Andrew Jupe , Kenneth Martin , Karena Chapman , 3 4 Peter Chupas , Peter Lee 1 1 1 1 2 3 Georgia Institute of Technology, Atlanta, GA, United States, Berry College, Mount Berry, 3 4 GA, United States, Argonne National Laboratory, Argonne, IL, United States, Department of Energy, Germantown, MD, United States The cubic “ReO3” framework structure is simple, and yet it has all the structural features necessary for negative thermal expansion (NTE) due to the transverse thermal motion of bridging anions. While ReO3 itself does not display negative thermal expansion at room temperature, materials with this structure display a variety of properties, ranging from pronounced NTE through to strong positive thermal expansion. We will discuss the thermal expansion and compressibility of several fluorides and oxyfluorides with this structure, and examine the role that O/F disorder plays in determining their properties. 2 07.15.1 Non-merohedral twin with partially known composition Ilia Guzei , Indika Arachchige , Sergei Ivanov 1 1 2 2 2 UW-Madison, Madison, WI, United States, MPA-CINT, Los Alamos National Lab, Los Alamos, NM, United States The hexagonal (space group P61) crystal structure of an ionic multinuclear gold cluster [Au6S2P(Ph2Cy)6][anion].(solvent) proved to be a non-merohedral twin. The twin components are related by a 180° rotation about the [110] axis with the minor compo nent contribution of 49.4(6)%. The composition of the cationic hexanuclear gold cluster was reliably established, however only the heavy atoms could be refined with anisotropic displacement coefficients. The Ph and Cy groups had to be refined with an idealized geometry taken from the newly created Idealized Molecular Geometry Library. There were several diffusely diffracting species in the lattice which could not be reliably identified and even their corresponding electron count could not be approximated with the PLATON/SQUEEZE option due to the twinning. Publication prospects for this structure remain unclear because the charge has not been balanced and possible solvents identified. 07.15.2 The cis-dioxo uranyl cation remains at large Christopher Cahill , Paula Cantos , Richard Wilson 1 1 1 2 2 The George Washington University, Washington, DC, United States, Argonne National Laboratory, Argonne, IL, United States Hexavalent uranium forms almost exclusively as the uranyl cation, [UO2] , a linear triatomic species. Although predicted to be stable, the cis- form of this species has not been formed reliably. Reports of its formation and subsequent crystallographic characterization have been scrutinized and labeled as suspect. A recent result from our group wherein the uranyl cation was reacted with 2,5-pyradinedicarboxylic acid appeared to yield such a cis-bearing material: UO2(C7H3O4N), P21, a = 7.0014(6), b = 8.1293(7), c = 7.8051 (7) Å, = 104.317(1)° R1 = , 2.92%. This was a relatively satisfactory refinement, save for one anomalous thermal parameter and a suspiciously long C-O bond. Standard CIF checks were not condemning, either. Careful reexamination (and subsequent re-collection of the data) revealed a P21/n cell: , a = 9.0141(11), b = 8.1137(10), c = 11.6729(14) Å, = 96.197(2) ° R1 = 2.55%. This larger cell did not display the anomalous features, nor did it contain a cis-oxo uranyl! The trans form continues to reign and we cite the need to exercise restraint when encountering such a combination of results and relatively minor red flags. Did we publish it? No way. The trans? It is in review. 2+ 07.15.3 Porphyrin Polymorphism … do we need more structures? Allen Oliver, W. Robert Scheidt University of Notre Dame, Notre Dame, IN, United States Chloro (octaethylporphyrinato)iron(III) [Fe(OEP)Cl] has two reported polymorphs; one of which is archived in the Cambridge Structure Database. A short discussion on a third polymorph and its merits to the literature will be presented. 07.15.4 The Best Way to Solve the Big R Problem? Kenneth Haller Suranaree University of Technology, Nakhon Ratchasima, Thailand A reportedly 1:1 cocrystal adduct of [SnCl2(C2H4COOCH3)2] and 1,10-phenanthroline that had remained on the shelf for 25 years was recrystallized from ethanol for an X-ray study. The space group was found to be triclinic P−1 with Z = 6. A strong vector in the Patterson function 2 1 1 of ( /3, /3, /3) suggested pseudo-translational symmetry, and a direct methods solution consistent with this suggested pseudo symmetry was obtained. However, the refinement failed, ending with R = 0.32. There is more than one way to skin a cat, and this structure has been published. 07.15.5 Is a 1.3Å resolution structure only useful for joining the dots? Gary Nichol The University of Arizona, Tucson, AZ, United States Crystallography is often used in organic chemistry to consider questions of conformation or chirality only after hours spent poring over various correlated nmr spectra have failed to point to a definitive answer. In many cases a perfectly reasonable set of crystals (they are usually excellent!) can be easily grown and a structure obtained in a timely fashion. Then there are cases where compounds stubbornly refuse to yield good crystals, but the crystals which are obtained can still be used in a diffraction experiment and provide useful information for the chemist. The question then is – what next? Two recent structures of poorly-diffracting (1.1Å & 1.3Å resolution) chiral compounds, along with the pros and cons of each structure, will be presented for audience debate and comment. 07.15.6 Common problems encountered with submissions to Acta Cryst. E Jim Simpson University of Otago, Dunedin, New Zealand As a section editor and co-editor of Acta Cryst. Section E, which in 2009 published 4116 papers, I get to see a reasonable cross-section of small molecule structures that fall within the proposed ambit of this session. I would like to take the opportunity to share information on the most common problems that we encounter in Acta E and how they are generally dealt with. It would be fair to say that problems – even some resulting in A alerts in the CheckCIF process – do not necessarily condemn a paper to rejection. The use and reliance on validation procedures as the ultimate determinant of the fate of a publication will also be discussed. 07.16.1 The National Synchrotron Light Source Facility at the Brookhaven National Laboratory Marc Allaire, et. al. Brookhaven National Lab, Upton NY, United States The National Synchrotron Light Source (NSLS) at the Brookhaven National Laboratory is located on Long Island, New York. The NSLS continues to be extremely active having more than 2200 users reporting nearly a thousand publications a year. The NSLS provides access to over 60 dedicated synchrotron beamlines with a wide range of applications targeting many areas of life, materials, and physical sciences. Synchrotron techniques as diverse as UV/IR/xray (micro)-spectroscopy and imaging, x-ray reflectivity and scattering, powder and single crystal diffraction, and x-ray footprinting are commonly available at the NSLS. The NSLS has a long tradition of strong user support. In macromolecular crystallography, a sea of photons is readily available, including two undulator-based MX beamlines X25 and X29, the latter being the second most productive beamline worldwide. A recent upgrade to beamline X26C allows correlated studies of single crystal diffraction with optical absorption and Raman spectroscopy. Responding to a growing demand, the newest beamline at the NSLS is the undulator-based beamline X9, which is dedicated to small/wide-angle x-ray scattering including the study of biomolecules in solution. Beamline X9 has been specially designed to provide simultaneous SAXS/WAXS data collection. In an effort to expand and educate the SAXS/WAXS user community, a hands-on training course is now frequently available to new users of this beamline. The is supported by ¡? ‒ ? ? are funded by the National Institute of Health. . Supports for specific user programs
07.16.2 Neutron Total Scattering and Powder Diffraction Capabilities at the Lujan Neutron Scattering Center Thomas Proffen Los Alamos National Laboratory, Los Alamos, NM, United States The Lujan Neutron Scattering Center at Los Alamos National Laboratory offers a number of diffractometers in the user program: NPDF is a dedicated and user friendly total scattering instrument allowing one to obtain the atomic pair distribution function (PDF) of disordered, nano-crystalline and amorphous materials. The PDF capabilities were recently extended with a series of successful PDF measurements on HIPD using the same user friendly web interface a NPDF. Data obtained on both instruments also allow Rietveld analysis of the materials – NPDF provides high resolution and HIPD high flux. The instrument HIPPO is a high flux instrument mainly used for high pressure and texture measurements but it can also be used as ‘regular’ powder diffractometer. Finally the instrument SMARTS is a engineering diffractometer at the Lujan Center. Curious about a specific instrument and experimental capability – come and join us at this special session and learn more. 07.16.3 Neutron Scattering Opportunities at the SNS and HFIR Dean Myles Oak Ridge National Laboratory, Oak Ridge, TN, United States With the United States' highest flux reactor-based neutron source for condensed matter research (the High Flux Isotope Reactor) and the world's most intense pulsed, acceleratorbased neutron source (the Spallation Neutron Source), ORNL is becoming one of the foremost center for neutron science. Research at these facilities encompasses the physical, chemical, materials, biological, and medical sciences and will provide opportunities for up to 2000 researchers each year from industry, research facilities, and universities all over the world. Opportunities for crystallography, small angle scattering and reflectometry at ORNL will be discussed. 07.16.4 Harness Advanced Photons at their Source to Further Your Research Brian Toby APS, Argonne National Labe, Argonne, IL, United States The Advanced Photon Source (APS) at Argonne National Laboratory is the country's brightest high-energy synchrotron. Each year over 5,000 scientists use the unique x-ray instrumentation at the APS. This presentation will provide a very brief overview of the diverse capabilities available to users and how to obtain more detailed information. Also to be discussed will be how scientists who have not used the APS can get access to the facility. 07.16.5 The General-Purpose Small Angle Neutron Scattering instrument on the High Flux Isotope Reactor HFIR at Oak Ridge National Laboratory Ken Littrell HFIR/NSSD Oak Ridge National Laboratory, Oak Ridge, TN, United States In May, 2007, the High Flux Isotope Reactor (HFIR) at Oak Ridge National Laboratory resumed routine operation and service to the scientific user community with a number of significant upgrades. Among the most important of these is a new supercritical hydrogen moderator (T ~ 20 K) that is the “brightest” cold source currently available. While this will eventually provide neutrons to a whole suite of scattering instruments through four cold neutron guides (CG1-4), the two flagship instruments, new small-angle neutron scattering (SANS) instruments on CG2 and CG3 have been installed and commissioned. The CG2 SANS (General Purpose SANS, funded by the Department of Energy (DOE) Office of Basic Energy Sciences) is a 40m maximum total flight path pinhole SANS instrument 2 5 2 variable wavelength and a large area (1m ) high count-rate, (> 10 Hz) high-resolution (5mm pixels) detector that can translate from 0 to 45 cm off-axis to increase the dynamic Q-range (< -1 7 2 0.001-1 Å overall). With a measured flux on sample of 10 /sec/cm and beyond in highthroughput configurations, this instrument is comparable to the best worldwide. This dramatically improves both the quantity and quality of data that we can collect from samples from a variety of systems, enabling us to better serve the neutron scattering community. This instrument has successfully operated and been fully available to users through an open, peerreviewed proposal system since December 2006. In this presentation we will discuss the present performance of the HFIR GP-SANS instrument, its available sample environment, gaining access to the instrument through the general user program (and what kind of support to expect), and our plans for upgrades to both sample environment and the instrument itself. 07.16.6 VIVALDI – the thermal-neutron Laue diffractometer for physics, chemistry and materials science at the Institut Laue-Langevin Garry McIntyre , Marie-Hélène Lemée-Cailleau , Clive Wilkinson 1 2 1,2 1 2 Institut Laue-Langevin, Grenoble, France, Department of Chemistry, University of Durham, Durham, United Kingdom The first eight years of operation of VIVALDI (Very-Intense Vertical-Axis Laue Diffractometer) [1] at the ILL have been a resounding success, producing spectacular neutron Laue diffraction patterns and exciting new science at a furious pace, with gains in data collection rate over conventional neutron diffractometers of up to 100 fold. In just a few hours VIVALDI has yielded detailed atomic structural information on large organic molecules [2], shown 2-D magnetic ordering in astounding clarity [3], revealed new magnetic structures [4], allowed full data collection from small crystals inside anvil pressure cells [5], followed variations of bond lengths with temperature in fine detail [6], detected the multiple adsorption sites of hydrogen in a metal-organic framework [7], and traced the kinetics of the continuous photo-induced transition in a spin-crossover compound [8]. With an intense and broad incident thermal-neutron waveband, a large-solid-angle imageplate detector, and fully automated readout and temperature variation, VIVALDI has proven to 3 be especially well suited to small crystals (0.1 mm is the norm!), rapid chemical crystallography, reciprocal-space surveys, and studies of structural and magnetic phase transitions. One copy of VIVALDI, called KOALA, is already in successful operation on the OPAL reactor in Australia, and another, IMAGINE, is planned for HFIR at ORNL. We briefly describe the unique technical aspects of VIVALDI and review the scientific highlights of recent years to show why neutron Laue diffraction at a reactor may be the technique of choice to solve your special crystallographic problem. [1] G.J. McIntyre et al, Physica B 385-386 (2006) 1055-1058. [2] M. Yousufuddin et al, J. Am. Chem. Soc. 130 (2008) 3888-3891. [3] E.M.-L. Chung et al, J. Phys.: Condens. Matter 16 (2004) 7837-7852. [4] P. Schobinger-Papamantellos et al, J. Phys.: Condens. Matter 16 (2004) 6569-6578. [5] G.J. McIntyre et al, J. Phys.: Condens. Matter, 17 (2005) S3017-S3024. [6] C. Dobe et al, J. Am. Chem. Soc. 126 (2004) 16639-16652. [7] E.C. Spencer et al. Chem Comm. (2006) 278-280. [8] F. Varret et al, Z. Krist. 223 (2008) 250-258. 07.16.7 MacCHESS: The Macromolecular Diffraction Facility at the Cornell High-Energy Synchrotron Source Richard Gillilan, Chae Un Kim, Ulrich Englich, Dave Schuller, Irina Kriksunov, Bill Miller, Scott Smith, Mike Cook, Qingqui Huang, Søren Nielsen, Marian Szebenyi Cornell University, Ithaca, NY, United States MacCHESS is an NIH Research Resource at the Cornell High-Energy Synchrotron Source in Upstate New York dedicated to providing researchers in the biological sciences with access to state-of-the-art facilities for x-ray diffraction. As a synchrotron source located on an academic campus, access to the facility is open to a very broad range of users by submitting a simple one-page online express-mode proposal. During running periods, requests for time are generally filled rapidly. Of the five beamlines available to users, A1 and F1 support the majority of protein crystallography. F1, with its large-area Q270 detector, finely-collimated beam, long sample-todetector distance, and BSL2 status is popular for large unit cell and virus work. A1 station is comparable to F1 but is tuned just above the selenium edge and may be used for SAD data collection. Automated sample mounting (based on ALS designs) is supported at F1 and is currently being designed for A1. Single-bounce monocapillary x-ray focusing optics developed at CHESS are routinely available at all crystallography stations on request when small (20 micron), higher-flux beams are desirable. F3 station provides a unique narrow band-pass (0.2%) multilayer system compatible with low-energy SAD crystallography. F2 station, originally a tunable MAD crystallography station, is in the process of being rebuilt as a high-flux multilayer-equipped beamline for high-throughput biological small-angle x-ray scattering (BioSAXS). MacCHESS also supports BioSAXS users part time on the CHESS G1 line, a high-flux (1.5% bandpass) multilayer system. MacCHESS currently provides the only high-pressure protein crystallography facility available for routine use. Protein crystals flash cooled at high pressure sometimes show significantly improved diffraction quality over those cooled at ambient pressure. High pressure conditions can also shift binding equilibria of ligands, improving crystallographic occupancy and may be valuable in trapping intermediates. MacCHESS engages in a wide range of collaborative research and teaching activities and encourages proposals for unusual experiments that would otherwise be difficult to do at dedicated crystallography beamlines where equipment cannot easily be reconfigured. A T-003 Five-Dimensional Crystallography Marius Schmidt , Tim Graber , Robert Henning , 2 Vukica Srajer University of Wisconsin-Milwaukee, Milwaukee, WI, 2 United States, BioCARS, The University of Chicago, Chicago, IL, United States 1 1 2 2 B A method for determining a comprehensive chemical kinetic mechanism for macromolecular reactions is presented. The method is based on five-dimensional crystallography [1], where in addition to space and time, temperature is also taken into consideration and an analysis based on singular value decomposition [2] is applied. We present first results of such a timeresolved crystallographic study. Temperature dependent time-resolved X-ray diffraction measurements were conducted on the newly upgraded BioCARS 14-ID-B beamline at the Right singular vectors from a Advanced Photon Source. The measurements aimed singular value decomposition of at elucidating a comprehensive kinetic mechanism of two complete time series of timethe photocycle of the photoactive yellow protein. resolved crystallographic data at o 0 Comprehensive time-series of crystallographic data (A) 20 C and (B) 30 C o o were collected at two temperatures, 20 C and 30 C. Relaxation times of extracted from these time-series exhibit measurable differences for the two temperatures, hence demonstrating that fivedimensional crystallography is feasible. [1] Schmidt, Graber, Henning, Srajer (2010) Five-Dimensional Crystallography, Acta Cryst A, 66, 198-206. [2] Schmidt, Rajagopal., Ren, Moffat (2003) Appli-cation of Singular Value Decomposition to the Analysis of Time-Resolved Macromolecular X-ray Data. Biophys. J. 84 2112-2129 AW.02 Roles of Carbon-Oxygen Hydrogen Bonds in Methyl Group Recognition and Catalysis in Protein Lysine Methyltransferases and Demethylases Raymond Trievel University of Michigan, Ann Arbor, MI, United States Protein lysine methylation has emerged as a prevalent post-translational modification associated with numerous biological functions. In eukaryotes, site-specific methylation of lysine residues occurs in histones, transcription factors, ribosomal subunits, chromatin modifying enzymes, and other protein substrates. Methylation of these targets has been implicated in diverse functions, including gene regulation, DNA damage response, protein turnover, and genome stability. The methylation status of protein lysine residues is dynamically regulated through the concerted activities of lysine methyltransferases (KMTs) and lysine demethylases (KDMs) and is biologically important because both the site and degree of methylation are critical for intermolecular recognition in cellular signalling pathways. Structural and functional studies of KMTs and KDMs have revealed key insights into their respective substrate specificities and catalytic mechanisms. In the course of studying the mechanisms of these enzymes, we have identified short-range interactions between the methyllysine methyl groups and active site oxygen atoms that are indicative of carbon-oxygen hydrogen bonds. This unusual type of hydrogen bonding can occur when a carbon atom and its hydrogens are polarized by an adjacent covalently bonded heteroatom that enables hydrogen bonding with a nearby oxygen atom. In KMTs, carbon-oxygen hydrogen bonds facilitate the alignment of the methyllysine substrate for multiple methyl transfer reactions, contributing to the product specificities of these enzymes. Similarly, hydrogen bonding to methyllysine substrates within the active sites of KDMs promotes demethylation and defines the methylation state specificities of these enzymes. These findings prompted us to examine whether carbon-oxygen hydrogen bonding represents a general mechanism by which polar methyl groups are recognized in biology. A survey of the PDB has revealed numerous examples of these interactions in the structures of different classes of methyltransferases and demethylases, highlighting the widespread nature of these hydrogen bonds in methyl group coordination and catalysis. 02.02.1 Structural Characterization of Active Site Residues involved in Proton Transfer in Catalysis by Carbonic Anhydrase II. Dayne West, Rose Mikulski, Katherine Sippel, Balendu Avvaru, Seungjin Jang, Chingkuang Tu, David Silverman, Robert McKenna University of Florida, Gainesville, FL, 32610, United States The reaction of physiological significance catalyzed by carbonic anhydrase (CA) is the hydration of carbon dioxide. This catalysis involves an attack on CO2 by zinc-bound hydroxide followed by a rate-limiting proton transfer from the active site to bulk solution to regenerate the zinc-bound hydroxide. In human CA II, the intramolecular proton transfer residue H64 accepts protons from the zinc-bound water through a network of active-site hydrogen-bonded 6 -1 waters at a turnover rate as great as 10 s and transfers them to solution. The unifying goal of this study is to expand our use of the CAs to understand such rate-limiting and long-range proton transfer steps in a way that can be extended to other proteins. The current goal is to understand the proton transfers in HCA II. We will present both structural and kinetic studies on the site directed mutants of HCAII: N62Q/N67Q, N62L/N67L, H64A/N62L/N67L, Y7N/N62L/N67L, and Y7F/N62Q. The structure-function studies of these mutations, when taken together, provide information on the importance of specific residues for proton transfer. These results provide a range of catalytic activities, geometries, and active-site environments. 18 Stopped-flow spectrophotometry, O exchange between CO2 and water measured by mass spectrometry, and solvent H/D isotope effects will be used to investigate rate constants for intramolecular proton transfer. Crystal structures of important mutants and the observed structural alterations will be discussed and correlated to the influence of distances, location, and environment on the rate of proton transfer and on CO2 binding at the active site. 02.02.2 Modeling Diffuse Scattering in a 1D Disordered Crystal Using a Probabilistic Layer Stacking Model Tara Michels-Clark , Hans-Beat Buergi , Jurg Hauser , Christina Hoffmann , Vickie Lynch 1 2 1,2 3,4 4 1 1 Oak Ridge National Laborator, Oak Ridge, TN, United States, University of Tennessee, 3 4 Knoxville, TN, United States, University of Zurich, Zurich, Switzerland, University of Bern, Bern, Switzerland We are reporting on a new approach for total scattering single crystal X-ray and neutron pattern analysis. Highly sensitive area detectors are now able to measure complete diffraction patterns from single crystal samples displaying local disorder. In many such cases interesting functional responses are dependent on the disordered rather than the ordered part of the structure. To elucidate the intricate interplay between local structure and overall structure quantitatively, new computational methods are needed. We are using a combination of Monte Carlo and Differential Evolution modeling techniques to simulate disorder and analyze diffuse scattering. Modeling diffuse scattering from modulated disordered crystal structures quantitatively proves to be a complex task with numerous optimization parameters. We have studied tris(bicyclo[2.1.1]hexeno)benzene, which exhibits hexagonal symmetry and 1D streaks of diffuse scattering along the hexagonal axis. It is composed of ordered layers of planar molecules stacked along the c-axis. The model takes into account layer-to-layer interactions up to the fifth layer. Initially a four-layer model was developed to interpret the diffuse intensity of fourteen of reciprocal lattice with four probabilistic stacking parameters [1]. The intensity distribution along c* (L) is given by I ( L) 2 M F S ( L) 2 F is the Fourier transform of a single P 6 2m layer of tris(bicyclo[2.1.1]hexeno)benzene molecules in the unit mesh a,b with thickness c that corresponding to four layers. S(L) is the interference function; its coefficients T, U, V, W, X, Y, and Z depend on the stacking probabilities and M is the number of layers. The probabilites have been optimized utilizing a differential evolutionary algorithm and parallel computing. Results of the four layer model of tris(bicylco[2.1.1]hexeno)-benzene will be presented comparing calculated intensities I(L) to experimental intensities. A five-layer model, which is expected to improve the model intensities, will also be presented. [1] H. B. Burgi, Marc Hostettler, H. Birkedal and D. Schwarzenbach, Z. Kristallography 2005, 220, 1066-1075. This research is supported by UT Battelle, LLC under Contract No. DE-AC05-00OR22725 for the U.S. Department of Energy, Office of Science and by a Swiss National Science Foundation Sinergia Grant W S L X cos L Y cos 2 L Z cos3 L T U cos L V cos 2 L 02.02.3 Disrupting a Bacterial Enzyme Alleviates Cancer Drug Toxicity. Bret Wallace , Hongwei Wang , Kimberly Lane , John Scott , Jillian Orans , Ja Seol Koo , 1 3 2 1 Christian Jobin , Li-An Yeh , Sridhar Mani , Matthew Redinbo 1 1 2 1 3 1 2 University of North Carolina at Chapel Hill, Chapel Hill, NC, United States, Albert Einstein 3 College of Medicine, Bronx, NY, United States, North Carolina Central University, BRITE, Durham, NY, United States 2 We show for the first time that a bacterial enzyme in human microbial symbiotes can be selectively inhibited to improve chemotherapeutic tolerance. The dose-limiting side effect of the colon anticancer chemotherapeutic CPT-11 is intense diarrhea caused by the GI reactivation of a primary drug metabolite. The enzymes responsible are bacterial glucuronidases present in the symbiotic gut microbiota. With the goal of reducing this side effect, we sought to selectively eliminate GI-specific drug reactivation without killing the commensal bacteria essential for human health. We identify potent bacterial -glucuronidase inhibitors through high-throughput screening that have no effect on the mammalian orthologue. Using crystal structures of E. coli -glucuronidase complexed with lead inhibitors, we demonstrate that selectivity is based on a loop unique to the bacterial -glucuronidases. We further establish that inhibitors are effective against the enzyme target in living bacterial strains grown under aerobic or anaerobic conditions, but do not kill the host bacteria or harm human colonic epithelial cells. Finally, we show that oral administration of an inhibitor lead protects mice from CPT-11-induced toxicity. Thus, undesirable enzyme activities in essential microbial symbiotes can be controlled to enhance chemotherapeutic efficacy. 02.02.4 Crystal structure of the engineered allosteric TEM-1 beta-lactamase fused to MBP reveals insights into the mechanism of its intended and non-intended allosteric regulation Wei Ke , Jing Liang , Marc Ostermeier , Focco van den Akker 1 1 2 2 1 2 Case Western Reserve University, Cleveland, OH, United States, Johns Hopkins University, Baltimore, Maryland, United States RG13 is a molecular switch created by recombining the genes coding for the Escherichia coli maltose binding protein (MBP) and the TEM-1 beta-lactamase (BLA). The beta-lactam hydrolysis activity of RG13 is positively regulated by the binding of maltose. In addition, RG13’s beta-lactam hydrolysis activity is switched off by zinc ion, which is serendipitously found to be a non-competitive inhibitor against RG13. To answer the question how the activity of TEM-1 beta-lactamase is heterotropically regulated by maltose and zinc ion, we determined the crystal structure of RG13 in complex with zinc. Our structure reveals that while the positions of catalytic S70, conserved active site residues Y105, S130, N132, E166 and N170 are relatively unchanged, the critical beta3-strand, one of the active site walls of Class A beta-lactamase, is completely stripped away from BLA active site and instead behaves as one of the loops connecting MBP domain and BLA domain. Consequently, new residues move close to the active site especially an Arg residue which sticks into the active site and makes hydrogen bonds with S130 and N132 and has the active site fully blocked. By careful examination of the position occupied by Zn ion, we conclude that this inhibition conformation is induced and stabilized by the zinc ion which is positioned near BLA active site and by residues from both MBP domain and BLA domain. Together with previous NMR insights of RG13 in complex with maltose, we propose that the maltose activation mechanism for RG13 entails that, in the absence of zinc, maltose binding causes a subtle lengthening of the linker region allowing the active site region to fully restore itself to render itself catalytically active. Molecular dynamics data will be presented to complement these crystallographic studies. 02.02.5 Resolution of pharmaceuticals via crystallization on chemically modified surfaces Pranoti Navare, John MacDonald Worcester Polytechnic Institute, Worcester, MA, United States Chirality is important in development of pharmaceuticals because more than half of commercial drugs are chiral, and the respective enantiomers often differ in their pharmacological and toxicological effects. The FDA requires that enantiomers be separated when racemic mixtures are produced during synthesis. Crystallization has been used to separate enantiomers in select cases where the two molecules resolve spontaneously, forming conglomerates. Unfortunately, crystallization energetically favors formation of racemic crystals for 95% of chiral compounds. We are investigating resolution of chiral drugs via crystallization on surfaces functionalized with chiral organic molecules as a means to bring about enantioseparation. Two goals of this work are to determine (1) whether chiral surfaces can act as templates that bias homochiral molecular aggregation at the surface, thereby inducing nucleation of conglomerates over racemic crystals, and (2) whether chiral templating can be used to control selective nucleation of one enantiomer leading to high enantiomeric excess. To demonstrate chiral discrimination on surfaces, we are investigating crystallization of the racemic 3-phenyllactic acid and N-acetylleucine on self-assembled monolayers (SAMs) of D- and L-cysteine and their derivatives. Important findings show that one enantiomer can be enriched over the other in up to 75% enantiomeric excess, and that the major enantiomer can be selected by switching the chirality of the SAM. We also observed oriented crystal growth and morphological changes indicating that diastereomeric interactions with chiral groups on the surface promotes both homochiral aggregation and face-selective nucleation of crystals of one enantiomer. 02.02.6 Evolution of Ligand Specificity in Steroid Hormone Nuclear Receptors Jennifer Colucci, Eric Ortlund Emory University, Atlanta, GA, United States Steroid hormone nuclear receptors (SRs) are ligand-regulated transcription factors that play critical roles in inflammation, development, and cancer progression. The SR family of proteins include the estrogen, androgen, mineralocorticoid, glucocorticoid, and progesterone receptors, which are activated by the endogenous hormones estrogen, testosterone, aldosterone, cortisol, and progesterone, respectively. Each member of the SR family is finely tuned to respond to its cognate hormone with high specificity. The SR family arose from a single evolutionary precursor that was activated by the 3-hydroxy steroid estrogen. Throughout a series of gene duplications, these proteins lost the ability to bind and be activated by 3-hydroxy steroids, and gained the ability to bind and be activated by 3-keto steroids. Currently, the molecular basis for hormone recognition is not well understood; this is illuminated by the fact that most pharmaceuticals targeting these receptors are crossreactive. In order to understand the molecular basis behind this functional switch, the ancestral precursor to the modern-day androgen, progesterone, mineralocorticoid, and glucocorticoid receptor, Ancestral Steroid Receptor 2 (AncSR2) was resurrected. This protein represents the first protein to have lost 3-hydroxy sensitivity and gained 3-keto specificity. We took a two-pronged approach to understanding ligand recognition and activation, using both biochemical and structural techniques. Using the crystal structure of AncSR2 in complex with progesterone, we made mutations to alter the specificity of the receptor from 3-keto steroids (progesterone) to 3hydroxy steroids (estrogen). We showed that with three amino acid mutations (Q353E, M384L, M387L) in the ligand binding pocket of AncSR2 that we can reverse steroid specificity from progesterone to estrogen, a feat that remained elusive using modern proteins. We then wanted to investigate the mechanism behind this functional switch. We hypothesized that the basis of the increased activation by estrogen in the Q353E M384L M387L mutant was due to the increased receptor-ligand complex stability. Chemical denaturation studies showed that the Q353E substitution conferred stability to the receptor. The M384L and M387L substitutions decreased stability, but increased specificity. Thus, in the forward evolutionary trajectory, this receptor gained specificity while losing stability. 02.02.7 Can Proper Selection of Data Reduction Program Optimize the Anomalous Signal for a Particular Set of Diffraction Images? James Swindell, John Rose, B.C. Wang University of Georgia, Athens Ga, United States To address the question as stated in the title, we have used two data sets from the same crystal collected at SER-CAT and processed with HKL2000, d*TREK, XDS, MOSFLM and PROTEUM2. We then compared the final results in terms of Rsym, I/σ I, heavy atoms position(s), anomalous signal contribution, and phase comparison. The use of the data processing programs and a comparison of results will be described and discussed. Work supported by SER-CAT Member Institutions, University of Georgia Research Foundation and Georgia Research Alliance. 02.02.8 Structural basis for nucleoside sugar discrimination in a thermostable DNA polymerase I Weina Wang, Eugene Wu, Lorena Beese Duke University, Durham, NC, United States DNA polymerases carry out accurate template-directed DNA synthesis by discriminating hundreds to thousands fold in favor of 2’-deoxyribonucleotide triphosphate (dNTP) compared to ribonucleotide triphosphate (rNTP). The two substrates differ only by the addition of 2’ hydroxyl group to the ribose sugar moiety, which makes it a larger substrate. Given the large number of nucleotides synthesized during genome replication and greater concentration of rNTP than dNTP in the cell, misincorporated ribonucleotides may compose the largest group of DNA lesions in the genome. Thus, it is critical to understand how DNA polymerases efficiently select the correct nucleoside sugar to incorporate. Site-directed mutagenesis and kinetic characterization have led to the identification of several residues involved in sugar discrimination. Here, we present five crystal structures of a high fidelity DNA polymerase I (pol I) large fragment from a thermostable strain of Bacillus stearothermophilus (Bacillus fragment, BF) complexed with DNA primer-template and dNTP, rNTP or ddNTP prior to chemistry. In addition, BF complexed with enzymatically incorporated rNTP or ddNTP into the DNA primer strand were captured after catalysis inside the crystals. The seven structures were determined at resolutions between 1.5 Å and 1.9 Å. Taken together, these structures increase our understanding of molecular mechanisms underlining sugar discrimination by DNA polymerase. 01.02.1 Structure and Mechanism of a Protein Disaggregating Machine Francis Tsai, Bernhard Sielaff, Jungsoon Lee, Sukyeong Lee Baylor College of Medicine, Houston, Texas, United States ClpB and Hsp104 are ring-forming AAA+ machines that recognize aggregated proteins as substrates, and remodel them in an ATP-dependent manner. The ability to disaggregate stress-damaged proteins is strictly dependent on the M-domain that is a hallmark of the ClpB/Hsp104 family. While the three-dimensional structures of ClpB and Hsp104 have been determined, the location of the M-domain is controversial and its exact function remains unclear. To provide an explanation for the observed structural differences, we determined the threedimensional structure of Hsp104 using a multi-pronged structural and biochemical approach. The mechanistic implication of our new structural insight will be discussed at this meeting. 01.02.2 Structural Genomics Approach to Macromolecular Assemblies and Motors Andrzej Joachimiak Argonne National Laboratory, Argonne, IL, United States Protein Structure Initiative (PSI) efforts are driven by genome sequence information and a focus on protein families that have no structural information available. Initially, at the Midwest Center for Structural Genomics (MCSG), as high-throughput methods were being adopted, refined and optimized, the majority of targets included single chains of small and medium size proteins. However, as technology matured and salvage pathways were implemented, MCSG targets included proteins that are considered “challenging,” e.g. larger, multi-meric and, more recently, multi-chain complexes as well as protein-NA complexes. Some of the selected protein families represent very distant sequence relatives of macromolecular assemblies and predicted macromolecular motors. These assemblies perform a variety of cellular roles, although some may have still poorly understood function. These novel structures provide new information and allow us to extract a common denominator, a function signature, or in some cases, an alternative solution for a particular functional requirement. These structures help to correlate function with structure and sequences, and generalize it for a set of proteins in a large family that share the same or similar function. This information can also help to transfer function from one protein to others, thereby, expanding functional coverage. The PSI is a discovery-based program that contributes to studies of the co-evolution of protein structure and function. It provides a wealth of ideas, concepts and understanding of mechanisms for the acquisition of novel biological function and the evolution of biological systems. Several examples will be provided, including tetrahedral protease, metal ion transporter, and a portal protein. This work was supported by NIH Grant GM074942 and by the U.S. DOE, OBER contract DEAC02-06CH11357. 01.02.3 Structure, mechanism, and regulation of a critical spliceosomal ATPase and helicase Brr2 Lingdi Zhang , Tao Xu , Corina Maeder , Laura-Oana Bud , James Shanks , Jay Nix , 2 3 1 Christine Guthrie , Jeffrey Pleiss , Rui Zhao 1 1 1 2 3 1 4 University of Colorado Denver, Aurora, CO, United States, University of California San 3 Francisco, San Francisco, CA, United States, Cornell University, Ithaca, NY, United States, 4 Lawrence Berkeley National Laboratory, Berkeley, CA, United States 2 Pre-mRNA splicing is an essential step in gene expression of all eukaryotes. Splicing of introns is carried out through two transesterification reactions catalyzed by the spliceosome, a huge RNA/protein complex composed of five snRNAs and over 100 protein factors. Structural and functional analyses are essential for our understanding of the molecular mechanism of pre-mRNA splicing. However, the structure and function of many spliceosomal components remain elusive. Among these are several DExD/H-box RNA helicases that play critical roles in the assembly and activation of the spliceosome. Brr2 is a large (>2,000 amino acids) and essential helicase in the spliceosome. It is responsible for U4/U6 unwinding, a critical step in spliceosomal activation. Brr2 has a unique domain structure, which contains an N-terminal domain (NTD, ~500 amino acids) and two tandem sets of a helicase domain followed by a Sec63 domain with unknown structure and function. Previous mutagenesis studies have shown that the first helicase domain is responsible for the ATPase and helicase activity of Brr2, but the function of all other domains is unclear. To understand the structure and function of this critical spliceosomal helicase, we recently determined the crystal structure of the second Sec63 domain, which unexpectedly resembles domains 4 and 5 of DNA helicase Hel308. In addition, the helicase domain upstream of Sec63 has clear sequence similarity with domains 1-3 of Hel308. We, therefore, hypothesize that Brr2 is composed of an N-terminal domain and two consecutive Hel308-like modules, providing our first glimpse of the overall structure of this unique spliceosomal ATPase and helicase. The structural similarity between Brr2 and Hel308 suggests a helicase mechanism for Brr2 that is consistent with our mutagenesis and biochemical studies. This mechanism is different from many DEAD-box RNA helicases and is likely responsible for Brr2’s unique ability to unwind the highly stable U4/U6 duplex. Furthermore, we demonstrated that the second Hel308 module of Brr2 interacts with Prp8 and Snu114 in vitro and in vivo, potentially serving as a mediator for the regulation of Brr2’s activity by Prp8 (an essential splicing factor known to stimulate Brr2’s helicase activity). This is the first example of a helicase-like module serving as a major protein-interacting domain. We further demonstrated that the C-terminal region of Prp8 (Prp8-CTR) facilitates the binding of the Brr2/Prp8-CTR complex to U4/U6, suggesting a potential role of Prp8-CTR as an auxiliary substrate binding and specificity domain for Brr2. In addition, we found that Brr2 NTD is required for yeast viability and have dissected the specific regions of NTD that are essential. We are currently investigating the specific function of this domain using a combination of genetic, biochemical, and structural approaches. Our results in general have important implications for the mechanism and regulation of Brr2’s activity. 01.02.4 Structure of a Membrane-associated AAA Machine Sukyeong Lee , Steffen Augustin , Takashi Tatsuta , Florian Gerdes , Thomas Langer , 1 Francis Tsai Baylor College of Medicine, Houston, Texas, United States, University of Cologne, Cologne, Germany AAA proteases are membrane-associated AAA machines that mediate the processing and turnover of soluble and membrane embedded proteins. We have determined the 12-Å resolution cryoEM structure of a detergent solubilized, full-length, hetero-oligomeric m-AAA protease hexamer, which reveals for the first time the structures of the twelve transmembrane spanning segments and six intermembrane space domains. Our fitted structure offers an explanation how m-AAA proteases dislocate and degrade membrane integral proteins, and provides the stereo-chemical framework for further biochemical and mechanistic studies. The structure and mechanism of m-AAA proteases will be discussed. 1 2 1 2 2 2 2 01.02.5 Crystal structure of the mammalian cytosolic chaperonin CCT in complex with tubulin at 5.5 Å resolution Ines G. Munoz , Hugo Yebenes , Min Zhou , Pablo Mesa , Marina Serna , Elisabeth 1 3 2 1 Bragado-Nilsson , Carol V. Robinson , Jose M. Valpuesta , Guillermo Montoya 1 1 2 3 1 2 Spanish National Cancer Research Centre (CNIO), Madrid, Spain, Centro Nacional de 3 Biotecnologia, Madrid, Spain, University of Cambridge, Cambridge, United Kingdom Protein folding in the cell is assisted by a large group of proteins termed molecular chaperones, one of the most important members being the chaperonins or Hsp60s (Heat Shock Proteins of 60 kDa). The eukaryotic cytosolic chaperonin CCT (chaperonin containing TCP-1, also know as TRiC) is the most complex of all chaperonins, an oligomeric structure built by two identical rings, each composed of single copies of eight different 60kDa subunits called α , β , γ , ζ , ε , δ , θ and η . This macromolecular complex of 1 MDa has crucial relevance in several essential biological processes, emerging as a key molecule due to its role in the folding of many important molecules including actin, α -, β - and γ -tubulins. Here we present the crystal structure of this protein machine in complex with tubulin. The structure of CCT trapping tubulin provides information about the molecular mechanism by which this macromolecular complex aids the tubulin folding process. The structure reveals the presence of one tubulin molecule in each CCT octamer showing the three-dimensional organisation of the different components in the presence of a substrate and providing new important insights into the function of this molecular chaperonin. 2 01.02.6 The structure of the phage T4 DNA packaging motor suggests a mechanism dependent on electrostatic forces Siyang Sun , Kiran Kondabagil , Bonnie Draper , Tanfis Alam , Valorie Bowman , Zhihong 2 2 1 1 2 Zhang , Shylaja Hegde , Andrei Fokine , Michael Rossmann , Venigalla Rao Purdue University, West Lafayette, IN, United States, The Catholic University of America, Washington, DC, United States Viral genomes are packaged into "procapsids" by powerful molecular motors. The crystal structure of the DNA packaging motor protein, gene product 17 (gp17), of bacteriophage T4 has now been determined. The structure consists of an N-terminal ATPase domain, which provides energy for compacting DNA, and a C-terminal nuclease domain, which terminates packaging. The structure showed that the function of the C-terminal domain also includes the translocation of the genome into the procapsid. The two domains are in close contact in the crystal structure, representing a "tensed state." A cryo-electron microscopy reconstruction of the T4 procapsid complexed with gp17 showed that the packaging motor is a pentamer and that the domains within each monomer are spatially separated, representing a "relaxed state." These structures suggested a mechanism, supported by mutational and other data, in which electrostatic forces drive the DNA packaging by alternating between tensed and relaxed states. Similar mechanisms may occur in other molecular motors. 1 2 1 2 2 2 1 01.03.1 The Use of Longer X-ray Wavelengths in Macromolecular Crystallography Manfred S. Weiss, B.-C. Wang 1 Helmholtz-Zentrum Berlin, Berlin, Germany, University of Georgia, Athens, United States 2 The use of longer X-ray wavelengths ( = 1.5-3.0 Å) in macromolecular crystallography has over the past few years almost become a routine tool for phase determination using the anomalous signal derived from the natively present sulfur and/or phosphorus atoms. Since the obtainable signal is very small, the experiment has to be conducted with great care. The challenges of the method are reviewed as well as some recent developments. Also, a survey about successful experiments carried out a beamlines around the world will be given. 01.03.2 Development of a beamline for low energy (4 keV) SAD experiments Soichi Wakatsuki, Naohiro Matsugaki, Yusuke Yamada, Leonard Chavas, Masato Kawasaki, Ryuichi Kato, Noriyuki Igarashi, Masahiko Hiraki Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), Tsukuba, Ibaraki, Japan SAD (Single-wavelength Anomalous Dispersion) phasing with sulfur or phosphor atoms is currently one of the most attractive methods to solve macromolecular crystal structures. The method is particularly important for a range of macromolecules for which heavy atom or selenomethionine derivative crystals are difficult to prepare. The Structure Biology Research Center at the Photon Factory, Tsukuba, Japan, is developing a new beamline dedicated to low energy (long wavelength) SAD experiments as part of the national project "Targeted Proteins Research Program (TPRP)". It is a nationwide structural biology effort which includes 35 target-oriented structural biology projects and 10 R&D projects in protein production, chemical library, structural analysis and bioinformatics. Each of the 35 target-oriented structural proteomics projects aims to solve structures of challenging targets in close collaboration with groups in cell biology, biochemistry, bioengineering, pharmacology, or medicine. Two synchrotron radiation facilities, SPring-8 and Photon Factory undertook to build two complementary micro-focus beamlines, and in collaboration with Hokkaido Univ., Osaka Univ., and Kyoto Univ. are developing techniques to facilitate user access and experiments at both synchrotron sites. At SPring-8 a micro-beam beamline, BL32XU, has been constructed to provide highly intense 1 micron by 1 micron beam while the Photon Factory has built a micro-focus beamline, BL1A, optimized for low energy SAD experiments. At PF BL1A beamline, an intense low energy beam at around 4 keV is provided using the first harmonic of an in-vacuum mini-gap undulator source to enhance anomalous signals from light atoms. A cryo freezing device designed by Isao Tanaka's group (Hokkaido Univ.) is available for capillary-top mounting of cryo-cooled crystals on site. The beamline optics consists of a cryo-cooled channel-cut monochromator and bimorph KB mirrors. The experimental hutch is equipped with a diffractometer specifically designed to minimize the attenuation of the lower energy beam and scattering by mother liquor and the air around the crystal. The beamline has been commissioned in spring 2010, and the user operation due to start in May. Initial results will be reported from small crystals and de novo structure determination using the 4 keV X-ray beam. 01.03.3 Solving structures with in-house long wavelength radiation Cheng Yang, Jim W. Pflugrath, Joseph Ferrara Rigaku American Corporation, Woodland, Texas, United States Recent advances in technologies for phasing with in-house long wavelength radiation has made it straightforward to solve protein structures by utilizing the intrinsic sulfur atoms and/or added selenium atoms in those proteins. In-house chromium Kα radiation (2.29Å) currently is the longest wavelength X-ray radiation which can be routinely used for data collection. This wavelength is capable of doubling the anomalous signal of many intrinsic elements or oftused heavy atoms for derivatization compared to Cu radiation. For example, the contribution to the anomalous terms of sulfur and selenium atom doubles to 1.14 and 2.28, electron equivalents, respectively. This report relates successful examples of protein structures solved by using chromium sulfur or selenium-SAD phasing. Some of these proteins were either difficult to synthesize in selenomethionine-substituted form or to produce more crystals. Furthermore, anomalous scattering collected with an in-house Cr X-ray radiation source can be used to determine protein bound ion positions, verify ligand orientation and help structure tracing. A properly collected Cr data set can also be used refine the protein structure. These results demonstrate that Cr radiation has become a routine phasing approach in the crystallographer’ s toolkit. Cr Kα makes it possible to solve a crystal structure with native crystals only or before synchrotron data collection when selenomethionine-substituted protein is available. This method improves the productivity of macromolecular structure determination, usage of the synchrotron beam time, and high throughput structure determination. 01.03.4 Crystallographic structure determination of an iron uptake regulatory protein (FeoA) by sulfur SAD in C2 space group Joseph Ng , Ronny Hughes , Yang Li , Zhi-Jie Liu , Bi-Cheng Wang 1 1 1 2 2 3 2 University of Alabama, Huntsville, Huntsville, AL, United States, Institue of Biophysics, 3 Beijing, China, University of Georgia, Athens, Athens, Georgia, United States The recombinant iron uptake regulatory protein (FeoA) derived from Thermococcus thioreducens was overexpressed in E. coli, purified and crystallized in the C2 monoclinic space group with unit cell parameters of a=93.8Å, b=68.4Å, c=68.97Å and β =132.67o. The asymmetric unit contained 4 molecules in which each monomer contained 6 sulfurs all contributed by methionines within 76 amino acids. The overall number of sulfur atoms within the asymmetric unit was 24. In the interest of obtaining phases by Sulfur Single-Wavelength Anomalous Diffraction (SAD), synchrotron X-ray data collection was performed at the SERCAT ID22 (Argonne National Lab, Chicago, IL). A 360-frame data set was taken using 1.9Å wavelength X-ray radiation with each frame consisting of a 1º wedge of data using a MAR300 CCD detector. Reflections extending out to 2.0 Å were recorded with an overall data redundancy of 22.5. The positions of 24 sulfur anomalous scattering atoms were identified and consequently phases were determined and refined. The final refined structure of FeOA was determined to 2.0Å with Rwork and Rfree to be 17.6 and 22.8 respectively. We show the anomalous signal of the sulfurs in FeOA crystals can be detected and used for phasing with sufficient sulfur content in monoclinic space group. 01.03.5 Getting the most out of weak phase information George Sheldrick University of Goettingen, Goettingen, Germany Longer wavelengths can achieve an increase in the ratio of anomalous to normal diffraction but some systematic errors are also enhanced at longer wavelength so careful data scaling is required and the attainable resolution is reduced. One approach to combat these problems is to combine the long wavelength data with data collected to higher resolution at a short wavelength, if possible from the same crystal. This also helps to reduce problems with the 1,2 location of heavy atoms using the program SHELXD when the low angle data are incomplete or contain errors. In the case of SAD phasing using naturally occurring sulphur 3 atoms or the iodine atoms in a partially occupied sticky magic triangle as anomalous scatterers, the known geometry of the disulfide units or the equilateral triangle of iodine atoms 4 should be useful in enhancing the heavy atom search. When the phase information derived from the heavy atoms is very weak, iterative density modification and tracing of the peptide 2,5 backbone with the program SHELXE is proving particularly effective at bootstrapping onto an interpretable structure; in borderline cases this procedure often requires several iterations before it locks in and traces most of the peptide backbone. 1. Schneider, T. R. & Sheldrick, G. M. (2002). Acta Cryst. D58, 1772-1779. 2. Sheldrick, G. M. (2008). Acta Cryst. A64, 112-122. 3. Beck, T., da Cunha, C. E. & Sheldrick, G. M. (2009). Acta Cryst. F65, 1068-1070. 4. Debreczeni, J. É., Girmann, B., Zeeck, A., Krätzner, R. & Sheldrick, G. M. (2003). Acta Cryst. D59, 2125-2132. 5. Sheldrick, G. M. (2010). Acta Cryst. D66, 479-485. 01.03.6 In-House SAD: experiences and structures. Elspeth Garman, Edward Lowe University of Oxford, Oxford, United Kingdom Over the last four years we have used our in house copper high brilliance X-ray source (Bruker Microstar generator) in conjunction with a kappa goniometer stage and a SMART 6000 CCD detector to great advantage for SAD phasing. Key structure solution determinations have been carried out using this method, utilising the high speed and sensitivity of the detector. These include the determination of a number of novel structures and complexes, such as domains CBEGF9-HYB2-CBEGF10 of fibrillin-1 in a calcium saturated form using a combination of molecular replacement and SAD methods which made use of anomalous signal from bound Ca ions and intrinsic sulphurs [1]. Other SAD structures solved have been the F1 F1 type 1 domains of human fibronectin (S-SAD) [2], the sulphur oxidising protein, SoxB (SAD from a bound Mn ion) [3], human HIF prolyl-hydroxylase, PHD2, in complex with an iodinated inhibitor (I-SAD) [4], the vonWillebrand factor (vWF) domain of the proteasomal component Pus1 (S-SAD) [5] and human PHYHD1 protein (SAD using iron and iodine derivatives). Systematic experiments have been carried out to optimise our current S-SAD data collection strategies, using crystals of insulin, lysozyme and glucose isomerase as test systems. In the case of glucose isomerase (GI) this involved phasing the structure of the 388 residue protein using the signal from only 8 sulphur atoms. The absence of manganese in the EDTA treated GI crystal was confirmed by microPIXE (proton induced X-ray emission) [6]. The results of these experiments and current protocols will be discussed. References: [1] Jensen, SA et al (2009) Structure, 17, 759-768 [2] Erat, M.C. et al (2009) PNAS, 106, 4195-4200 [3] Sauvé, V et al (2009) J Biol Chem, 284, 21707-21718 [4] McDonough, MA et al (2006) PNAS, 103, 9814-9819 [5] Riedinger, C. et al (in press) [6] Garman, EF & Grime, GW Progress in Biophysics and Molecular Biology (2005) 89/2, 173-205. 8 9 01.03.7 The upgrade programme of the ESRF and a new structure. daniele de sanctis, Christoph MUELLER-DIECKMANN ESRF, Grenoble, France Structural Biologists are tackling ever more ambitious projects, for example more complex membrane proteins and larger macromolecular assemblies. Such systems often show considerable inter- and intra- crystal variation in diffraction quality. Sample evaluation prior to data collection, already widespread in Macromolecular Crystallography (MX), will thus become more crucial as will data collection facilities optimised for the collection of diffraction data at long wavelengths or from crystals that are very small and/or diffract to low resolution (i.e. dmin > 5 Å). As part of the Upgrade Programme of the European Synchrotron Radiation Facility (ESRF; http://www.esrf.fr/AboutUs/Upgrade) its Structural Biology Group will develop a unique resource, based on 2nd generation automation, designed to maximise the chances of a successful conclusion to MX experiments. The hub of this resource will be a sample evaluation and sorting facility, MASSIF. The most suitable crystals from which to collect data will be distributed from this hub to upgraded MX data collection facilities that also form an integral part of the ESRF’s upgrade plans. The ESRF’s Structural Biology Group’s upgrade plans will be presented, with particular emphasis on a refurbished ID29 which will be optimised to enable data collection from crystals of macromolecules using X-rays of lower energies (E = 5 keV, = 2.5 Å). Recent successes in the solution of a macromolecular crystal structure exploiting only the small anomalous signal from sulphur atoms innate to protein amino acid sequences will be also be described. 01.03.8 The MDS Approach: A Data Collection Strategy for Routine Sulfur Phasing and Other Applications that Require High-Quality and Higher Resolution Data B.C. Wang , Z.-J. Liu , L. Chen , W. Zhou , H. Xu , H. Zhang , J.T. Swindell II , J.P. Rose , 1 1 1 2 G. Rosenbaum , Z.-Q. Fu , J. Chrzas , M. Benning 1 1 1 1 1 1 1 1 1 University of Georgia, Athens, Georgia, United States, Bruker AXS, Madison, Wisconsin, United States A fundamental question facing the crystallographer is "For a given X-ray dose, what is the best strategy for collecting a data set from a crystal that will increase structure solvability?" A common answer to this question is to increase the exposure time for each diffraction image, so that we may better “visualize” the recorded reflections to get better data. 2 Interestingly, we have found that a significantly better data set may be produced for a given crystal and X-ray dose by using shorter exposures (i.e. reduced X-ray dose) and collecting the complete data set multiple times such that the total X-ray dose the crystal receives remains the same. This is the MDS (Multiple-Data-Set) approach. In a recent case, a total of 8,600 images were collected using the MDS approach, from a single protein crystal with an estimated (visual inspection) diffraction limit of around 2.5Å, using a copper home X-ray source. The resulting merged and scaled data set gave good statistics to better than 2.0Å resolution. Thus, this unlikely candidate crystal for sulfur phasing has now produced a S-SAD structure from its MDS data collected with a highly sensitive detector. Both the theoretical and practical aspects of the MDS method as well as the future perspectives of the MDS approach will be discussed Work supported by NIH, SER-CAT, University of Georgia Research Foundation, Georgia Research Alliance, and Bruker AXS * Current Address: Institute of Biophycs, Chinese Academy of Sciences, Beijing, China † Current Address: The College of Life Sciences, Nankai University, Tianjin, China. 02.03.1 Larry R. Falvello University of Zaragoza - C.S.I.C., Zaragoza, Spain Methods for obtaining undistorted views of the reciprocal lattice are described. The principle of de Jong and Bouman for diffraction photography is presented along with a brief description of a camera based on it. The Buerger precession method is described, including the role of the de Jong - Bouman principle in the design of the "Mach 2" precession camera. The use of undistorted reciprocal lattice views has been facilitated in recent years by the routine availability of extensive digital data from contemporary diffractometers, together with the advent of rapid pixel harvesting and layer reconstruction techniques. These are summarized, and applications of these reciprocal lattice views to topical crystallographic problems are also presented. 02.03.2 When the Structure is Difficult Look at the Whole Diffraction Pattern Carolyn Brock Department of Chemistry, University of Kentucky, Lexington, KY, United States Synthetic “precession photos”, i.e., reciprocal-lattice (or, RL) slices, can be calculated easily from the pixel values in the many frames typically collected with a CCD diffractometer. A quick look at these images, which show the non-Bragg as well as the Bragg scattering, often reveals the reason for a problem refinement. If a second set of Bragg reflections is observed then the crystal is not single. Structured diffuse scattering between the Bragg peaks indicates short- or medium-range order. If the Bragg peaks are very elongated then the crystal either had a large mosaic spread or moved during data collection. If the Bragg peaks seem a little large and seem to have surprisingly similar intensities then there is reason to suspect pseudo-merohedral twinning. If large numbers of Bragg peaks are very weak the structure may be modulated. RL slices for a number of problem structures from our lab will be shown and discussed. 02.03.3 Electron-density maps. What insight can we gain from them? Jenny Glusker Fox Chase Cancer Center, Philadelphia, United States Electron-density maps result from attempts to represent the material in a crystal that has scattered X rays and given a diffraction pattern. In a similar way, neutron diffraction gives a picture of nuclear density. However, since the first diffraction patterns were obtained in 1912, it has been clear that experimentally measured intensities of diffracted beams will not give all the information necessary for an electron-density map. The relative phases of the diffracted beams have been lost because an appropriate X-ray lens is not currently available. This is the “phase problem,” the most important part of a course in X ray crystallography. It is solved in a variety of ways to give a picture of the scattering matter. Analyses of the preliminary maps obtained, however, need care and attention to detail. Methods for finding good atomic coordinates and identifying missing atoms, or movements or disorder of atoms, will be described. Finally omit maps, deformation-density maps, nuclear-density maps and anomalous dispersion effects will be discussed. The primary experimental data are the intensities, directions, and orders of diffraction of the diffracted beams. Their relationship to the required electron-density or nuclear-density maps is the subject of this talk. I thank the National Institutes of Health (CA-10925 and CA-06927) for support through the years. 02.03.4 Topotaxy HOWTO: Following Two-Phase Solid-State Reactions in the 1970s and Today with a Modern Diffractometer Aaron R. Gell, Shai R. Posner, Chun-Hsing Chen, Bruce M. Foxman Brandeis University, Waltham, MA, United States Topotactic reactions are single crystal-to-single crystal reactions (SCSCRs) where the daughter phase(s) is(are) aligned in an explicit three-dimensional fashion with respect to the 1 mother phase. The largest class of SCSCRs are class T1, where the space group of the original unit cell does not change, and the lattice parameters change smoothly over time. The presentation will review a complex T1 example, followed by a number of T2 (two-phase) cases. In the 1970’s, topotaxy was established using film techniques; today, the elegant 2 approach of Gougoutas and coworkers has been neglected, and it is time for a return to an era where the alignment of mother and daughter crystals is firmly established in every experiment. IT’S EASY! Using a modern diffractometer, a single crystal reaction may be followed, and the alignment established in a relatively simple fashion: truly, only a few 3 minutes’ work. Examples from the 1970s will be compared with modern-day measurements, 4 and protocols for the future set out. Examples will be taken from the ancient and new work on the topotactic transformations of coordination compounds as well as polymorphic phase transformations. 1. Lotgering, F. K. J. Inorg. Nucl. Chem. 1959, 9, 113-23; Dent Glasser, L. S.; Glasser, F. P.; Taylor, H. F. W. Quart. Rev. 1962, 16, 343-60; Shannon, R. D.; Rossi, R. C. Nature 1964, 202, 1000-1001. 2. Gougoutas, J. Z. Isr. J. Chem. 1972, 10, 395-407. 3. http://www.nonius.com/cad4/manuals/user/chapter08.html Cheng, K.; Foxman, B. M. J. Am. Chem. Soc. 1977, 99, 8102-8103. 02.03.5 Structure Analysis. Why do we always ..........? David Watkin Oxford University, Oxford, United Kingdom In the last decade X-ray Crystal Structure Analysis has become increasingly automated, with a growing number of crystallographic decisions being made automatically by the software. In addition, a number of crystallographic Cook Books have been published which give instructions for how to deal with more complicated tasks. Taken together, these aids enable chemists with quite modest training to undertake routine analyses. While this broadening of the crystallographic community is almost certainly a good thing (because it makes X-ray crystallography more accessible), it carries the risk of misuse of the technique, and potentially the failure of analyses which, with a little more experience, could have been resolved. The very high levels of automation now available reduce the exposure of novices to the actual processes of structure analysis. This leaves them ill-equipped to deal with unusual situations. This talk will aim to explain why we always do a selection of things, such as aim at high completeness, aim at high redundancy, find a fancy weighting scheme for the refinement etc. Once one knows why these things are done, one is in a position to judge when things might be done differently, when the "rules" can be broken. A "hand-waving" (as opposed to a deep mathematical) understanding of the physical background to structure analysis enables one to carry out analyses more effectively. 02.03.6 The Structure of Fe3(CO)12 Revisited Charles Campana , Ilia Guzei , Evgueni Mednikov , Lawrence Dahl 1 2 1 2 2 2 Bruker AXS Inc., Madison, Wisconsin, United States, Department of Chemistry, University of Wisconsin - Madison, Madison, Wisconsin, United States Triiron dodecacarbonyl was one of the first metal carbonyl clusters synthesized. The molecular structure of the Fe3(CO)12 molecule has been a subject of interest since the 1950’s. Dahl and Rundle proposed that Fe3(CO)12 consists of a triangle of iron atoms surrounded by 12 CO ligands. Ten of the CO ligands are terminal and two span an Fe---Fe edge. By contrast, Ru3(CO)12 and Os3(CO)12 adopt D3h-symmetric structures, wherein all 12 CO ligands are terminally bound to the metals. However, the elucidation of the detailed structure of Fe3(CO)12 has proved to be a challenging crystallographic problem. The original crystal structure by Wei and Dahl was done at room temperature, using data collected on Weisenberg and precession cameras with visual estimation of intensities. Subsequent datasets were collected with scintillation detectors on Syntex P-1 and Nonius CAD-4 diffractometers. We report on the latest datasets collected with a Mo I S micro-focus source and a two-dimensional CCD detector at 100K. As new instrumentation and software have become available, it is now possible to re-examine this old structure to obtain a much more precise structure. The details of the modelling of this disordered structure will be discussed. 03.01.1 New techniques in fiber diffraction illuminate the supramolecular organization of the mammalian Extracelluar Matrix Joseph Orgel , Olga Antipova , Irit 1 2 Kalyanasundaram , James San Antonio 1 1,3 1 Sagi , 3 Yujia Xu , 2 4 Sandra Bishnoi , 1 Aruna Illinois Institute of Technology, Chicago, IL, United States, Orthovita, Inc, Malvern, PA, 3 4 United States, Weizmann Institute of Science, Rehovot, Israel, Hunter College, New York, NY, United States The fibrous collagens are the fundamental constituents of the Extracellular Matrix (ECM) of animals, forming the structural basis of all known mammalian connective tissues and organ systems (1). Yet, despite the fundamental biological importance of collagen, many of us are perplexed by the complexity of the assemblies that the collagens form. This is particularly true at what may be the most significant aspect of collagen structure from a cellular point of view, at the intermediate sub-fibrillar and at fibril surface levels (i.e. collagens molecular packing) where many important biological processes occur in growth, development and disease (1, 2). These include but are not limited to: fibrillogenesis, tissue remolding and in forming the scaffolding upon which organ systems, bones, cartilage, etc., i.e. the animal body, are built upon. Clearly, obtaining an unambiguous and contextualized visualization of collagen molecules would be of significant value to the scientific community. We have recently determined the structure of the type I collagen microfibril (3) and fibril (2) at the molecular level from whole intact rat-tail tendons and produced an initial one (and now two) dimensional structure for type II collagen from lamprey notochord (4). Using these data, it is possible to map the amino acid chemistry, ligand binding data and other observations onto the defining shape modality of the fibrillar collagen ECM. In so doing, we have been able to propose the first fibrillar based mechanism of collagenolysis and provide a number of illuminating observations regarding other collagen fibril - ligand interactions involving cell adhesion, hemostasis and matrix organization (1-5). Here we expand on these observations using new technical developments in fiber diffraction and complimentary methods such as TEM and AFM. 1. Sweeney S et al. (2008. J Biol Chem 283:21187-97; 2. Perumal S, Antipova O, Orgel J (2008). Proc Natl Acad Sci U S A 105:2824-9; 3. Orgel J et al., (2006) Proc Natl Acad Sci U S A 103:9001-5; 4. Antipova, A., and Orgel, J.P.R.O. (2010) In Press, Journal of Biological Chemistry; 5. Joseph P.R.O. Orgel et al., (2009) Public Library of Science ONE. 4(9), e7028. 03.01.2 Using Micro- and Nano-beams for Scanning Diffraction Experiments on Fibrous Materials Manfred Burghammer, Sebastian Schoeder, Richard Davies, Aurelien Gourrier, Christian Riekel European Synchrotron Radiation Facility, Grenoble, France X-ray diffraction on single fibres, instead of multi-fibre assemblies, has been enabled more than a decade ago when highly focused X-rays became available at third generation synchrotron radiation sources. Today the structure of fibrous materials can be probed with unprecedented spatial resolution using x-ray beams in the range of one micron down to a few hundred nanometres. Evidently, there is a high interest to combine such measurements with in situ sample environments e.g. allowing to control temperature, humidity, and mechanical deformation. However, in order to benefit from these possibilities challenging problems like mechanical stability issues, high throughput data analysis, and, above all, radiation damage issues have to be addressed. The Instrumentation dedicated to high resolution scanning diffraction experiments at the ESRF Microfocus Beamline (ID13) will be presented. Examples from the fields of life science and polymer research will be discussed. 03.01.3 The Molecular Basis for Stretch Activation in Insect Flight Muscle Thomas Irving , RJ Edwards , Michael Reedy 1 1 2 1 2 Illinois Institute of Technology, Chicago, IL, United States, Duke University Medical School, Durham NC, United States Deciphering the molecular mechanism of muscle contraction and its regulation using smallangle fiber diffraction has been a driving force for the development of x-ray technology since the beginning of this field in the 1950’s. In fact, the very first diffraction experiment using synchrotron radiation (on anything) was done on the indirect flight muscle (IFM) of the giant water bug Lethocerus indicus nearly 40 years ago in Hamburg by Rosenbaum, Witz and Holmes. This muscle system is an attractive model system because of its high degree of crystallinity which allows more detailed structural analysis than muscles from mammals. When slightly calcium-activated glycerinated Lethocerus insect flight muscle (IFM) can be 2+ mechanically stretch-activated at constant [Ca ] to give a delayed active rise to peak force, a response typical of asynchronous IFM that allows efficient flight. The structural mechanism underlying stretch activation has long been elusive. We oscillated demembranated muscle at 2 HZ, and collected continuous 8ms time frame movies of x-ray fiber patterns, using a Pilatus 100K pixel array detector, reflecting structural changes within the muscle as it performs oscillatory work, much as it would in the living insect. The results showed that stretch induces movement of tropomyosin alternatively allowing attachment of crossbridges with every wing beat. This movement appears to ce due to persistent mechanical linkages between the thick and the thin filaments at the level of the troponins on the thin filament. In addition, we found clear evidence for twisting and untwisting of the myosin containing thick filaments as the muscle is stretched and released. During stretch, this would result in placing myosin heads closer to their “target zones”, i.e. stereospecific binding sites on the actin-containing thin filament. This, combined with stretch induced recruitment of myosin heads may finally provide an explanation for stretch activation in these muscles. Since stretch activation is also a feature of human cardiac muscle, this may have relevance to understanding this feature of cardiac function. Supported by NIH 5R37AR014317 and RR08630. 03.01.4 Fibre diffraction flexes its muscles. Carlo Knupp Cardiff University, UK, United Kingdom Muscle sarcomeres, the repeating structural units in muscle, contain two sets of fibrous proteins namely actin and myosin filaments. In the sarcomere these filaments run parallel to each other. The interaction of the actin filaments with globular domains projecting from the myosin filament backbone (myosin heads) makes the two sets of filaments slide past each other. This process ultimately result in muscle contraction, but the exact details of how force and movement are produced at the molecular level in the sarcomere are still unknown. X-ray fibre diffraction offers the possibility of probing the molecular events involved in muscle contraction by recording and analysing time-resolved diffraction patterns from live contracting muscles. Here we will review some of the computational techniques that can be used to understand the changes in the diffraction patterns (including interference effects arising between adjacent myosin head arrays) and to reconstruct, at different times within the contracting cycle, the overall arrangement of interacting myosin and actin filaments in the sarcomere. ORMOPMT d¢¢¡¦ ? ›¢? f·\‹ g¡ ¦¡ ? ‹ ·«? h›‹ ? ›‹? ⁄¡? s⁄‒¡¡L «¡‹ ›‹\ ? n‒ ¡‒ ‹£? ›¢? h› \L? \‹ ? j\fifi\Lb\‒‒\£¡¡‹\‹ ¨¨¨ ¨ ¨¨ ¨¨¨ §¦¢¥£¢ ¢¤ £¢¡ v⁄ ¡‒? b¡‹ ¡‒? ¢›‒? b\‒ ›⁄„ ‒\ ¡? q¡ ¡\‒¦⁄K? c¡fi\‒ «¡‹ ? ›¢? e›› ? r¦ ¡‹¦¡K? o·‒ ·¡? t‹ ¡‒ „K? v¡ k\¢\„¡ ¡K?hmK?t‹ ¡ ?r \ ¡ Carrageenans represent a special class of biopolymers extracted from marine algae and are utilized in food applications as thickeners, gelling agents, syneresis inhibitors and binders, to name a few. They are recognized as GRAS materials and FDA approved the food usage. Their anti-coagulant, anti-therapeutic, anti-tumor and anti-HIV activities resulted in the pharmaceutical utilization as well. Further, their regular intake in human diet is proven to reduce blood cholesterol and lipid levels. These hydrocolloids are made up of a disaccharide galactan backbone with variable amounts of sulfation at different hydroxyl positions. Depending on their source of extraction, presence or absence of a sulfate ester as well as anhydro group fifteen carrageenans are known to date. However, only kappa-, iota- and lambda-carrageenans have so far been exploited industrially and are subjected to extensive x-ray and rheological studies for understanding their structure-function relationships. Insights about structural organization and solution properties are very much needed for delineating their functional behavior, especially in the presence of other biopolymers. In this set, iota-carrageenan is well characterized with precise atomic details and insights about the pertinent roles of metal ions in stabilizing packing structure. On the other hand, no such detailed information exists for kappa-carrageenan. Among the two known hydrogen bond disrupters urea and guanidine hydrochloride (GH), the later is reported to enhance gelation in kappa-carrageenan. Our recent fiber diffraction results on guanidinium salt of iotaand kappa-carrageenan yield interesting observations. The pattern of iota-carrageenan, having one sulfate group on each sugar residue of its disaccharide repeat, contains three well defined layer lines within 4.3 Å resolution. The polymer prefers a three fold, parallel, halfstaggered double helical structure. The stability of the helix is gained through two interchain O-6H O-2 and O-2H O-5 hydrogen bonds, and the peripheral sulfate groups promote interhelix association via cations and ordered water molecules. In contrast, kappacarrageenan, with only one sulfate group per disaccharide repeat, diffracts to generate six layers lines within the same resolution period, suggesting a novel molecular structure. Analysis reveals that non-half-staggered as well as anti-parallel double helical arrangements are viable options. In the second case, the two chains are held together by three strong interchain O-6H O-6, O-2H O-2 and O-2H O-2 hydrogen bonds two more than the former indicating the preferred molecular structure for kappa-carrageenan might be an anti-parallel double helix. Although both of them have similar backbones, absence of a sulfate group on alternate residues seems to bestow additional freedom and a novel molecular assembly to kappa-carrageen chains. These results unequivocally attest to the observed differences in solution behavior between the two carrageenans. ¨¨ ¨ ¨¨¨ 03.01.6 Structural Insights into the Interactions of Amines with Cellulose B. Leif Hanson , Yoshiharu Nishiyama , Masahisa Wada , Paul Langan 1 2 1 2 3 4,1 University of Toledo, Toledo, OH, United States, Joseph Fourier University of Grenoble, 3 4 Grenoble, France, University of Tokyo, Tokyo, Japan, Los Alamos National Laboratory, Los Alamos, NM, United States We have studied structural changes during the treatment of highly crystalline microfibers of Cladophora cellulose with ethylenediamine (EDA) using time-resolved X-ray microprobe diffraction methods. EDA molecules penetrate the cellulose crystals converting the naturally occurring crystalline cellulose I phase to a crystalline complex of cellulose with EDA, called EDA-cellulose I. The (200) direction of cellulose I is resistant to EDA penetration, with EDA penetrating most effectively at the hydrophilic edges of the hydrogen bonded sheets of cellulose chains. Most of the cellulose chains in the initial crystals of cellulose I are incorporated into crystals of EDA-cellulose I, and there is no evidence of any gradual structural transition from cellulose I to EDA-cellulose I involving a continuously changing intermediate phase. Instead a rapid transition to EDA-cellulose I occurs in regions of the microfibrils that have been penetrated by EDA. The size of the emerging EDA-cellulose I crystals are limited to about half the size of the cellulose I crystals, due to strains introduced by the penetrating EDA molecules. 03.01.7 Fiber diffraction, auto-florescence, and Raman microscopy studies of the timedependent solubilisation of lignocellulosic biomass with ionic liquids. Paul Langan , Kirk Rector , Marcel Lucas , Greg Wagner , Indira Samayam , Lefi Hanson , 1 3,1 4 Indira Samayam , Yoshiharu Nishiyama , Masahida Wada University of Toledo, Toledo, OH, United States, Los Alamos National Laboratory, Los 3 4 Alamos, NM, United States, CNRS, Grenoble, France, University of Tokyo, Tokyo, Japan Lignocellulosic biomass, the fibrous material derived from plant cell walls, is a potential clean and renewable, non-food feedstock for future biorefineries. The components of lignocellulosic biomass can serve as a source of carbon based feedstock for fuel and chemical production in much the same way as crude oil serves as the carbon feedstock in petrochemical refineries. In particular, the sugars derived from the cellulosic and hemicellulosic portions of biomass can be converted to biofuels or other value added products using current technologies. The deconstruction of lignocellulosic biomass into simple sugars constitutes a core barrier for producing products from the sugar platform. Cellulose, a fibrous material biogenerated from the linear polymer poly(1-4) b-D glucan, is difficult to break down into glucose monomers because of its crystalline nature. Furthermore, cellulose fibers are encrusted in the branched heterogeneous polymers of hemicellulose and lignin. Lignin adds structural rigidity to plant cell walls, but it also protects the cellulosic component from hydrolyzing enzymes that can release glucose. Recently, Ionic Liquids (ILs) have been investigated by several groups as a promising new approach for the pretreatment of lignocellulosic biomass so that its complex architecture can be disrupted, thereby making it accessible to water and enzymes for an accelerated conversion to glucose. We have shown that pretreatment with ILs not only disrupts the plant cell wall and separates its cellulosic, hemicellulosic, and lignin components, but it also disrupts the crystallinity of cellulose making it more rapidly digestible by hydrolyzing enzymes. Some advantages of this approach are that it is non-derivatizing, that it does not produce fermentation inhibitors, and that it is amenable for "easy recovery" of the ILs employed in the pretreatment. However, further optimization of the use of ILs as a pretreatment will require a detailed chemical-level understanding of the composition and structure of lignocellulosic biomass and the time dependant impact of pretreatment. Because of the compositional and structural complexity of the cell wall and its interaction with ILs, no one experimental technique can provide this understanding. Therefore we have taken the approach of combining several experimental techniques including fiber diffraction, autoflourescence and Raman microscopy to provide a program that can be used to characterize the intact plant cell wall at multiple length scales. The experimental techniques were chosen not only for the complementarity of the information that they provide, but also because they are non-invasive and non-destructive and therefore avoid interference from traditional staining, embedding, and processing chemicals that can sometimes alter the material under study. We will report on the application of our experimental program to investigate the time dependent solubilization of lignocellulosic biomass from poplar trees by the IL 1-n-ethyl-3methylimidazolium acetate. 1 2 2,1 2 2 2 1 1 07.13.1 The Magic of Spin Ice and Emergence of Magnetic Monopoles Jeffrey Lynn National Institute of Standards and Technology, Gaithersburg, Md 20899, United States Cubic pyrochlores such as Dy2Ti2O7 have a structure where the Dy sites form a cornersharing tetrahedral network, which is the prototype geometry to frustrate the magnetic interactions that can lead to novel magnetic ground states that are fundamentally different than conventional long range magnetic order. For this material the magnetic anisotropy of the 3+ Dy spins requires them to point along a local <111> axis, such that on each tetrahedron the moment can only point into the center, or in the opposite direction. The ground state then turns out to be where two spins on each tetrahedron point inward, and two point outward. But you don’t know which two are in and which two are out, giving rise to six equivalent configurations for any particular tetrahedron, and a macroscopic degeneracy and finite entropy for the ground state. This “two-in two-out” description of the spin system is identical to the proton configurational disorder for hexagonal ice discussed by Pauling [1], inspiring the name “spin ice” [2]. Recently it was realized that the magnetic excitations of spin ice can then be described as magnetic monopoles [3], and such excitations have now been observed experimentally [4]. We will describe the nature of magnetic frustration, spin-ice, and how magnetic monopoles emerge from the ground state of this very special magnetic material. [1] L. Pauling, J. Am. Chem. Soc. 57, 2680 (1935). [2] J. S. Gardner, M. J. P. Gingras, and J. E. Greedan, Rev. Mod. Phys. 82, 53 (2010). [3] C. Castelnovo, R. Moessner, & S. L. Sondhi, Nature 451, 42 (2008). [4] H. Kadowaki, N. Doi, Y. Aoki, Y. Tabata, T.J. Sato, J. W. Lynn, K. Matsuhira, and Z. Hiroi, J. Phys. Soc. Japan 78, 103706 (2009). 07.13.2 Two ACuCl3(H2O) Compounds Containing Unusual Aggregation of the Copper(II) Complexes. Marcus Bond Southeast Missouri State University, Cape Girardeau, MOI, United States The structures of (N,N-dimethylpiperidinium)CuCl3(H2O (I)):monoclinic P21/c, a = 10.1958(3) Å, b = 7.6080(3) Å, c = 16.3281(5) Å, β = 102.927(2)° V = 1234.46(7) Å 3, Z=4; and of , (1,2,3-trimethylpyridinium)CuCl3(H2O) (II): monoclinic C2/m, a = 15.6948(6) Å, b = 9.2431(4) Å, c = 8.5103(3) Å, β = 103.488(2)°, V = 1200.53(8) Å 3, Z = 4; are reported. The structures consist of distorted tetrahedral (CuCl3(H2O) complexes separated by organic cations. The distorted geometry of the complexes precludes the stacking found for similar compounds with quasi-planar complexes, in which copper(II) ions of one complex complete ... their coordination spheres by forming long, semi-coordinate Cu Cl bonds to neighboring complexes. In these compounds these complexes aggregate in more unusual ways. In (I) the water molecule of a complex forms hydrogen bonds to chloride ions on neighboring complexes to organize the complexes in stacks in which the water molecules are sequestered into parallel channels within the crystal. In (II) the complexes form pairs in which Cu...Cl semi-coordinate bond are shared to form weakly bound dicopper complexes which then aggregate into sheets via O-H...Cl hydrogen bonding. 07.13.3 Unexpected and Incomplete Super-Triangles and Rectangles: When Designed SelfAssembly Does Not Go According to Plan. Louise Dawe , Konstantin Shuvaev , Santokh Tandon , Laurence Thompson 1 2 1 1 2 1 Memorial University, St. John's, Newfoundland, Canada, Kent State University at Salem, Salem, Ohio, United States The targeted synthesis of [4x4]M16 grids involves designing polytopic ligands with coordination pocket composition that is complimentary to a metal ion’s coordination ‘ algorithm’. These grids exhibit interesting, and well characterized magnetic properties, and have attracted [1] much attention for their possible nanotechnological applications. Incorporating large, quinoline or bipyridine-type ligand end-pieces has been targeted, in an attempt to achieve long-range ordering, and extended magnetic communication between grids. While several [1-4] synthetic successes with M = Mn, Cu, and Co have been achieved, subtle changes in [5] ligand design has also lead to unanticipated results. A Mn(II)12 rectangle, and a Cu(II)11 super-triangle, both with available coordination pockets will be presented, and their intermolecular - interactions highlighted. 1. Dawe, L.N., Shuvaev, K.S., Thompson, L.K., Inorg. Chem., 2009, 48, 3323-3341. 2. Dey, S.K., Abedin, T.S.M., Dawe, L.N., et al., Inorg. Chem., 2007, 46, 7767-7781. 3. Dey, S.K., Thompson, L.K., Dawe, L.N., Chem. Commun., 2006, 4967-4969. 4. Dawe, L.N., Thompson, L.K. Angew. Chem., 2007, 46, 7440-7444. 5. Shuvaev, K.V., Tandon, S.S., Dawe, L.N., Thompson, L.K., Chem. Comm., 2010, Submitted. ©© 07.13.4 Crystal Structures of BaSrR4Zn2O10, R = La, Nd, Sm, Eu James Kaduk , Winnie Wong-Ng 1 1 2 2 Poly Crystallography Inc, Naperville IL, United States, NIST, Gaithersburg MD, United States The crystal structures of BaSrR4Zn2O10 have been determined using synchrotron powder diffraction data collected at 11-BM at the Advanced Photon Source at Argonne National Laboratory. BaR2ZnO5 crystallize in the tetragonal space group I4/mcm for R = La and Nd, and in orthorhombic Pbnm for smaller lanthanides. All four structures are tetragonal. The Nd, Sm, and Eu compounds crystallize in I4/mcm, and the trends in lattice parameters follow those of the BaR2ZnO5. Four weak peaks in the BaSrLa4Zn2O10 pattern could not be attributed to any impurity phase, but corresponded to the 102, 122, 124, and 128 peaks of the tetragonal unit cell. The body centering condition was thus violated, and the true space group is P4/ncc. The 10-coordinate sites in all four compounds are occupied by Ba and Sr. The 8coordinate sites are occupied primarily by the lanthanide cations, but small concentrations of Sr are apparently present at this site for R = La and Nd. The tetrahedral Zn sites are similar in all four compounds. The larger size of the R = La cell apparently results in movement of the Ba/Sr off the center of the 10-coordinate cage. The largest errors in the Rietveld plots are in the tails of the peaks, and the errors are not random. Density functional quantum chemical geometry optimizations for Ba-only and Sr-only structures help understand local microstructural distortions and the potential effects of local compositional variations. 07.13.5 Charge density study of NaI3O8: Origin of the non linear optics properties CLAUDE LECOMTE , CHRISTIAN JELSCH , EMMANUEL WENGER , ISABELLE 3 3 4,1 GAUTIER LUNEAU , YAN SUFFREN , PIERRE FERTEY NANCY UNIVERSITE, NANCY, France, CNRS UHP UMR7036, NANCY, France, CNRS 4 UPR 2940, GRENOBLE, France, SOLEILSYNCHROTRON, SACLAY, France NaI3O8 is a promising material for infrared parametric generation [1]. It crystallises in an acentric space group P-4 , prerequisite for quadratic nonlinear properties. NaI3O8 is among the rare materials having a large domain of transparency from visible to the beginning of the far IR (12.5 μ m) allowing applications in the atmospheric transparency windows. It is not hygroscopic, has a good thermal stability (up to 350° and shows high optical damage C) -2 thresholds on powder (4.2 GW.cm ). Moreover, NaI3O8 could be grown as millimetric single crystal which is a key point to study its NLO properties and further for the development of device systems. The crystal structure reveals a novel oxo anion, [I3O8] which is the first polynuclear anion of pentavalent iodine. This anion is formed from the condensation of three iodate anions in concentrated acidic solution, and contains three polarisable lone electron pairs (LEP) favouring high nonlinear susceptibilities. In previous calculations of NLO polarisabilities coefficients in iodate compounds, the contribution of LEP of iodine atom was neglected, or has been considered to be equal to the I-O bond influence. In order to establish relations between the crystal structure of NaI3O8, and its optical properties, as done for KTiO(PO4) [2],a charge density study must be performed. Modeling the electron density of inorganic materials containing heavy elements is very far from a routine work: accurate absorption and extinction corrections are the key for a realistic electron density modeling. Low temperature multiple wavelength diffraction data have been collected on the CRISTAL beamline at SOLEIL from 30KeV to 8 KeV in order to extrapolate the extinction correction to zero wavelength before collecting the 30KeV ultra high resolution data set used for the electron density model [1] with the program suite MOPRO [1] This communication will describe the preliminary electron density results concentrating on both methodological aspects and NLO properties . [1] D. Phanon & I. Gautier-Luneau, Angew. Chem. Int. Ed., 46, 8488-8491, 2007. "Promising Material for Infrared Nonlinear Optics: NaI3O8 salt containing a New Octaoxotriiodate(V) Anion formed from Condensation of [IO3] Iodate”[2] Hansen N K, Protas J P and Marnier G; C. R. Acad. Sci., Paris 307, 475, 1988 “Structure atomique, densité électronique et propriétés optiques non linéaires de KTiOPO4.” ; Hansen N K, Protas J P and Marnier G; Acta Cryst., B47, 660-672, 1991. “The Electron Density Distribution in KTiOPO4.”[3 C.Jelsch, B.Guillot, A.Lagoutte & C.Lecomte, J. Appl. Cryst., 38 , 38-54,2005. " Advances in proteins and small molecules charge density refinement methods using software MoPro" 1 2 3 1,2 1,2 1,2 07.13.6 A Non-Isostructural Series of Lanthanide complexes that puts the “Fun” in Dysfunctional. Christine Beavers, Guoxin Tian, Linfeng Rao Lawrence Berkeley National Lab, Berkeley, CA, United States In an attempt to observe the lanthanide contraction, a series of lanthanide complexes (and the occasional actinide) was synthesized and crystallized. To the eager crystallographer, these compounds seemed destined for the esteemed realm of “isostructural series.” However this series has proved to be much more difficult than imagined, but also much more complex and intriguing. Identical stoichiometry did not lead to identical structures. Phase changes, twinning and a possible modulated structure all found their way into this collection of complexes. Asymmetric unit of Pr-TMDA with hydrogen sites omitted for clarity 07.13.7 Weird Structures A Moving Framework Structure, a P1 Compound with Unusual Bonding Correlations and a Disappearing Molecule. Abraham Clearfield, Aaron Celestian, Houston Perry, Paul Zhang CUNY Queens College, New York, United States A. Clearfield, Aaron Celestian, Houston Perry, and Paul Zhang Chemistry Department, Texas A&M University College Station, TX 77842 The compound Na2Ti3O3(SiO4)•2H2O has a tunnel structure and is highly selective for Cs+. It is able to remove cesium ions from highly alkaline solutions in the presence of 6M Na+ and therefore is under consideration to remove the cesium from nuclear waste solutions. The structure of this titanium silicate and to its Cs+ form have been determined from x-ray powder data. However, treatment of the Cs+ phase with HCl fails to remove the cesium. In situ time resolved x-ray and neutron diffraction revealed the mechanism of exchange which showed that the framework can rotate to narrow the tunnel and change the shape of the tunnel to trap the Cs+. A video of the movement of the framework will be displayed. A second structure of a strange nature is the copper complex of 1,3,5-benzene tris(methylphosphonic)acid and 4,4'-bipyridyl. This compound was refined in space group and found to be a dimer of copper square pyramids linked into a supermolecular array. However, there were indications that violations of centrosymmetric symmetry occurred in the placement of the protons. Refinement in P1 resolved the symmetry problems. However it revealed an unusual correlation between the positioning of oxygen atoms in a sixth (or octahedral) position of the square pyramids and the Cu-O bond distances of the water molecules in the apex position of the pyramids. If time permits we will include the structure of a Zr PMIDA derivative that self exfoliates and reconfigures as a function of pH. T-006 EVIDENCE FOR CONFORMATIONAL CHANGES UPON INHIBITOR BINDING IN SERINE RACEMASE Myron Smith, Volker Mack, Andreas Ebneth, Isabel Moraes, Brunella Felicetti, Michael Wood, Dorian Schonfeld, Owen Mather, Andrea Cesura, John Barker Evotec UK Ltd, Oxfordshire, United Kingdom Serine racemase is responsible for the synthesis of D-serine, an endogenous co-agonist for N-methyl-D-aspartate receptor-type glutamate receptors (NMDARs). This pyridoxal 5’phosphate-dependent enzyme is involved both in the reversible conversion of L- to D-serine and serine catabolism by α ,β -elimination of water, thereby regulating D-serine levels. Since D-serine affects NMDAR signalling throughout the brain, serine racemase is a promising target for the treatment of disorders related to NMDAR dysfunction. To elucidate the reaction mechanism and provide a molecular basis for rational drug design the X-ray crystal structures of human and rat serine racemase were determined at 1.5 Å and 2.1 Å resolution respectively, and in the presence and absence of the orthosteric inhibitor malonate. The structures revealed a fold typical of β -family PLP-enzymes, with both a large domain and a flexible small domain associated into a symmetric dimer, and indicated a ligand-induced rearrangement of the small domain that organises the active site for specific turnover of the substrate. T-009 Trapping and structurally characterizing true catalytic intermediates in a target enzyme Ronald Viola University of Toledo, Toledo, Ohio, United States The biosynthetic pathway derived from aspartic acid is unique to plants and microorganisms, producing four of the essential amino acids required for protein synthesis. In addition, intermediates in this pathway are involved in bacterial cell wall cross-linking, sporulation in Gram-positive bacteria, and quorum sensing in Gram-negative bacteria. ASA dehydrogenase, a core enzyme in this pathway, has been targeted for inhibitor design and drug development. Two proposed enzyme-bound intermediates in the catalytic cycle have been trapped and structurally characterized. These structures have helped delineate the mechanism of this key metabolic enzyme and provided guidance for selective inhibitor design. Structures have also been determined for this target enzyme isolated from Gram-negative and Gram-positive infectious bacteria and from a fungal species. The structural differences between these mechanistically identical enzymes are being exploited for the development of species-specific antibiotics. T-012 A Joint X-ray and Neutron Diffraction Study on a Copper Protein Reveal the Role of Protein Dynamics in Electron Transfer Narayanasami Sukumar , Scott Mathews , Paul Langan , Victor Davidson 1 1 2 3 4 NE-CAT and Dept. of Chemistry and Chemical Biology, Cornell University, , Argonne 2 National Laboratory, Argonne, IL 60439, Dept. of Biochemistry and Molecular Biophysics, 3 Washington Univ. School of Medicine, St. Louis, MO 63110, , Bioscience Div., Los Alamos 4 National Laboratory, Los Alamos, NM 87545, Dept. of Biochemistry, Univ. of Mississippi Medical Center, Jackson,MS 39216. Amicyanin from Paracoccus denitrificans contains a single copper site and mediates the electron transfer (ET) from methylamine dehydrogenase (MADH) to cytochrome C551i. The protein has a molecular mass of about 12.5 kDa and folds as a β - sandwich, with nine β strands forming two mixed β -sheets. Though several X-ray crystallographic studies in combination with spectroscopic and kinetic studies have been carried out on amicyanin, a detailed understanding of its electronic properties has been hampered by lack of information on the precise positions of hydrogens. In order to determine precisely the position of hydrogen atoms, the extent of hydrogen/deuterium exchange (HDX), and the flexibility and dynamic nature of the protein, a joint X-ray/neutron (XN) diffraction study was performed with amicyanin. The amide hydrogen atoms of ~86% residues including the residues that provide the copper ligands are either partially or fully exchanged, indicating that the structure of amicyanin is highly dynamic. An analysis on the crystal structure of the tertiary complex of MADH, amicyanin, and cytochrome c-551i indicated that the likely point of inter-protein ET from amicyanin to cytochrome c-551i is Glu31. Further ET analysis in solution yielded a value for -1 electronic coupling, HAB of 0.3 cm which was unusually large, given the ET distance of ~23 Å. Subsequently a study of this same ET reaction in crystals of the protein complex yielded a -4 -1 much slower rate and a much smaller value for HAB of 7.3 ´ 10 cm . The experimentally determined reorganization energies for the reactions in solution and in the crystal state were identical; only the HAB was affected. The dynamic motion of the residues during the ET was not considered when the calculations were performed using X-ray crystal structures. In the present XN structure, the dynamic nature of individual residues could be ascertained based on HDX. The difference in the ET studies between solution and crystal state may be explained by the finding in this present study that the amicyanin molecule, particularly the portion of the protein through which ET occurs, is highly dynamic. Despite this dynamic nature, all of the conventional H-bonds around ~8Å of copper site that are predicted to occur in the X-ray structure are directly observed in the XN structure. A careful analysis of N-H...X and C-H...X types of hydrogen bonds reveals that five of the CH....X bonds involve the copper ligand-His95 are absent in the reduced structure, which undergoes a pH-induced conformation change in the reduced state. The results reveal previously unknown role of these weaker C-H...X bonds as they, like conventional H-bonds, collectively influence the structure, redox and electron transfer properties of amicyanin. T-015 Crystallization and structure determination of a transient electron transfer complex Miki Senda , Shigenobu Kimura , Tetsuo Ishida , Toshiya Senda 1 2 3 1 2 3 4 Japan Biological Informatics Consortium, Tokyo, Japan, Ibaraki University, Ibaraki, Japan, 4 Shiga University of Medical Science, Shiga, Japan, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan Electron transfer complexes of redox proteins are characterized by weak affinity, transient interaction, and redox-dependent affinity regulation. These characteristics are critical to fast and efficient electron transfer. We have studied an electron-transfer system for a multicomponent dioxygenase, BphA, derived from Acidovorax sp. strain KKS102. BphA3 and BphA4 are a Rieske-type [2Fe-2S] ferredoxin and an FAD-containing NADH-dependent ferredoxin reductase, respectively (1, 2). To reveal the electron-transfer mechanism from BphA4 to BphA3, crystal structure analysis of the BphA3-BphA4 complex is indispensable. However, oxidized BphA3 and oxidized BphA4 hardly form a stable complex in solution, because the Kd value for their interaction was too large, 294 M, to form a stable complex in solution. The Kd value, however, changed significantly upon their reduction (Kd = 14.6 M); BphA3 and BphA4 can form a stable complex under the reduced conditions. This complex seems to resemble a productive complex for the electron transfer reaction. We have developed an anaerobic chamber to crystallize the BphA3-BphA4 complex under anaerobic conditions and have succeeded in crystallizing the BphA3-BphA4 complex (3). This system has also been used to crystallize free BphA3 and free BphA4 in their reduced form. We have so far determined the crystal structures of the BphA3-BphA4 complex (productive electron transfer complex); free BphA4 in oxidized, hydroquinone, and semiquinone forms; and free BphA3 in oxidized and reduced forms. Comparison of these crystal structures revealed that (i) BphA4 undergoes conformational changes upon reduction and (ii) BphA4 shows conformational changes while forming a complex with BphA3. Since these conformational changes found in BphA4 are similar to one another, they seem to be essential to the formation of an electron-transfer complex; they are likely to contribute to the formation of a high-affinity binding site for BphA3 on BphA4 (1, 2). References: 1. Senda, T. et al., (2009). Antioxid. Redox. Signal., 11, 1741-1766; 2. Senda, M. et al., (2007). J. Mol. Biol. 373, 382-400; 3. Senda, M. et al., (2007). Acta Cryst. F63, 520-523. ª ª T-018 Redox-dependent conformational changes of NADH-dependent ferredoxin reductase under various pH conditions Toshiya Senda , Miki Senda , Shigenobu Kimura , Tetsuo Ishida 1 1 2 3 4 2 National Institute of Advanced Industrial Science and Technology, Tokyo, Japan, Japan 3 4 Biological Informatics Consortium, Tokyo, Japan, Ibaraki University, Ibaraki, Japan, Shiga University of Medical Science, Shiga, Japan BphA4, which is an FAD-containing NADH-dependent ferredoxin reductase, receives two electrons from NADH and delivers one electron each to ferredoxins (BphA3). To elucidate the molecular mechanism underlying the electron-transfer reaction and the redox-dependent interaction between BphA3 and BphA4, we determined the crystal structures of the productive BphA3-BphA4 complex as well as of free BphA3 and BphA4 in all redox states occurring in the catalytic cycle. Our results revealed that (i) FAD in BphA4 changes its conformation depending on the redox state, (ii) BphA4 also changes its conformation depending on the redox state of FAD, and (iii) the conformational changes of BphA4 are required to form a highaffinity binding site for BphA3. These results suggested that the conformational change of FAD could be one of the main reasons for the conformational changes in the overall structure of BphA4 and affinity regulation between BphA3 and BphA4. However, it has been known that FAD changes its conformation and chemical properties depending on pH and environment. Since BphA4 was crystallized at pH 5.4, there might be some artifacts in the FAD conformations found in the crystal structures. We therefore analyzed the effects of pH on the structures of FAD and the overall structure of BphA4 in oxidized and semiquinone (BphA4NAD complex) forms. BphA4-NAD crystals were prepared by the soaking method as reported earlier. The crystal structures of the BphA4-NAD complex and oxidized BphA4 have been determined at higher than 1.5 Å resolution under various pH conditions (pH 5.4–8.5). These crystal structures showed that semiquinone FAD in BphA4 adopts different conformations in a pH-dependent manner. However, in the pH range from 5.4 to 8.5, no significant differences were found in the bound NAD conformation and in the overall structures of the BphA4-NAD complex. In addition, BphA4 formed a high-affinity binding site for BphA3 throughout the pH range 5.4-8.5. Our biochemical analysis demonstrated that reduced BphA4 has an approximately 100-fold higher affinity for NAD than the oxidized BphA4 at pH 5.4-8.5. These results suggested that the binding of the nicotinamide moiety of NAD to reduced BphA4 facilitates the formation of the high-affinity site to BphA3. This mechanism seems to contribute to the efficient electron transfer from BphA4 to BphA3 under various pH conditions. T-021 Structural Basis for Allosteric Activation of Ubiquitylation Mediated by Ube2g2 and gp78 RING Finger Yuhe Liang , Ranabir Das , Jess Li , Jennifer Mariano , Allan Weissman , Andrew Byrd , 1 Xinhua Ji 1 1 2 2 3 3 2 Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD 21702, 2 United States, Structural Biophysics Laboratory, National Cancer Institute, Frederick, MD 3 21702, United States, Laboratory of Protein Dynamics and Signaling, National Cancer Institute, Frederick, MD 21702, United States Ube2g2 is an E2 ubiquitin-conjugating enzyme and gp78 is an endoplasmic reticulumassociated E3 ubiquitin ligase with the RING finger. Distinct from the RING finger, gp78 recruits Ube2g2 with its G2BR domain. The Ube2g2-G2BR interaction is specific with high affinity, induces significant conformational changes near the active site of Ube2g2, causes a 50-fold increase in the affinity between Ube2g2 and the RING finger, and results in markedly increased ubiquitylation by Ube2g2 and the gp78 RING finger (Molecular Cell 34, 674-685, 2009). Here, we report the crystal structure of the ternary Ube2g2-G2BR-RING complex at 2.3-Å resolution. The crystal belongs to space group P41212, with unit cell parameters a = b = 58.25 and c = 158.43 Å. The structure shows that the G2BR and RING finger of gp78 bind to the opposite sides of the Ube2g2 molecule. Comparative analysis of the ligand-free Ube2g2 (PDB entry 2CYX), Ube2g2-G2BR (3H8K), and Ube2g2-G2BR-RING (this work) structures reveals structural basis for the allosteric activation of ubiquitylation mediated by Ube2g2 and the gp78 RING finger. The Ube2g2-G2BR-RING structure presented here is the first of its kind, shedding lights on the mechanism of other E3 ubiquitin ligases with the RING finger. This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. X-ray diffraction data were collected at beamline 22-ID of SER-CAT, Advanced Photon Source, Argonne National Laboratory. T-024 Crystal Structures of Human Exonuclease I/DNA Provide Insight Into Catalytic Mechanism Jillian Orans, Elizabeth McSweeney, Paul Modrich, Lorena Beese Duke University Medical Center, Durham, NC, United States Human exonuclease I (hEXOI) plays a significant role in human mismatch repair through the 5’ to 3’ excision of the non-template DNA strand, as well as possessing additional roles in DNA replication and recombination. Here we present crystal structures of the hEXOI catalytic domain as both the wild-type protein and an inactive mutant (D173A) in complex with a 5’ recessed DNA substrate. The initial structure was determined using selenomethionine SIRAS phasing and refined to a resolution of 2.5 Å. The model shows a common core of a sevenstranded β -sheet surrounded by flanking helices which is similar to those seen in a variety of homologous flap endonuclease (FEN1) structures; however, certain commonly conserved nucleotide binding motifs have altered secondary structural elements and are significantly repositioned in our structure. We have also identified three metal ion-binding sites, two in the active site surrounded by conserved acidic residues, and another in close proximity to the DNA phosphate backbone. The active site metals are appropriately positioned relative to each other and the scissile phosphate such that we can propose a two-metal ion catalytic mechanism analogous to that observed in the 3’ to 5’ exonuclease sites of DNA Polymerase I Klenow fragment. This structure of hEXOI is the first known structure of a Class III member of the RAD2 nucleases, a family with highly divergent sequences and DNA structure specificities. Structural knowledge of hEXOI will greatly assist in our understanding of disease-associated mutations, as well as aid in the modeling and rationalization of similar mutations in additional RAD2 nucleases such as XPG. Furthermore, analysis of this structure in conjunction with previously determined DNA-complexed homologs and recently acquired SAXS data will allow identification of possible protein binding surfaces and aid in the elucidation of a mechanism for substrate specificity. T-027 Structural Analysis of Glucokinase Activators and Inhibitors Christine Lukacs , Joseph Grimsby , Nancy-Ellen Haynes , R. Ursula Kammlott , Robert 1 2 1 1 Kester , Paige Mahaney , Frank Podlaski , Ramakanth Sarabu Roche, Nutley, NJ, United States, Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT, United States Glucokinase is an enzyme of the glycolytic pathway which converts glucose to glucose-6phosphate. It plays an essential role in the maintenance of glucose levels in the liver and in controls glucose stimulated insulin release in pancreatic beta cells, making it an attractive target for the development of small molecules to treat type II diabetes. 1 2 1 1 1 1 Roche has previously published the discovery of small molecule glucokinase activators (GKAs). These compounds do not bind at the enzyme active site (glucose binding site), but rather at a distant allosteric site 20Å away. Modulating the kinetic binding properties of GKAs in turn affect the kinetics of the glucokinase enzyme. We have been studying these properties via biochemical and cellular assays, kinetic assays, and biophysical methods, including X-ray crystallography. We have identified compounds within our phenylacetamide series which have either activating, inhibitory, or no effect on the glucokinase activity. We have solved the crystal structure of GK in its closed conformation with several of these compounds bound. This poster will show details of these structures and correlate the binding features of these small molecules with their effect on GK activity. T-031 Communication Between Thiamin Cofactors in the E. coli Pyruvate Dehydrogenase Multienzyme Complex E1 Component Active Centers Palaniappa Arjunan , Krishnamoorthy Chandrasekhar , Natalia Nemeria , Frank Jordan , 1,2 William Furey 1 1,2 1 3 3 University of Pittsburgh, Pittsburgh, PA 15261, United States, VA Healthcare System, 3 Pittsburgh, PA 15240, United States, Rutgers University, Newark, NJ 07102, United States 2 Structural, spectroscopic and kinetic analyses tested the hypothesis that a chain of residues connecting the 4’-aminopyrimidine N1’-atoms of thiamin diphosphates (ThDPs) in the two active centers of the E. coli pyruvate dehydrogenase complex (PDHc) E1 component provides a signal transduction pathway. Substitution of the three acidic residues (E571, E235, E237) and R606 resulted in impaired binding of the second ThDP, once the first active center was bound to the cofactor suggesting a pathway for communication between the two ThDPs. Titration of the E235A and E237A variants with methyl acetylphosphonate (MAP, an analog for the substrate pyruvate), monitored by circular dichroism suggested that only half of the active sites were filled with the related, covalently bound, predecarboxylation intermediate analog. We have determined the crystal structures of the active site variants E571A in complex with cofactor ThDP, and E235A in complex with ThDP as well as with MAP, and compared the structural changes with biochemical activity data. Crystal structures of E235A and E571A in complex with ThDP revealed the structural basis for the spectroscopic and kinetic observations and showed that either substitution affects cofactor binding, despite the fact that E235 makes no direct contact with the cofactor nor does it block entrance to the active site. The structural results also support the idea of non-equivalence of active sites within the E1 dimer. While there are general similarities between the native and these two mutant structures, there are significant differences in the active sites, and the role of the conserved E571 residue in both catalysis and in defining cofactor orientation was revealed by the structural results. Details and implications of ThDP binding on catalytic activity for the mutant structures will be presented. T-034 Crystal Structure of Human Prethrombin I at 1.66 ä Resolution Zhiwei Chen, Leslie Bush-Pelc, Enrico Di Cera Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Mo 63104, United States Prethrombin I is an inactive precursor of thrombin along the prothrombin activation pathway. Prethrombin I consists of a single chain composed of fragment 2 (F2), A and B chains. A single peptide bond cleavage between R15 and I16 results in generation of the active intermediate meizothrombin and further cleavage between F2 and the A chain produces thrombin. Here we report the first structure of prethrombin I carrying the double mutation W215A/E217K. Comparison with the recent structure of meizothrombin reveals that F2 has shifted by at least 10 Å and the rms deviation is 3.65 ä. The region around residues 215-223 is partially collapsed as seen in the inactive E* form of thrombin, presumably as a result of the double substitution W215A/E217K. Interestingly, the region around residues 186-193 is also collapsed and assumes a conformation never before observed in thrombin structures. The side chain of R15 from a second molecule in the lattice is bound to the active site. The side chain of residue D194 that in the active enzyme H-bonds to the amino terminus of the B chain, I16, interacts with the backbone N atoms of residues 141 and 142. The structure suggests a pathway of activation from prethrombin 1 to thrombin that takes into account the allosteric equilibria between the E* and E forms of the enzyme. T-037 Structural studies of enduracididine biosynthetic enzyme MppR Nicholas Silvaggi, Tyler Voegtline University of Wisconsin-Milwaukee, Milwaukee, WI, United States Antibiotic-resistant pathogens are a serious and persistent threat to public health. The recent isolation of methicillin-resistant Staphylococcus aureus (MRSA) with significant resistance to vancomycin, the current last line of defense against this and other antibiotic-resistant pathogens, is particularly ominous and underscores the need for new, effective antibiotics. The nonribosomal peptide antibiotics mannopeptimycin and enduracidin are promising candidates for development into chemotherapeutic agents for the treatment of MRSA, vancomycin-resistant enterococci (VRE), and penicillin-resistant Streptococcus pneumoniae. Mannopeptimycin is a lipoglycopeptide with a cyclic hexapeptide core of alternating D- and Lamino acids. Enduracidin is a 17-residue cyclic lipopeptide. Both compounds contain the rare nonproteinogenic amino acids beta-hydroxy L-enduracididine or L-enduracididine, respectively. In mannopeptimycin biosynthesis, L-enduracididine is produced from L-arginine through the action of three enzymes, MppP, MppQ, and MppR. The details of this transformation are currently unknown. The success of a semi-synthetic mannopeptimycin analog, AC98-6446, has prompted much interest in producing additional derivatives of these promising natural products. These efforts will be facilitated by a ready supply of enduracididine and its analogs for use in combinatorial biosynthesis or semi-synthetic approaches. Herein we present the X-ray crystal structure of MppR (32.2 kDa) from Streptomyces hygroscopicus at 1.6Å resolution (Rcryst=0.152, Rfree=0.179). The structure was determined by SAD from a single data set collected from a SeMet-derivitized crystal at LS-CAT beamline 21ID-D at the Advanced Photon Source. The overall fold of the protein is nearly identical to acetoacetate decarboxylase (PDB ID 3BH2; SSM RMSD over 218 Cα atoms = 1.78Å). In addition, despite low sequence identity (<10%), the active site residues in these two enzymes are very similar, suggesting that MppR is also a decarboxylase. T-040 Mechanism of catalysis and structure of pharmaceutically relevant enzymes in the biosynthetic pathways of NAD and pyrrolobenzodiazepines (PBDs) Watchalee Chuenchor , Melissa Resto , Kaiti Chang , Tzanko Doukov , Barbara Gerratana University of Maryland, College Park, United States, Lightsource, Menlo Park, United States 1 2 1 1 1 2 1 Stanford Synchrotron Radiation We are studying the mechanisms and structures of biosynthetic pharmaceutically relevant enzymes. The first project aims at elucidating the mechanism and structure of NAD synthetase, an essential NAD biosynthetic enzyme in M. tuberculosis for the design of TB structure- and mechanism-based inhibitors. The structure of wild type NAD synthetase in complex with DON and NaAD, previously solved by the Gerratana’s laboratory, is homooctameric and features a 40 angstrom long inter-subunit ammonia tunnel for transport of ammonia from the glutaminase active site to the synthetase active site. An 180-fold activation of the glutaminase active site was measured when the synthetase intermediate complex is formed. We hypothesize that ordering of loop P2 at the synthetase active site induces a conformational change that activates the glutaminase active site and we reasoned that inhibitors that will trap the enzyme in this closed activated conformation will have high affinity. TB To obtain the NAD synthetase bound to ligands that stabilize the disordered loop P2 at the synthetase active site, we solved several structures of different ligands complexes in the resolution range of 2.4 to 3.0 angstroms. The analysis of these structures is reported in this poster. We have also expressed the human NAD synthetase in insect cell and purified for kinetic and structural characterization. With the characterization of this ortholog, we will be able to clarify the basis of substrate specificity and design high affinity inhibitors selective for TB NAD synthetase . The second project is focused on characterizing novel enzymes of the biosynthesis of pyrrolo[1,4]benzodiazepines (PBDs). A glycosylated member of this class, sibiromycin, has remarkable potency against cancer cells (pM range). We have solved the apo structure of SIbS from Se-Met and native crystals at 2.3 and 2.1 angstrom resolutions, respectively. SibS is hypothesize to catalyze an unusual C-C hydrolase reaction and shows very little sequence homology to the phenazine biosynthesis-like proteins. This is the initial step in the characterization of the enzymes involved in the unique transformation of L-tyrosine to hydropyrrole identified in PBD biosynthesis. T-043 Structural and Functional Characterization of Outer-Sphere Mutations of Toluene 4Monooxygenase Lucas Bailey, Justin Acheson, Brian Fox Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, United States Diiron hydroxylases are multicomponent enzyme complexes that catalyze the oxidation of a wide range of hydrocarbons. Toluene 4-monooxygenase (T4moH) is a four-component hydroxylase consisting of two electron transfer components, T4moF and T4moC, responsible for the reduction of the diiron hydroxylase (T4moH). Additionally, a small cofactorless effector protein, T4moD, forms a high affinity complex with T4moH and is responsible for the modulation of a number of catalytic phenomena associated with enzymatic function. A series of crystal structures of the T4moH-T4moD complex have revealed how residues outside of the first coordination sphere rearrange to create an active site pocket poised for turnover. These include Asn202 and Gln228, which form a hydrogen-bonding network with conserved Thr201 and a newly ordered HOH5. T4moH variants N202A, Q228A and Q228E all had lower enzyme activity as determined by a combination of steady state and transient kinetic approaches. High-resolution structures of the T4moH variant in complex with T4moD provide new insight into the role of these residues in catalysis. Funded by NSF MCB 0843239 to BGF. T-046 Hydroxymethylglutaryl-CoA lyase (HMGCL): Insights into the Reaction Mechanism ++ ++ from Structures of HMGCL-Mg -hydroxyglutaryl-CoA and HMGCL R41M-Mg -HMGCoA Ternary Complexes Jennifer Runquist , Zhuji Fu , Henry Miziorko , Jung-Ja Kim 1 1 1 2 1 2 Medical College of Wisconsin, Milwaukee, WI, United States, University of Missouri-Kansas City, Kansas City, MO, United States HMGCL is crucial to ketogenesis and inherited human mutations result in potentially fatal disease. Detailed understanding of the HMGCL reaction mechanism as well as the molecular basis for correlating recently reported human mutations with enzyme deficiency have been limited by the lack of structural information for enzyme bound to an acyl-CoA substrate or inhibitor. Soaking crystals of wild-type HMGCL with the competitive inhibitor 3hydroxyglutaryl-CoA (HG-CoA) or of the R41M HMGCL mutant with substrate HMG-CoA has supported determination of X-ray structures for complexes of wild type with inhibitor (2.4 Å) and R41M with substrate (2.2 Å). Comparison of these β /α barrel structures with those of unliganded HMGCL and R41M reveal substantial differences for positioning of the flexible loop containing the conserved “signature” sequence of HMGCL. In the ternary complex ++ formed with substrate, Mg , and R41M, loop residue C266 (implicated in active site function by mechanistic and mutagenesis observations) is more closely juxtaposed to the catalytic site ++ than in the case of either unliganded enzyme or the complex of wild-type enzyme with Mg and inhibitor HG-CoA. In both ternary complexes, the S-stereoisomer of substrate or inhibitor is specifically bound, in accord with the observation that oxygens from both the C3 hydroxyl ++ and the C5 carboxyl groups are Mg ligands. In addition to H233 and H235 imidazoles, other ++ Mg ligands are the D42 carboxyl oxygen and an ordered water molecule. This water is positioned between D42 and the C3 hydroxyl of bound acyl-CoA substrate/inhibitor and may function as a proton shuttle. Results also display the interaction of R41 with the acyl-CoA’ s C1 carbonyl oxygen, in agreement with the observed effects of R41 mutation on reaction product enolization. This substrate enzyme interaction offers an explanation for the drastic enzyme 5 deficiency (10 –fold) seen in human R41 mutation. T-049 Structural insights into allosteric activation of pyruvate carboxylase: the asymmetric tetramer organization of Rhizobium etli PC is independent of acetyl coenzyme A. Martin St. Maurice, Adam Lietzan, Sudhanshu Kumar Marquette University, Milwaukee, WI, United States o„‒· \ ¡? ¦\‒ ›‚„ \ ¡? GobK? db? UMSMPMPH? ? \? «· L¢·‹¦ ›‹\ K? › ‹L ¡fi¡‹ ¡‹ ? ¡‹ „«¡? ⁄\ ? ¦\ \ „ ¡ ? ⁄¡ ¦\‒ ›‚„ \ ›‹? ›¢? fi„‒· \ ¡? ›? ›‚\ ›\¦¡ \ ¡? • ⁄? ⁄¡? ¦›‹¦›« \‹ ? ¦ ¡\ \£¡? ›¢? l£`soM? s⁄¡? ‒¡\¦ ›‹? \¤¡ fi \¦¡? ¡fl·¡‹ \ „K? ‹? •›? ‹ ¡fi¡‹ ¡‹ ? \‹ ? ‒¡«› ¡? \¦ ¡? ¡ ? \‹ ? ? · ¡¦ ? ›? \ › ¡‒ ¦? \¦ \ ›‹? „ \¦¡ „ ? ¦›¡‹ „«¡? `M? r¡ ¡‒\ ? ‒¡¦¡‹ ? wL‒\„? ¦‒„ \ ? ‒·¦ ·‒¡ ? ›¢? ⁄¡? ¡‹ „«¡? ¢‒›« q⁄ › ·«? ¡ ? \‹ ? ¢‒›« r \fi⁄„ ›¦›¦¦· ?\·‒¡· ?⁄\ ¡?‒¡ ¡\ ¡ ? ⁄¡?‒¡ \ ¡?fi› ›‹?\‹ ?›‒ ¡‹ \ ›‹?›¢? ⁄¡ ‹ ·\ ? ›«\ ‹ ? ‹?ob?\‹ ⁄\ ¡? ¡¢ ‹¡ ? ⁄¡? ‹ ‹£? ¡?¢›‒? ⁄¡?\ › ¡‒ ¦?\¦ \ ›‒K?\¦¡ „ Lb›`M?s⁄¡ ¡? ‒·¦ ·‒¡ ?⁄\ ¡?\ ›?‒¡ ¡\ ¡ £‹ ¢ ¦\‹ ? ¢¢¡‒¡‹¦¡ ? ‹? ⁄¡?› ¡‒\ ?›‒£\‹ \ ›‹?›¢? ⁄¡? ¡ ‒\«¡‒ ¦?¡‹ „«¡Y?ob?¢‒›« rM?\·‒¡· ? ?\?£¡‹¡‒\ „ „««¡ ‒ ¦? ¡ ‒\«¡‒? ‹? ⁄¡?fi‒¡ ¡‹¦¡?\‹ ?\ ¡‹¦¡?›¢?\¦¡ „ Lb›`K?•⁄ ¡? ⁄¡?ob?¢‒›« qM?¡ ? ?\‒‒\‹£¡ ?\ ?\ ⁄ £⁄ „? \ „««¡ ‒ ¦? ¡ ‒\«¡‒? ‹? ⁄¡? fi‒¡ ¡‹¦¡? ›¢? ¡ ⁄„ Lb›`M? h ? ‒¡«\ ‹ ? ·‹¦ ¡\‒? •⁄¡ ⁄¡‒? ⁄¡? ¢¢¡‒¡‹¦¡ ¡ •¡¡‹? ⁄¡ qM?¡ ob? ‒·¦ ·‒¡?\‹ ? ⁄¡ rM?\·‒¡· ?ob? ‒·¦ ·‒¡ ?‒¡ · ?¢‒›«? ¢¢¡‒¡‹¦¡ ? ‹? ›·‹ ? £\‹ K ¦‒„ \ \ ›‹?¦›‹ ›‹ ?›‒?¢·‹ \«¡‹ \ ? ¢¢¡‒¡‹¦¡ ? ‹? ⁄¡? ¡ ‒\«¡‒?›‒£\‹ \ ›‹?›¢?¡‹ „«¡ ? › \ ¡ ?¢‒›« •›?·‹‒¡ \ ¡ ? \¦ ¡‒ \ ? fi¡¦ ¡ M?s›?\ ‒¡ ? ⁄ ?fl·¡ ›‹K?•¡?⁄\ ¡? ¡ ¡‒« ‹¡ ? ⁄¡?wL‒\„?¦‒„ \ ? ‒·¦ ·‒¡ ›¢ qM?¡ ?ob? ‹? ⁄¡?fi‒¡ ¡‹¦¡?\‹ ?\ ¡‹¦¡?›¢?\¦¡ „ Lb›`K? ›?QMU?\‹ ?QMX?ä?‒¡ › · ›‹K?‒¡ fi¡¦ ¡ „M?h‹? › ⁄ ›¢? ⁄¡ ¡? ‒·¦ ·‒¡ ? ⁄¡? ¡ ‒\«¡‒? ‒¡«\ ‹ ? ⁄ £⁄ „? \ „««¡ ‒ ¦M? s⁄ ? ·££¡ ? ⁄\ ? ¢·‹ \«¡‹ \ ? ¢¢¡‒¡‹¦¡ ¡‚ ? ¡ •¡¡‹? ⁄¡ rM? \·‒¡· \‹ qM? ¡ ob? ¡‹ „«¡ ? ‹? ⁄¡ ‒? ? › ¡‒\ ? ¡ ‒\«¡‒ ¦? \‒‒\‹£¡«¡‹ ? \‹ ? ‹? ⁄¡ › ¦⁄ ›«¡ ‒„?›¢?\¦¡ „ Lb›`? ‹ ‹£M T-052 Domain Movement in NADPH Cytochrome P450 Oxidoreductase: Effect of an Engineered Disulfide Bond between the FAD- and FMN Domains Chuanwu Xia , Djemel Hamdane , Anna Shen , Vivian Choi , Sang-Choul Im , Haoming 2 3 2 1 Zhang , Charles Kasper , Lucy Waskell , Jung-Ja Kim 1 2 1 2 3 3 2 Department of Biochemistry, Medical college of Wisconsin, Milwaukee, WI, United States, The University of Michigan and Veterans Affairs Medical Research Center, Ann Arbor, MI, 3 United States, McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI, United States NADPH-cytochrome P450 oxidoreductase (CYPOR) is a multidomain microsomal diflavin protein that transfers electrons from NADPH to cytochromes P450 via FAD and FMN. It has been suggested that interflavin domain movement must occur, resulting in a more open conformation than that seen in the crystal structure, to form a productive electron transfer complex with the redox partners. In order to directly evaluate whether this hypothesis is structurally relevant in solution, we constructed a mutant CYPOR (D147C-R514C) in which a disulfide bond has been engineered between residue 147 of the FMN domain and 514 of the FAD domain. The crystal structure of the mutant determined at 2.2 Å resolution reveals that the two flavin domains are indeed joined by the disulfide linkage and that the distance between the two flavins is ~5 Å (wild type, ~4 Å) and the two flavin rings are twisted ~20º from that of wild type. The steady state kinetic characterization of this mutant showed that the affinity of NADPH (and NADP+) is slightly decreased by 2-4 fold. Although the hydride transfer from NADPH to FAD proceeds normally, electron transfer from FADH2 to FMN is significantly impaired (~2% of wild type). This slow interflavin electron transfer does not, however, explain the dramatically decreased ability of the crosslinked D147C- R514C mutant -3 to reduce ferric cytochrome P450 (10 of wild type). The reduction of cytochrome P450 by the mutant is slow and incomplete, indicating that the FMN domain is no longer competent to transfer electrons to cytochrome P450 when the FMN and FAD domains are locked in a closed conformation. These results, together with our previous studies showing that a CYPOR variant with an open conformation is capable of reducing cytochrome P450 (Hamdane et al., J Biol Chem. 2009,284(17):11374-84), directly demonstrate that CYPOR adopts large conformational changes during its catalytic cycle. The enzyme adopts a closed conformation for electron transfer from NADPH to FAD to FMN and an open conformation when it interacts with cytochrome P450, its electron transfer partner. T-055 Product specificity and the role of active site water molecules in the methyltransfer reaction of SET7/9. Paul Del Rizzo , Jean-Francois Couture , Lynnette Dirk , Bethany Strunk , Marijo Roiko , 4 3 1 Joseph Brunzelle , Robert Houtz , Raymond Trievel University of Michigan, Ann Arbor, MI, United States, University of Ottawa, Ottawa, ON, 3 4 Canada, University of Kentucky, Lexington, KY, United States, Northwestern University Center for Synchrotron Research, Argonne, IL, United States SET domain lysine methyltransferases (KMTs) are responsible for the methylation of specific lysine residues in histone proteins as well as other substrates found in cellular signaling pathways. One such enzyme is human SET7/9, a monomethyltransferase that transfers a methyl group from S-adenosylmethionine (AdoMet) onto target lysine residues in histone and non-histone substrates. In the work presented here, we have characterized the mechanism underlying the product specificity of SET7/9, and two active site mutants, Y305F and Y245A, that convert the enzyme into a di- and trimethyltransferase, respectively. High resolution structures of the enzymes in complex with the product S-adenosyl-homocysteine (AdoHcy) and a series of unmodified and methylated peptide substrates (mono-, di- and tri-methylated) were obtained. Comparisons between these complexes reveal the role of active site residues, as well as the coordinating water molecules, that help to position the lysine epsilonamine group for successive rounds of methylation. These results provide the first molecular snapshots of the mono-, di- and trimethyltransfer reactions catalyzed by SET domain enzymes. 1 2 1 1,2 3 1 1 T-058 Crystal Structure of Chicken Muscle Lactate Dehydrogenase in the Presence of Oxalate, NAD and Pyruvate. Milan Draganov, L Grant, E Greiner, J Warfel, N Polder, G Watanabe, C Smith, B Rupp, X Ouyang, S Herron, C Meyer, C Srinivasan, K Kantardjieff CSU Fullerton, Fullerton, CA, United States Lactate dehydrogenase (LDH) is an essential enzyme in carbohydrate metabolism. The structure of chicken muscle LDH-A has not been reported previously, although coordinate data for orthologs are available. Protein crystallography has been used for a comprehensive structure determination and analysis of LDH-A in the upper division biochemistry laboratory and subsequent student research at Cal State Fullerton, providing insights into structural features that regulate LDH kinetic and stability properties. LDH-A was isolated and purified using established protocols. Enzymatic activity was assayed spectrophotometrically. LDH-A was co-crystallized with substrate, cofactor and inhibitor by vapor diffusion methods using commercially available screens. Diffraction data were collected from flash cooled crystals at SSRL BL9-2. Model building and refinement was achieved using the CCP4 program suite. The 1.9Å native structure has been determined by molecular replacement, using porcine LDH-A as probe, in space group P212121, a = 84.04, b = 126.78, c = 252.74, two tetramers with 222 pseudo-symmetry in the asymmetric unit, R = 0.22, freeR = 0.27. The 1.9Å structure of LDH-A complexed with pyruvate has been solved by molecular replacement, using the native chicken structure as probe, in space group C2, a = 90, b = 92.77, c = 90, β = 93.5, with one tetramer in the asymmetric unit, R= 0.19, freeR =0.24. A 1.9Å structure of the LDH-A + complexed with oxalate and NAD has been determined by molecular replacement using the native structure as probe, in space group P1 21 1, a= 72.665, b= 147.836, c= 139.642, two tetramers each with 222 pseudo-symmetry in the asymmetric unit, R = 0.21 and freeR = 0.26. A 3Å structure of the LDH-A complexed with pyruvate has been determined by molecular replacement using the native structure as a probe, in space group P1 21 1, a = 72.019, b = 146.518, c = 139.469, two tetramers each with 222 pseudo-symmetry in the asymmetric unit. Model building and refinement are in progress. Overall topology in each of the structures is consistent with crystal structures of orthologs, although secondary structure active site flexible loop rearrangements (residues 96-112) result from ligand binding and crystal contacts. The flexibility in the active site entails an “extended unit” involving much of the structure. T-067 Correlated Single-Crystal Spectroscopy and X-ray Crystallography at Beamline X26-C of the NSLS and Plans for Spectroscopy + MX Beamlines at the NSLS-II Allen Orville, Deborah Stoner-Ma, John Skinner, Dieter Schneider, Robert Sweet Brookhaven National Laboratory, Upton, NY, United States Understanding the complex relationships among atomic structure, electronic structure, and chemistry is crucial for obtaining fundamental insights into biological processes. Inspiration and breakthroughs will come from new tools developed to probe biological processes with several complementary techniques. To achieve these goals, we are accelerating the construction of an integrated beamline to enable the measurement of spectroscopic and high resolution crystallographic data. Beamline X26-C of the National Synchrotron Light Source (NSLS) is the only user facility in the US to support correlated measurements of up to three types of complementary data -- X-ray diffraction to high resolution, optical absorption spectroscopy, and Raman spectroscopy -- from the same sample and under nearly identical experimental conditions. Single-crystal electronic absorption spectra correlated with X-ray diffraction data are routinely collected from a 25µm diameter region of the crystal that intersects the X-ray beam during the readout time of each X-ray detector image. Raman spectra are also collected with either 785nm or 532nm laser excitation from the same 25µm region of the crystal that intersects the X-ray beam and the electronic absorption optical path. Integration of the controls for these spectroscopic techniques into the X-ray beamline operations software yields fully correlated atomic and electronic structures for deposition to the PDB. A complementary off-line laser spectroscopy laboratory immediately adjacent to the beamline will support additional spectroscopic techniques (e.g. fluorescence, time-resolved fluorescence). Some recent findings will be presented including correlated studies of hemebased and flavin-based macromolecules. NSLS-II is a new 3 GeV, 500 mA storage ring currently under construction at BNL. The 1nm 15 spatial resolution, 0.1meV energy resolution, flux density ( 10 ph/s/0.1%BW) and brightness 21 2 2 (2 keV - 10 keV; 10 ph/mm /mrad /s/0.1%BW) will be state-of-the-art. Several MX and Spectroscopy+MX beamlines are being designed now, with construction to commence in 2011, and full operations by 2015. Some of these plans will be discussed. Supported by the NIH National Center for Research Resources and the US Department of Energy, Office of Biological and Environmental Research. « « T-070 Human UDP-Glucose Dehydrogenase Reveals a Bifunctional Active Site: The Pin in Fischer’ s Lock Renuka Kadirvelraj , Stephen Weitzel , Nicholas Sennett , Samuel Polizzi , Zachary Wood 1 2 1 2 1 1 1 University of Georgia, Athens, GA, United States, University of Oregon, Eugene, OR, United States UDP-glucose dehydrogenase (UGDH) oxidizes UDP-glucose to UDP-glucuronic acid using + two molecules of NAD . UGDH is regulated in vivo by another nucleotide sugar, UDP-xylose, which acts as a cooperative feedback inhibitor. Using sedimentation velocity studies, we show that the substrate UDP-glucose or the inhibitor UDP-xylose induce human UGDH (hUGDH) to form a hexameric complex, while the unliganded enzyme favors the dimeric state. The available crystal structures of active hUGDH reveal a hexameric complex with 32 symmetry, best described as a ‘trimer of dimers’. We have solved four different crystal structures of the inhibited UDP-xylose:hUGDH complex, revealing a horseshoe-shaped ‘broken hexamer’ conformation. These crystal structures show that UDP-xylose binds in the active site, causing a buried loop to repack. The restructured loop alters the oligomerization interface to favor the + broken hexamer conformation. The NAD -binding site is occluded in the broken hexamer structure, suggesting a link between the oligomeric state of the enzyme and the observed cooperative inhibition kinetics. Our results show that hUGDH has evolved a bifunctional active site that can either facilitate catalysis or respond to a heterotropic effector to promote the formation of an inactive enzyme complex. T-073 Structure Determination and Characterization of Klebsiella pneumoniae HpxO, a FADDependent Urate Oxidase Katherine Hicks , Sean O’Leary , Tadhg Begley , Steven Ealick 1 2 1 2 2 1 Cornell University, Ithaca, NY, United States, Texas A&M University, College Station, TX, United States HpxO is involved in a novel purine catabolism pathway in Klebsiella pneumoniae, a multi-drug resistant pathogen. This pathway consists of five enzymes that convert hypoxanthine to allantoic acid. HpxO is responsible for the third step in the pathway, the hydroxylation of uric acid to 5-hydroxyisourate. Here we report the structural and biochemical characterization of HpxO. Our detailed biochemical studies have illustrated that, in contrast to most urate oxidases, HpxO requires a FAD cofactor for catalysis and is catalytically similar to the wellcharacterized enzyme p-hydroxybenzoate hydroxylase. Here we present the crystal structure of HpxO at 2.3 Å resolution which was solved by SeMet SAD phasing and a 2.0 Å resolution structure in complex with its substrate, uric acid. To gain further mechanistic detail, we have biochemically characterized a number of active site mutants including R204Q HpxO, which appears to uncouple the uric acid hydroxylation and FAD reduction reactions. Recently, a 2.7 Å dataset of this mutant has been collected. Based on these structures, we are able to propose a mechanism for the HpxO hydroxylation reaction. T-076 Toward understanding an unusual His-Tyr ligated c-heme: X-ray crystal structures of MauG point mutants. Lyndal Jensen , Nafez Abu Tarboush , Victor Davidson , Carrie Wilmot 1 2 1 2 2 1 University of Minnesota, Minneapolis, MN, United States, The University of Mississippi Medical Center, Jackson, MS, United States MauG is a di-heme enzyme responsible for the post-translational modification of two tryptophan residues to form the tryptophan tryptophylquinone cofactor (TTQ) of methylamine dehydrogenase (MADH). MauG converts preMADH, containing monohydroxylated-β Trp57, to mature MADH by catalyzing the insertion of a second oxygen atom into the indole ring and covalently linking β Trp57 to β Trp108. We recently reported the X-ray crystal structure of MauG from Paracoccus denitrificans in complex with its substrate, preMADH (PDB code 3L4M). The structure revealed that the two c-hemes of MauG and the nascent TTQ site of preMADH are separated by long distances over which electron transfer must occur to achieve catalysis. One of the c-hemes has an atypical His-Tyr axial ligation involving residues His205 and Tyr294. While cytochrome c peroxidases (CCPs) have the highest sequence similarities with MauG (up to ~30% homology), they exhibit His or Met axial ligands at the MauG Tyr294 position. A key distinction for MauG is that its 6-coordinate c-heme iron attains the Fe(IV) state during enzyme turnover (i.e. TTQ catalysis), with concomitant formation of an Fe(IV)=O species at its other c-heme. As such, we are interested in understanding the coordination and stabilization of this new member of the c-heme family. We present here the X-ray crystal structures of MauG-Tyr294X mutants in their diferric resting states, complexed with preMADH. T-079 Twisting of the DNA Binding Surface By A Beta-Strand-Bearing Proline Modulates DNA gyrase Activity Nei-Li Chan , Tung-Ju Hsieh , Tien-Jui Yen , Te-Sheng Lin , Hsun-Tang Chang , Shu-Yun 2 1 3 Huang , Chun-Hua Hsu , Lynn Farh 1 3 1 1,2 1 1,2 2 National Taiwan Univ., Taiwan, Taiwan, National Chung Hsing Univ., Taiwan, Taiwan, National Pingtung Univ. of Education, Taiwan, Taiwan 2 DNA gyrase is the only topoisomerase capable of introducing (-) supercoils into relaxed DNA. The C-terminal domain of the gyrase A subunit (GyrA-CTD) and the presence of a gyrasespecific “GyrA-box” motif within this domain are essential for this unique (-) supercoiling activity by allowing gyrase to wrap DNA around itself. Here we report the crystal structure of Xanthomonas campestris GyrA-CTD and provide the first view of a canonical GyrA-box motif. This structure resembles the GyrA-box-disordered Escherichia coli GyrA-CTD, both adopting a non-planar -pinwheel fold composed of 6 seemingly spirally arranged -sheet blades. Interestingly, structural analysis revealed that the non-planar architecture mainly stems from the tilted packing seen between blades 1 and 2, with the packing geometry likely being defined by a conserved and unusual β -strand-bearing proline. Consequently, the GyrA-boxcontaining blade 1 is placed at an angled spatial position relative to the other DNA-binding blades, and an abrupt bend is introduced into the otherwise flat DNA-binding surface. Mutagenesis studies support that the proline-induced structural twist contributes directly to gyrase’ s (-) supercoiling activity. To our knowledge, this is the first demonstration that a β strand-bearing proline may impact protein function. Potential relevance of β -strand-bearing proline to disease phenylketonuria is also noted. ¬ ¬ T-082 Structural Basis of the Nucleotidase Activity for HAD Super Family Member P4 from Haemophilus influenzae. Harkewal Singh, Thomas J. Reilly, John J. Tanner Univ. of Missouri, Columbia, MO, United States Class C non-specific acid phosphatases catalyze the transfer of phosphoryl group from phosphomonoesters to water at acidic pH using an active-site aspartate residue and represent a major subgroup of haloacid dehalogenase (HAD) superfamily. The acid phosphatase e (P4) from Haemophilus influenzae is a major component of outer membrane of the organism, and it is highly conserved among H. influenzae strains, making it an attractive vaccine candidate. The main biological role of e (P4) is to catalyze the conversion of + nicotinamide mononucleotide (NMN) to nicotinamide riboside (NR) as part of vestigial NAD utilization pathway. We show that e (P4) is promiscuous enough to catalyze the hydrolysis of various 2’, 3’ and 5’ mononucleotide phosphates. Furthermore, we used high resolution X-ray crystallography to understand the basis of substrate promiscuity. An active site mutant (Asp to Asn) of e (P4) was engineered using site directed mutagenesis and structure of NMN, 5’AMP, 2’AMP and 3’AMP enzyme-substrate complexes were obtained at 1.35 Å, 1.55 Å, 1.90 Å and 1.85 Å resolution respectively. We also obtained a high-resolution crystal structure of e (P4) complexed with its very potent inhibitor adenosine 5′ -O-thiomonophosphate (AMPS) at 1.35 Å. The e (P4) –AMPS complex structure shows a different conformation of AMPS in the active site as compared to the binding of its natural substrate NMN. The difference is due to the larger size and lower electro negativity of sulfur (S) compared to oxygen (O) Finally, steady state kinetics parameters of P4 with its substrates NMN, 5’AMP, 2’AMP, 3’AMP and inhibitor AMPS, were determined using discontinuous colorimetric assay. T-085 Structural basis for molecular recognition in the mouse AKR1C13-NAD complex Debanu Das, Ashley Deacon JCSG-SSRL, SLAC National Accelerator Laboratory, Menlo Park, CA, United States Proteins in the Aldo-Keto Reductase (AKR) superfamily are involved in metabolism and human disease, including cancer. Subfamily C of AKR family 1 (AKR1C) has 25 members that are all mammalian proteins (AKR1C1-C25) and they share more than 60% sequence identity. Of these, only 7 members C1-C5, C9 and C21 have been structurally characterized. AKR1C13 is believed to play an important role in detoxification in the mouse stomach. Biochemical characterization by others has shown that AKR1C13 has some key differentiating features compared to its subfamily members: a broader substrate specificity and preference for non-steroidal alcohols; and the use of NAD as cofactor instead of NADP. We have determined an ultra-high resolution 1.18 Å crystal structure of the mouse AKR1C13NAD complex in the JCSG that now provides a 3D basis for explaining some of the biochemical differences. The structure reveals that Glu276 is the determinant for selecting NAD as a cofactor, compared to Arg276 in family members that use NADP, which requires additional interactions with the phosphate moiety. Tyr55 and His117 interact with the nicotinamide ring and are conserved in family members. Residue 54, a determinant of substrate specificity, is present as Ala (Leu, Val or Phe in C1-C5 and C9) and nearby an MPD-like molecule in the active site. Tyr24, Asp128 and Phe129 that are known to be important in substrate interactions are poised around the MPD. Lys27 and Ser137, located near the substrate entry point, may play a role in initial substrate recognition. The JCSG is funded by NIGMS/PSI, U54 GM074898. SSRL operations are funded by DOE BES, and the SSRL SMB program by DOE BER, NIH NCRR BTP and NIH NIGMS. T-091 Kinetic and Structural Charaterization of the GTP-Dependent Aminoglycoside Phosphotransferases from the APH(2”) family Clyde Smith , Marta Toth , Hilary Frase , Laura Byrnes , Sergei Vakulenko 1 2 1 2 2 3 2 Enterococcal SSRL, Menlo Park, CA, United States, University of Notre Dame, Notre Dame, IN, United 3 States, Cornell University, Ithaca, NY, United States The aminoglycoside (2”) phosphotransferases (APH(2”)s) are a family of four protein kinaselike enzymes (APH(2”)-Ia, APH(2”)-IIa, APH(2”)-IIIa, and APH(2”)-IVa) which are a major cause of acquired resistance to the aminoglycoside antibiotics in enterococci and staphylococci. The APH(2”)-Ia enzyme is the best studied and is capable of phosphorylating virtually all known aminoglycosides including 4,5-disubstituted drugs such as neomycin, and 4,6-disubstituted drugs such as kanamycin and gentamicin. The enzyme exists as the Cterminal half of a bifunctional molecule, the N-terminal domain comprising an aminoglycoside acetyltransferase, AAC(6’)-Ie. The other three APH(2”) enzymes are all monofunctional, single domain enzymes which react with a smaller subset of the drugs, and appear to be selective for the 4,6-disubstituted aminoglycosides. The APH(2”)-IIa, APH(2”)-IIIa and APH(2”)-IVa enzymes phosphorylate only the 4, 6-disubstituted aminoglycosides at the 2” 3 6 -1 -1 position with kcat/Km values in the range 10 - 10 M s . The nucleotide substrate specificity for each enzyme has been analyzed and we have found that GTP is a substrate for all four enzymes and that furthermore, two of the enzymes have a preference for GTP over ATP (APH(2”)-Ia has a 250-fold preference and APH(2”)-IIIa has a 400-fold preference in terms of Km). This is unprecedented amongst the phosphotransferase enzymes, and in the protein kinases in general. The crystal structures of all four APH(2”) enzymes have been solves in order to ascertain the structural determinants of substrate binding, specificity and selectivity. Analysis of the nucleotide binding site shows the presence of two overlapping hydrogen bonding templates responsible for the selective binding of GTP and ATP. We feel that the understanding we now have of the ways in which these enzymes interact with nucleotides and aminoglycoside antibiotics may lead to novel ways of inhibiting these enzymes. T-094 Atomic resolution structure of the cytoplasmic domain of Yersinia pestis YscU, a regulatory switch involved in type III secretion George Lountos, Brian Austin, David Waugh Macromolecular Crystallography Laboratory, National Cancer Institute at Frederick, Frederick, MD 21702, United States Yersinia pestis and many other gram-negative bacterial pathogens use a type III secretion system (T3SS) as a protein transport apparatus to inject a number of effector proteins through a hollow needle that penetrates the cytosol of eukaryotic cells, thereby enabling the pathogen to defeat the immune response of the host. The export apparatus consists in part of cytoplasmic and inner-membrane proteins that identify T3SS substrates and control the switching of substrate specificity during morphogenesis and host-cell contact. YscU, an essential component of the secretion apparatus, is composed of a transmembrane N-terminal domain and a C-terminal cytoplasmic domain that are separated by a long, conserved linker region. The cytoplasmic domain undergoes auto-cleavage of the N263-P264 peptide bond at a conserved NPTH motif, resulting in N- and C-terminal fragments that remain tightly bound. The N263A mutant inhibits auto-cleavage and blocks the export of pore-forming translocators but not effector proteins. Therefore, it has been proposed that cleavage results in a conformational change that triggers the recognition and export of translocators during assembly of the T3SS. Crystal structures of the noncleaved and cleaved YscU cytoplasmic domain were solved at 1.5 and 1.1 Å resolution, respectively, by SAD phasing. The structure of the noncleaved YscU-N263A mutant consists of a four-stranded, mixed -sheet that is surrounded by five -helices. The NPTH motif is located on a -turn between strands 1 and 2. The structure of cleaved YscU reveals that the core protein structure remains essentially the same, however, cleavage of the N263-P264 scissile bond results in a rearrangement of the NPTH loop that exposes residues that were buried by the -turn, thus forming a new surface that may be a recognition site for other T3SS proteins. Additionally, there is a conformational change that also occurs in the N-terminal linker region in which the N-terminal helix switches into a mostly disordered conformation that likely has a significant impact on the orientation of the cytoplasmic domain with respect to the membrane-bound domain. This conformational change may imply a regulatory role for this region which influences substrate specificity. ® T-097 The crystal structure of AroE from Pseudomonas putida and insights into substrate binding in the shikimate dehydrogenase superfamily James Peek , Sasha Singh , Kay Chen , Dinesh Christendat University of Toronto, Toronto, Ontario, Canada, Massachusetts, United States 1 2 1 2 1 1 Harvard Medical School, Boston, AroE catalyzes the reversible NADPH-dependent reduction of dehydroshikimate to shikimate. This reaction represents the fourth step in the shikimate pathway, the common route for the biosynthesis of the aromatic amino acids in plants, fungi, bacteria and apicomplexan parasites. The absence of this pathway in metazoans makes the enzymes in the pathway attractive targets for herbicides and antimicrobials. AroE belongs to the shikimate dehydrogenase (SDH) superfamily which consists of at least five members that share similar overall structures, but bind a range of substrates. Through comparative analysis, we are exploiting the limited structural variability among SDH homologs to understand modes of substrate discrimination used by the enzymes. The results of this study may be relevant to the rational design of drugs targeting the SDH homologs. We have recently solved the crystal structure of one member of the SDH superfamily, AroE-Like1 (Ael1) from Pseudomonas putida. Here, we present the structure of AroE from the same species. Interestingly, both enzymes are capable of accepting shikimate, but Ael1 binds the substrate with a dramatic reduction in Km. Comparison of the AroE and Ael1 structures reveals a high level of conservation among substrate binding residues. However, subtle variations in active site architecture appear to result from differences in residues in a secondary active site layer. These variations may contribute to the observed difference in the binding affinity of the two enzymes. T-100 A pair-based approach to structural homology using quaternion SLERP averaging Herbert J. Bernstein , Lawrence C. Andrews 1 1 2 2 Dowling College, Oakdale, NY, United States, Micro Encoder, Kirkland, WA, United States We present an atom-pair-based alternative to the Kabsch algorithm [Kabsch 1978] for measuring structural homology between commensurate molecular fragments that is able to reproduce the results of the Kabsch algorithm for fragments that can be mapped into each other by a single rigid body motion. In addition, the pair-based algorithm is able to illuminate cases in which different rigid body motions are required to bring various substructures into the alignment. The Kabsch algorithm is the gold standard for measuring structural homology between two molecular fragments with the same numbers of atoms in each of two fragments with the same connectivity. The Kabsch algorithm is based on first centering each of the two fragments on their centroids and then computing the covariance matrix of the first fragment against the other. Our pair-based approach takes pairs of atoms, one atom from the first fragment and the matching atom from the second fragment, and computes the plane dividing the line between them. Then, taking an appropriate sampling of pairs of pairs yields an axis, and, most importantly, an angle of rotation from the intersections of the planes separating the pairs of atoms. Each axis plus an angle determines a quaternion. If the quaternions are averaged using Spherical Linear Interpolation (SLERP), the result agrees with the results of the Kabsch algorithm. As noted by Ye and Godzik [Ye Godzik 2003], “When flexible molecules in different conformations are compared to each other as rigid bodies, even strong structural similarities can be missed and significant errors in alignments can occur because such algorithm compensate global rearrangements with local alignment shifts.” Their “FATCAT” algorithm works in terms of rigid body movements of fragments. Our pair-based algorithm can be used in a similar manner, but is not dependent on the identification of fragments, nor on the identification of translations, going directly to the identification of the rotations needed. [Kabsch 1978] Kabsch, W. (1978), “A discussion of the solution for the best rotation to relate two sets of vectors”, Acta Cryst. A34:5, 827 – 828. [Ye Godzik 2003] Ye, Y., Godzik, A. (2003), “Flexible structure alignment by chaining aligned fragment pairs allowing twists,” Bioinformatics 19, suppl. 2., ii246 -- ii255. T-103 The Relevance of H-Bonding to the Relative Thermodynamic Stability of Polymorphs Alicia Ng, Chiajen Lai, Feng Qian, Baoqing Ma, Qi Gao Bristol-Myers Squibb, New Brunswick, NJ, United States Relative thermodynamic stability between crystalline polymorphs can be established in most cases, however, the phenomenon is not usually well understood. Nevertheless, insight into this important property can be obtained by examining the crystal structure, which offers a wealth of information about the intrinsic nature of a crystalline solid. In this presentation, the contribution of H-bonding to relative thermodynamic stability of true polymorphs is evaluated. The compounds showcased in this discussion offer a good system for comparing structures containing strong H-bonding to their polymorphic counterparts which have only weak or no Hbonding. In essence, the effects of H-bonding to 3-dimensional molecular packing is compared to molecular packing that relies mainly on van der Waals forces and/or stacking interactions, and how these differences translate into thermodynamic stability of true polymorphs. The correlation between molecular conformation and H-bonding characteristic is also henceforth discussed. T-106 PSI:BIOLOGY: High-Throughput-Enabled Structural Biology Ward Smith, Ravi Basavappa, Jean Chin, Charles Edmonds, Paula Flicker, Peter Preusch, Janna Wehrle, Catherine Lewis, Jeremy Berg National Institute of General Medical Science, National Institutes of Health, Bethesda, MD, United States The National Institute of General Medical Sciences has announced PSI:Biology (Protein Structure Initiative:Biology) to continue the development of high-throughput structural biology methods and apply them to important biological problems. This will be accomplished by establishing a network of collaborations between centers for structure determination and biologists with interests in problems involving particular proteins or collections of proteins that would benefit from structural information. The collaborations will be established through separate awards to investigators outside the structure determination centers. These awards are named Consortia for High-Throughput-Enabled Structural Biology Partnerships and successful applicants will help to define targets for structure determination by the centers and will receive funds for functional studies in the applicants’ laboratories. This mechanism provides an on-going opportunity for the wider biomedical research community to obtain funding to participate in the PSI through collaboration with the high-throughput structure determination centers and with the centers for membrane structure determination. The PSI:Biology Network will include Centers for High-Throughput Structure Determination, Centers for Membrane Protein Structure Determination, The PSI:Materials Repository and The PSI:Biology Knowledgebase and the Consortia investigators. In addition, R01 and P01 applications are solicited from individual investigators or groups of investigators to participate in the PSI:Biology Network. The applicants may propose to further develop high-throughput structural biology methods, novel methods for comparative molecular modelling or additional biological partnerships as member of the PSI:Biology Network Individual researchers may also suggest proteins for structure determination by the PSI Structure Determination Centers on the PSI Knowledgebase Community Nomination site. T-109 Crystallographic studies of human Kynurenic Acid Transaminase II (hKAT II), a novel schizophrenia target. Marie Anderson , Artem Evdokimov , Jay Pandit , Kim Fennell , Patrick Verhoest , Brian 1 1 Campbell , Jim Valentine 1 1 2 1 1 1 Pfizer, Groton, CT, United States, Monsanto, St.Louis, United States 2 KATII has emerged as a potentially attractive target for the treatment of Schizophrenia. KATII, a pyridoxal phosphate dependant enzyme catalyzes the conversion of L-kynurenine to kynurenic acid (KYNA), and is part of the tryptophan metabolism pathway. KYNA can act as a competitive antagonist at the glycine site of NMDA receptors. Reduced NMDA receptor function is predicted to contribute to symptoms of Schizophrenia. It is hypothesised that reducing KYNA blockade of the glycine co-agonist site should increase NMDA receptor function, which could lead to a novel antipsychotic agent. A high-throughput screen of our corporate sample collection led to the discovery of several potent inhibitors of KAT II activity. In this report we describe the x-ray structure of one such inhibitor co-crystallized with KAT II, which can serve as the starting point for structure-based optimization. T-113 Use of the ScreenMachine as a tool to augment graduate-level Biophysics education Bryan Sutton Texas Tech University Health Sciences Center, Lubbock, TX, United States For biologically-oriented students, X-ray crystallography is a daunting aspect of their graduate education. They are typically ill-prepared for a comprehensive mathematical treatment of the subject, and they are usually not familiar with the basic principles of optical physics; however, it is crucial that these students gain a working knowledge of the strengths and weaknesses of the technique. At Texas Tech University, we are experimenting with an innovative concept in graduate education to bridge between the familiar discipline of biochemistry and the less familiar physics of X-ray crystallography. Our Deep Saturation Mutagenesis (DSM) course seeks to study all possible mutations at all possible sites of one enzyme. Each student will be assigned a single point mutation to introduce into the Synechocystis glutaredoxin gene. Analysis of each mutation will range from the enzymatic consequences of the mutagenesis to the X-ray crystal structure of the soluble, mutated enzyme and management of the data through LIMS systems. Throughout the course, documentation of all results will be maintained with the intention of publication once the entire DSM space has been covered (~8000 mutations). Our aim is to make this a viral course by pre-packaging lecture material as YouTube-style lectures and tasks for the course to accommodate a wide variety of potential students. The X-ray diffraction aspects of the DSM course will center on the ScreenMachine. This is a non-threatening, robust instrument that does not require specialized personnel or extensive instruction. Further, it does not present as great of a radiation hazard for students relative to a full-sized x-ray instrument, so it is well-suited for this task. Our objective is to provide these students with a workable knowledge of x-ray crystallography, with the hope that they will pursue a more in depth study of the discipline afterward. Further, this concept links real-world biophysical research with graduate education to accomplish useful science for the community at large. T-116 Study of Serine Dehydrogenase from Mycobacterium tuberculosis Dana Hogan , William L. Duax 1 1 1,2 2 Hauptman Woodward Medical Research Inst., Buffalo, NY, United States, State Univ. of New York at Buffalo, Buffalo, NY, United States Mycobacterium tuberculosis, the etiologic agent of tuberculosis in humans, is responsible for the global resurgence of the disease affecting more than 30 million people worldwide. Current preventative drugs have been effective at inhibiting the expression of the bacterium; however several antibiotic-resistant strains are starting to emerge. This study was undertaken to identify and characterize the structure of a putative L-Serine Dehydrogenase (SerDh), an important enzyme involved in the metabolic pathway of at least 8 other amino acids. SerDh belongs to the superfamily of proteins known as the Short Chain Oxidoreductase Enzymes (SCORs), which are important in growth and development of all organisms. The cofactor predicting sequence XR, * occurs 19 residues from the Gly-rich N-terminal motif, indicting NADP binding . Substrate binding in SCOR proteins occurs within 3 loops. There are 5 quasi-conserved amino acids in 2 of those loops that can predict a substrate for the enzyme. SerDh contains 4 of the 5 identical positions with the known serine dehydrogenase fingerprint (AG-YGG represents 164 proteins). The 4 positions are the amino acids [G]G-YGG with the bracketed G replacing the A in the fingerprint. A deeper understanding of the proteins that are critical to the function of Mycobacterium tuberculosis could lead to the design more effective inhibitors that will interrupt the expression of the disease. Support in part by: Mr Roy Carver, Stafford Graduate Fellowship, Caerus Forum Fund and The East Hill Foundation. * Proteins. 2003 Dec 1;53(4):931-43. T-119 A Fast And Fully Automated Solution For Lipidic Cubic Screening (LCP) using Mosquito LCP Joby Jenkins, Patricia Edwards, Rob Lewis, Joanne Franklin TTP LabTech Ltd., Melbourn, United Kingdom Membrane proteins such, as G-protein coupled receptors, are known to be much more difficult to purify and crystallise than soluble proteins due to their native environment within the lipid bilayer of the cell membrane. As a result aqueous solutions are unsuitable for their reconstitution as they require lipids or detergents to retain their structural integrity. The in meso crystallisation technique revolutionised the process of crystallising membrane proteins. This method utilises highly viscous lipid mesophases to contain the membrane proteins for crystallisation. However, there are a number of technical difficulties associated with the LCP method which makes this process difficult to perform and challenging to automate. One problem is the viscous nature of the lipids which can be almost solid at room temperature. As a result the addition of protein to the lipid and subsequent reconstitution can be hard to achieve. In addition, the accurate dispensing of LCP, required for efficient miniaturisation, and the precise positioning of drops required for efficient imaging of membrane crystals present two other challenges. TTP LabTech have solved this problem by developing mosquito LCP, a dedicated instrument that offers a fully automated solution to LCP screening. This instrument offers fast throughput, high precision and unrivalled reproducibility. Here we describe the benefits of the instrument and how the renowned and reliable positive displacement tip technology ensures that the LCP screening preparation is performed to the highest standard with the minimum amount of effort. ® sLPQQ b›«fi· \ ›‹\ ?«› ¡ ?›¢?¦‒„ \ ‹¡?fi‒› ¡ ‹? „‹\« ¦ M c¡« \‹?q ¦¦\‒ K?p \‹£?b· K?f¡›‒£¡?mM?o⁄ fi K?i‒ In this study, the variance-covariance matrix of protein motions is used to compare several elastic network models within the theoretical framework of X-ray scattering from crystals. A set of 33 ultra-high resolution structures is used to characterize the average scaling behavior of the vibrational density of states and make comparisons between experimental and theoretical temperature factors. Detailed investigations of the vibrational density of states, correlations, and predicted diffuse X-ray scatter are carried out for crystalline Staphylococcal nuclease; correlations and diffuse X-ray scatter are also compared to predictions from the TLS (translation, libration, screw) model and a liquid-like dynamics model. We show that elastic network models developed to best predict temperature factors without regard for the crystal environment have relatively strong long-range interactions that yield very short-ranged atom-atom correlations. From atom-atom correlations, which are found to require more modes to converge than is typically assumed, the diffuse X-ray scatter is computed to explore practical implications for such models; for this we use a novel approach that decomposes the intensity into contributions from different types of correlated motions. ¶¼»¹» ¼»±° µ °³¶±º¹¸ °±¶°³·¶±µ ´³ ²±°¯ K? K? hK?t ?r T-125 Fiber diffraction, electron microscopy, and molecular modeling in studies of complex assemblies Wen Bian, Amy Kendall, Michele McDonald, William Wan, Gerald Stubbs Vanderbilt University, Nashville, TN 37235, United States Fiber diffraction has been used to determine the structures of filamentous macromolecular assemblies that are not amenable to conventional crystallography or nuclear magnetic resonance methods. However, many such assemblies are highly disordered and diffract poorly even with the best available sample preparation and diffraction techniques, so that their structures cannot be solved using fiber diffraction alone. We have obtained fiber diffraction data for two important types of assembly, amyloids (including prions) and flexible filamentous plant viruses, and have used a combination of fiber diffraction, electron microscopy, and molecular modeling to derive important structural features and produce low to medium resolution models for these systems. We are continuing to develop computational methods to make use of the data provided by fiber diffraction and electron microscopy, providing important constraints for helical reconstruction and molecular modeling. Supported by NIH grant P01 AG010770 and NSF grant MCB-0743931 T-128 The Absolute Intensity Calibration of a Small-Angle X-ray Scattering Instrument with a Laboratory X-ray Source Lixin Fan , Mike Degen , Scott Bendle , Nick Grupido , Jan Ilavsky 1 1 1 1 1 2 2 Rigaku Innovative Technologies Inc., Auburn Hills, MI, United States, Argonne National Laboratory, Argonne, IL, United States Advanced Photon Source, Absolute calibration of small-angle scattering data (in units of differential cross-section per unit sample volume per unit solid angle) is necessary for the determination of molecular weights, the number density of particles, the scattering-length densities of phases in multiphased systems, volume fraction, the specific surface area of the scatters and to restrict the parameters of a given model to the set which reproduces the observed intensity. It is also a useful means for the detection of artifacts in SAS experiments. Absolute intensities from the same sample also allows intercalibration among different instruments. This work details the absolute calibration procedure of a small-angle X-ray scattering instrument, the Rigaku S-Max3000. Absolute calibration was achieved by using two standards: homogeneous and stable glassy carbon and water. The scattering intensity of glassy carbon is calibrated by comparison with the absolute-calibrated measurements taken on the USAXS instrument located at the 32ID beamline of the Advanced Photon Source in Argonne National Laboratory. This instrument has primary calibration capability. The scattering from water is angle-independent and only depends on the physical property of isothermal compressibility. The absolute calibrations using two standards were compared. The agreement of scale factors obtained using two standards suggests that precalibrated glassy carbon can serve as a convenient standard for all type materials under study. T-131 High-Efficiency SAXS/GISAXS/WAXS instrument for the Laboratory: Rigaku S-Max3000 Nick Grupido, Lixin Fan, Michael Degen, Scott Bendle Rigaku Innovative Technologies, Inc., Auburn Hills, MI, United States The Rigaku S-MAX-3000 instrument provides excellent SAXS, GISAXS and simultaneous WAXS capabilities while maintaining maximum flexibility in controlling sample environment [1]. This instrument utilizes a high brilliance X-ray microfocus source running at 40W of power. Its state-of-the art design concentrates the applied power into a tiny spot which, when coupled with a confocal graded multilayer focusing optic, yields a high intensity x-ray beam comparable to conventional laboratory sources operating at kilowatt power. Combining this intense beam with three-pinhole collimation, a fully evacuated beam path and a photoncounting MWPC detector, this instrument is capable of making highly sensitive measurements from both isotropic and anisotropic materials without neeeding desmearing corrections. A photodiode embedded inside the beamstop allows continuous monitoring of the beam intensity yielding a direct measurement of the sample transmission. A second optional sample chamber allows exploration of a middle Q range without moving the detector or realigning the beam. High throughput SAXS measurements can be performed by running a user friendly script which automatically controls sample movement and environment. Simultaneous WAXS measurements can be collected on image plates at scattering angles up to 68° An automated . high weight capacity stage for GISAXS provides better than 5 arc second angular precision o o and motion ranges of 8 in plane, 10 out of plane and 12.5mm vertical. The high weight capacity can be utilized to support in-situ vessels or samples of all types. An alternative high precision stage is available with sub-arc second motion but lower weight capacity. Both stages are fully automated with automatic determination of the zero incident angle as well as automatic collection of the scattering data. The Rigaku S-MAX-3000 is capable of characterizing a large variety of materials, ranging from colloids of all types, cements, nanoparticles, oils, polymers, plastics, proteins, surfactants, foods and pharmaceuticals. In this presentation, we demonstrate Rigaku GISAXS capability by determining structural morphology from polymer thin films. http://www.rigaku.com/saxs/index.html ½ ½ ½ T-134 Case studies from the structural genomics of infectious disease Anna Gardberg , Thomas Edwards , Jan Abendroth , Michelle Dietrich , Becky 1,4 1,4 1,4 1,4 1,4 Poplawski , Jameson Bullen , Jeff Christensen , Eric Smith , Nathan Ng , Taryn 1,4 1,4 1,4 1,4 3,4 Haffner , Amy Raymond , Don Lorimer , Bart Staker , Alberto Napuli , Wes 3,4 2,4 2,4 1,4 VanVoorhis , Robin Stacy , Peter Myler , Lance Stewart 1 1,4 1,4 1,4 1,4 Emerald BioStructures, Bainbridge Island, WA, United States, Seattle Biomedical Research 3 Institute, Seattle, WA, United States, University of Washington, Dept of Medicine, Seattle, 4 WA, United States, Seattle Structural Genomics Center for Infectious Disease, Seattle, WA, United States The Seattle Structural Genomics Center for Infectious Disease (SSGCID) is one of two consortia funded by NIAID to apply genome-scale approaches in solving protein structures from biodefense organisms, as well as those causing emerging and re-emerging disease. In its first two years, the SSGCID has submitted ~170 protein structures to the Protein Data Bank (PDB) and is on track to solve a further 100 per year going forward. For several organisms, this represents the majority of PDB submissions during this time, including 100% of the structures for Ehrlichia, Anaplasma, and Burkholderia. SSGCID’s target selection strategy has focused on drug targets, essential enzymes, virulence factors and vaccine candidates from a number of bacterial (Bartonella, Brucella, Ehrlichia, Anaplasma, Rickettsia, Burkholderia, Borrelia and Mycobacterium) and eukaryotic (Babesia, Cryptosporidium, Toxoplasma, Giardia, Entamoeba, Coccidioides and Encephalitozoon) pathogens, as well as ssDNA and negative-strand ssRNA viruses. More than 3000 targets have been selected to date, with >700 proteins being purified for crystallization trials. Crystallization screening and analysis of X-ray diffraction datasets for structure solution are performed at Emerald BioStructures. We present a selection of protein crystal structures solved at Emerald as part of its work with the SSGCID. Individuals or groups of investigators interested in proposing a target for structure determination at the SSGCID are requested to submit a “Target Selection Proposal”. 2 T-137 The c-AMP Receptor-Like Protein CLP is a Novel c-di-GMP Receptor Linking Cell-Cell Signaling to Virulence Gene Expression in Xanthomonas campestris Shan-Ho Chou , Ko-Hsin Chin 1 1,2 1 National Chung-Hsing U., Taichung, Biotechnology Center, Taichung, Taiwan Taiwan, 2 National Chung Hsing University C-di-GMP controls a wide range of functions in eubacteria, yet little is known about the underlying regulatory mechanisms. In the plant pathogen Xanthomonas campestris, expression of sub-set of virulence genes is regulated by c-di-GMP and also by the CAP-like protein XcCLP, a global regulator in the CRP/FNR superfamily. Here, we report structural and functional insights into the interplay between XcCLP and c-di-GMP in regulation of gene expression. XcCLP bound target promoter DNA with sub- M affinity in the absence of any ligand. This DNA-binding capability was abrogated by c-di-GMP, which bound to XcCLP with M affinity. The crystal structure of XcCLP showed that the protein adopted an intrinsically active conformation for DNA binding. Alteration of residues of XcCLP implicated in c-di-GMP binding through modeling studies caused a substantial reduction in binding affinity for the nucleotide and rendered DNA binding by these variant proteins insensitive to inhibition by cdi-GMP. Taken together, the current study reveals the structural mechanism behind a novel class of c-di-GMP effector protein in the CRP/FNR superfamily and indicates that XcCLP regulates bacterial virulence gene expression in a manner negatively controlled by the c-diGMP concentrations. ¾ ¾ T-143 Structure Determination and Biochemical Analysis of TR4, a Retinoid-activated Nuclear Receptor Ross Reynolds , X. Edward Zhou , Kelly Suiino-Powell , Yong Xu , Schoen Kruse , Eric Xu 1 2 1,2 2 2 2 2 2 Grand Valley State Univ., Allendale, Michigan, United States, Van Andel Institute, Grand Rapids, Michigan, United States The atomic structure of the apo form of the LBD of the orphan nuclear receptor TR4 has been determined. Similar to the structure of COUP II TF the protein crystallizes in the autorepressed inactive apo form. Evidence is presented for the active form of the receptor to be a homodimer as that present in the crystal. Biochemical assays and mutagenesis studies have been performed which show that the ligand is likely to be a retinoic Acid derivative and that a cofactor similar to SRC-1 is necessary for ligand binding and activation. Molecular 3 modeling indicates an active site pocket in the 600 – 700 Å range, consistent with a retinoid ligand and results of binding experiments of over 60 retinoid related ligands are presented. Based on this lab’ s studies one can describe TR2/TR4, the COUP-TF’ s, a the RXR’ s as a nd group of retinoid-activated nuclear receptors. T-146 Structural Proteomics Effort with Protein Tyrosine Phosphatases : Experience & Perspective Dae Gwin JEONG , Tae-Sung YOON 1 1,2 1,2 2 KRIBB, Daejeon, Korea, Republic of, University of Science & Technology, Daejeon, Korea, Republic of Protein tyrosine phosphatases (PTPs) consisting of 103 human genes encoding the conserved catalytic domains with CXXGXXR motifs [Alonso A et al, (2004). Protein Tyrosine Phosphatases in the Human Genome, Cell 117:699-711] are an important family of signal transduction proteins, together with other protein phosphatases and protein kinases, controlling cellular protein phosphorylation which plays an important role in human disease conditions. We have participated in an ‘in-house’ proteomics project, of which resources are now commercially available as more than 80 purified proteins (www.bioneer.com) and several monoclonal antibodies (www.younginfrontier.com). Along with this line of effort, we have contributed in the structural elucidation of more than sixteen catalytic domains of PTPs. Among them, we have deposited eight structures of dual-specifity protein phosphatises (DUSPs) at PDB (3EZZ, 2NT2, 2GWO, 2G6Z, 1ZZW, 2ESB, 1YZ4, 1XM2). Complementary with the world-wide efforts of structural genomics consortium, we have focused on the structural elucidation of catalytic domains of DUSPs, which consist of more than half of PTPs and ‘relatively’ less understood compared to ‘classical’ PTPs. We believe that the development of specific ‘modulators’ for DUSPs is of significant importance to further elucidate their physiological roles in molecular contexts. Together with the progress of structural elucidation of DUSPs, we hope that the proteomics research better to understand the physiological role of DUSPs in molecular context will be leveraged with the ‘better’ development of specific modulators among DUSPs. T-149 Franck Borel , Isma Hachi , Andres Palencia , Marie-Claude Gaillard , Jean-Luc Ferrer 1 2 1 1 2 3 1 Intitut de Biologie Structurale, Grenoble, France, Europeen Molecular Biology Laboratory, 3 Grenoble, France, Institut de Biologie et de Technologies de Saclay, Gif-sur-Yvette, France Mu-Crystallin (or CRYM) was first described as a major structural component of the eye lens in Australian marsupials. This cytoplasmic protein was identified, although in much lower quantities, in other mammals where it has been found in eye, ear, heart, kidney, brain, muscle, skin. CRYM is structuraly close to bacterial ornithine cyclodeaminase (OCD) and alanine dehydrogenase (alaDH) but do not display any of their enzymatic activities. To date there is no enzymatic activity identified for CRYM. However CRYM has been characterized as an NADPH-dependent cytosolic T3 thyroid hormone binding protein. Thyroid hormones are produced by the thyroid gland and play important regulatory roles in process such as growth, metabolism, homeostasis or development. They are secreted under two forms T4 (thyroxine or 3,5,3',5'-tetraiodo-L-thyronine) and T3 (triiodothyronine or 3,5,3'-triiodo-L-thyronine); the most abundant T4 is later converted, by a selenium deiodinase, into the less abundant but more active T3 form. Thyroid hormones exert their action by interacting with their cognate nuclear receptor to regulate the transcription of target genes. Currently the mechanism of CRYM action involves its dimerisation in the cytoplasm followed by the binding of NADPH. NADPH activated CRYM binds T3 and induces an increase of hormone concentration in the cytoplasm. Whereas the action of CRYM-bound T3 is suppressed, dissociation of NADPH enables the release of free T3 that can transactivate genes expression. Abnormal CRYM expression have been linked to syndromes as diverse as hyperglycemia, muscular dystrophy, deafness or prostate cancer. In the our study we compare three crystal structures of mouse CRYM. We solved the structure of the apo form, the structure of the complex with NADPH and also the structure of the ternary complex with NADPH and T3; the first one of a NADPH-dependent cytosolyc T3 binding protein containing the two ligands. This structural analysis coupled to in silico docking experiments, thermodynamic and kinetic parameters determination provide new insight into the sheltering of T3 hormone by CRYM protein. T-152 Structural Characterization of Inositol Catabolic Enzymes Karin van Straaten, Ryan Stubbing, David Palmer, David Sanders University of Saskatchewan, Saskatoon, SK, Canada Our research is focused on inositol catabolic enzymes. Inositol dehydrogenase from Bacillus subtilis (BsIDH) is the first enzyme in the myo-inositol catabolic pathway, a primary carbon + source for soil bacteria. BsIDH catalyses the NAD -dependent oxidation of myo-inositol to scyllo-inosose. BsIDH is able to oxidize other substrates, including the mono-saccharides α D-glucose and α -D-xylose but does not oxidize β -D-glucose, D-mannose or D-galactose. IDH also oxidizes the α -(1,6)-linked disaccharides melibiose and isomaltose. Scyllo-inositol, the equatorial stereoisomer of myo-inositol is neither a substrate nor an inhibitor for BsIDH. These observations indicate that an axial hydroxyl group is required for the substrate and that the active site of BsIDH can selectively discriminate between structural variations in substrate. IolG1 from L. plantarum is annotated as a putative inositol dehydrogenase (structural genomics consortia). It shows 24% sequence identity to BsIDH. However, our primary biochemical data on IolG1 indicates that this enzyme is not an inositol dehydrogenase but shows activity towards inosose. Understanding the structural basis of inositol dehydrogenase substrate selectivity and the residues involved in catalysis form the basis for our structural studies of BsIDH. We are also using X-ray crystallography to probe potential substrates for IolG1 to understand its role in inositol metabolism. So far BsIDH crystal structures have been solved for apo, holo and ternary complex with inositol and inosose. These results allowed us to identify key residues involved in cofactor and substrate binding. Recently, we have solved the crystal structures of the ternary complex of IolG1 with an inosose product, scyllo-inositol and myo-inositol. Although both enzymes share the same tetramer arrangement, their substrate recognition site is different. T-155 Biological Implications of Trimer Self-assembly in Solution State and in Crystal Structure of Mtb UreA Jeff Habel , Li-Wei Hung 1 1 1,2 2 Lawrence Berkeley National Laboratory, Berkeley, CA, United States, Los Alamos National Lablratory, Los Alamos, NM, United States Crystal Structure of the Urease γ subunit, UreA, from Mycobacterium tuberculosis (Mtb) was determined previously (RCSB: 2FVH), and revealed a homotrimetric arrangement akin to the UreA trimers in the Urease structures from Klebsiella aerogenes (RCSB: 2KAU), and from Bacillus pasteurii (RCSB: 1UBP). Analysis of the inter-molecular contacts strongly suggests that the Mtb UreA self-organizes into such trimeric assembly. The oligomeric state in solution together with a low-resolution envelop of Mtb UreA calculated from Small Angle X-ray Scattering data further support this hypothesis. The homotrimer formation in addition to the 3 gene organization within the Mtb genome support the Mtb Urease composition of (α β γ ) . While not having any known associated catalytic activity, the Mtb UreA has the potential to be the driving force of the trimer of trimers formation seen with known bacterial Urease structures. The need of oligomerization beyond catalytic efficiency might play a role in Mtb Urease’ s extreme tolerance to environmental challeng es. T-158 High Throughput Screening Identifies Ligands that Disrupt Exonuclease-SSB Interactions Kenneth Satyshur , Duo Lu , James Keck 1 1,2 1 1 University of Wisconsin, Department of Biomolecular Chemistry, Madison, Wisconsin, United 2 States, University of Wisconsin, Small Molecule Screening Facility, Madison, Wisconsin, United States Exonuclease I (Exo1) is a DNA repair enzyme whose action is mediated by the interaction of single strand DNA binding proteins (SSB). The far C-terminal end of SSB contains the evolutionary conserved MDFDDDIPF sequence that binds in two separate hydrophobic sites on Exo1 stimulating Exo1 activity. A process of chemical High Throughput Screening (HTS) has identified several compounds that disrupt this interaction and compete for the SSB binding site on Exo1. Crystal structures of 2 of these compounds bound to the secondary (B) site of Exo1 reveal the mechanism of this binding. (Lu, et.al.). The hydrophobic modified phenyl groups of the ligands bind in a deep pocket while the charges on the core of the ligands bind to the electropositive surface near the pocket. Thru an in-silico HTS drug docking process, we have identified more potential ligands for these binding sites. Phase I of the in-silico HTS enriches the 340,000 compound Life Sciences chemical database using the docking program Surflex Dock (Prof. Ajay N. Jain, UCSF) as implemented in the Sybyl (Tripos Crop) molecular modeling program. In Phase II, the top 1% of Surflex Dock results were further docked with Autodock4 (Garrett A. Morris, David Goodsell, Scripps Inst.) and re-scored. The best scoring compound was further docked using receptor flexibility for 5 charged groups in the binding pocket, and resulted in substantially increased binding energy and a docking similar to that of the crystal structure. Efforts are ongoing to identify more ligands that disrupt this vital interaction of DNA repair proteins. Lu, D, Bernstein, D.A., Satyshur, K.A., and Keck,J.L., (2010), Proc.Nat.Acad.Sci., 107, 2, 633–638. T-162 Structural and Biochemical Characterization of Seasonal Influenza Virus Hemaggluinin Ki Joon Cho , Ji-Hye Lee , Seokha Kang , Yi Ho Park , Jun Young Lee , Joo Yeon Lee , 2 1 Chun Kang , Kyung Hyun Kim Korea University, Seoul, Korea, Republic of, Korea National Institute of Health, Seoul, Korea, Republic of Influenza is one of the most important respiratory infectious diseases causing seasonal epidemics or pandemics. It was reported that structural feature of antigenic region of 2009 pandemic influenza hemagglutinin (HA) is different from that of seasonal influenza HA, but similar to that of 1918 pandemic influenza HA. We determined the crystal structure of seasonal H1N1 HA protein and compared with that of 1918 pandemic influenza HA. Biochemical properties of both seasonal and 2009 pandemic HA proteins were also investigated. In order to find a universal antiviral drug for influenza virus, various plant extracts and compounds were screened, and KC2002, SC2740A, GA007, GA1002, and TY10 were screened to show affinities to seasonal HA, but not to 2009 pandemic HA. 1 2 1 1 1 1 1 2 T-165 Structure of the catalytic domain of glucuronoyl esterase from Hypocrea jecorina P. Raj Pokkuluri , Stephen Wood , Norma Duke , Michael Cotta , Xin-Liang Li , Peter Biely , 1 Marianne Schiffer 1 1 1 1 2 2 3 Argonne National Laboratory, Lemont, IL, United States, USDA-ARS, Peoria, IL, United 3 States, Slovak Academy of Sciences, Bratislava, Slovakia 2 We have determined the structure of the catalytic domain (Cip2S) of glucuronoyl esterase, Cip2 from Hypocrea jecorina (formerly known as Trichoderma reesei). This is the first structure of the recently established carbohydrate esterase family 15 (http://www.cazy.org/fam/CE15.html). The catalytic activity of the enzyme is important in degradation of lignocellulosic material. The crystals of Cip2S have three independent molecules and diffracted X-rays to 1.9 Å resolution. The structure was determined by the conventional “heavy-atom soaking” method followed by a SAD experiment at the Structural Biology beam line, 19BM (APS). The structure was refined to a crystallographic R-factor of 19.5% and R-free of 23.4%. The catalytic domain Cip2S has 375 amino acids and its structure has an α /β hydrolase fold. Inspection of the structure revealed a triad arrangement of Ser – His – Glu residues on the surface of the protein suggesting a putative active site. To confirm the active site and to understand the substrate binding site we have obtained crystals of Cip2S in presence of a serine inhibitor, phenyl methyl sulfonyl fluoride (PMSF) and a synthetic substrate, methyl ester of 4-O-methylD-glucuronic acid. The structure of the native enzyme and the results obtained from cocrystallization of the enzyme with the above compounds will be presented and discussed. T-167 Small-angle neutron scattering study of Sindbis virus produced from vertebrate and invertebrate hosts Lilin He , Amanda Piper , Flora Meilleur , Dean Myles , Raquel Hernandez , Dennis 2 1 Brown , William Heller Oak Ridge National Laboratory, Center for Structural Molecular Biology, Oak Ridge, TN, 2 United States, North Carolina State University, Department of Molecular & Structural 3 Biochemistry, Raleigh, NC, United States, Oak Ridge National Laboratory, Neutron Scattering Sciences Division, Oak Ridge, TN, United States Understanding the life cycle of viruses that are vectored between in nature different hosts, such as insects and mammals, presents many challenges, yet this knowledge is crucial for addressing some of the most devastating mosquito-transmitted infectious diseases. The Sindbis virus, an Arbovirus and prototypic alphavirus, transitions between insect and vertebrate hosts. It has inner protein and outer glycoprotein shells separated by a lipid membrane. Host-specific differences in the composition of Sindbis virus have been observed, but not structurally characterized. Here, we present the results of a small-angle neutron scattering (SANS) investigation of mammalian- and insect-grown Sindbis virus that provide the first evidence of host-derived differences in virus structure. The non-destructive nature of SANS allowed for the characterization without decreasing the infectivity of the Sindbis virus particles studied. The results demonstrate that the radial position of the lipid membrane does not change significantly, but the lipid membrane of the mammalian-grown virus contains significantly more cholesterol. Additionally, the outer glycoprotein coat of the mammalian Sindbis virus is more extended. The SANS data also indicate that the inner nucleocapsid protein and the RNA of Sindbis virus interact more closely in the mammalian-grown virus than in Sindbis virus grown in insect cells. Research sponsored by the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory (F.M.). Dennis Brown and Raquel Hernandez are supported by The Foundation for Research, Carson City, Nevada. This research at Oak Ridge National Laboratory's Center for Structural Molecular Biology was supported by the Office of Biological and Environmental Research, using facilities supported by the U. S. Department of Energy, managed by UT-Battelle, LLC under contract No.DE-AC05-00OR22725. This manuscript has been authored by UT-Battelle, LLC, under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher by accepting the article for publication, acknowledges that the United States Government retains a nonexclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. 1 1 2 2,3 1,3 2 T-171 Crystal structure of the human IQGAP1 calponin homology domain. Vinodh Kurella, David Worthylake LSU Health Sciences Center, New Orleans, Louisiana, United States The 190 kDa scaffold protein IQGAP1 reversibly binds to a variety of cellular proteins including active forms of Cdc42 and Rac1, calmodulin, and actin; the latter activity requiring the IQGAP1 amino terminus which contains a calponin homology domain. In cells, IQGAP1 forms homodimers that bind to and cross-link filamentous actin and cross-linking activity is 2+ reduced in the presence of Ca -calmodulin. Recently it has been shown that amino terminal fragments of IQGAP1 bind to both actin and calmodulin and that calmodulin competes with actin for binding to these fragments. In this study, we have determined the crystal structure of the human IQGAP1 calponin homology domain (CHD). The overall structure of the protein is very similar to other type -3 calponin homology domains such as those found in calponin, Vav-3 and the yeast IQGAP ortholog Rng2. In the crystal, the CHD has associated into a parallel homodimer that displays a high degree of surface-shape complementarity at an interface that is predominantly hydrophobic in nature. Gel filtration experiments verify the presence of a dimer in solution. Isothermal titration calorimetry indicates that the CH domain 2+ binds to Ca -calmodulin but not apo-calmodulin, via a two-site-sequential mode of binding and that mutation of two adjacent lysine residues within the CHD significantly reduces this interaction. Using an actin-pelleting assay and SDS-PAGE, we find that both wild-type and 2+ mutant CHD bind to filamentous actin and that pre-incubation with Ca -calmodulin does not reduce actin binding. Interestingly, we do not detect even small amounts of calmodulin in the pellet fraction. These results suggest that full-length IQGAP1 may associate as a parallel homodimer and that the binding sites for calmodulin and actin on the CHD overlap to a large 2+ degree with actin able to out-compete Ca -calmodulin for binding. T-176 The Tautomerase Superfamily and its Structural Relatives - New Insights into Possible Functions Marvin Hackert, Youzhong Guo, Hector Serrano, William Johnson, Jr., Christian Whitman The University of Texas at Austin, Austin, Tx, United States The tautomerase superfamily has been divided into five families represented by 4oxalocrotonate tautomerase (4-OT), 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), cis-3-chloroacrylic acid dehalogenase (cis-CaaD), malonate semialdehyde decarboxylase (MSAD), and macrophage migration inhibitory factor (MIF). 4-OT and many of its homologues are homo- or heterohexamers composed of small (60-75 a.a. residue) subunits while CHMI, MSAD, cis-CaaD and MIF are nearly twice that size and form trimers. The subunits of this family share two distinguishing features – one or two beta-alphabeta structural motifs and a catalytically important N-terminal Pro residue. Several different catalytic activities are known to utilize this same structural motif - tautomerase, isomerase, decarboxylase, dehalogenase, etc. The smaller members of the 4-OT family had been previously categorized into five subfamilies and a representative member from each group has been crystallized and its X-ray structure determined. In spite of knowing the X-ray structures, several members of this family have defied attempts to identify their biological / catalytic activities. However, it is observed that several other proteins with similar folds, but some lacking an N-terminal proline, have now been implicated in binding and regulation. While MIF has an N-terminal proline and can function as a phenylenolpyruvate tautomerase, its major role is immunosuppression and MIF is known to bind to the receptors CD74, CXCR2 and CXCR4. Thus many members of the tautomerase superfamily may play roles in receptor-based regulation instead catalysis. A summary of these findings and a comparison of the representative structures will be presented. This work is supported in part by The Welch Foundation (F 1219, F1334). T-182 Structural basis for neuropilin ligand binding. Craig Vander Kooi, Matthew Parker, Hou-Fu Guo, Ping Xu University of Kentucky, Lexington, KY, United States Neuropilin is an essential cell surface receptor that function in VEGF dependent angiogenesis and semaphorin dependent axon guidance. Accumulating evidence indicates that neuropilin may mediate cross-talk between the two pathways but the mechanism is unclear. We demonstrate that both VEGF and semaphorin binding to neuropilin requires a C-terminal arginine residue. The crystal structure of the core ligand binding domains of neuropilin bound to ligand derived peptides reveals the structural basis for this interaction. The C-terminal residue is tightly bound in a pocket on the b1 domain of neuropilin. Further, proteolytic processing of semaphorin is found to regulate competition of the two classes of ligands for binding to neuropilin. T-185 Solution Studies of DNA bound Gyrase Nicole Baker , Steven Weigand , Sarah Maar-Mathias , Alfonso Mondragón 1 2 1 2 1 1 Northwestern University, Evanston, IL, United States, DND-CAT Synchrotron Research Center, Argonne, IL, United States DNA gyrase, a type II topoisomerase, is unique amongst topoisomerases due to its ability to introduce negative supercoils into DNA. While many details of its mechanism are still not completely understood, it is known to involve the assembly of a large gyrase/DNA complex and coordinated combination of DNA strand movements and protein rearrangements modulated by ATP hydrolysis. Although structures of gyrase domains have been elucidated, structures of the intact GyrA2-GyrB2 heterotetramer or complexes with DNA are still unknown. To establish the arrangement of its domains during the binding of a DNA substrate that directs the reaction towards negative supercoiling, gyrase complexes with large DNA fragments representing the starting conformational state of the catalytic cycle were characterized. Purified Escherichia coli and Deinococcus radiodurans gyrase bound to 137 or 217 base pair DNA fragments were characterized by sedimentation velocity and small angle x-ray scattering (SAXS) experiments and revealed elongated complexes with hydrodynamic radii of ~70Å. Molecular envelopes calculated from these SAXS data show elongated, twofold symmetric molecules with the carboxy-terminal domain (CTD) of the A subunit and the ATPase domain of the B subunit at opposite ends of the complexes. This domain placement is supported by experiments using a mutant gyrase lacking the CTD, by DNA footprinting analysis, as well as SAXS and AUC simulations. All SAXS models suggest an initial arrangement where the CTDs are found near the exit gate of the protein and with the DNA wrapping along the sides of the molecule and around the CTDs. Overall, this arrangement is consistent with mechanisms previously proposed for gyrase, but with a different arrangement of the CTDs. T-188 Structure-based Antibody Engineering: Mechanism of Action and Binding Affinity Determinants for anti-IL13 Antibodies Derived from Co-structures with Antigen Thomas Malia, Alexey Teplyakov, Galina Obmolova, Raymond Sweet, Gary Gilliland Centocor R&D, Inc., Radnor, PA, United States The three-dimensional structures of antibodies and antibody/antigen complexes provide detailed insight into epitope and mechanism of action and are valuable for guiding engineering. Therapeutic antibodies derived from mouse sources require humanization to minimize their immunogenicity and often subsequent affinity maturation to restore or improve binding. We determined the structures of a set of antibody Fab fragments from various stages of engineering in complex with their target IL-13 in order to understand their mechanism of action and to evaluate the process of engineering that was employed (1). IL13 is a pro-inflammatory cytokine produced in Th2 immune cells and has been implicated in asthma and allergy pathogenesis. IL-13 mediates its action by engaging with receptors IL4Rα and IL-13Rα 1 to form a heterotrimeric signaling complex. A high affinity neutralizing antibody against human IL-13 was isolated from mouse hybridoma (m836), which was then human framework adapted into h826 and affinity matured into am836. Using a combination of high-throughput screening and manual optimization with microseed-matrix screening (MMS), we crystallized the anti-IL-13 Fabs from these three stages of antibody engineering in complex with IL-13 and determined their structures. The structures of the complexes indicate that the epitope and paratope were preserved throughout humanization and affinity maturation. We describe the crystallization process, how the antibodies block binding of IL-13 to its receptors, and the structural features of the engineered antibodies that contribute to their changes in binding affinity. Fransson, J., et al. Human Framework Adaptation of a Mouse Anti-human IL-13 Antibody. J. Mol. Biol.(2010), doi:10.1016/j.jmb.2010.03.004 T-191 De novo structure of a putative GAF-domain protein from S. aureus by SeMet SAD. Kevin Battaile1, Rob Lam2, Kathy Johns2, Jean Brawn2, Vlad Romanov2, Emil Pai2,3, Nickolay Chirgadze2,4 1 IMCA-CAT/Hauptman Woodward Medical Research Institute, Argonne, IL, United States, 2Division of Cancer Genomics and Proteomics, Ontario Cancer Institute, University Health Network, Toronto, ON, Canada, 3Departments of Biochemistry, Molecular Genetics and Medical Biophysics, University of Toronto, Toronto, ON, Canada, 4Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, Canada It is imperative to the survival of a cell, be it a unicellular organism or a part of a multicellular organism to be able to sense the chemicals in it surroundings and respond to them. This could be through chemotaxis for a motile microorganism, or as part of the metabolic control of an individual cell in a larger organism. GAF domains (named for cGMP-phosphodiesterases, adenylate cyclases and FlhA) represent one of a class of chemical sensors. Here we describe the structure of a hypothetical GAFdomain protein (SA1058) from Staphylococcus aureus. SA1058 is a 150 amino acid protein that whose structure was determined by SeMet SAD. SAS1058 crystallized in space group P21212 (a=109, b=36, c=38) and diffracted to 2.1Å. Three methionines are present in the sequence but only two were identified in density with the positions of both confirmed by anomalous difference maps, of which the position of one was well defined while the other appeared to assume two conformations. The asymmetric unit contains one peptide, of which residues 8-133 were sequenced in the map. The final model has an R=0.209 and Rfree=0.288. The general structure of SA1058 is of a 5-stranded β sheet with the N-terminal α helix and C-terminal strand on one face and a grouping of several smaller helical regions on the other face of the sheet. The secondary structure is similar to domains found in other proteins in the PDB including phosphodiesterase 5, profiling 1 and bacteriophytochrome. The wide role in chemical sensing of the GAF domain in various organisms could make it an interesting target for antimicrobial therapy. T-194 Towards the crystal structure of a lectin purified from the marine sponge Cinachyrella Pamela Focia , Caleb Smith , Yuka Nakamura , Bryan Copits , Martin Gill , Ryuichi Sakai , 1 Geoffrey Swanson 1 1 1 2 1 1 2 Northwestern University, Chicaog, IL, United States, Hokkaido University, Hakodate, Japan 2 Marine sponges represent a rich source of natural products, some of which have already been characterized and found to be medically interesting. Analysis of lectins from marine sponges can illuminate a deeper understanding of their biologically relevant role, as well as how they might be used as valuable tools in biomedical research. Lectins are proteins that bind carbohydrates and agglutinate cells. The sponge in this study was collected in the waters off of Iriomote Island in Japan. It is a yellow ball sponge of the genus Cinachyrella, however the species is not yet known. Its purified galectin, BaL, was determined to be an allosteric modulator of glutamate-gated ion channels of the mammalian AMPA and kainate receptor families in physiology studies. The functional protein appears to be a ~49 kDa trimer comprised of two ~18 kDa subunits and one ~16kDa subunit, each having a unique but conserved N-terminal sequence. As this protein was purified from the marine sponge, rather than expressed, very limited amounts were available. We have thus set up two commercial crystallization screens (192 conditions) using a drop size of 200nL:200nL with 10 mg/mL protein, and obtained 6 conditions with protein crystals, and 4 crystals that allowed datasets to be measured and processed with good statistics. Among those crystals we have identified 3 different space groups; the crystal which diffracts to the highest resolution, 2.1Å, takes the space group P21, with one trimer expected in the asymmetric unit. There are two structures in the PDB that are likely to have a similar fold as BaL, and after superimposing them, removing the parts of the structures that are different, and creating a polyalanine search model, we have initiated molecular replacement to solve the phase problem. However, all crystals are held in LN2 storage in case the need for derivatives arises. That we have only the N-terminal sequence of 20 amino acids for each monomer makes this an exciting project of sequencing by X-ray crystallography! T-197 Diffuse scattering in scanning x-ray nanodiffraction as a probe for high-resolution mapping of strain distribution around epitaxial nanostructures Tao Sun , Zixiao Pan , Xujing Xie , Zhonghou Cai , Jin Wang , Vinayak Dravid Argonne National Laboratory, Argonne, IL, United States, Evanston, IL, United States 1 2 1 2 2 1 1 2 Northwestern University, In the recent decade, the unexpected behavior and enhanced properties of functional materials, induced by the spatial confinement and dimensional constraints, have been extensively reported. Such constrained nanostructures are believed to be one of the central themes in materials science, because of their significance in potential applications and fundamental scientific underpinning. Although considerable efforts have been spending on the fabrication/synthesis and function assessments of nanopatterned systems, quantitative structural characterization has remained elusive, despite its importance for elucidating the microstructure-property relationship. Strain issue in electronic and magnetic materials is a good manifestation of confinement effects, and possessing powerful tools for measuring strain is prerequisite for strain engineering and property controlling. Scanning x-ray nanodiffraction (SXND) is one of the very few techniques that can be used to investigate the local strain distribution of nanostructures. However, in the traditional SXND experiment, strain information is obtained by evaluating Bragg diffraction signal from either substrate or nanostructures. It faces great challenges when applied for small-strain systems, because the strong Bragg diffraction from the un-strained lattices of singlecrystalline substrates can easily shadow the small perturbation induced by the structural imperfection. In order to solve this problem, we developed a novel approach based on SXND technique for probing spatially varying and small values of strain at/around individual epitaxial nanostructures. By presenting an example of CoFe2O4/MgO system, i.e. single-crystalline epitaxial CFO nanolines on (100) MgO substrate, we demonstrate that diffuse scattering obtained by setting the incident angle slightly off the Bragg angle can be used to quantitatively reveal the nanostructure-induced lattice imperfection in the system. The results indicate an edge-induced strain distribution, which is consistent with the strain simulation based on the edge-force model. Moreover, the shape parameter in the scattering intensity line-fit can differentiate the contribution of mosaic structure and elastic residual strain in a quantitative manner. We show the strain map around an oxide-on-oxide nanostructure, which has extremely small strain values and cannot be effectively characterized by other techniques. We believe that a thorough understanding of strain issues in such spatially and dimensionally confined (oxide) nanostructures will not only facilitate the elucidation of their size-dependent behaviors, but also guide for development of novel devices based on strained functional oxides for advanced applications. T-203 Flurbiprofen Tris polymorph I: the slog to R = 11% Carl Schwalbe, Miren Ramirez Aston University, Birmingham, United Kingdom We have systematically studied a series of salts of the anti-inflammatory drug flurbiprofen, starting with the t-butylammonium salt and progressively changing methyl to hydroxymethyl groups, ending up with the Tris salt. Me COO F + H3N CH 3 CH 3 CH 3 + to H3N CH 2 OH CH 2 OH CH 2 OH This salt was prepared by mixing equimolar amounts of flurbiprofen and Tris in acetonitrile solution and harvesting the precipitate that formed. Single crystals grew from different solvents as different polymorphs. Recrystallization from methanol gave polymorph I, but methanol:acetonitrile 40:60 yielded polymorph II. Although crystals of polymorph II diffracted well, polymorph I gave small poorly diffracting crystals with Z’=2. Data collected by the National Crystallography Service on a powerful conventional small molecule diffractometer revealed the molecular connectivity, but the R factor never went below 20%. Next, we raised the stakes by requesting re-collection of data on beamline I19 of the Diamond synchrotron. The specimen crystal measured 0.1 x 0.01 x 0.01 mm. These data gave a stable refinement, 3 but unfortunately it converged at R>15%, and numerous peaks around 1 e/Å remained in the difference map. Some disorder was apparent: the fluorine atom could be either side of its benzene ring, and enantiomer discrimination was imperfect with some switching of H and Me. Other peaks were uninterpretable. Eventually, inspection of the coordinates revealed that the two independent cations and COO were related by a pseudo-glide plane while the biphenyl units were related by a pseudo-translation! The extra peaks on the difference map arose from applying the “opposite” pseudosymmetry operation, i.e. the translation, to the cations. 3 Now R=11% and the largest difference peak is 0.40 e/Å . We thank Drs. S. Callear and R. W. Harrington and Prof. W. Clegg for data collection and Bristol-Myers Squibb for support. T-206 Seeing is Believing? The first structurally characterized [4×4] Ni Self-Assembly. Louise Dawe, Konstantin Shuvaev, Laurence Thompson Memorial University, St. John's, Newfoundland, Canada Tritopic and tetratopic bis-hydrazone ligands have been prepared, and produce [3x3]M9 [1-4] and [4x4]M16 grids respectively, by self-assembly reactions with metal salts (M = Mn(II), Cu(II), Co(II)). These molecules have attracted much attention for their possible nanotechnological applications, and with molecular footprints of ~ 3 nm x 3 nm, they could eventually lead to the production of magnetic nanoparticles. Until now, however, there has been no report any [nxn] Ni(II) grid with nuclearity exceeding n = 3, and the one report where [5] n = 3, did not yield a satisfactory single crystal X-ray structure . The structural and magnetic characterization of a novel [4x4]Ni(II)16 grid (Fig. 1) will be presented. While this is a very exciting result, details have yet to be published, and, as will be discussed, beg the question, should they be published? [1,2] II 16 Grid by Designed 1. Dawe, L.N., Shuvaev, K.S., Thompson, L.K., Inorg. Chem., 2009, 48, 3323-3341. 2. Dey, S.K., Abedin, T.S.M., Dawe, L.N., et al. Inorg. Chem., 2007, (46), 7767-7781. 3. Dey, S.K., Thompson, L.K., Dawe, L.N. Chem. Commun. 2006, 4967-4969. 4. Dawe, L.N., Thompson, L.K. Angew. Chem., 2007 (46), 7440-7444. 5. Niel, V., Milway, V., Dawe, L.N., Grove, H., et al. Inorg. Chem., 2008, 47, 176-189. Fig.1: [4x4] Ni II 16 grid. T-209 The HB2A High Resolution Powder Diffractometer at the High Flux Isotope Reactor Ovidu Garlea, Clarina dela Cruz Neutron Scattering Science Division, Oak Ridge National Laboratory, Oak Ridge, TN, United States Neutron powder diffraction is increasingly recognized as one of the most powerful techniques for studying the structural and magnetic properties of advanced materials. We are presenting an overview of the HB2a diffractometer that has recently been installed at the High Flux Isotope Reactor in Oak Ridge. The instrument has been designed to provide an optimum balance between high neutron flux and high resolution. Due to its versatility the diffractometer can be employed for a large variety of experiments, but it is particularly adapted for refinements of structures with large interplanar spacings as well as of complex magnetic structures. Instrument capabilities will be illustrated by recent studies undertaken on various materials ranging from rare-earth iron oxyarsenides to zeolites. T-212 An Advanced Small-Angle X-ray Scattering Station for Structural Biology: Automated High Throughput Solution Scattering, Time-Resolved Studies and Beyond. Thomas Weiss, Ping Liu, Anne Martel, Marc Niebuhr, Hiro Tsuruta Stanford University, Menlo Park, CA, United States Beamline 4-2 at the Stanford Synchrotron Radiation Lightsource (SSRL) is a small angle xray scattering/diffraction facility dedicated to structural studies on mostly non-crystalline biological systems. The facility recently received an extensive array of optics and in-hutch instrumentation upgrades to take full advantage of the high brightness beam produced by the third generation storage ring SPEAR3. The instrument features a pin-hole geometry camera configurable in one of seven sample-to-detector distances ranging from 0.3m to 3.5m, -1 providing access to the Q-range 0.003-4.2 Å (at 11keV). It is equiped with a highsensitivity/stability CCD detector as well as a silicon pixel array detector, both of the latest generation. The latter achieves high frame rates up to 300Hz, suitable for time-resolved studies. The instrument and all experiments are controlled by a version of Blu-Ice software customized for non-crystalline diffraction experiments. We have developed several sample handling devices specific to each distinctive class of experiments such as a stopped-flow rapid mixer for time-resolved solution x-ray scattering and a humidity controlled sample chamber for lipid/fiber studies. Our high-throughput solution x-ray scattering data collection system integrates an automatic sample changer in the 96-well microplate format with the BluIce data collection tab SolSAXS. The system achieves high throughtput without compromizing high reliability in data collection. Our data processing software SasTool keeps up with the high throughtput of data collection, generating fully processed data in real time. Remote data collection is currently in trial. The high level of beam and detector stabilities enables routine use of dilute protein solutions in the sub-mg/ml range. The multilayer monochromator option 14 provides an extremely high beam flux level of approx. 10 photons/s for time-resolved studies, achieving sub-millisecond time resolution in single trigger events. This presentation will discuss relevant characteristics of the instrumentation on BL4-2 and a few recent scientific applications in structural molecular biology, complementing crystallographic studies. T-215 Capabilities at GM/CA CAT Beam Lines at the APS Nagarajan Venugopalan , Michael Becker , Stephen Corcoran , Mark Hilgart , Oleg 1 1 1 1 1 Makarov , Craig Ogata , Sudhirbabu Pothineni , Ruslan Sanishvili , Sergey Stepanov , 1 1 1,2 1 Shenglan Xu , Derek Yoder , Janet Smith , Robert Fischetti Argonne National Laboratory, Argonne, IL, United States, University of Michigan, Ann Arbor, MI, United States GM/CA CAT operates two independent undulator beamlines, 23ID-B and 23ID-D, at the APS. Both beamlines are rapidly tunable for MAD and are equipped with ALS-style sample automounters. Several features of the beamlines have been developed or improved over the past year, all controlled within the Bluice-EPICS interface. Over the past three years we have provided Micro-diffraction capabilities at both beamlines through versatile collimator systems. Recently we have developed a robust quad-collimator monolith containing user-selectable beam-sizes of 5 μ m, 10 μ m, 20μ m or "full beam" (~25 mm (V) x ~75 mm (H) FWHM). Several groups have used these mini-beams to solve structures that otherwise would not have been possible. For challenging samples, such as, invisibly small membrane protein crystals in lipidic cubic phases, larger inhomogeneous crystals or multiple crystals, we developed a semi-automated rastering procedure that records diffraction images over a user-defined region of the sample on a 2-D grid. Automated analysis, or visual inspection, of each diffraction pattern indicates the best position to collect data. A similar semi-automated rastering feature based on fluorescent signal is also available for locating metallo-proteins, Se-Met derivatives or back soaked heavy atom derivatives. Also, to facilitate data collection on radiation sensitive crystals, we recently developed an automated procedure to collect data along a user-defined 3D vector. The automounter is used by nearly two-thirds of GM/CA CAT users to screen and collect data. The Web-ice package has been implemented to facilitate strategy calculations and scoring of samples. Remote access is available to experienced GM/CA CAT users. GM/CA CAT is supported by NIGMS and NCI within the NIH. 1 2 1 1 1 1 T-218 The Gulf Coast Protein Crystallography Consortium Beamline at the Center for Advanced Microstructures and Devices. Henry D. Bellamy , Robert O. Fox 1 1 2 2 Louisiana State University, Baton Rouge LA, United States, University of Houston, Houston TX, United States The Gulf Coast Protein Crystallography (GCPCC) beamline is a fully equipped tunable MAD experimental station at the CAMD synchrotron in Baton Rouge LA. The beamline has been in operation since 2002. Construction was jointly funded by the NIH and NSF through a Major Research Instrumentation grant to a consortium of Universities in LA, TX and OK. The beamline is open to general users as well as GCPCC consortium members. Access is free for non-proprietary users. The detector is a MAR (Rayonix) 165 mm CCD mounted on a MAR dtb goniostat. The beamline is equipped with a fluorescence counter and all standard equipment and software required for MAD data collection. Data collection uses the marccd program supplied by the detector vendor and the beamline is controlled through the MX software package, which has been customized and extended to meet our requirements. Users have access to a small wet lab next to the beamline with an anaerobic chamber, a cold room, and incubators for sample storage. The beamline has an active “FedEx” data collection program. Because of the relatively low energy of the CAMD ring (1.3 GeV) the beamline uses a 7 T single-pole superconducting wavelength shifter as its source. We have recently received a NSH MRI grant to replace the wavelength shifter with a 7.5 T 11-pole wiggler which will increase the flux about 8 fold. The new wiggler will become operational in the fall of 2011. We will describe the beamline and the associated equipment and software. We will also briefly describe CAMD and its ring and the other beamlines at CAMD. Finally we will discuss our future plans for the beamline and for structural biology at CAMD. M-219 The PSI Structural Biology Knowledgebase – Search Online for Protein Seqeunces, Structures, Models, Methods, and More John Westbrook , Margaret Gabanyi , Wendy Tao , Raship Shah , Andrei Kouranov , Torsten 2 2 2 3 3 Schwede , Konstantin Arnold , Lorenza Bordoli , Paul Adams , Lester Carter , Wladek 4 1 Minor , Helen Berman 1 1 1 1 1 1 Rutgers, the State University of New Jersey, Piscataway, NJ, United States, Swiss Institute 3 of Bioinfomatics & Biozentrum, Basel, Switzerland, Lawrence Berkeley National Laboratory, 4 Berkeley, CA, United States, University of Virginia, Charlottesville, VA, United States 2 ? ? ? ? ? j‹›• ¡ £¡ \ ¡? 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Duke, Gofron Kazimierz, Youngchang Kim, Krzysztof Lazarski, Jack Lazarz, Mike Molitsky, Bogi Nocek, Jurek Osipiuk, Soon Ok Park, Gerd Rosenbaum, Frank J. Rotella, Kemin Tan, Rongguang Zhang, Andrzej Joachimiak Argonne National Laboratory, Argonne, IL, United States The Structural Biology Center (SBC) at Argonne National Laboratory operates two beamlines - one insertion device (ID) and one bending magnet (BM) - at Sector 19 of the Advanced Photon Source as a national user facility for macromolecular crystallography. These beamlines continue to be one of the most powerful, capable and productive X-ray sources for structural biology in the US. The beamlines can deliver very low angular divergence X-ray micro-beams onto micrometer-size crystal samples mounted using robotic systems, thereby permitting structural biologists to study the structures of large and complex molecular systems at atomic resolution. Diffraction from these crystals is recorded on large, fast, and efficient CCD area detectors, and is processed on high-performance, integrated computing systems with advanced control and data analysis software designed specifically for the SBC. Presentation will highlight new and upgraded advances to the SBC beamlines including: on axis crystal viewing, beam visualization, point and click sample alignment, auto-loop alignment, adjustable mini beam with apertures to 5m, ACTOR crystal mounting robotic using either Rigaku or Uni-Puck/ALS sample pucks, remote data collections options, new fluorescence scanning, auto energy changes, data bases and interfaces and advances found in HKL3000 a program suite for data collection and processing, structure solution and model building in near real time. Some recent important SBC developments and research highlights from the sector 19’s PDB deposits will be presented. The SBC beamlines offer the most efficient worldwide data collection and structure determination systems currently available for protein crystallography and have demonstrated record productivity (2727 PDB deposits (on average 389 per year in the past 3 years) and 1006 publications). In 2009, the Nobel Prize in chemistry was awarded for research on ribosomes, a major part of which was performed at the SBC 19-ID beamline. Beamtime on the sector 19 beamlines is available to the crystallographic research community via the APS peer reviewed proposal system. The proposal evaluation is based upon the projects’ scientific merit, need for synchrotron time, feasibility of conducting the experiments at the SBC, and the probability for success of the project. The highest rated proposals will receive beamtime first. Information on the user program and the sector 19 beamlines will be provided and can also be obtained from the SBC web site (http://www.sbc.aps.gov). This work is supported by the U.S. Department of Energy, Office of Biological and Environmental Research, under Contract DE-AC02-06CH11357. T-224 Laser Induced Protein Crystallization Neela Yennawar, Sava Denev, Venkataraman Gopalan, Hemant Yennawar Pennsylvania State University, University Park, United States Screening of proteins for crystallization under laser irradiation was investigated with six proteins ribonuclease B, glucose dehydrogenase, lysozyme, sorbitol dehydrogenase, fructose dehydrogenase and myoglobin. Shining 532nm green circularly polarized laser light with picosecond pulse and 6mW power for 30 seconds, on newly setup protein drops, showed marked improvement in the number of screen conditions amenable for crystal growth as compared to the control drops under identical conditions but without laser exposure. In some proteins bigger and better quality crystals were formed. The speed of crystallization increased in most. During laser irradiation, the amount of precipitation in the screened drops increased indicating a transient decrease in protein solubility. The resolution of x-ray diffraction of crystals grown in drops with laser induced nucleations improved in some examples. At the optimised laser settings, there was no deleterious effect of the laser on crystal growth. Crystal structure solution confirmed that the protein had not degraded due to the laser radiation. T-227 High-Brilliance Home-Lab X-Ray Sources: Status and Future Carsten Michaelsen, Jürgen Graf, Jörg Wiesmann Incoatec GmbH, Geesthacht, Germany Modern microfocus X-ray sources define the state-of-the-art for a number of applications such as protein crystallography and small-angle scattering in the home lab. These sources have small source sizes of 100 µm or smaller. They are usually combined with multilayer mirrors as beam-shaping devices that image the source spot onto the sample position, magnified to a suitable size, and deliver a parallel or focused monochromatic beam. Microfocusing rotating anode systems deliver flux densities in the range of 10 photons/(s 2 2 mm ) at power loads of up to 20 kW/mm when combined with synthetic multilayer mirrors. However, these sources are expensive and need regular and, sometimes, time-consuming maintenance. Low power microfocus sealed tube sources such as the Incoatec Microfocus Source “IµS” represent an interesting low-maintenance alternative to rotating anode generators. Power 2 loads of several kW/mm in anode spot sizes of 50 µm deliver a small and highly brilliant 10 2 beam. The IµS delivers a flux density of up to 10 photons/(s mm ) in a focused beam (FWHM = 0.11 mm, 7.6 mrad) suitable for most protein crystals. Emerging microfocus X-ray sources based on liquid-metal-jet technologies show even higher 2 power loads up to 500 kW/mm , an order of magnitude higher than possible with solid target 12 2 sources, and intensities up to 10 photons/(s mm ) together with a relatively low power consumption and reduced maintenance. We will present selected results from several microfocus source systems to demonstrate their potential for crystallography and small-angle scattering. 11 Ò T-230 Complementary Technology To The Synchrotron Pierre Le Magueres, Angela Criswell, Joseph Ferrara Rigaku Americas Corporation, The Woodlands, Texas, United States X-ray diffraction data collection at synchrotron beam lines is a critical tool for crystallographers to resolve protein crystal structures. The characteristics of the x-ray beam (high intensity, low divergence, very small size) and its tuneable wavelength are features required for anomalous diffraction-based phasing methods, for high-resolution structure refinements and for data collection on weakly diffracting samples or samples with long unit cell parameters. In addition, the proliferation of synchrotron beam lines in many countries and the increased availability beam time has made synchrotron facilities accessible to virtually every crystallographic laboratory in the world. To use the synchrotron most effectively, it is absolutely essential that crystallographers arrive prepared with samples whose quality as well as cryo-conditions have been previously tested and optimized at home. To address this, Rigaku has developed new instruments that will help researchers screen large numbers of samples in their own lab and recover those suitable for synchrotron data collection. We will first present the new ‘ScreenMachine’, a simple and selfcontained x-ray diffractometer optimized for fast and easy screening of macromolecular samples. We will also report on the improvements made to the automatic sample changer ACTOR , as well as on the latest technologies now offered to the home lab in the area of hybrid pixel array detectors (Pilatus) and multilayer optics with a smaller beam size. T-233 X-ray compatible microfluidic platforms for membrane protein crystallization Sudipto Guha, Sarah Perry, Paul Kenis Department of Chemical and Biomolecular Engineering, Univerity of Illinois at Urbana Champaign, Urbana, IL, United States Membrane proteins play a crucial role in many important biological processes including energy and material transduction across cellular membranes, molecular recognition and immune response. Efforts into understanding the function of these proteins have been severely hampered by difficulty in obtaining high quality crystals. These proteins are amphiphilic in nature and extremely sensitive to the surrounding environment. To obtain crystals, the proteins have to be isolated from the cellular membrane into artificial membrane 1 like environments without denaturing them. We use a technique called in meso crystallization which uses lipids to create mesophases in which proteins are stabilized. We have created microfluidic platforms which allow creation of these mesophases by mixing aqueous protein solution with highly viscous lipids. Addition of salt and precipitant leads to nucleation and growth of crystals. We have successfully validated a polydimethylsiloxane 2 (PDMS) based microfluidic platform to crystallize Bacteriorhodopsin in meso. However PDMS attenuates X-rays; hence on chip analysis of crystals is not feasible. We present here an X-ray compatible chip comprising of cyclic olefin copolymer (COC) and a thin PDMS layer needed for valve actuation. As proof of concept we have crystallized soluble proteins on-chip and obtained a full data set for the same. We are currently working on validating this platform with membrane proteins. Further uses of such a platform include onchip screening using minute quantities of protein, studying phase behaviour and interaction between various lipids and using a cryocooled chip to minimize radiation damage to crystals. References: 1. Cherezov V. et al., 2007, Science, 318, 1258–1265 2. Perry S.L. et al., 2010, CG&D, 9, 2566–2569 T-236 Testing Protein Crystals with X-rays in Crystallization Plates. Tadeusz Skarzynski Oxford Diffraction, Yarnton, Oxfordshire, United Kingdom Protein crystals are usually difficult to grow and can suffer damage on their way from the crystallization drop to the X-ray beam. The damage may be caused by manual handling, change of environment during harvesting, adverse effects of cryo-protection solutions and the dramatic change of temperature due to cryo-cooling. Traditional X-ray experiments to test crystals take time and effort and are often inconclusive. Characterization of protein crystals with X-rays, in-situ, without needing to extract crystals from the crystallization plate allows establishing a “base line” for crystal quality, and evaluating resolution limits before crystals are subjected to any manipulation. The in-situ testing also allows quickly distinguishing between salt and protein crystals, test harvesting, soaking and cryoprotectant conditions and selecting the best crystals for data collection. We will show how the in-situ testing, both at synchrotron beam lines and using the Oxford Diffraction PX Scanner system for home labs can be used as a powerful tool providing valuable feedback at various stages of crystal handling. Several examples of significant variation of diffraction properties of crystals grown from the same conditions will be shown and discussed, highlighting the importance of critical assessment of crystal quality at room temperature in some cases. Initial results of using the in-situ diffraction to detect ligand and heavy-atom binding will be presented and discussed as well. T-239 New micro-beam beamline at SPring-8, targeting at protein micro-crystallography Masaki Yamamoto , Kunio Hirata , Go Ueno , Yoshiaki Kawano , Takaaki Hikima , Atsushi 1 1 1,2 1,2 1 Nisawa , Hironori Murakami , Nobutaka Shimizu , Takashi Kumasaka , Takashi Tanaka , 1,2 1,2 1,2 1,2 Sunao Takahashi , Tomoyuki Takeuchi , Hirokatsu Yumoto , Haruhiko Ohashi , Shunji 1,2 1 Goto , Hideo Kitamura 1 1 1 1 1 1 RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5198, Japan, SPring-8/JASRI, 1-1-1 Kouto, Sayo, Hyogo 679-5198, Japan 2 In order to collect proper diffraction data from outstanding micro-crystals, a brand-new data collection system should be designed to provide high signal-to noise ratio in diffraction images. SPring-8 and KEK-PF are currently developing two micro-beam beamlines for Targeted 1) Proteins Research Program by MEXT of Japan. At SPring-8, a new undulator beamline dedicated for protein micro-crystallography, named RIKEN Targeted Proteins Beamline (BL32XU), is under construction, which will start user operation from May 2010. The beamline is designed to provide the stabilized and brilliant micro-beam to collect highquality data from outstanding micro-crystals. A small sized and highly brilliant X-ray beam with size of a micrometer will be providing high S/N data by both increasing reflection intensities and reducing background scattering. An in-vacuum undulator and K-B mirrors fabricated with 2) Elastic Emission Machinery technique will be equipped for the light source and the microfocusing optics, respectively. The beam size is variable from 1 to 20 m with high-precision slits at virtual light source according to designed experiments. The initial result of beamline 2 commissioning showed the minimum beam size at sample position corresponds to 1 x 1 m 2 10 with 6 x 10 photons/sec/ m . At end station, R&D for high-precision diffractometer, high-efficiency area detector, sample auto-changer, and sample environment suppressing background scattering are in progress. Support of real-time damage monitoring system for radiation sensitive micro-crystals is also being planned. We will present the current status and the future prospects of protein micro-crystallography at SPring-8. This study was supported by Targeted Proteins Research Program from the Ministry of Education, Science and Culture (MEXT) of Japan. [1] http://www.tanpaku.org/e_index.html [2] Mimura H. et.al, JAPANESE J. Appl. Phys. 44, L539-L542 (2005). Ó Ó Ó T-242 Polymorphism in molecular crystals via charge density distribution Mikhail Antipin , Konstantin Lyssenko , Yulia Nelyubina , Tatiana Timofeeva 1 1 2 2 1 2 New Mexico Highlands University, Las Vegas, NM, United States, Organoelement Compounds RAS, Moscow, Russian Federation Institute of To consider different factors affecting the stabilization of a particular polymorph we used the analysis of the electron density distribution function (r) in crystal within the “Atoms in Molecules” (AIM) theory. This experimental X-ray diffraction approach allows comparing the topological characteristics of chemical bonds, atomic charges and effective atomic volumes. Such way of description is substantially more sensitive to the difference in the intermolecular interaction patterns in comparison with the classical approach based on the analysis of interand intramolecular geometrical parameters. The usage of the AIM approach allows distinguishing the bonding interactions from all other contacts in a crystal by means of bond critical points, and estimating their energy values with high accuracy and, thus, to obtain the energy of a crystal lattice. It should be noted that the difference between the crystal lattice energies obtained from the X-ray diffraction data and sublimation enthalpy measured experimentally in many cases are as small as 0.2 kcal/mol. In the current presentation the results of experimental charge density analysis in the series of polymorphs of acetaminophen (paracetamol), p-dichlorobenzene, triphenylphosphine oxide and sulfide and other will be discussed and the benefits of this approach for the analysis of different factors, governing stabilization of particular form, will be demonstrated. Data on relative stability of polymorphs are important to define active pharmaceutical ingredient that satisfy drug manufacturers with their high solubility, processability, and biological action. Ô T-245 More flux – Less background: New improvements in low power microfocus beam delivery systems for diffraction and SAXS experiments Vincent Roger, Sergio Rodrigues, Peter Hoghoj XENOCS, Sassenage, France Microfocus sealed tube systems are increasingly used in single crystal applications replacing more and more traditional high power rotating anode sources for small crystal analysis. These solutions out-perform traditional x-ray generators with higher brilliance x-ray beam despite low power and benefit from low maintenance and low facilities requirements. Nevertheless so far microfocus sealed tube systems’ performances remained significantly lower compared to new generation of microfocus rotating anode sources. We will present Xenocs new developments in the field of beam delivery and beam conditioning systems enabling the optimum use of low power high brightness microfocus sources. These developments include both new aspheric multilayer optics with increased capture angle and improved focusing properties as well as new collimation devices for reduced background signal. Overall performance in terms of useful intensity is thus increased by a factor two or more compared to previous generation of microfocus sealed tube systems narrowing the gap with microfocus rotating anode generators. Comparative data, for single crystal diffraction and SAXS applications, acquired in collaboration with our academic partners will be shown to illustrate improved beam properties impact. T-248 Development and Performance of Microfocusing Source and Multilayer Optics based Beam Solution for Crystallography Bonglea Kim, Boris Verman, Doug Wilcox, Roman Samokyszyn, Mike Young, Licai Jiang Rigaku Innovative Technologies, Auburn Hills, United States Beam solutions based on microfocusing source and multilayer optics technology was first developed and applied to protein crystallography and small angle x-ray scattering by Rigaku more than ten years ago. The technology developed at Rigaku offers the best performance in this product category, which includes the highest resolution and intensity while lowering cost and making operation easier for users. Yet the technology is still evolving, and the performance continues to improve. In this presentation, we will review major issues in the development of this technology and illustrate the major system parameters which are key to excellent performance. These issues include fundamental principles, major engineering issues, past achievements and current status. Particularly, we will discuss the development of x-ray sources, x-ray optics and the close integration of these two key technologies. These discussions will offer some essential methodologies in evaluating the performance and avoiding confusion. Applications to macro and small molecule crystallography and SAXS will be discussed. T-252 API crystallogenesis probed by second harmonic generation microscopy Garth Simpson, Duangporn Wanapun, Umesh Kestur, Lynne Taylor Purdue University, West Lafayette, United States Second order non-linear optical imaging of chiral crystals (SONICC) is investigated as a selective probe for characterizing crystallinitiy in active pharmaceutical ingredient (API) formulations. Second harmonic generation, or the frequency doubling of light, is symmetry forbidden in amorphous media, but allowed for all crystals with a chiral unit cell. Consequently, SONICC provides excellent selectivity for trace crystallinity of APIs and can be performed rapidly over large fields of view for diverse samples. Studies with model compounds (griseofulvin and chlorpropamide) demonstrate detection limits of SONICC for crystallinity better than 1 part in 100 billion by volume, corresponding to a >9 order of magnitude improvement in % crystallinity compared to existing commonly used conventional methods (e.g., x-ray diffraction). The absence of a background response from disordered media allows the development of simple image analysis algorithms for automated quantification of nucleation rates, crystal growth rates, and activation energies for nucleation from a single set of measurements. Studies with powdered samples demonstrate the ability to easily quantify the residual 0.05% crystallinity remaining after exhaustive cryo-milling (S/N >1000). T-258 X-RAY POWDER DIFFRACTION STUDY FOR THE Cu2Cd1-zFezSnSe4 ALLOY SYSTEM Jose Henao , Mario Macias , Miguel Quintero , Ekadink Moreno , Manuel Morocoima , 2 2 2 2 Eugenio Quintero , Pedro Grima , Rafael Tovar , Pablo Bocaranda Universidad Industrial de Santander, Bucaramanga, Colombia, Universidad de Los Andes, Merida, Venezuela Room temperature X-ray powder diffraction (XRPD) measurements were carried out on polycrystalline samples of the Cu2Cd1-zFezSnSe4 alloy system, in steps of approximately 0.1 in z. The diffraction patterns were used to show the equilibrium conditions and to derive crystallographic parameters values. In each case, the XRPD reflections were indexed and the calculated lattice parameters were refined, and then, the initial values were estimated. Afterward, the XRPD patterns were refined by the whole pattern fitting using the Rietveld method. The results confirmed that the tetragonal stannite α (I-42m) structure occurs across the whole composition range at room temperature. From analysed data, was found that line splitting, viz. [(220), (204)], [(312), (116)], etc., due to tetragonal distortion c/a < 2 of the stannite structure, is observed across the whole composition range, and the separation of the splittings decreases as the composition z is increased. Furthermore, were found that the values of c/a increase nonlinearly from about 1.955 for z=0 to 1.975 for z=1. The deviation of the crystallographic parameter c from the Vegard law was related to the nonlinear variation of the internal distortion parameter σ with z. In the observed tetrahedrally coordinated stannite structure, each Se anion is surrounded by four cationic sites, i.e. two Cu, one Sn and one M, where the mixed cation M is given as M=(1-z)Cd + zFe, and each cation is similarly coordinated by four Se atoms. The results shows that as z is increased, the size of the mixed cation M is reduced and this resulting in an overall increase of the Se-M-Se and a reduction of the Se-Sn-Se angles. Additionally, the magnetic measurements showed that the amounts of extra phases were found to decrease considerably for samples which were re-melted in compressed form. 1 2 1 1 2 2 2 T-267 Diffraction Data from Liquid Crystal Elastomers: Versatile Display and Analysis Techniques John Konnert, Christopher Spillman, Jeffrey Deschamps, Jawad Naciri, Banahalli Ratna Naval Research Laboratory, Washington DC, United States Diffraction patterns of liquid crystal elastomers may contain broad features due both to short range order and to rotational disorder of domains. Techniques have been developed for interpolating, compressing or expanding the 3D diffraction data obtained with a CCD detector and placing the resulting scaled diffraction pattern into either a (256,256,256) or a (512,512,512) array. One then applies a rotation matrix to bring the pattern into the desired orientation for analysis. Viewing and analyzing the properties of constant Intensity surfaces, spherical half shells, and planes of data has proved useful. It is not necessary to rotate the entire array of data ,x , by the rotation matrix A, x'=Ax to obtain the intensity values , x', of the subset of data to be examined. The inverse of A need only multiply the elements x' of the -1 subset, A x'=x, in order to retrieve the elements of x, to be placed in x'. Below is shown a spherical half shell and a 3D surface plot with intense core of the 40A layer data for a LC elastomer. T-270 CRYSTAL STRUCTURE OF CCM3, A CEREBRAL CAVERNOUS MALFORMATION PROTEIN CRITICAL FOR VASCULAR INTEGRITY Xiaofeng Li, Rong Zhang, Haifeng Zhang, Weidong Ji, Wang Min, Titus Boggon Yale Univ., New Haven CT, United States CCM3 mutations are associated with cerebral cavernous malformation (CCM), a disease affecting 0.1-0.5% of the human population. CCM3 (PDCD10, TFAR15) is thought to form a ‘CCM complex’ with CCM1 and CCM2, however, the molecular basis for these interactions is not known. We have determined the 2.5Å crystal structure of CCM3. This structure shows an all alpha-helical protein containing two domains, an N-terminal dimerization domain with a fold not previously observed, and a C-terminal focal adhesion targeting (FAT)-homology domain. We show that CCM3 binds CCM2 via this FAT- homology domain and that mutation of a highly-conserved FAK-like hydrophobic pocket (HP1) abrogates CCM3-CCM2 interaction. This CCM3 FAT-homology domain also interacts with paxillin LD-motifs using the same surface, and partial CCM3 co-localization with paxillin in cells is lost on HP1 mutation. Disease-related CCM3 truncations affect the FAT-homology domain suggesting a role for the FAT-homology domain in the etiology of CCM. T-273 Influence of Glycerol addition to Dispersed Liquid Crystalline Phases – A Small Angle X-Ray Study Heiner Santner , Sandra Engelskirchen , Reinhard Maurer , Otto Glatter 1 2 3 2 1,3 1 1 Karl-Franzens University, Graz, Austria, Anton Paar, Graz, Austria, University of Stuttgart, Stuttgart, Germany Phytantriol is a hydrophobic surfactant comprising a highly branched phytanyl-chain with a tri-hydroxy headgroup. The binary Phytantriol – H2O system shows a complex phase behavior featuring a variety of lyotropic liquid crystalline phases (lamellar, inverse hexagonal and cubic structures) and a fluid isotropic phase (L2 phase). Above a certain composition these phases coexist with an excess water phase, which allows dispersing the respective nanostructure in a continuous water phase. The resulting sub-micrometer sized particles are stabilized by the addition of the tri-block copolymer Pluronic F127. Internally self-assembled dispersions represent self-assembly in confinement. Inside the sub-micrometer sized particles liquid crystalline material is confined, which consists of water and oil domains separated by an amphiphilic monolayer. While the internal nanostructure was found to be in thermodynamic equilibrium the whole particle is kinetically stabilized [1, 2]. The confined nanostructure is capable of solubilizing hydrophilic, hydrophobic or amphiphilic substances offering many advantages as carrier systems for active substances. Bridging the gap between basic research and technical application one often faces the need to improve the properties of the respective formulation. In the present contribution we focus on stability against cold through the addition of glycerol to the continuous water phase of the internally self-assembled dispersion. The effect of glycerol on the confined nanostructure was determined via Small Angle X-Ray Scattering revealing that the addition of glycerol induces phase transitions from inverse hexagonal phases to water-in-oil microemulsions. In this respect the addition of glycerol has a similar effect like raising temperature. zP| ¡?b\«fi›K?kM?¡ ?\ M k\‹£«· ‒ GQOOSH QOK?TQTSLTQUPM zQ| x\£⁄«·‒K?`M¡ ?\ M k\‹£«· ‒ GQOOTH QPK?TUXLTVVM T-276 SWAXS Analysis on Multicompartment Micelles formed by The Newly Designed Ion Pair Hybrid Surfactant Semra Ide , Hande Unsal , E.Hilal Soylu , Nihal Aydogan 1 1 2 3 2 Hacettepe University , Faculty of Engineering, Department of Physics Engineering 06800 2 Beytepe, Ankara, Turkey, Hacettepe University , Faculty of Engineering, Department of 3 Chemical Engineering, 06800 Beytepe, Ankara, Turkey, Karadeniz Technical University, Faculty of Science & Literatur, Department of Physics 61080, Trabzon, Turkey Multicompartment micelles are aggregates of surfactants composed of a hydrophilic shell and a multidomain hydrophobic core which makes it possible to cosolubilize and transport several different and immiscible materials in different subdomains selectively and preventing any undesired interactions before reaching the target. Hence, multicompartment micelles have high potential to be used in controlled drug delivery, imaging technology, selective entrapment and release of dyes,gene therapy agents, etc.Because of molecular structure, there is the possibility of being arranged one after another for hydrocarbon and fluorocarbon-based compartments in some of these aggregates, especially for segmented worms resulting in small-volume micellar subdomains .Therefore, a novel hydrocarbon-fluorocarbon ion pair + + hybrid surfactant CH3(CH2)11(OCH2CH2)23N (C2H5)3SO3-(CF2)7CF3 (C12E23N SO3-F8) was designed in order to minimize some problems faced in previous studies such as low content of hydrophobic groups in the aggregate, absence of common interface between two distinct hydrophobic cores, low solubilization capacity, and overlapping of hydrophobic subdomains. + Having high solubilization capacities of C12E23N SO3-F8 for hydrocarbon-based and fluorocarbon based probes both separately and simultaneously leads to the deduction of achieving efficient compartmentalization inside the micellar core. SAXS study can be used for yielding valuable structural information about nanostructured aggregations built by these type surfactants. Depending on the molecular structure of the ion paired segments, diverse morphologies can be expected for the formed compartments e.g. spheres in/on spheres, open sandwich bread and mace shapes which may be composition of several rods / sphere and rod. SAXS experiments were performed on the samples (with mM of 3%, 7% and 10%) by using a Hecus SWAXS system (installed in the content of Hacettepe BAB Project fund: 06A602012) with CuK , = 1.54 Å. To define the possible multicompartment structure, various models for the form factor were considered for fitting the SAXS patterns. Ö Õ T-279 Structural Chemistry of Mono–substituted Nitrobenzenes With Pendant Ethylamino Substituents Philip Squattrito , Dillip Mohanty , Thomas Payne , Chad Thurman , Hao Yu , Qian Sun , 2 2 2 Kristin Kirschbaum , Mark-Robin Giolando , Chris Brue Central Michigan University, Mount Pleasant, Michigan, United States, University of Toledo, Toledo, Ohio, United States As part of our continuing study of model compounds for intermolecular interactions in polyamine polymers, a series of mononitrobenzenes with one ortho ethylamino substituent and either a para ethylamino substituent or a bridging sulfonyl have been synthesized and structurally characterized. The compounds 4-nitro-N,N’-diethylbenzene-1,3-diamine (I) and 2,6-(bisethylamino)-3-nitrobenzonitrile (II) differ only in the absence or presence of a cyano group in between the two ethylamino groups on the ring, allowing for an analysis of the effect of the cyano group on the intermolecular interactions and crystal packing. The primary interaction in (I) is an intermolecular N-H…O hydrogen bond between an amino hydrogen atom and a nitro O atom that links molecules into one-dimensional chains. By contrast, molecules of (II) are joined into dimers by bifurcated N-H…O hydrogen bonds between the amino H atom and both a nitro O atom on the ortho nitro group of the same molecule and a nitro O atom on the neighboring molecule. The third compound, di(4-ethylamino-3nitrobenzene)sulfone (III), contains the same ortho nitro/ethylamino pairing as in (I) with the position para to the nitro group occupied by the sulfone instead of a second ethylamino group. Molecules of (III) are linked into zigzag double chains through N-H…O hydrogen bonds between the amino H atoms and sulfonyl O atoms. The detailed intermolecular interactions and packing of (III) will be analyzed in relation to those observed for (I) and (II). 1 2 1 1 1 1 1 1 T-282 Functional group recognition in carboxyalkylammonium salts Melanie Rademeyer, Belinda van der Westhuizen University of Pretoria, Pretoria, South Africa The carboxyalkylamine 4-aminobutanoic acid (GABA) is a commercially available supplement 1 against anxiety , while the longer chain 6-aminohexanoic acid is marketed as a treatment for 2 bleeding disorders . In the pharmaceutical industry the salts of active ingredients often show improved physicochemical properties compared to the neutral ingredient. This study investigated the structures and functional group recognition occurring in halide and oxoanion salts of 4-aminobutanoic acid and 6-aminohexanoic acid, with a number of novel structures reported. Monovalent counter anions including chloride, bromide, iodide, nitrate and perchlorate were chosen. Packing trends were identified and non-covalent interactions and the role of the anion highlighted. Emphasis was also placed on hydrogen bonding interactions and recognition occurring between terminal functional groups and anions. A number of permutations of hydrogen bonding donors and acceptors are possible. It was found that for the anhydrous halide salts all three hydrogen bonding groups (anion, ammonium group and carboxylic acid group) interact to form a complex, two-dimensional hydrogen bonding network. However, in the case of the hydrated halide salts and the oxoanion salts the carboxylic acid functional groups only interact with other carboxylic functional groups, while the ammonium groups exclusively hydrogen bonds to the anions (and water molecules when present). Thus, in the case of the hydrated halides and the oxoanion salts a certain degree of “recognition” is displayed. 1. Abdou, A. M., Higashiguchi, S., Horie, K., Mujo, K., Hatta, H., Yokogoshi, H. 2008, 26, 201-208. 2. Thomas, D. C., Wormald, P. J., Am J Rhinol., 2008, 22, 188-191. BioFactors, T-285 The influence of crystallographic symmetry elements on the observation of a uniform stacking motif in ¼-filled one dimensional molecular systems. Eric Reinheimer , Marc Fourmigue , Patrick Batail , Claude Coulon , Kim Dunbar 1 2 1 2 3 4 1 Texas A&M University, College Station, TX, United States, Universite Rennes 1, Rennes, 3 4 France, Universite d' Angers, Angers, France, Centre de Recherches Paul Pascal (CRPPCNRS), Pessac, France The study of the structures and physical properties of conducting molecular solids have spawned many fascinating discoveries in the realms of crystallography, chemistry and solid state physics. Seminal discoveries such as TTF-TCNQ (TTF = tetrathiafulvalene; TCNQ = tetracyanoquinodimethane) and the TMTSF (TMTSF = tetramethyltetraselenafulvalene) family of electrocrystallized salts have been the subject of intense study and debate since their initial syntheses more than thirty years ago. One unifying principle has dominated interdisciplinary debate on these materials; that they suffer from a confluence of multiple physical phenomena which serve to inhibit the complete understanding of each individual phenomenon. In order to gain a greater understanding of the novel behavior that these materials display, physicists and theoreticians have suggested that simpler systems be prepared. Among the approaches advocated is to prepare charge transfer materials based on oxidized chalcofulvalene moieties that exhibit equivalent intermolecular spacing between the moieties along the stacking axis. These one dimensional, non-dimerized, systems are ¾-filled with electrons (1/4- filled with holes) and are essential for testing theoretical work that predicts that such systems will be Mott insulators. To date, systems with uniform non-dimerized chains have been quite rare. Initial examples with DMtTTF (DMtTTF = dimethyltrimethylene-tetrathiafulvalene) as well as its selenium analogue were reported to show uniform stacking when first crystallized via electrochemical methods with the tetrahedral anions [ClO4] and [ReO4] in the 1980s. More recent examples of exhibiting a uniform stacking motif were found to exist in samples containing the non-centrosymmetric tetrathiafulvalene donors EDT-TTF-CON(CH3)2 and oMe2TTF when crystallized with [AsF6] and the halide anions respectively. Closer inspection of each salt’s solid state structure reveals that the individual crystallographic symmetry of each of the materials described above is critical for defining the uniform stacking motif deemed necessary by theoreticians. This talk serves to illustrate the contribution that X-ray crystallography has made in characterizing these chalcofulvalene salts as unique solid state materials. In addition current understanding of their physical properties will also be discussed. T-289 Experimental and Theoretical Determination of the Electron Density Distribution of Methyl- -Cellobioside Edwin D. Stevens , Ryuta Sasabayashi , Michael K. Dowd , Glenn P. Johnson , Alfred D. 2 French Department of Chemistry, University of New Orleans, New Orleans, LA 70148, United 2 States, Southern Regional Research Center, USDA, New Orleans, LA 70124, United States -Cellobiose is a disaccharide containing two glucose residues joined by a 1,4 glycosidic linkage and therefore it provides a model for the basic, structural repeat unit of cellulose. The crystal structure of the methanol solvate of methyl- -cellobioside shares a remarkable number of structural similarities with the structure of cellulose IIII. High-resolution single-crystal x-ray diffraction measurements of methyl- -cellobioside collected at 120 K have been used to determine the experimental electron density distribution of the molecule in the crystal. The electron density has also been obtained from large basis set DFT calculations of the molecular wavefunction using the experimental geometry. The crystalline environment has been simulated in the theoretical calculations by including fragments of surrounding molecules in a cluster calculation. A topological analysis of both the experimental and theoretical electron distributions has been performed using the Atoms in Molecules approach, and the results are compared with recent low temperature studies of the electron density , -trehalose (Stevens, distributions of Dowd, Johnson, and French, Carbohydrate Research 2010, in press) and sucrose (Jaradat, Mebs, Chęcińska and Luger, Carbohydrate Research 342 2007, 1480– 1489). 1 1 1 2 2 × ØØ × × × sLQXP g\ ›£¡‹Lg\ ›£¡‹?m›‹ ›‹ ¡ ?h‹ ¡‒«› ¡¦· \‒?h‹ ¡‒\¦ ›‹ ? ‹?l› ¡¦· \‒?b‒„ \ ¢ Ýø ÿõ äøúÚøÞÝú ßóßÞÝ÷ÞÝÞ öÝ óøÚßÿõ ÷ó£ þÞãßõùúÞã ¢ óäÚóùóÙ á¡ü óôø Ý äóôúöÚó¡ äøúÚøÞÝú óôø Ý Ûøãùã÷ÚãöÚâ úãöøóÜÝóþ óôø ÞÝ äãäÚôÿÜó ôøãû äÞóþÝùÚô ùÚý ü ôøãû äÞÝãøúÚöóøÞã öÚùõúóùÝÜöóøÞã ßóßÞÝ÷ÞÝÞ ßóÛóâöõä óâÚô ó çå òñ ïîêíìçëêéè çæå v ? ? ? ? äãâÚá à ßÞÝÜÛÚÙ ? ? ‚\ ?\ ?`· ‹K?`· ‹K?sw?VWVPQK?t‹ ¡ ?r \ ¡ ? ? ? ? L K? ? ? ? ? ? ? M? ? ? ? ?G ? MRP? ? ? QOPO? H ? ? ? ? ? OM ä?£‒¡\ ¡‒? ⁄\‹? ⁄¡? \‹? ¡‒?v\\ ? ·«?£\ ¡? ⁄¡?¢› ›• ‹£?‹·« ¡‒ ?›¢ ‒¡¢¦› ¡?⁄ ?G‹› ‹ ·\ ?¦›‹ \¦ HY ▁ ? ? h ▁ a‒ ▁ b ▁ e bLh▁ bLa‒▁ bLb ▁ bLe▁ PKSTX WW TQS RUW WX SKXWO PKPQV SRO QQW WSS PXKWOU QKTVR RST RXQ RKTSP PWKTWS f¡›«¡ ‒ ¦? ‒ · ›‹ ? ›¢? ⁄¡? ‒¡ · ‹£? ‹›‹ ›‹ ¡ ? ¦›‹ \¦ ? ⁄\ ¡? ¡¡‹? · ¡ K? • ⁄? fi\‒ ¦· \‒ ¡«fi⁄\ ?›‹?¦›‹ \¦ ? • ⁄? ⁄¡?⁄¡\ ¡‒?⁄\ ›£¡‹ ?h \‹ ?a‒? ›‹ ¡ ? ›?¦\‒ ›‹M?n ⁄¡‒?¢\¦ ›‒ ?¦›‹ ¡‒¡ ‹¦ · ¡ ? •⁄¡ ⁄¡‒? ¦›‹ \¦ ? •¡‒¡? • ⁄? bG\ fi⁄\ ¦H♠g\ ? M? bG\‒›«\ ¦H♠g\ ? \‹ ? ‹¡· ‒\ ? M? ›‹ ¦ ‹›‹ ›‹ ¡ ?g\ ?¦›‹ \¦ ? ›?b♠g\ M T-295 Synthesis, Characterization, and X-ray Determination of Monoitaconate Esters. 1 1 and Synchrotron 1 Radiation 2 Structure Graciela Diaz de Delgado , Belkis Ramirez , William Velasquez , Maren Pink 1 2 Universidad de Los Andes, Merida, Merida, Venezuela, Indiana University, Bloomington, Indiana, United States Monomers derived from itaconic acid are widely used in the preparation of polymer complexes of potential use in biomedicine, agriculture, as hydrogels, in drug-delivery systems, fabrication of contact lenses, among other applications. In this work, the structure of several itaconate monoesters is presented. The esters were prepared by reaction of itaconic acid with alcohols in the presence of acetyl chloride. The materials prepared include the methylitaconate, ethylitaconate, benzylitaconate, dodecylitaconate, among others. For example, Methylitaconate crystallizes in the orthorhombic system, space group Pca21, with 3 unit cell parameters a=11.305(4), b=5.156(2), c=23.659(7) Å, V=1379.1(8) Å , Z=8. The refinement converged to R=0.0753, wR=0.1878, S=1.057. In this structure, typical cyclic dimer hydrogen bonds are observed between molecules in the bc plane. Data from synchrotron radiation studies on other monoitaconates will also be presented. Funding for this work was provided by CDCHT-ULA and by FONACIT, grant LAB-97000821. T-299 The Molecular Structure Of The First Benzoindenone Compound Isolated From The Roots of Psychotria prunifolia Jose Ricardo Sabino , Christopher Ceccarelli , Laryssa Campos Ribeiro , Cecilia Maria Alves 1 1 de Oliveira , Lucilla Kato Univ. Federal de Goias, Goiania GO, Brazil, Agilent Technologies, Blacksburg, VA, United States As part of our program to assess the chemical and biological diversity of native plants of the Brazilian Cerrado, we have examined promising active extracts of Rubiaceae species concerning their antitumoral potential. In this work, leaves, barks and roots from Psychotria prunifolia was subjected to ethanolic extraction and successive chromatographic separation to provide an unpolar compound which have not been identified before. The molecule, shown below, is described as a benzoindenone, with no reference in CAS or other public database sources. A crystal suitable for x-ray study was obtained by evaporation of a solution of methanol-chloroform (1:1). The molecule crystalizes in the P21/c space group with cell parameters: a = 9.7355(2) Å, b = 7.3271(2) Å, c = 19.6875(5) Å, β = 102.676(2)° volume , 3 1370.14(6) Å . Data collection were performed with a Varian Gemini Ultra diffractometer controled by CrysAlisPro (Oxford Diffraction Ltd., Version 1.171.33.55), using Cu-Kα radiation at 100 K. 16063 data points were collected of what 2447 are symmetry independent (Rint = 0,038). Structure solution was achieved with Direct Methods using Olex2 (J. Appl. Cryst. 2 (2009). 42, 339–341). Model refinement was performed with full matrix least squares on F with final residuals R1 = 0.034, wR2 = 0.095 for observed data with I>2σ (I), and R1 = 0.042, wR2 = 0.10 for all data. 1 2 1 2 1 T-309 Synthesis and Activity of Oxynitride Nanoparticles Craig Bridges, Mariappan Paranthaman Oak Ridge National Laboratory, Oak Ridge, TN, United States Metal oxides have played a key role in the development of modern science and technology, in large part due to the ability to manipulate chemical and electronic structures through chemical doping and substitution. The position that mixed anion phases can occupy in the development of materials with novel properties has recently been well demonstrated, through the flurry of activity stimulated by the discovery of superconducing oxypnictide and oxychalcogenide phases. While oxyanion materials have been well investigated in the bulk, they are relatively underdeveloped as nanoparticle phases. The development of the chemistry of nanophase oxyanion synthesis is an important area, as this may expand the role these materials play in the future for both energy storage and conversion, as well as more fundamentally providing insight into the impact of particle size on anion substitution. The synthesis of oxynitride phases has typically required the formation of an amorphous or small particle precursor, due to the relatively slow diffusion of the nitride anion at typical synthesis temperatures. Here we examine in detail the correlation between particle size and crystallinity on the resulting synthesis conditions in the formation of oxynitrides. Nanoparticles have been prepared through a variety of solution phase methods to obtain well defined precursors for ammonolysis. In situ diffraction results illustrating the important role of nanocrystal precursors size on oxyanion nanoparticle formation will be presented, and the impact of particle size on the resulting catalytic properties will be discussed. This work provides insight into the underlying factors controlling anion transport for this particularly difficult class of oxyanion synthesis. T-313 Practical use of a bent perfect Si monochromator on a neutron four-circle diffractometer at the HFIR. Bryan Chakoumakos , Huibo Cao , Alexandru Stoica , Mihai Popovici† 1 1 1 1 2 2 Oak Ridge National Laboratory, Oak Ridge, TN, United States, Research Reactor, Columbia, MO, United States Missouri University The design of doubly-bent perfect crystal monochromators has steadily improved, making them competitive with mosaic crystal monochromators. A multi-wafer neutron † monochromator, designed by M. Popovici and A.D. Stoica, has been commissioned on the HB-3A Four-Circle Diffractometer at the HFIR, ORNL. The unit is made from a bent packet of silicon wafers of almost [110] orientation with the <1-10> zone axis vertical. The reflection planes of practical interest are (220)/(440), (331), and (111)/(333), accessible by rotation of o the unit, which give lambda = 1.56, 1.01, and 2.5 Å, respectively at the fixed 48 take-off angle -1 for the instrument. The horizontally curvature, rho = 1/R (m ), of the monochromator is -1 variably adjustable, from essentially flat to a curvature of 0.7 m . The neutron flux incident on the sample and the Bragg peak width at the detector are highly dependent on rho, such that the incident flux on the sample passes through a maximum, increasing by ×1.8 for 1.01 Å and by ×3.3 for 1.56 Å, as compared to the flat condition. The flux increase is due to the deltalambda/lambda increasing, but eventually the incident beam mask cannot handle the increasing divergence and the intensity drops off. The Bragg peak width increases and the width versus scattering angle flattens as rho increases Given these effects, rho be adjusted to deliver high intensity primarily for crystal structure refinements, or high resolution for resolving symmetry changes, e.g., charge order with lattice distortion and complex magnetic orders. Traditional step scanning and more efficient continuous scanning modes are possible. This research is supported by UT Battelle, LLC under Contract DE-AC05-00OR22725 for the U.S. Dept. Energy, Office of Science. ©¥¨§¥¦¥¤ 2 T-317 X-ray studies of the photoexcitation of Zn[4Cl-PhS]2phenanthroline and Zn[4-Me-PhS]2bathocuprine Mette Schmoekel , Jason Benedict , Radoslaw 1,3 1 Kaminski , Philip Coppens University at Buffalo, SUNY, Buffalo, NY, United 2 States, Aarhus University, Aarhus C, Denmark, 3 University of Warsaw, Warsaw, Poland Photo-crystallographic studies of light-induced short-lived species in molecular crystals are part of a relatively unexplored but rapidly increasing area of crystallography. It is a powerful tool for increasing the understanding of the processes that occur when crystals are exposed to laser light. Depending on the type of compound, time scale, and the lifetimes of the metastable species in question such studies can be performed by either in-house pseudosteady state or synchrotron-based pump-probe experiments using either monochromatic or 1 polychromatic sources. Here we present photo-crystallographic liquid-helium temperature 2 studies of two ~100 μ s lifetime Zn-complexes, Zn[4-Cl-PhS]2phen and Zn[4-MePhS]2bathocuprine. The in-house experiments used a high rep-rate Nd-vanadate laser at both 100 and 20 kHz. The structural changes are analyzed with photo-difference plots showing the changes in electron density due to laser exposure, and subsequent least-squares refinement 3 4 with the program Laser05. The refinement is based on the RATIO method in which the ratios, R(h)=Ilight ON(h)/Ilight OFF(h) rather than the absolute intensities are refined. The results are interpreted in terms of both a molecular change and a slight motion of the unconverted ground state molecules. Parallel theoretical calculations suggest the excitation to correspond to formation of a weak S-S bond in the excited triplet states of both complexes. 1 1,2 1 1 W. K. Fullagar, G. Wu, C. Kim, L. Ribaud, G. Sagerman and P. Coppens, J. Synchr. Rad. 7, 2 229-235 (2000). R. G. Highland, J. G. Brummer, and G. A. Crosby, J. Phys. Chem., 90, 3 1593-1598 (1986). Y. Ozawa, S. Pillet, I. Vorontsov, R. Kaminski, University at Buffalo, 4 Crystallographic Programs. P. Coppens, M. Pitak, M. Gembicky, M. Messerschmidt, S. Scheins, J. B. Benedict, S. Adachi,T. Sato, S. Nozawa, K. Ichiyanagi, M. Chollet and S. Koshihara, J. Synchrotron Rad., 16, 226-230 (2009). Acknowledgements: Research supported by the National Science Foundation (CHE0843922). T-331 Characterization of the crystal of protein CTB and protein -environmental ligand complexes by using a X-ray diffraction Huong Tran, Jeffrey Wolt, Edward Yu Iowa State University, Ames, IA, United States Transgenic plants may play an important role in the cost-effective large scale production of biopharmaceutical proteins and industrial enzymes. The introduction of novel plant-made proteins in agro-ecosystems poses concerns about the potential harm of crop residuals to the surrounding ecosystem. Few studies have considered how the environmental fate and activities of transgenic proteins may impact soil processes. Protein reactivity with environmental ligands are investigated through crystallization and x-ray diffraction as a means for understanding environmental degradation and persistence of transgenic protein. Vibrio cholera enterotoxin subunit B (CTB) is studied as a model system under laboratory conditions. In order to have a full understanding of changing conformation of protein with humic acid, three quinones [lawsone (2-hydroxy-1, 4- naphthoquinone), juglone (5-hydroxy-1, 4 –naphthoquinone) and AQDS (anthraquinone-2, 6- disulfonate)] were used to model reactive sites found in humic acid. The crystals of protein –ligand complex will be analyzed by X-ray diffraction at Agronne National Lab with detector ADSC Q315. The molecular replacement is used to obtain phase information for determining the structure. The results from this research will provide the novel insight into the mechanism of interaction between protein and environmental ligands. T-200 Cathepsin K and the regulation of bone resorption Michael James , Martin Kienetz , Maia Cherney , Zhenqiang Li , Dieter Bromme 1 1 1 1 2 2,3 2 Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada, Department 3 of Human Genetics, Mount Sinai School of Medicine, New York, NY, United States, Faculty of Dentistry, University of British Columbia, Vancouver, BC, Canada The regulated interplay between bone resorption and bone formation leads to a complete replacement of the human skeleton every seven to ten years. The inorganic demineralization stage is accomplished by the release of HCl from specialized cells called osteoclasts. These cells also secrete large quantities of the papain-like lysosomal cysteine peptidase, cathepsin K. The organic matrix of bone consists of > 90% type I collagen and cathepsin K is the predominant peptidase that degrades collagen. The lack of a cathepsin K activity leads to a severe bone resorption defect known as pycnodysostosis, a disease that Henri ToulouseLautrec was believed to have suffered from. Excessive cathepsin K activity leads to orteoporosis. Collagen consists of three polypeptide chains each ~ 1000 amino acids in length. At the N- and C-termini of each chain there are ~50 amino acids that form the globular telopeptides; the remaining ~900 amino acids of each chain are twisted into left-handed helices that intertwine to form a right-handed triple helix. The triple-helical region of each chain is especially resistant to general peptidase degradation. The unique triple-helical degrading activity of cathepsin K depends on the formation of complexes with glycosaminoglycans such as chondroitin 4-sulfate (C4-S). We have determined the structure of the 1:n complex between cathepsin K and C4-S to 1.8Å resolution. The C4-S molecule (17 kDa) adopts a cosine wave-shaped oligomeric conformation and each cathepsin K molecule interacts tightly with three disaccharides (an alternating copolymer of β -D-glucuronic acid and 2′ -deoxy-2′ -acetamido-β -D-galactose-4-sulfate) of the C4-S. The binding sites of C4-S are located in the R-domain of cathepsin K and are distant from its active site. Our poster will present the structure this interesting complex between cathepsin K and the oligosaccharide of chondroitin 4-sulfate. T-332 Crystal Structure of the Catalytic Domain of Rlf Peng Wei , Xu Jiwei , Sun Yao , Liu Ian , Rao Zihe , Li Xuemei 1 2 1 1 1 1 1,2 1 3 Chinese Academy of Sciences, Beijing, China, Nankai Univ., Tianjin, China, tsinghua Univ., Beijing, China Ral, the subfamily of the small GTPase Ras superfamily, plays essential role in many different cellular processes including cell growth, differentiation and vesicular transport. Ral family includes two members in human, named RalA and RalB. As other members of the Ras family, Ral cycles between its activated GTP-bound form and inactivated GDP-bound form in cells, controlling by the guanine nucleotide exchange factors (GEF), which catalyse the substitution of Ral bound GDP by GTP, and the GTPase activating proteins(GAP), which enhance the GTPase activity of Ral, leading to hydrolysis of the Ral bound GTP to GDP. Six different GEFs for Ral GTPase have been identified up-to-date; they are Rlf, RalGDS, Rgl, Rsc, RalGPS1 and RalGPS2. All those GEF share a common cdc25 domain responsible for their GEF activity and bear different regulatory domain. RalGDS, Rgl, Rlf, Rsc have a Ras-GTP binding domain(RBD) and their GEF activity are promoted by Ras-GTP, while RalGPS1 and RalGPS2 lack the RBD but have a GRB2 binding PXXP motif, suggesting their different activation pathway. We have determined the three dimension structure of the cdc25 domain of Rlf. The cdc domain of Rlf shows high similarity with that of Son Of Sevenless (SOS) but differ in the conformations of a long loop close to the active site. Unlike that of SOS, Rlf cdc25 domain alone has GEF activity when it separated from the whole protein, and this structure variety between Rlf and SOS may cause their functional differences. T-333 Structural Diversity of the Proline Utilization A Family Revealed by SAXS, Light Scattering, and Analytical Ultracentrifugation Ranjan K. Singh, Travis A. Pemberton, John J. Tanner Univ. Of Missouri, Columbia, Mo, United States The proline catabolic enzymes catalyze the 4-electron oxidation of proline to glutamate. The 1 reaction involves two enzymes, proline dehydrogenase and ∆ -pyrroline - 5-carboxylate dehydrogenase. Some bacterial organisms have both of these enzymes fused together, and the fused bifunctional enzymes are called Proline utilization A (PutA). In addition to these bifunctional enzymes, some PutAs are trifunctional, because they moonlight as transcription repressors of their own gene. Our lab recently reported that the quaternary structure of the bifunctional PutA from B. japonicum is a ring-shaped tetramer [1]. However, the structural organization of PutAs from other organisms is still unknown. In particular, there are no structures available for moonlighting trifunctional PutAs. This poster will focus on an examination of the diversity of the quaternary structure within the PutA family. Small angle X-ray scattering, analytical ultracentrifugation, and light scattering experiments have been done to reveal the oligomeric states and shapes of several bifunctional and trifunctional PutAs. Our current data suggest that bifunctional and trifunctional PutAs have substantially different quaternary structures. We suggest that these structural differences are related to the specific set of functions that PutAs must fulfill in utilizing proline in different organisms. [1] Srivastava D, Schuermann JP, White TA, Krishnan N, Sanyal N, Hura GL, Tan A, Henzl MT, Becker DF, Tanner JJ. Crystal structure of the bifunctional proline utilization A flavoenzyme from Bradyrhizobium japonicum. Proc. Natl. Acad. Sci. U. S. A. (2010) 107(7):2878-83. This research is supported by NIH grants GM065546 and GM061068. SP.03 From the Structure of the Ribosome to New Antibiotics Thomas Seitz Yale Univ., New Haven CT, United States Over the past decade, we have determined the structures of the Haloarcula marismortui large ribosomal subunit and many of its complexes with substrate analogues and antibiotics, which have led to an understanding of the mechanism of peptide bond formation and its inhibition by antibiotics. More recently, we have established the structure of the Thermus thermophilus fmet 70S ribosome complexed with tRNA in the P site and elongation factor P (EF-P) bound next to it between the E and P sites which suggests that EF-P stimulates the formation of the fmet first peptide bond by properly positioning the fmet-tRNA . The structures of the 70S ribosome and three bound tRNA molecules and either the anti-TB antibiotics viomycin or capreomycin show how they inhibit protein synthesis and suggest how new anti-TB drugs might be designed. Further progress is being made in our pursuit of the structures of the 70S ribosome with arresting polypeptide captured in the exit tunnel. SP.04 The Amazing Ribosome, Its Tiny Enemies And Thoughts About Its Origin Ada Yonath Weizmann Inst., Rehovot, Israel Ribosomes, the universal cellular machines, possess spectacular architecture accompanied by inherent mobility, allowing for their smooth performance as polymerases that translate the genetic code into proteins. The site for peptide bond formation (PTC) is located within a universal internal symmetrical region connecting all of the remote ribosomal features involved in its functions. The elaborate architecture of this region positions ribosomal substrates in appropriates stereochemistry for peptide bond formation, for substrate-mediated catalysis, and for substrate translocation. The high conservation of the symmetrical region implies its existence irrespective of environmental conditions and indicates that it represents the ancient ribosome. The PTC is located above an elongated tunnel along which nascent chains progress until they emerge out of the ribosome. This tunnel may also be involved in chaperoning function, provides the binding site of the first cellular chaperone that encounters the emerging nascent chain, and hosts a major family of antibiotics. Crystallographic analysis of complexes of ribosomes and antibiotics targeting them revealed the structural bases for synergism, selectivity, induced fit; remote interactions, secondary conformational rearrangements and cross-resistance to ribosomal antibiotics; the common and specific pathways of resistance and cross resistance; the minute chemical differences that can turn competition into synergism; and the factors leading to resistance acquired by secondary conformational alterations. 05.01.1 Solid-State Conformational Differences Between “Bridge-Flipped” Isomeric Organic Molecules William Ojala , Jonathan Smieja , Dana Newman , Jeremy Leavell , Anthony Gerten , 2 Charles Ojala 1 1 1 1 1 1 University of St. Thomas, St. Paul, Minnesota, United States, Normandale Community College, Bloomington, Minnesota, United States 2 We have designated as “bridge-flipped” isomers those organic molecules related by a reversal of a bridge of atoms connecting two major parts of the molecule. Families of compounds in which this isomerism is found include the benzylideneanilines, for which the isomerism is Ar-CH=N-Ar’ vs. Ar-N=CH-Ar’ , and the phenylhydrazones, for whi the ch isomerism is Ar-CH=N-NH-Ar’ vs. Ar-NH-N=CH-Ar’ (Ar = aryl In these two families, steric ). differences between the isomers are minor regardless of bridge orientation, raising the question of whether the two isomers might assume the same molecular packing arrangement in the solid state and be readily co-crystallized. We have found such isostructuralism to be rare, but several examples do exist. In a continuing effort to identify more isostructural pairs and to examine structural factors such as hydrogen bonding or molecular conformation that would facilitate or discourage isostructuralism, we have been examining pairs in which one of the isomers has been found to be nearly planar, preparing the bridge-flipped isomer and determining its crystal structure to find out whether the isomer is planar as well and the packing arrangements thus potentially isostructural. We have determined and describe here the crystal structures of two benzylideneanilines, 4-methoxy-N-[(3nitrophenyl)methylene]benzenamine and N-[(3-bromophenyl)methylene]-4chlorobenzenamine, nonplanar molecules for which the isomer has been found to be planar in each case by previous workers. We also describe the structures of 4-bromo-N(phenylmethylene)benzenamine and N-[(4-bromophenyl)methylene]benzenamine, compounds for which cell constants but not coordinates have been reported in the literature previously. Here as well, one isomer is nearly planar in the solid state, but the other is significantly nonplanar and the compounds are not isostructural. We discuss here also the crystal structures of two bridge-flipped, non-isostructural phenylhydrazones, 4chlorobenzaldehyde-4-nitrophenylhydrazone (nearly planar) and 4-nitrobenzaldehyde-4chlorophenylhydrazone (nonplanar). Finally, we describe the structures of two bridge-flipped symmetrical bis-benzylideneanilines, both of which would be capable of occupying a crystallographic inversion center by assuming a centrosymmetric molecular conformation but only one of which actually does. 05.01.2 To bridge or not to bridge? Reversible bridging and terminal carbonyl ligand configurations in some unusual tricobalt carbon cluster derivatives of a tripodal phosphine ligand. Jim Simpson, John McAdam, Brian Robinson, Roderick Stanley University of Otago, Dunedin, New Zealand In simple methinyltricobaltnonacarbonyl clusters, RCCo3(CO)9, six of the carbonyl ligands occupy equatorial sites, close to the plane of the triangle of cobalt atoms with the other three carbonyl groups approximately orthogonal to that plane in axial positions. Monodentate or bidentate phosphine or phosphite ligands almost invariably substitute carbonyl groups from equatorial sites [1,2]. Furthermore, the remaining equatorial carbonyl ligands generally adopt terminal conformations. A search of the Cambridge Database [3] reveals that carbonyl bridging of the Co—Co bonds is unusual and is generally found only in situations where the apical substituent R, or the substituting ligands, significantly increase the electron density on the CCo3 cluster core. Me We have prepared a number of derivatives of methinyltricobaltnonacarbonyl clusters with the potentially tridentate 1,1,1- OC Co tris(diphenylphosphinomethyl)-ethane, CH3C(CH2PPh2)3, (triphos) Co ligand. In all cases, the resulting compounds have bridging carbonyl OC OC ligands. This paper will report the structure of these complexes and P examine the factors that influence carbonyl bridging in these and Ph P Ph Ph related cluster systems. In particular, remission of the build-up of H C H C electron density on the cluster core by oxidation of the triphos derivatives returns the carbonyl configurations to fully terminal. 2 2 CO Co CO CO Ph Ph P Ph CH2 CH3 Acknowledgements: We thank the New Economy Research Fund; Grant No. UOO-X0808 for support of this work and the University of Otago for purchase of the diffractometer. [1] Matheson, T.W., Robinson, B.H. & Tham, W.S. (1971) J Chem Soc A 1457-1464. [2] Downard, A.J., Robinson, B.H. & Simpson, J. (1986) Organometallics 5, 1122—1131. [3] Allen, F. H. (2002). Acta Cryst. B58, 380—388. 05.01.4 Crystallography of Complexes of the [Re6(μ -Se)8] Gary Nichol, Xiaoyan Tu, Zhiping Zheng The University of Arizona, Tucson, AZ, United States Cluster complexes which contain an [Re6(μ -Se)8] core attract interest from synthetic and computational chemists due to their facile chemical transformations, ease of control of 1 stereochemistry and notable electrochemical and luminescent properties. In almost all cases single crystal X-ray diffraction is used to characterize these complexes, with supporting information provided by spectroscopic techniques. However, such cluster complexes frequently present a challenge to the crystallographer. Disorder, twinning, highly solvated structures, weak diffraction and unstable crystals are all common problems encountered in the course of this research. The dominance of the 2+ diffraction pattern by the [Re6(μ -Se)8] core, and the use of counter ions which are synthetically suitable but crystallographically horrific, often make resolving some of these problems a serious challenge. Here, problems, challenges and solutions will be discussed in 2+ the context of our recent work on the use of [Re6(μ -Se)8] core-containing clusters in so2 called “click” chemistry and the resulting unexpected formation of imino complexes . 1. 2. X. Tu and Z. Zheng. CrystEngComm, 2009, 11, 707—719. X. Tu, E. Boroson, H. Truong, G. S. Nichol and Z. Zheng. Inorg. Chem. 2010, 49, 380–382 2+ 2+ Core-Containing Clusters 05.01.3 X-ray Crystal Structure of [(As6V IV V 912V 3O51) ]∞ . Carla Slebodnick, Victoria Soghomonian, Elinor Spencer Virginia Tech, Blacksburg, VA, United States A number of recently reported anionic 3D oxo-vanadium arsenate frameworks have shown promising electronic properties. Expanding on this effort, a new anionic framework, IV V 9[(As6V 12V 3O51) ]∞ , is reported. The material crystallizes in the cubic space group Im3m. Original efforts to solve the structure yielded what appeared to be ten edge-shared VO5 square pyramids to form V10O26 'balls' with bidentate cornersharing AsO4 tetrahedra linking two separate V10O26 balls to form a 3D network. Unfortunately, a satisfactory refinement of this structure model was not achieved after trying many space groups and twinning models. Constraining specific vanadium and oxygen occupancies to instead form disordered 'half-balls', however, gives a model that is both chemically and crystallographically reasonable. In addition to summarizing the 3-year effort to achieve this satisfactory model, the title structure will be compared and contrasted with a number of very similar structures from the literature. 05.01.5 Be Careful What You Wish For: A Service Crystallographer’ s Lament Patrick Carroll University of Pennsylvania, Philadelphia PA, United States This is the tale of a “bored” service crystallographer who wished for some interesting problems with which he could entertain his colleagues at the ACA meeting. The result is a series of twinned structures, disordered structures, unexpected and difficult-to-solve structures and other forms of crystallographic misery. 05.01.6 Investigating Hexafluroacetylacetone (HFAA) by X-ray Diffraction – Solid-State and Theoretical Studies of a Volatile Liquid Christopher Incarvito , Chandrima Chatterjee , Lori Burns , Patrick Vaccaro 1 2 1 1 2 1 Yale University, New Haven, CT, United States, Georgia Institute of Technology, Atlanta, GA, United States Despite numerous efforts to unravel the ground-state structure of HFAA, its detailed geometry is yet to be ascertained. While most theoretical endeavors point to the Cs structure as the global minimum configuration, gas-phase electron diffraction studies [2,3] have suggested a symmetric (C2v) enol tautomer. Motivated by such contradictory conclusions, the groundstate manifold of HFAA has been re-examined using low-temperature single-crystal X-ray diffraction techniques. HFAA has a normal melting point of 177K and, therefore, exists as a liquid at room temperature. After introducing the liquid in a glass capillary, it was mounted vertically on a Rigaku R-AXIS SPIDER diffractometer and cooled to 93K by a cryogenic stream of nitrogen vapor. A single crystal was grown 'in situ' by the zone-melting method [1], with a heated filament producing a molten zone that was slowly translated along the length of the capillary. The HFAA crystal structure emerging from collected diffraction data clearly favors an asymmetric H-bond. In addition, the wider separation of 2.683 Å between the donor and acceptor oxygen atoms implies a weaker hydrogen bond compared to acetylacetone. Our X-ray analysis has been corroborated by high-level quantum chemical calculations, a detailed discussion of which may be presented in this paper. [1] R. Boese, M. Y. Antipin, D. Blaser, and K. A. Lyssenko, J. Phys. Chem. B, 102, 8654 (1998). [2] K. IIjima, Y. Tanaka, and O. Shigeki, J. Mol. Struc., 268, 315 (1992). [3] A. L. Andreassen, D. Zebelman, and S. H. Bauer, J. Am. Chem. Soc., 93, 1148 (1971). 05.01.7 Similar Shapes and Isomer Disorder in the Structure of C86•Ni(OEP)•2toluene Marilyn Olmstead , Alan Balch , Ziyang Liu , Hua Yang , An Jiang , Zhimin Wang 1 2 1 1 2 2 2 2 University of California, Davis, United States, Zhejiang University, Hangzhou, China Since the structure of the fullerene with 60 carbons, Ih-C60 was determined 18 years ago, the structures of few pristine empty cage fullerenes have been successfully characterized by X13 ray crystallography. In most cases their structures have been inferred from C NMR and IR measurements or by derivatization.. The largest higher fullerene characterized by X-ray 1 crystallography to date is that of D5h-C90, reported by us earlier this year . Some of the reasons for the paucity of these structures stems from the increasing number of possible isomers as the number of carbon atoms increases and the difficulty in the separation of the isomers that are preferentially formed in the carbon soot of the vaporized graphite. Other reasons are the very small amount of material isolated, together with the propensity for disorder in the structures of these nearly spherical molecules. We recently succeeded in obtaining the structure of an empty cage C86 fullerene, obtained by co-crystallization with Ni(OEP) in toluene. In this structure, the eight ethyl arms of the porphyrin molecule encapsulate the fullerene and enable the determination of the structure with less rotational disorder. However, in this instance, we were surprised to be able to detect the presence of two concomitant isomers of C86 that reside in the same crystallographic position. The two isomers, Cs(16)-C86 and C2(17)-C86, differ by a 90 ° rotation of a set of two pentagons and two hexagons, the so-called Stone-Wales transformation. The presence of both isomers is in agreement with theoretical calculations. Structural similarities between the two isomers are compared in this report. 1 H. Yang, C. M. Beavers, Z. Wang, A. Jiang, Z. Liu, H. Jin, B. Q. Mercado, M. M. Olmstead and A. L. Balch. Angew. Chem. Int. Ed., 2010, 49, 886. 06.01.1 40 Years Synchrotron X-Radiation in Biology Gerd Rosenbaum University of Georgia, Athens, GA, United States, Argonne National Laboratory, Argonne, IL, United States 2010 marks the 40th anniversary of the recording of the first x-ray diffraction with synchrotron radiation [1]. The small angle diffraction pattern of insect flight muscle proved that the calculated 100-fold gain in flux from the DESY synchrotron over a state-of-the-art fine focus rotating anode X-ray generator could, indeed, be achieved. Time resolved diffraction of the cross-bridge cycle in muscle, the driving force behind the synchrotron adventure, would be a big step closer to reality. 1 2 The first part of the presentation will commemorate this event. The main part will trace the tremendous advances synchrotron radiation has enabled in biology: from small angle diffraction and solution scattering to macromolecular crystallography, spectroscopy, microscopy and imaging. References: 1. G Rosenbaum, K C Holmes, J Witz: “Synchrotron radiation as a source for X-ray diffraction (a preliminary report)”, Nature, 230, 434-37, 1971 06.01.2 Femtosecond Protein Nanocrystallography at the LCLS John Spence ASU, Tempe Az, United States First results will be reported from experiments at the world's first hard X-ray laser (the Linac Coherent Light Source) at Stanford using membrane protein nanocrystals. X-ray pulses at 2 kV of femtosecond duration were used to read out 30 diffraction patterns per second from a liquid jet of protein nanocrystals sprayed across the beam. We have evaluated the idea that a sufficiently short pulse will terminate before radiation damage begins, yet contain sufficient photons to produce a useful diffraction pattern. Details of the protein-beam injector, which must provide full hydration, will be given, and the method of data analysis discussed. This involves merging diffraction data from millions of patterns from sub-micron nanocrystals of photosystem one , of varying size and in random orientations. Many terrabytes of data were collected over several days. Plans for pump-probe experiments will also be outlined. This approach allows structure analysis of proteins which do not produce large crystals, possibly without radiation damage, directly from solution and without need for cooling. This project is a large international collaboration, involving the CAMP group from three Max Plank Institutes and ASU physics. PIs include H. Chapman, P. Fromme, I. Schlichting, B. Doak, U. Weierstall, J. Uhlich, A. Barty, L. Struder, D. Rolles, the LCLS staff and the ASG team. 06.01.3 Aligning Molecules with Polarized Lasers for X-ray Diffraction Analysis Linda Young Argonne National Laboratory, Argonne, IL, United States Single molecule imaging with atomic resolution endures as a dream for crystallographers who often have difficulties producing crystals of suitable size and quality. Initial successes with the world's first x-ray free-electron laser in imaging nanometer-sized crystals of biomolecules are a step toward this dream. Such nanocrystallography studies retain the N^2 Bragg intensity enhancement and extending coherent diffractive imaging to the single-molecule limit will be challenging. One limitation is the unknown orientation of the molecule at the instant of x-ray diffraction. In this talk, I will describe laser-based techniques to align molecules that can constrain the rotational degree-of-freedom to form an effective laser-based goniometer. From a fundamental perspective, the molecule will be aligned by the presence of a strong laser field and understanding the ensuing structure distortion is of interest. 06.01.4 Coherent X-ray diffraction from sub-micron protein crystals Francesco Gramiccia , Céline Besnard , Sebastian Basso , Cameron Kewish , Phil 4,1 5,3 1 Pattison , Franz Pfeiffer , Marc Schiltz 1 1 2,1 1 3 Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland, UNIversité de GEnève, 3 4 Geneva, Switzerland, Paul Scherrer Institut, Villigen, Switzerland, European Synchrotron 5 Radiation Facility, Grenoble, France, TU München, Münich, Germany 2 X-ray diffraction is the most widely-used technique to investigate the structure of biologically important molecules. However, the growing of crystals of sufficient size and quality for diffraction experiments remains one of the major bottlenecks. Crystals of sizes of about 20-30 microns have been studied successfully on dedicated microfocus beamlines, but radiation damage is thought to be the ultimate factor that puts a lower limit on the size of crystals that can be used. With future X-ray Free Electron Laser sources, the high flux provided by a single pulse may be sufficient to record the scattering of the object before the onset of radiation damage effects. Moreover, in a diffraction pattern of very small crystals, the complex and coherent maxima in between Bragg peaks may be used to solve the phase problem by oversampling. Using existing third generation synchrotron sources which provide sufficient coherent flux to investigate macromolecular nanosized crystals, we have recorded coherent diffraction images on D-xylose isomerase in order to explore the effects of coherence in the diffraction patterns. The diffraction pattern recorded on micron-sized crystals of D-xylose isomerase reveals starshaped aspects around the Bragg spots. They are interpreted as crystal-shaped truncation features which are characteristic effects in coherent scattering. Simulated data, computed by using an analytical expression for the shape amplitude, agree well with the observed patterns. 06.01 Temperature- and cooling rate-dependence of protein conformation: the active site flap of urease as a model system. Robert Thorne , Matthew Warkentin , Andrew Karplus 1 2 1 1 2 Cornell University, Ithaca, NY, United States, Oregon State University, Corvallis, OR, United States Urease from Klebsiella aerogenes is an ~240 kDa ( )3 heterotrimer, with the -subunits having a TIM-barrel domain that contains a bi-nickel active site. The unit cell is cubic I213, with ~178 Å edges (Jabri et al., 1995). Comparison of structures at room temperature and at T=100 K shows a dramatic change in the ~20 amino acid "flap" covering the active site (Pearson, MA & PAK, unpublished).. At room temperature the flap is more mobile than the rest of the molecule, but has an average position placing a key residue, His320, in the active site properly positioned to act as a catalytic acid. At T=100 K half of the flap becomes a more highly ordered -helix that pulls His320 out of the active site; the other half (starting at His320) becomes so disordered that it cannot be modeled. ð ð ð We have examined how these changes develop as a function of temperature by solving the structure at 11 temperatures between T=300 K and 100 K. The conformational transition occurs between 270 and 240 K. The disorder in the flap goes through a maximum at T~210 K, and at still lower temperatures remains larger than at room temperature. We have also investigated how the cooling rate affects the behavior of the flap at T=100 K. For conventional flash cooling (~100 K/s) and slow cooling (~0.1 K/s), the T=100 K structure is as previously described. However, for very rapid cooling (~10,000 K/s) using the "hyperquenching" protocol (Warkentin et al., 2006), the T=100 K structure more closely resembles the 270 - 300 K structure: the B-factors are lower and the conformation is more "uncoiled" than for the smaller cooling rates. This suggests that biologically relevant information lost during cooling for urease (and in principle other proteins) might be made available by increasing cooling rates to 10,000 K/s or larger. Jabri, E., M. B. Carr, R. P. Hausinger, and P. A. Karplus. 1995. The crystal structure of urease from Klebsiella aerogenes. Science 268:998-1004. Warkentin, M., Berejnov, V., Husseini, N. S. & Thorne, R. E. (2006). Hyperquenching for protein cryocrystallography. J. Appl. Cryst. 39, 805-811. 06.01.6 Liquid metal jet micro-focus x-ray source: Highest brilliance for home lab instrumentation Christoph Ollinger, Lutz Bruegemann Bruker AXS, Karlsruhe, Germany For a wide variety of x-ray applications the depth of accessible information is limited by the brilliance of the x-ray source. Recent break throughs in x-ray source technology push the limits further. By using liquid metal jet targets (e.g. a Gallium alloy) instead of fast spinning solid metal targets power loads of the order of 500 kW/mm^2 can be provided. Enabling focal spot sizes below 20 microns at an x-ray energy of 9.2 keV pave the way for applications with a brilliance comparable to bending magnet sources in a home-lab instrument. Combining such a source with state-of-the-art multilayer mirrors, allows to transport the brilliance, enabling highest flux-densities at the sample suitable for e.g. diffraction and scattering investigations on very small samples or with very high spatial resolution. During the course of the presentation dedicated data will demonstrate the potential of such a source integrated into a laboratory x-ray diffraction instrument. The investigations will be compared with results obtained with common micro-focus x-ray diffraction instrumentation. 07.17.1 Charge-density-wave crystals and low-dimensional magnets Sander van Smaalen University of Bayreuth, Bayreuth, Germany Long-range order in solid matter can be achieved without three-dimensional translational symmetry [1]. Such incommensurate crystal structures have been observed in all classes of compounds, from the elements to protein crystals. Key to the understanding of aperiodic order is competing interactions, which individually would favor mutually incommensurate periodicities. Metals and insulators supporting low-dimensional valence bands are prone to the formation of superstructures due to electron-phonon interactions. Charge-density-waves (CDWs) and low-dimensional magnetic interactions may lead to incommensurate and commensurate superstructures, depending on the values of the Fermi wavevector. Here, the implications are discussed of these superstructures for understanding the phase transitions and physical properties of CDW materials and other low-dimensional electronic crystals. References [1] S. van Smaalen: Incommensurate Crystallography, Oxford University Press (2007). 07.17.2 2D-noncommensurate Modulated Misfit Layer Structures of Franckeite and Cylindrite Emil Makovicky , Václav Petříček , Michal Dušek , Dan Topa 1 2 1 2 2 3 University of Copenhagen, Copenhagen, Denmark, Czech Academy of Sciences, Prague, 3 Czech Republic, University of Salzburg, Salzburg, Austria Crystal structures of these triclinic Pb-Sn-Sb-Fe-S compounds have a pronounced 1D transversal wave-like modulation and a non-commensurate layer match in two dimensions. They consist of alternating pseudohexagonal (H) and pseudotetragonal (Q) layers and form a homologous pair: franckeite has Q layers of double thickness compared to cylindrite. Franckeite from San José, Bolivia, is Pb5.2Ag0.2Sn2.4Sb2.2Fe1.0S14.5, Q layer is an MS layer (M 2+ 2+ 3+ = Pb ,Sn ,Sb ..) four atomic planes thick, with a 5.815 Å, b 5.873 Å, (the layer-stacking o o o * vector) c 17.366 Å, α 94.98 , β 88.43 , γ 89.97 ; the modulation vector q = -0.001 a + 0.1282 * * 4+ 2+ 3+ b - 0.0295 c . H layer is a single-octahedron MS2 layer (M = Sn , Fe , Fe ..) with a 3.672 Å, o 0 o * b 6.275 Å, c 17.447 Å, α 95.26 , β 95.45 , γ 89.97 ; the modulation vector is q = -0.001 a + * * 0.1374 b - 0.031 c . The modulation wave has λ = 45.80 Å; the match of centred (sub)cells in this b direction, 15.5 Q : 14.5 H, occurs at 91.01 Å, what makes 2 λ minus a structurally important Δ = 0.59 Å. The a and b vectors of both subsystems are parallel; the stacking c vectors diverge; divergence between modulation wave-front and the stacking c directions is present as well. 5D superspace refinement was performed in the superspace group C-1, using 7397 observed reflections; overall R(obs) = 0.094. The Q layers are composed of two tightly-bonded double-layers; their interspace hosts lone electron pairs. Average composition for the outer surface is Pb0.74(Sn,Sb)0.26S; for the interspace lining it is (Sn,Sb)0.74Pb0.26S. Local variations in the Pb:(Sn,Sb) ratios produce the transversal modulation of the Q layer. It re-establishes a one-dimensional commensurate contact along [010] between the curved Q and H surfaces to the greatest extent possible. Layer-stacking disorder is important and omnipresent. Cylindrite forms large cylindrical aggregates, franckeite contorted crystals because of the increased thickness of the Q layer. The modulated b direction becomes cylinder axis and the unmodulated a direction of non-commensurate mismatch becomes cylinder tangent. The existence of these structures depends on the radius ratios for the cations involved. They cannot exist for a pure Pb-Bi combination that results in the structure type of cannizzarite. 07.17.3 Electron microscopy and its application to the structural characterization of incommensurate, compositely modulated and 'disordered' crystal structures. Ray Withers The Australian National University, Canberra, A.C.T, Australia Very many crystalline materials are sensibly inflexible - locked into a fixed stoichiometry, a conventional 3-D space group symmetry and in a unique dominant free energy minimum. However, not all materials like to be nailed down! Close inspection of a large and ever increasing number of materials has shown that many do not in fact fit into such a neat pigeon hole and are modulated in one form or another. These modulations can be short or long range ordered, of large or small amplitude while the associated occupational and displacive Atomic Modulation Functions (AMF’s) required for complete structural characterization in superspace can be essentially sinusoidal, inherently square wave or saw tooth in form. Whatever the particular characteristics, an understanding of the local crystal chemistry as well as the associated physico-chemical properties of such phases can not be had until such modulations are recognized and properly taken into account. The Transmission Electron Microscope (TEM) is an extremely well-adapted instrument for the detection as well as the symmetry and structural characterization of such modulated structures as a result of the sensitivity of electron diffraction to weak subtle features of reciprocal space, the ability to obtain such information from small local regions as well as the capacity to image in various modes with excellent spatial resolution and over a considerable range of temperature. In this contribution, the application of electron microscopy to the study of interface, composite, compositionally and/or displacively modulated structures (including nominally ’disordered’ structures) will be discussed. The characteristic diffraction signatures associated with these different types of modulated structure will be highlighted along with the practical application of transmission electron microscopy to problems such as pseudosymmetry and twinning, to indexation in (3+D)-dimensional superspace and to overall superspace symmetry and structural characterization. 07.17.4 A new table of (3+d)-dimensional superspace groups Harold T. Stokes , Branton J. Campbell , Sander van Smaalen 1 1 1 2 2 Physics & Astronomy, Brigham Young University, Provo, UT, United States, Laboratory of Crystallography, University of Bayreuth, Bayreuth, Germany The symmetry of an incommensurately modulated structure is described by a superspace group in (3+d)-dimensional space, where d is a positive integer that gives the number of independent modulation waves. Superspace groups are a subset of the general (3+d)dimensional space groups that is restricted to point symmetries that do not interchange or mix the three external dimensions with the d internal dimensions. Here, we present a table of superspace groups for (3+1), (3+2) and (3+3) dimensions that resolves errors in previous tables and also contains a variety of previously unpublished symmetry data. After discussing the use of alternative superspace-group settings, we will demonstrate tools for comparing two sets of operators to determine whether they correspond to distinct superspace groups or to two different settings of the same superspace group, which make it possible to quickly identify symmetry and setting given an arbitrary set of operators. The focus will be on practical examples. 07.17.5 Pr2(MoO4)3 and Nd2(MoO4): Partially Disordered Scheelite-Like Structures? Cristina González-Silgo, Candelaria Guzmán-Afonso, Manuel E. Torres, Antonio D. LozanoGorrín, Javier González-Platas, Ulises R. Rodríguez-Mendoza, Juan Rodríguez-Carvajal Universidad de La Laguna, La Laguna, Tenerife, Spain Due to a large number of possible cationic substitutions and polymorphisms, the spectrum of physical properties displayed by scheelites is very broad. They are important host crystals for a variety of inorganic phosphors-converted light emitting diodes [1], tunable solid state laser materials [2] and nonlinearities for second harmonic generation and stimulated Raman scattering [3]. Several authors have recently published new ordered scheelite superstructures and partially disordered scheelite-like modulated structures. The effects of the different cation order and the superstructures symmetry, in what concerns to the optical properties analysis, present some puzzeling challenges [4]. Very recently, the structure of Pr2(MoO4)3 (where 1/3 of the A-site positions are vacant) has been solved, with an incommensurate structure from synchrotron data [5]. In this work Pr2(MoO4)3 and Nd2(MoO4)3 have been prepared by solidstate synthesis. We have collected neutron and X-ray powder diffraction data, at room temperature, in the D2B diffractometer at ILL and in our laboratory. All the structures have been obtained by multipattern Rietveld refinement using a new symmetry modes procedure: by AMPLIMODES [6] and FULLPROF [7] programs; and using the advantages of the neutron 3+ 3+ radiation. We have found that Pr and Nd cations are not completely ordered within the La2(MoO4)3 superstructure with possible new ordering similar to the Eu2(MoO4)3 structure. [1] Su, Y.; Li, L; Li, G.; Chem. Mater. 2008, 20, 6060-6067. [2] Méndez-Blas, A.; Rico, M.; Volkov, V.; Zaldo, C.; Cascales. C.; Phys. Rev. B, 2007, 75, 174208-174222. [3] Zverev, P.G.; Basiev, T.T.; Prokhorov, A.M.; Opt. Mater. 1999, 11, 335-352. [4] Arakcheeva, A.; Chapuis, G.; Acta Crystallogr. B, 2008, 64, 12-25. [5] Logvinovich, D.; Arakcheeva, A.; Pattison, P., Eliseeva, P.; Tomes, P.; Marozau, I.; Chapuis, G.; Inorg. Chem. 2010, 49, 1587-1594. [6] Orobengoa, D., Capillas, C., Aroyo, M. I., Perez-Mato, J. M.; J. Appl. Cryst. 2009, 42, 834845. [7] Rodríguez-Carvajal, J. (1993). FULLPROF. Program for Rietveld analysis of X-ray and Neutron powder diffraction patterns. ( http://www.ill.eu/sites/fullprof/). [] 07.17.6 Positional average structure from an incommensurately modulated crystal of profilin:actin Jason Porta , Jeff Lovelace , Antoine Schreurs , Gloria Borgstahl 1 1 1 2 1 2 University of Nebraska Medical Center, Omaha, NE, United States, Utrecht University, Utrecht, Netherlands Modulation of protein crystals is seldom reported, mainly due to a lack of methods for solving these unique structures. We report here the incommensurately modulated average structure of profilin:actin and the methods that were used to carry out the analysis. Crystal modulation is characterized by a loss of short-range translational symmetry, where a single unit cell is no longer sufficient to accurately describe the structure. Such a loss of periodicity is often caused by dynamic processes within the crystal arising from, for example positional modulations. Experimentally, the incommensurately modulated state is characterized by the appearance of distinct satellite reflections surrounding the main Bragg reflections on the diffraction pattern that cannot be indexed with a supercell. In order to fully describe a modulated structure, and hence the dynamic processes within the crystal, one must explore higher-order space over multiple unit cells. By careful examination of atomic positions over higher dimensional space, a modulation function can be calculated that traces the atomic disorder. Such a function is periodic, but incommensurate with the crystal lattice. As a first step to solving this function for PA crystals, we have determined the average structure of the modulated state. Main and satellite reflections were integrated with Eval15 and scaled with SadAbs to 3.0 Å. The average structure was then solved using the Phenix crystallographic software suite. Fourier electron density maps indicate the whole structure moves with major motion in actin subdomains 2 and 4. Superposition with the periodic profilin:actin structure and analysis by DYNDOM confirm these observations by showing the rotation of these domains. The modulation is in the b direction, which corresponds to the ribbon of actin molecules along this crystallographic axis. Analysis of these domain movements give insight into the long sought after globular (G) to fibrous (F) actin transition. 07.17.7 Extending the 14-electron rule beyond the Nowonty Chimney Ladder phases: a 14electron series based on defect structures of the MoSi2 structure type. Daniel Fredrickson , Magnus Bostroem , Yuri Grin , Sven Lidin 1 1 2 3 4 2 University of Wisconsin-Madison, Madison, WI, United States, Sandvik Materials 3 Technology, Sandviken, Sweden, Max-Planck-Institut fuer Chemische Physik fester Stoffe, 4 Dresden, Germany, Lund University, Lund, Sweden The Nowotny Chimney Ladder phases (NCLs) provide one of the clearest examples of electron count control of structure stability in intermetallic phases. The NCLs are compounds formed between transition metal (T) and main group elements (E) with stoichiometries of the form TE2-x. The structures of these phases are based a beautiful variation on the TiSi2 structure type in which E atom helices thread through T atom helical channels. For NCLs with late transition metals (groups 7 and higher), the E atom deficiency, x, is tuned (often quite precisely) so that the total number of valence electrons divided by the number of T atoms is fourteen -- a criterion for stability known as the fourteen electron rule. In this presentation, we will explore the possibility of extending this rule beyond the TiSi2 defect structures. Electron microscopy investigations of the Re4Si7 phase and ternary derivatives by a number of researchers have revealed complex and incommensurate superstructures based on another transition metal disilicide structure type: the MoSi2 type. The superstructures appear to be correlated with electron count, suggestive of a close connection to the fourteen electron rule of the NCLs, but the details in terms of both geometry at the atomic level and the electron structure remain unresolved. In an effort to complete this picture, we have synthesized Re4Si7 and several Os- or Al- substituted derivatives, and analyzed their crystal structures with single crystal X-ray diffraction. These phases exhibit complex diffraction patterns that can be resolved into strong reflections arising from a MoSi2 basic cell, and weaker satellite reflections. Structure solution and refinement using superspace methods reveal that satellites arise from Si vacancies occurring as edgedeletions, with the neighboring Si atoms relaxing to smooth out the deletions. The result resembles a crystalline ordering of Si edge-dislocations in a MoSi2-type crystal. Electronic structure calculations on these phases, at both the ab initio and semi-empirical levels, show features analogous to the band structures of the NCLs, strongly suggestive of a common origin of stability for both families of phases. These results suggest that domain of the fourteen electron rule extends beyond the NCL phases to incorporate defect structures of the MoSi2 type. 07.17.8 To What Extent Does the Zintl-Klemm Formalism Work? The Eu(Zn1-xGex)2 Series Tae-Soo You , Sven Lidin , Olivier Gourdon , Yaqiao Wu , Gordon Miller 1 2 1 2 3 4 5 University of Delaware, Newark, DE, United States, Stockholm University, Stockholm, 3 4 Sweden, Oak Ridge National Laboratory, Oak Ridge, TN, United States, Ames Laboratory, 5 Ames, IA, United States, Iowa State University, Ames, IA, United States The series of ternary polar intermetallics Eu(Zn1-xGex)2 (0 ≤ x ≤ 1) has been investigated and characterized by powder and single crystal X-ray diffraction as well as physical property measurements. For 0.50(2) ≤ x < 0.75(2), this series shows a homogeneity width of hexagonal AlB2-type phases (space group P6/mmm, Pearson symbol hP3) with Zn and Ge 3 atoms statistically distributed in the planar polyanionic 6 nets. As the Ge content increases in this range, a decreases from 4.3631(6) Å to 4.2358(6) Å, while c increases from 4.3014(9) Å to 4.5759(9) Å, resulting in an increasing c/a ratio. Furthermore, the Zn-Ge bond distance in the hexagonal net drops significantly from 2.5190(3) Å to 2.4455(3) Å, while the anisotropy of the displacement ellipsoids significantly increases along the c direction. For x < 0.50 and x > 0.75, respectively, orthorhombic KHg2-type and trigonal EuGe2-type phases occur as a second phase in mixtures with an AlB2-type phase. Diffraction of the x = 0.75(2) sample shows incommensurate modulation along the c direction. Temperature-dependent magnetic susceptibility measurements for two AlB2-type compounds show Curie-Weiss behavior above 40.0(2) K and 45.5(2) K with magnetic moments of 7.98(1)μ B for Eu(Zn0.48Ge0.52(2))2 and 7 7.96(1) μ B for Eu(Zn0.30Ge0.70(2))2, respectively, indicating a (4f) electronic configuration for Eu 2+ atoms (Eu ). The Zintl-Klemm formalism accounts for the lower limit of Ge content in the AlB2-type phases, but does not identify the observed upper limit. 07.17.9 Charge-Density Compounds Waves and Structural Modulations in Layered Polytelluride Christos Malliakas, Mercouri Kanatzidis Northwestern University, IL, Evanston 60201, United States The generation of charge density waves (CDW) produces modulated structures and is believed to be a competing process to unconventional superconductivity and other quantum ground states. Incommensurate to commensurate or near-incommensurate phase transitions associated with CDW can give rise to unusual and often unexplained phenomena. CDW states can be created through a Fermi surface nesting effect and the creation of a new ground state with broken translational symmetry. As a result of the new periodicity, a band gap opens at the Fermi surface and an overall electron energy lowering is achieved. In this presentation, a systematic study of layered polytelluride CDW compounds will be reported in order to understand and elucidate their structural modulations. Since these structural distortions are generally incommensurate with respect to the underlying crystal sublattice the use of multidimensional crystallographic methods in superspace is necessary for solving their structures. The general approach for solving aperiodic crystals and details for the crystallographic refinement and solution in (3+1) and (3+2) dimensions will be also discussed. The family of compounds RETe3 (RE = Rare-Earth), revealed for the first time that the nature of the CDW in these compounds varies subtly but significantly with RE element. Furthermore, investigation of AMRETe4 (A = Na, K; M = Cu, Ag; RE = La, Ce) that is structurally related to RETe3 showed that cross-plane interactions play an important role in defining the CDW distortions in these materials. Additionally, RE2Te5-x that is composed of the substructures of RETe2-x and RETe3 represents a rare example of composite structure with two different Te nets which exhibit a hybrid CDW distortion with two independent modulation vectors. Finally, a unique double modulated Te net in Cu0.66EuTe2 will be also presented. CDWs in these materials originate from the extended hypervalent Te∙ ∙ ∙ Te bonding that occurs in the planar square nets of tellurium. 07.17.10 Ga square nets – Modulation and Twinning, Oh My! Danielle Gray, Mercouri Kanatzidis University of Illinois, Urbana, IL, United States, Northwestern University, Evanston, IL, United States Compounds with electrons confined to low dimensional structures are prone to distortions known as charge density waves (CDWs). A series of RENi1-xGa3Ge (RE = Tb, Dy, Ho, Er, Tm; 0.04 ≤ x ≤ 0.16, but is fixed +/- 1% for a given RE composition) compounds with 2-D Ga square nets exhibit CDWs. Examination of the average tetragonal (I4/mmm) structure for the RENi1-xGa3Ge materials revealed structural problems despite very good fitting statistics. The Ga in the square nets had large thermal parameters as compared to the rest of the atoms, and the Ni atoms had occupational disorder on one site that induced a positional disorder on the Ge sites. Upon close inspection of reciprocal space, 4 pairs of additional reflections are visible around each main reflection. A (3+1)D superspace approach was used with application of 4-fold pseudomerohedral twinning in order to model the incommensurate structure. RENi1-xGa3Ge compounds crystallize in the superspace group I2/m(α β 0)0s with the c-axis as the unique axis. The structural models show coupling between the CDWs in the Ga nets and the Ni/Ge site occupancy waves. 1 2 07.18.1 Snapshots into HIV-1 capsid maturation: structural insights derived from mutations in the HIV-1 Capsid Assembly Inhibitor binding site Luis R. Castillo , Vanda Lux , Sebastien Igonet , Felix Rey , Hans-Georg Kräusslich 1 2 1 1,2 3 3 1 University Hospital Heidelberg, Heidelberg, Germany, Universität Duisburg-Essen, Essen, 3 Germany, Institut Pasteur, Paris, France HIV-1 maturation from a non-infectious into an infectious agent is accompanied by morphological changes in its capsid shell which undergoes a protease-mediated conversion from a spherical into a conical shape. We previously identified a Capsid Assembly Inhibitor (CAI) that blocks immature and mature HIV-1 capsid assembly in vitro. CAI binds to a hydrophobic pocket in the C-terminal domain of the capsid protein (C-CA), which promotes a conformational change. X-Ray structures from mutants in the C-CA binding pocket showed that while some keep the unliganded C-CA conformation, others adopt the conformation of CCA in complex with CAI(C-CA/CAI). More extensive mutagenesis work has allowed us to identify two C-CA mutants that adopt an intermediate phenotype between the C-CA and CCA/CAI conformations. Recently, cryo-electron tomography of the HIV-1 immature capsid lattice showed that a C-CA/CAI conformation mutant fits very well in the immature shell. Besides, an NMR structure of a C-CA with two amino acids extension at the N-terminus showed a distinct quaternary conformation from the X-Ray structures, but which fitted better into a cryo-electron reconstruction of a mature-like capsid assembled particle. X-ray structures from the construct used in NMR showed the N-terminus extended C-CA conformation in the crystal is the same as that found in the unliganded C-CA. Nevertheless, the C-CA NMR structure showed a similar conformation of the mutants with an intermediate phenotype. Since more than one C-CA construct structure could be classified in a defined state based on the CAI-induced conformational change and based on the fact that each defined conformation could fit into EM reconstructions from capsid shell particles; these structures might represent snapshots of a conformational change pathway that the HIV-1 capsid undergoes during maturation. 07.18.2 Receptor Recognition Mechanisms of Coronaviruses Fang Li University of Minnesota, Minneapolis, MN, United States Coronaviruses recognize a variety of receptors and infect many hosts. NL63 coronavirus (NL63-CoV), a prevalent human respiratory virus, is the only group-I coronavirus known to use angiotensin-converting enzyme 2 (ACE2) as its receptor. Curiously, ACE2 is also used by group-II SARS coronavirus (SARS-CoV). Defined receptor-binding domains (RBDs) on the spike proteins of NL63-CoV and SARS-CoV bind ACE2 with high affinity. We have determined the crystal structures of NL63-CoV RBD complexed with human ACE2 and of SARS-CoV RBD complexed with human ACE2, revealing for the first time how two different viruses can recognize the same receptor protein. Specifically, NL63-CoV and SARS-CoV RBDs have no structural homology in cores or receptor-binding motifs (RBMs) that directly contact ACE2, but recognize the same “virus-binding hotspot” on ACE2. Among group-I coronaviruses, RBD cores are conserved, but RBMs are variable, explaining how these viruses recognize different receptors. Moreover, we have also determined the crystal structures of the RBDs from various SARS-CoV strains complexed with ACE2 proteins from humans and palm civets, revealing the structural mechanisms whereby SARS-CoV transmitted from palm civets to humans and caused the worldwide SARS epidemic in year 2002-2003. Specifically, SARS-CoV strains from palm civets became adapted to human ACE2 through stepwise mutations in their RBMs, gaining affinity for human ACE2 and infectivity in human cells. Overall, our studies provide the molecular and structural basis for understanding viral evolution, virus-receptor interactions, viral host ranges and cross-species infections. They also guide the development of novel antiviral strategies against coronavirus infections. 07.18.3 The crystal structure of E. coli fimbrial tips at 2.7 Angstroms resolution: Structural views of fimbrial assembly, donor strand complementation, and force-mediated cell adhesion Ronald Stenkamp, Isolde Le Trong, Pavel Aprikian, Brian Kidd, Wendy Thomas, Evgeni Sokurenko University of Washington, Seattle, WA, United States Fimbriae and pili are macromolecular structures on the surface of Gram negative bacteria that are important for cellular adhesion. We have solved a 2.7 Å resolution crystal structure of a complex of E. coli fimbrial proteins containing FimH, FimG, FimF, and FimC. This provides the most complete model to date for how subunits assemble into these non-flagellar adhesive appendages. The first three subunits form the tip of the fimbriae while FimC is the chaperone protein involved in the usher-chaperone assembly process. The fimbrial subunits form an extended structure in the two complexes per asymmetric unit that can serve as a model for native fimbrial tips. The subunits are held together through donor strand complementation where a β -strand from one subunit completes the β -sandwich structure of the subunit closer to the tip of the fimbria. FimC provides a surrogate donor strand before delivery of each subunit to the FimD usher and the growing fimbria. Structures of several of the subunits in complex with FimC have been seen before. Comparison of the subunits in this structure with those complexes show that the lectin and pilin domains of FimH change their relative orientation and position in forming the tip complex. The pilin domain also compresses one end of the lectin domain β -sandwich which loosens the mannose-binding pocket at the opposite end. This provides a model for FimH’ s force mediated mannose binding properties. In addition to this structural change, the non-chaperone subunits undergo a conformational change in their first β -strand when the chaperone is replaced by the native donor strand. Some residues differ by as much as 14 Å between their positions in the tip complex and their positions in their FimC complexes. Donor strand complementation is used in the assembly of many bacterial sub-structures, and this structural shift has not been described elsewhere. The structure of the tip complex provides new views into the allosteric effects of mechanical force on receptor-ligand interactions and into how multiple subunits can bind the same chaperone and still bind specifically to particular subunits in the larger fimbrial structure. 07.18.4 Crystallographic and cryo-EM studies of the 750-kD coenzyme A carboxylase 1 1 1 6 6 holoenzyme of propionyl2 2,3 Christine Huang , Kianoush Sadre-Bazzaz , Yang Shen , Binbin Deng , Z. Hong Zhou , 1 Liang Tong Columbia University, New York, NY, United States, University of Texas Medical School at 3 Houston, Houston, TX, United States, University of California, Los Angeles, Los Angeles, CA, United States Propionyl-CoA carboxylase (PCC) is essential for the catabolism of several amino acids, cholesterol, and odd-carbon fatty acids. Deficiencies of PCC activity in humans are linked to propionic acidemia, an autosomal recessive disorder that can be fatal in infants. The PCC holoenzyme is an 6 6 dodecamer, with a molecular weight of 750-kD. The subunit contains the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains, while the subunit supplies the carboxyltransferase (CT) activity. We describe the crystal structure at 3.2Å resolution of a bacterial PCC 6 6 holoenzyme as well as cryo-EM reconstruction at 15Å resolution demonstrating a similar structure for human PCC. The structure defines the overall architecture of PCC and reveals unexpectedly that the subunits are arranged as monomers in the holoenzyme, decorating a central β 6 hexamer. A hitherto unrecognized domain in the α subunit, formed by residues between the BC and BCCP domains, is crucial for interactions with the subunit. We have named it the BT domain. The BC and CT active sites in the holoenzyme are separated by approximately 55Å, indicating that the entire BCCP domain must translocate during catalysis. The BCCP domain is located in the active site of the subunit in the current structure, providing insight for its involvement in the CT reaction. The structural information establishes a molecular basis for understanding the large collection of disease-causing mutations in PCC, and also has important relevance for the holoenzymes of other biotin-dependent carboxylases, especially MCC and eukaryotic ACC. 1 2 07.18.5 Crystal structure of the Head module of Mediator in gene expression, and its interactions with the RNA polymerase II transcription machinery Yuichiro Takagi , Tsuyoshi Imasaki , Gang Cai , Kuang-Lei Tsai , Guillermo Calero , Kentaro 1 1 4 3 2 Yamada , Francesco Cardelli , Imre Berger , Roger Kornberg , Francisco Asturias 1 1 1 2 2 3 Indiana University School of Medicine, Indianapolis, IN, United States, The Scripps 3 Research Institute, La Jolla, CA, United States, Stanford University School of Medicine, 4 Stanford, CA, United States, EMBL Grenoble, Grenoble, France 2 Mediator is a large multi-protein complex essential for eukaryotic gene expression by transducing the regulatory DNA information through DNA-bound transcription activators to RNA polymerase II (pol II). Mediator from yeast is complex composed of 21 subunits that are organized into three distinct modules, termed Head, Middle/Arm and Tail. The Head module (7 subunits, 223 kDa) is an essential sub-complex of Mediator as it controls Mediator-pol II and Mediator-promoter interactions in vivo. Thus, understanding structural basis of the Mediator Head module, and the Head-RNA pol II machinery is required to elucidate the mechanism of transcription initiation. We have determined the structure of the Head module of Mediator at 4.5 Å resolution by X-ray crystallography. A use of Tantalum cluster SIRAS, as well as Se-Met SAD was the key to obtain significant phase information. Combined with biochemical and EM studies, the X-ray module of the Head reveals three distinct domains: fixed jaw, movable jaw, and handle domain. The three domains are predominantly rich in apha helices. They are connected to the less-well ordered central hinge domain, which appears to provide flexibilities to the three domains. To reveal how the Head module interacts with the pol II machinery, using single particle cryo-EM, we have determine the structure of the Head-the minimum preinitiation complex (Head-mPIC) composed of Head, pol II, IIF, IIB, TBP, and promoter DNA. In the EM structure, the fixed and movable jaws clearly interact with Rpb4/7 subunits of pol II Notably, the clamp of pol II appears widely open in the presence of the Head module, providing the structural evidence to support our model in which Mediator modulates access of promoter DNA to the pol II cleft in part through the Head-Rpb4/7 interaction. 07.19.1 Combined Ultrasmall-Angle Spectroscopy X-ray Scattering and X-ray Photon Correlation Studies of Nonequilibrium Dynamics in Polymer Composites Fan Zhang , Andrew Allen , Lyle Levine , Jan Ilavsky , Gabrielle Long 1 2 1 1 1 2 2 National Institute of Standards and Technology, Gaithersburg, MD, 20877, United States, Argonne National Laboratory, Argonne, IL, 60439, United States While scattering and imaging techniques have enjoyed great success in studies of the microstructure over the full nanometers-to-micrometers size range in advanced materials such as composites and alloys, the dynamics of these materials, especially the response to abrupt changes in the sample environment, largely remains elusive. Knowledge of the incipient dynamical behaviour inevitably leads to a better understanding of the processing– structure–property relationships, and to a consequent improvement in material design and performance. Recently, we have developed combined ultrasmall-angle X-ray scattering / X-ray photon correlation spectroscopy (USAXS/XPCS) studies to probe the slow equilibrium and nonequilibrium dynamics of optically opaque materials with prominent scattering features in a q range between that of dynamical light scattering and conventional XPCS. Using USAXS/XPCS, we have studied the dynamical, nonequilibrium structural variation of polymer composites to an extent beyond what has been currently available. Upon heating, these polymer composites, synthesized as advanced dental materials, undergo irreversible dynamical structural change that is insensitive to either small angle X-ray scattering or X-ray diffraction. On exploiting the coherent beam properties of the Advanced Photon Source in combined USAXS/XPCS studies, a distinctive variation in the coherent scattering patterns is observed. Detailed analyses show systematic changes under different starting conditions, which we attribute to polymer creep arising from effects such as thermal mismatch on heating, or a change in particle density associated with an incipient amorphous-to-crystalline transformation in the “amorphous” calcium phosphate (ACP) filler material used in advanced dental nanocomposites. With the polymer composites as a prototype for a future class of materials study, we hope to establish the USAXS/XPCS technique as a unique tool to follow slow dynamics in disordered materials. 07.19.2 Nanocrystalline sodalites and intermediate structures between sodalite and cancrinite grown under high NaOH and NaOH/TEA concentrations at 333K Josef-Christian Buhl, Sandra Cramm, Karsten Schuster Leibniz University Hannover, Hannover, Lower Saxony, Germany The systems Na2O-SiO2-Al2O3-H20-NaCl/NaNO3 were investigated within reaction periods of 1 - 96 h under superalkaline low temperature conditions at 333 K. Mixtures of aluminosilicate gels with NaOH and NaCl or NaNO3 were used as educts. Nanoparticles of NaCl-sodalite (SOD) or nanocrystalline nitrate enclathrated intermediate phases (INT) between the structures of SOD and cancrinite (CAN) were successfully synthesized already after 3 h reaction time. Triethanolamine (TEA) was added in further series of syntheses to study the influence on formation of SOD, CAN and INT, because deceleration of nucleation rate is well known in the zeolite A and X system due to the complexing effect of TEA on the reactive aluminium-species in the solution [1]. The products were characterized by X-ray powder diffraction, scanning electron microscopy, FTIR spectroscopy and thermal analysis in comparison with the microcrystalline samples NaCl-SOD and NaNO3-CAN. Both were synthesized at 473 K and 48 h from kaolinite, i.e. the common conditions of hydrothermal formation of microcrystalline SOD and CAN. Analyses data show formation of nanoparticles in each case already after 3 hours with sizes not exceeding 50 nm and total batch compositions of about 50% nanocrystalline material beside 50% amorphous parts. Whereas the experiments with NaCl yielded the SOD-structure nitrate addition favours nanocrystalline intermediates (INT) between the structures of SOD and CAN as previously found in the carbonate system under common synthesis conditions from kaolinite [2]. INT is characterized by one dimensional stacking disorder of aluminosilicate layers along hexagonal c-axis. In CAN framework these layers show AB-stacking and SOD exhibits cubic ABC-sequence [2]. In the reaction series with TEA in principle the same phases were observed but SOD appeared in form of unusual big board-like aggregates of small spheres which in turn consist of numerous nanocrystaline particles. INT was found as big spheres formed by countless nanocrystalline rods. [1] Charnell, J.F.: J. Crystal Growth 8 (1971) 291 – 294. [2] Hermeler, G., Buhl, J.-Ch., Hoffmann, W.: Catalysis Today 8 (1991) 415 – 426. XMR ·‒¡?›‹?lneLT P )#(#&' &!%$#"! P s⁄¡?d¢¢¡¦ ?›¢?o‒¡ ? K?r ¡fi⁄¡‹?l›££\¦⁄ K?c\ P ?` ¡‹ Q Q t‹ ¡ „? ›¢? d ‹ ·‒£⁄K? d ‹ ·‒£⁄K? l › ⁄ \‹K? t‹ ¡ ? j ‹£ ›«K c \«›‹ ? k £⁄ ? r›·‒¦¡K c ¦› K?n‚¢›‒ ⁄ ‒¡K?t‹ ¡ ?j ‹£ ›« Porous materials are the subject of intense research. The most familiar porous materials are zeolites, which have numerous applications including molecular sieves for selective absorption (e.g. for gas storage or water purification) and catalysis. Although zeolites are well known, they form only one class of porous material. Molecularbased porous materials form an important growth area in crystal engineering; one class of these materials are metal organic frameworks (MOFs). These are an attractive alternative to zeolites because they are not restricted to tetrahedral network topologies, and are much more amenable to incorporation of chemical modifications, such as chirality. (MOFs) contain metal ions linked by rigid organic linkers. One of the first MOFs to be studied was (Zn4O(C8H4O4)3), otherwise known as MOF-5 (Fig. 1). MOF-5 is the parent structure of a series of iso-reticular MOFs (IRMOFs) whereby the overall repeating structure is conserved while allowing changes in the pore size and functionality by alteration of the organic linker. Fig. 1 MOF-5 structure shown as ZnO4 tetrahedra joined by benzene dicarboxylate linkers. The large sphere represents the largest van der Waals sphere that would fit in the cavity. 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qcehUVp eXaVUT ihYgfUVe ihYgfUVe ? ?d SR Q P H P P ‒? ¡‒‹ ¡ „ r ‒?s\ \ «› ›‒¤¡ ‹ ‹ ?o\ ¡ ? £ ‒ ‹ › ⁄ ‹ K?d K? ? ›?⁄? ¢¢¡¡?⁄ „?¦ •‹\¡? ?«› › ‹?› · ‹ ? \ ¦‹ ¦ £ ¡ «\ ? \ fi‒‒⁄ „›„ «¡«¡ \¦ ·‒£› ¦ ·‒£ ? ‒› ? \ 6E97GDCDB9DC DC6E 6F9EC6 7 AC @7DDCB 3F79BC 6 DC6 E 78 66DAE7B 66 A @9 dc baV`YXWVUT 87 ⁄ ? ·? 6 431 O MP 6 5 I H u 0 ? ⁄ ⁄? ‒ ·? ‒ ‚? ? ‒ ‚ ‒ ‒ „ ‒ ⁄„? ? ‚ ? ? „ ‚ ¦ ¡ ⁄ ‹ fl·¡ ♠ 07.19.5 Combined neutron- and X-ray diffraction study of borosilicate glasses relevant to the immobilization of nuclear waste Margit Fabian , Thomas Proffen , Uta Ruett , Erzsebet Svab 1 1 2 3 1 2 Research Institute for Solid State Physics and Optics, Budapest, Hungary, Los Alamos Natl. 3 Lab., Lujan Neutron Scattering Ctr, Los Alamos, United States, Deutsches ElektronenSynchrotron (DESY), Hamburg, Germany Borosilicate based glasses are generally accepted as proper high-level radioactive waste isolating media [1]. In spite of their importance, relatively few diffraction investigations have been performed because of the large number of constituent elements. Here we report our results on three borosilicate based glassy series prepared under the same melt quenching preparation technique [2], starting with the 3-component material up to the 6component one, with the composition of (75-x)SiO2•xB2O3•25Na2O, (65-x)SiO2•xB2O3•25Na2O•5BaO•5ZrO2 [2] and 70wt%[(65-x)SiO2•xB2O3•25Na2O•5BaO•5ZrO2)]+30wt%UO3 (x=5,10,15,20 mol%). For the study of the short- and intermediate range order we have performed neutron- and X-ray diffraction measurements. Neutron diffraction measurements were carried out at the 10 MW Budapest research reactor using the PSD diffractometer [3] and the time-of-flight NPDF instrument at the LANSCE/Los Alamos pulsed neutron source [4]. The high-energy X-ray diffraction measurements were performed at the BW5 experimental station at Desy/Hamburg using 109,5 keV radiation energy [5]. Both the traditional Fourier transformation technique and the reverse Monte Carlo (RMC) simulation of the experimental data have been applied to get structural information. Simultaneous RMC simulation of the two data sets was applied to generate reliable 3-dimensional atomic configurations and to calculate the partial atomic pair correlation functions and coordination number distributions. It was established that the basic network structure consists of tetrahedral SiO4 units and of mixed tetrahedral BO4 and trigonal BO3 units for the three investigated glassy systems. The multi-component glasses proved to be stable and capable of hosting uranium. For the U-O first neighbour correlations two distinct peaks were resolved at 1.84 Å and 2.24 Å, and for higher distances intermediaterange correlations were observed. Significant correlations have been revealed between uranium and the network former Si and B atoms, indicating that uranium ions take part in the network forming. Details of the structural characteristics will be presented. [1] Chun K S, Kim S S and Kang C H 2001 Journal of Nuclear Materials 298 150 [2] Fábián M, Sváb E, Proffen Th and Veress E 2008 J. Non-Cryst. Solids 354 3299 [3] Sváb E, Mészáros Gy and Deák F 1996 Materials Science Forum 228 247 [4] Proffen Th, Billinge S J L, Egami T and Louca D 2003 Zeitschrift für Kristallographie 18 132 Poulsen H, Neuefeind J, Neumann H B, Schneider J R and Zeidler M D 1995 J. Non-Cryst. Solids 188 63 07.19.6 Advances in Deep Space Nuclear Reactor Design Boris Udovic Private, Sezana, Slovenia Lightweight thermo-emissive nuclear reactor hot cathode bodies with thick shells of highly 12 3 thermally conductive monoisotopic C sp CVD diamond and high-density tetrahedral amorphous ta-C CVD carbon phases are projected to acquire severe long-life properties with high temperature self-repairing sealant capabilities over encapsulated nuclear fuel kernels in deep space flight missions. The unavoidable leakage of the white spectrum of striking neutrons, fissile particles and the continuous emission of and rays gives rise to fission spikes by cascades of primary, secondary and higher order displacements of ballistically knocked-on atoms via gradual transitions from “Rutherford” to “hard sphere” collisions inside the monoisotopic matrix of the whole onion carbon envelopment. As the thermo-emissive current sharply intensifies with the squared value of the absolute temperature, the operative regime targeted near 2500 K matches to a considerable extent some multiple requirements: to avoid greater effects of sublimation phenomena at the diamond surfaces, -to promote structural changes that can overcome the threshold energy barrier of the settlingdisplacements inside the superheated solid phase because of localized excitations produced 3 12 by hot thermal spikes. Once formed, the C sp ta-C CVD carbon phase network prevents any re-hybridization of the whole diamond unit cell and makes impossible the relaxing states to graphite allotrope backwards. Namely, the compressive shrinkage of the carbon matrix in 3 the fashion of a self-pressing nutcracker jaw, which squeezes every displaced sp carbon atom out from its tightly strained inter-atomic cage, hinders any size enlargement of the disordered ta-C phases by self organization phenomena, raising a higher degree of symmetry of the whole tetrahedral network bone. The high temperature and low pressure working gas of free caesium atoms Cs saturates the nearest inter-space around the hot cathode boundary layer. Congruently, the driving force of the electron transfer reaction from the nearest thermo12 2 ionized caesium atom Cs to each C sp carbon atom at the top of the ta-C coating epi-layer promptly promotes the quick rehybridization change of the appearing planar carbo-olefinic units into pyramidalized free radicals, which rebuild and keep safe the diamontoid 3D network. f f f 07.19.7 Evolution of copper-rich precipitates in reactor pressure vessel steels under irradiation Mikhail Sokolov , Michael Miller , Randy Nanstad , Ken Littrell , Lee Robertson , Enrico 2 Lucon 1 1 1 1 1 1 ORNL, Oak Ridge, United States, SCK-CEN, Mol, Belgium 2 Current fleet of nuclear power plants is poised for operating life time extension. It means that main structural components, including reactor pressure vessel, will be subject to higher neuron exposure than originally planned. This raises serious concerns regarding our ability to predict reliability of reactor pressure vessel steels at such high doses. In this study, several representative reactor pressure vessel steels (RPVS) were irradiated at high doses to study degradation of mechanical properties and related microstructural changes of RPVS. It is well known that copper-rich precipitates are key microstructural futures that are responsible for radiation hardening of RPV steels. In this study, the evolution of copper-rich precipitates (CRP) is studied by means are small-angle neutron scattering and atom-probe tomography. These techniques are used to measure number density, volume fraction, and radius of precipitates. Evolution of these microstructural features is cooperated to degradation of fracture toughness and hardening of these steels. 07.19.8 Correlating Small Angle Scattering with Electrical Resistivity Changes in the NickelBase Superalloy Waspaloy Ricky Whelchel , V. S. K. G. Kelekanjeri , Rosario Gerhardt , Jan Ilavsky , Ken Littrell 1 2 2 2 2 3 1 HFIR/NSSD Oak Ridge National Laboratory, Oak Ridge, TN, United States, School of Materials Science and Engineering, Georgia Institute of Technology, Atlanta, GA, United 3 States, 2X-ray operations division, Argonne National Laboratory, Argonne, IL, United States Waspaloy is a nickel-base superalloy used primarily in disc rotors in gas turbine engines. Waspaloy is ideal for this purpose due to the material's increased high temperature strength and creep resistance due to the formation of nanometer-scale precipitate phases (γ ') within a solutionized matrix phase (γ ) via heat treatment. The pre-existing precipitate phases formed via heat treatment can evolve with in-service thermal exposure in gas turbine engines, resulting in evolving mechanical properties of the bulk superalloy components. Consequently, it is desirable to monitor this evolution in mechanical properties non-destructively. Electrical resistivity is one method by which the precipitate microstructural evolution may be sensed. Electrical resistivity is sensitive to the formation of small phases and the removal of precipitate phase solute [1]. Small precipitates, on the order of 1nm, have significant electron scattering ability and are the dominant features affecting electrical resistivity changes at early stages of the aging process. Solute removal due to precipitation results in a more pure, and thus more conductive, matrix phase. Waspaloy specimens aged at 800° from 0.5h to 88.5h were evaluated via small angle C neutron scattering (SANS), ultra small angle X-ray scattering (USAXS), electrical resistivity, and SEM. The average γ ' precipitate size and volume fraction obtained from modeling the small angle scattering data was used to calculate a figure of merit of electron scattering. This figure of merit is designed to correlate the electron scattering ability of the material with the precipitate microstructure. 07.21.1 A Career is What You Make of It Joseph Ferrara Rigaku Americas Corp, The Woodlands, TX, United States My professional odyssey started while I was an undergraduate at Case Institute of Technology. For my senior research project I used ion cyclotron resonance to study collision quenching of photoexcited iodobenzene ions. This is where I started my love of instrumentation; I worked in a lab with very old equipment, some handmade and even some I made myself. I then continued on at Case for my graduate degree thinking I would finish quickly continuing my undergraduate research. That was not to happen. I heard a new faculty mumble something about room temperature superconductors (this was 5 years prior to the discovery of cuprate superconductors). That phrase took me down a path of synthetic organic chemistry and organometallic chemistry, physical chemistry and finally, my true calling, crystallography, Upon completing my dissertation in 1987, my wife and I moved to College Station, TX, where I started as a service crystallographer for Molecular Structure Corporation. In 1996, Rigaku bought MSC lock, stock and barrel. Currently, I am currently CSO of Rigaku Americas Corporation, VP of the X-ray Research Laboratory in Tokyo, and a Rigaku Innovative Technologies board member. Over the years I have solved some pretty tough structures, written and rewritten lots of FORTRAN, dealt with European compliance issues, started a quality assurance department, managed software and hardware development teams, learned some Japanese and watched some people blossom. I get to meet lots of very interesting people and really do have fun. 07.21.2 Beamline Scientist James Holton University of California, San Francisco, CA, United States, Lawrence Berkeley Laboratory, Berkeley, CA, United States Probably the best thing about working at a beamline is the sheer number of “collaborators” you work with (we call them “users”). Most crystallographers will only work directly with a few dozen other scientists in their entire career, but a typical beamline scientist works side-by-side with more than a hundred different investigators each year. Each user has their own favourite tools, techniques and software, but it is only by seeing lots of different approaches that one can fully understand what works and what doesn’ t in protein crystallography. This is not just a good way to accumulate expertise, but also a good way to become widely recognized as an expert. Even if your career goals eventually lead you away from the synchrotron, you can hardly do worse than having hundreds of crystallography labs recognize you as that one person who was really helpful and knowledgeable in their time of need. Becoming an expert in methods is not just a luxury of beamline science, it is a job requirement, and although there are those who regard methods development as secondary to “real science”, there are few who do not recognize the importance of data. There is nothing quite like beautiful data. Even the most cantankerous reviewers can’ t argue with it nor the conclusions that follow obviously from it. But, the trick to cutting edge science is knowing what you can and cannot conclude from “decent” data (the kind you get early in a project), and making these calls requires someone who knows the method inside and out. 1 2 1995 2001 2001 2005 B. S. in Biology, Caltech Ph. D. Molecular and Cell Biology – Tom Alber, UC Berkeley Staff Scientist, Beamline 8.3.1 ALS Assistant Adjunct Professor, UCSF 07.21.3 An adventure: Odyssey or Oddity? David Rose University of Waterloo, Waterloo, Ontario, Canada Well, it certainly has been an adventure. After a “traditional” training (BA, Univeristy of Pennsylvania; DPhil, Oxford; Postdoc, MIT), my career has been divided between three research sectors: government, research institute, academia. They each have somewhat different cultures, plusses and minuses. I would be happy to share with the audience my perspectives on these, as well as other issues such as: making your own breaks, what size “pond”, how to be an attractive candidate for research positions, etc. I would also encourage young scientists to be “citizens of science” both within your institutions and through international societies like the ACA. 07.22.1 Multiple Conformations of human glucokinase in solution: Insights into enzyme cooperativity and activation of glucokinase Shenping Liu, Mark Ammirati, Xi Song, Xiayang Qiu Pfizer Inc, Groton, CT 06385, United States Glucokinase (GK) plays an important role of glucose sensor in controlling plasma glucose level. To fulfill this role, the kinetic properties of GK are essential: it shows low affinity for glucose and displays positive enzyme cooperativity. Inactivating GK mutations cause diabetes, while activating GK mutants have decreased cooperativity and cause hypoglycemia, underscoring GK's importance in glucose homeostasis. The positive cooperativity of glucoskinase, a monomeric enzyme with one active site, was hypothesized to be caused by either a mneumonica or a slow-transition mechanism, both of which involve two conformations with different glucose affinity. Using small angle X-ray scattering (SAXS) method, we determined the conformations of wild type GK and activating mutants in solution, in presence of glucose and GKA. Our results show that glucose dose dependently shift the population of GK from inactive to an active conformation, clearly supporting the mneumonic model of GK's coorperativity. Compared to wild type GK, activating mutants requires less glucose concentration to be activated. GKAs decrease the level of glucose required for GK activation, and different GKAs demonstrated different GKA activation profiles. Our findings provide an insights in GK’s coorperativity, activation as well as design GKA with different profiles. 07.22.2 From biochemical and structural studies of soluble guanylate cyclase (sGC) toward drug design Emmanuelle Laffly, Jane Macdonald, Franziska Seeger, Elsa Garcin UMBC, Baltimore, MD, United States The overall goal of my research project is to understand the structural basis for assembly and activation of soluble guanylate cyclase (sGC). sGC is the direct sensor and mediator of nitric oxide (NO) signal transduction via cyclic GMP (cGMP). NO-induced vasodilation depends on the activation of sGC. Compounds activating cGMP production by sGC have outstanding clinical potential for treating cardiovascular diseases. Structural information on sGC is limited to x-ray structures of homologous single domains. Despite these pieces of information, a detailed understanding of the events leading to activation in full-length sGC (Fl-sGC) is missing. To elucidate the structural details of Fl-sGC assembly and determine the dynamic events associated with NO-induced Fl-sGC activation, I have initiated a multidisciplinary approach. The combination of biochemical and mutagenesis studies with low- (SAXS), medium- (DXMS) and high-resolution (X-ray crystallography) structural methods will yield critical information to design novel sGC activators. I have developed the first heterologous bacterial overexpression system for bovine Fl-sGC, expressed and purified soluble and active Fl-sGC, and collected preliminary SAXS data. I am now docking high-resolution x-ray structures from homologous single domains into the calculated ab initio low-resolution SAXS envelopes to build a 3D model for Fl-sGC. To pursue this work, we plan further developments including purification under a controlled atmosphere to prevent oxidation damage and the use of Baculovirus or Pichia pastoris that may be better suited to obtain larger quantities of Fl-sGC necessary for our structural characterizations. 07.22.3 Crystal structure and thermodynamic analysis of Fab 106.3 complexed with BNP 5-14 reveal molecular details of mAb for clinical diagnosis. Kenton Longenecker, Qiaoqiao Ruan, Elizabeth Fry, Sylvia Saldana, Susan Brophy, Paul Richardson, Sergey Tetin Abbott, Abbott Park, United States Plasma concentration of the B-type natriuretic peptide (BNP) is a clinically recognized biomarker for cardiovascular disease. Diagnostic immunoassays can measure BNP levels using the monoclonal IgG1 antibody 106.3, which binds with high affinity to an epitope spanning residues 5-13 of the mature bioactive peptide. To understand this molecular recognition, we crystallized the Fab fragment of mAb106.3 complexed with the peptide epitope, and determined the structure with X-ray diffraction to 2.1 Å resolution. The crystal structure reveals detailed interactions that five of the complementary-determining regions (CDRs) make with the partially folded peptide. Thermodynamic analysis of fluorescence spectroscopy measurements suggests the interaction is driven by enthalpy changes with an overall free energy of binding, Δ G = -54 kJ/mol. We interpret parameters based on the structural information, and the kinetics suggest a rapid diffusion-limited mechanism of binding. Comparative analysis with alanine-scanning studies of the epitope explains the basis of mAb106.3 selectivity for BNP over other related natriuretic peptides. 07.22.4 Structural and Functional Insights into a Cardiac Specific Histone Methyltransferase Nualpun Sirinupong , Joseph Brunzelle , Jun Ye , Ali Pirzada , Lindsey Nico , Zhe Yang 1 2 1 2 1 1 1 1 Wayne State University, Detroit, MI, United States, Advance Photon Source, Argonne, IL, United States SmyD1, a histone H3K4 methyltransferase, was found specifically expressed in heart and skeletal muscle, important for cardiac development and related to heart diseases. The unique domain structure characterized by a split SET domain, a conserved MYND zinc finger, and a novel C-terminal domain (CTD) distinguishes SmyD1 from other SET domain methyltransferases. Here we report the crystal structure of full-length SmyD1 in complex with AdoHcy at 2.3 Å. The crystal structure reveals that SmyD1 folds into five distinct structural domains, and the shape of the protein resembles an open-ended wrench, where the two thick grips are separated by a large, deep concave opening. Substantial structural differences exist between SmyD1 and other SET proteins, which reflect the unique structural and functional properties of this protein. The crystal structure reveals that SmyD1 does not contain the preSET domain that is necessary for methylation by other SET proteins. In addition, SmyD1 has a nearly buried cofactor binding site due to the bulky SET-I domain, which appears to restrict the exchange during catalysis between cofactor and its product as suggested by our mutation studies. Moreover, the structure reveals an unusually spacious target lysine binding site, which provides structural basis for the low histone binding affinity and weak methylation activity of SmyD1. The remarkable feature of the SmyD1 structure is the presence and location of the CTD domain, whose function was unknown. The structure reveals that the CTD domain is located adjacent to the histone binding site and contributes to the formation of a unique Y-shaped binding cleft. The structural and functional analysis suggest that the CTD domain may be involved in the direct interaction with histone H3, and form an extended substrate binding site that is likely to recognize the residues far from the C-terminal side of target lysine 4. The structure determination of SmyD1 also offers important functional implications of SmyD1 in cardiac development. The structure reveals that the proline-rich peptide binding site in the MYND domain is fully exposed and can be readily accessed by skNAC, a cardiac transcription factor. The structure and function studies suggest that the MYND domain may primarily serve as a protein interaction module, and cooperate SmyD1 with skNAC to regulate cardiomyocyte growth and maturation. In addition, the structure explains why both the MYND domain and the S-sequence are required for the interaction with skNAC, and whether this interaction can affect SmyD1 structure and function, which in turn may alter the histone methylation profile in heart and influence gene expression in cardiac development. Overall, the crystal structure of SmyD1 provides structural insights into the novel mechanism of SmyD1 regulation, and also provides structural basis for further understanding the role of SmyD1 in heart development and cardiovascular diseases. 07.22.5 DPPIV Inhibitors – A Structural Biologists Perspective Sridhar Prasad G. CalAsia Pharmaceuticals, Inc., San Diego, CA 92121, United States Dipeptidyl peptidase IV (DPPIV) is a member of the prolyl oligopeptidase family of serine proteases. DPPIV removes dipeptides from the N terminus of substrates chemokines, neuropeptides and peptide hormones including Glucagon-like peptide-1 (GLP-1). GLP-1 enhances the glucose-dependent secretion of insulin from pancreatic beta-cells following the ingestion of food. Infusion of exogenous GLP-1 in type 2 diabetes patients has shown to lower the plasma glucose and improve the beta-cell function. However, the rapid clearance of GLP-1 in vivo by DPPIV has diminished the prospect of exogenous GLP-1 as a potential therapy. An alternate approach has been to reduce the activity of the GLP-1 processing enzyme, DPPIV, by small molecule inhibitors. A number of small-molecule DPPIV inhibitors have shown to have beneficial effects in animal models and additionally proven clinically benefits in human trials. As a result, DPPIV inhibitors have now become marketed drugs. Given the importance of this new class of drugs, a large number of pharmaceutical and academic groups have been pursuing to discover more safe and efficacious compounds using multiple approaches, including fragment and structure-based hit identification and lead optimization methods. The availability of the three-dimensional structural information of the enzyme DDPIV has played an important role in the discovery and development of safe, potent and novel clinical compounds in record time. These “drug candidates” have now successfully entered human trials and are awaiting approval of the regulators. A comprehensive analysis of the inhibitors discovered using different approaches will be presented, with an emphasis on programs that involved the use of three-dimensional structural information. 07.22.6 Inhibition of recombinant maltase-glucoamylase by acarbose, salacinol, kotalanol, and de-O-sulfonated kotalanol Kyra Jones , Lyann Sim , Buford Nichols , B. Mario Pinto , David Rose 1 2 1 2 4 3 1 University of Waterloo, Waterloo, Ontario, Canada, University of Toronto, Toronto, Ontario, 3 4 Canada, Simon Fraser University, Burnaby, British Columbia, Canada, Baylor College of Medicine, Houston, Texas, United States Inhibition of intestinal α -glucosidases and pancreatic α -amylases is an approach to controlling blood glucose and serum insulin levels in individuals with type II diabetes. An important target enzyme is maltase-glucoamylase, a Family 31 glycoside hydrolase responsible for the final step of starch hydrolysis releasing free glucose in the small intestine. Here we examine the inhibition of the C-terminal catalytic subunit of maltase-glucoamylase by salacinol, acarbose, kotalanol, and de-O-sulfonated kotalanol and present the inhibition profile of the catalytic domain with respect to these compounds. We determined enzymatic activity using a coupled assay measuring glucose hydrolyzed from substrate in the presence of each inhibitor. The results facilitate comparison of the active site requirements of the N- and C-terminal subunits, as the N-terminal catalytic domain has previously been characterized. The structure of the Nterminal domain has been used as a basis for understanding these results, enhancing the understanding of the role of each catalytic subunit in starch digestion. Ultimately, this will help to guide the development of new compounds with anti-diabetic activity. Further, this research can be applied to nutritional diseases such as obesity and cardiovascular disease. 07.22.7 Intestinal Glucosidases: Structure/Mechanistic studies towards clinical applications for diabetes and obesity. David Rose , Lyann Sim , Kyra Jones 1 1 2 1 2 University of Waterloo, Waterloo, Ontario, Canada, University of Toronto, Toronto, Ontario, Canada The human system for digesting starch to nutritional glucose has evolved to handle diverse sources of starch. Initial processing by salivary and pancreatic amylases breaks starch polymers down into “limit dextrins”, which include both -1,4 and 1,6 linked glucose saccharides. These are further processed by the small intestinal glucosidases, maltaseglucoamylase (MGAM) and sucrase-isomaltase (SI). This presentation will review our results on investigating the crystal structures of the 4 glycoside hydrolase family 31 enzymes that make up MGAM and SI. We are participating in an international collaboration to understand the structural basis for the inhibitory properties of these enzymes, the development of novel inhibitors, and the investigation of the complementary roles of MGAM and SI in the processing of limit dextrins of starch. Our hypothesis is that the enzymes each play key roles in different circumstances of diet, starvation and gorging. Anti-diabetic compounds on the market currently are broadly specific inhibitors of glucosidases. We suggest that an improvement of their affinities and specificities for the most relevant glucosidase activities, through structural and synthetic studies, will improve their efficacy and reduce side effects. The action of these compounds to control blood glucose levels has relevance not just to diabetes but also to nutritional disorders such as obesity, and to cardiovascular diseases. Our progress on testing novel compounds in a diabetic rat model will be presented. g See also Poster presentation by Kyra Jones on the inhibition profiles of MGAM domains. 07.22.8 Structural and Functional Studies of Gα 1 1 q-mediated 1 Signaling 3 1,2 Angeline Lyon , Valerie Tesmer , Krishna Suddala , John Northup , John Tesmer 1 2 Life Sciences Institute, University of Michigan, Ann Arbor, MI, United States, Department of 3 Pharmacology, University of Michigan, Ann Arbor, MI, United States, Laboratory of Cell Biology, National Insitutes of Health, Bethesda, MD, United States G protein-coupled receptors (GPCRs) are the largest family of membrane receptors and serve to integrate extracellular signals with intracellular responses. Upon activation by hormone, neurotransmitter, or other agonist binding, GPCRs facilitate nucleotide exchange on G proteins (Gα β γ ), which in turn signal through downstream effectors. The Gq-coupled class of GPCRs are linked to platelet activation and heart development, as well as pathologic processes such as the onset and maintenance of arrhythmias, hypertrophy, and heart failure. In an effort to develop a structural understanding of these pathways, we have pursued studies of Gα q with GRK2 (a G-protein coupled receptor kinase) and p63RhoGEF (a guanine nucleotide exchange factor for RhoA). New results in our lab on other Gα q effectors, such as phospholipase Cβ (PLCβ ), indicate that these effector interactions occur through highly conserved structural elements. Through biochemical and structural studies, we are defining the mechanisms of Gα q-mediated activation of effector. 07.22.9 Surprises and explications in two projects: Discovery of high quality dual thrombin/factor Xa inhibitors and renin inhibitors Zsolt Bocskei , Gary McCort , Steinhagen Henning , Scheiper Bodo , Matter Hans , Thiers 2 4 4 2 2 Bérangère , Lassalle Gilbert , Meneyrol Jerome , Altenburger Jean-Michel , Petit Frederic , 4 3 4 3 Herault Jean-Pascal , Wehner Volkmar , Alet Nathalie , Schreuder Hermann , Bono 4 Françoise sanofi-aventis research, Strasbourg, France, sanofi-aventis research, Paris, France, sanofi4 aventis research, Frankfurt, Germany, sanofi-aventis research, Toulouse, France, 5 Grunenthal, Aachen, Germany Two cardiovascular drug discovery projects will be presented with considerably different scopes: 1 2 3 1 2 5 3 3 Thrombin-Factor Xa dual antithrombotic inhibitors were developed on the basis of the expectation that simultaneous targeting of multiple coagulation enzymes may offer an improved efficacy and therapeutic index. Optimization started from an internally established selective thrombin inhibitor series and crystal structures helped to explain the SAR and to achieve increased affinity on FXa, while keeping activity low on other serine proteases like trypsin. Crystal structures explained how it was possible to maintain high FXa activity despite a bulky P2 group on the inhibitor. Replacement of basic P1 groups with neutral groups was also followed and rationalized. In the renin project most interesting hits of an HTS as well as backscreening campaigns were characterized by co-complex protein crystallography and these crystal structures contributed to the selection of hits and the optimization strategies. While studying these early hits we met a number of unexpected structural features and binding modes due to the high flexibility and plasticity of the renin active site. These structures and their use will be described along with the optimization of two chemical series (acyl-guanidines and indol piperazines) into highly specific drug-like renin inhibitors using structure assisted drug design. 07.23.1 Coordination Polymers with Surface Areas exceeding 5000 m /g: Should Traditional Sorbents Worry? Adam Matzger University of Michigan, Ann Arbor, United States Adsorbents play a critical role in a variety of industrial, laboratory and consumer applications. Materials such as silica gel, zeolites, and activated carbon have been investigated for centuries and represent the most commonly used adsorbents. In the last decade new high surface area materials based on coordination chemistry have emerged. These inorganicorganic hybrid materials are porous crystals that promise to redefine the types of processes and applications that can be enabled by adsorption. Synthetic challenges and novel approaches to the synthesis of crystalline microporous coordination polymers (MCPs) will be discussed. Recent progress with coordination copolymerization will be discussed in the context of producing structurally defined ultrahigh surface area materials. Application of MCPs for gas separations and storage will be briefly discussed. 2 07.23.2 Metal organic frameworks as CO2 capture materials and as fuel cell electrolytes George Shimizu, Ramanathan Vaidhyanathan, Jeff Hurd, Simon Iremonger University of Calgary, Calgary, Alberta, Canada Metal organic frameworks (MOFs) represent a tunable molecular scaffolding that can be adjusted for a breadth of applications. This presentation will concern our efforts towards tailoring the properties of MOFs towards two globally relevant energy challenges. The first concerns our efforts to make MOFs with amine lined pores for CO2 capture. In contrast to liquid amines which chemisorb CO2 and have high energy costs for regeneration, the MOF approach gives physisorbed gases and hence more facile release. Despite the weaker binding mode, we will show that high selectivities are possible owing to heats of adsorption over 40 kJ/mol. We will also present molecular level insights to the CO2 capturing ability of these solids.[1] The second topic concerns new electrolyte membranes for PEM and direct methanol fuel cells. A major hurdle in these technologies is an electrolyte capable of conducting protons above 100˚C. Higher operating temperatures will enhance electrode kinetics and decrease electrode poisoning among several critical operational benefits. In contrast to the macromolecular approaches typically employed towards these electrolytes, we have used a MOF strategy to generate crystalline networks with acidic pores. These MOFs can include amphoteric N-heterocycles, to act as non-volatile proton carriers in their pores to give proton -4 -3 -1 conduction from 10 -10 Scm at 150˚C without humidification. Moreover, we show that PCMOF materials can be incorporated into gas-tight membrane electrode assemblies to give open circuit voltages > 1.0 V at 100˚C in a H2/air fuel cell.[2] [1] R. Vaidhyanathan et al. “An amine-functionalized metal organic framework for preferential CO2 adsorption at low pressures,” Chem. Commun. 2009, 5230. [2] J. A. Hurd et al. “Anhydrous proton conduction at 150˚C in a crystalline metal organic framework,” Nature Chem. 2009, 1, 705. 07.23.3 Material-based hydrogen and methane storage: Understanding the storage mechanism using x-ray/neutron diffraction and computational modeling Wei Zhou National Institute of Standards and Technology, Gaithersburg, United States The storage of energy carrier gases (e.g., H2 and CH4) based on novel materials has attracted much research attention in recent years. For physisorptive systems (such as porous metal-organic frameworks), it is important to identify the gas adsorption sites and elucidate the gas-host interaction mechanism. For chemisorptive systems (such as complex hydrides), obtaining accurate structural information and understanding the reaction pathway are critical. In this talk, I am going to present some of our recent work on both metal-organics frameworks and complex hydrides. Combing the strength of x-ray/neutron diffraction and computational modeling, we are able to understand the storage mechanism of fuel gases in several new material systems. Rational strategies are proposed to improve the material storage performance. 07.23.4 Polyhedra Stabilized Metal-Organic Frameworks and Their Applications in Hydrogen, Methane, and Carbon Dioxide Storage Daqiang Yuan , Dan Zhao , Daofeng Sun , Hong-Cai Zhou 1 1 1 1,2 1 2 Texas A&M University, College Station, Texas, United States, Shandong University, Jinan, China A previously described approach towards stable metal-organic frameworks (MOFs) with high surface area by incorporating microwindows within mesocavities was advanced by using longer hexatopic ligands. One of the generated MOFs (PCN-68) has a BET surface area of 2 -1 5109 m g , which is among the highest so far. Their hydrogen, methane and carbon dioxide storage for clean energy applications were systematically studied. 07.23.5 Structure and Dynamics of Proton-Conducting Metal-Organic Frameworks Jamie Ford , Jason Simmons , Taner Yildirim 1 1 2 1,2 2 University of Pennsylvania, Philadelphia, PA, United States, NIST Center for Neutron Research, Gaithersburg, MD, United States Vehicles powered by polymer electrolyte membrane (PEM) fuel cells are an exciting alternative to current fossil fuel technology. The membranes in these cells serve as both charge transporter, ferrying protons from the anode to the cathode, and gas diffusion barrier, preventing the backflow of oxygen to the anode. Currently, hydrated sulfonated polymers are the preferred material for these membranes. The presence of water, however, limits the operating temperature to 100 C, reducing the electrode kinetics and CO tolerance of the entire system. In an effort to increase the efficiency and operating temperature of these fuel cells, we are investigating the proton conductivity of new host/guest materials based on metal-organic frameworks (MOFs) loaded with proton conducting small molecules. Through choice of organic linker and inorganic metal salts, the geometry, size, chemistry and interconnectivity of the MOF frameworks can be tuned. These thermally stable structures provide well-defined pores for the guest molecules to form proton-conducting pathways. Here, we will discuss the crystallographic structures of the bare and loaded frameworks as well as the dynamics of the guest molecules and protons within in the systems. 07.23.6 Mn2-xFexP1-yGey , a potential system used for room temperature magnetic refrigeration Qingzhen Huang , J.W. Lynn , Danmin Liu , Ming Yue 1 1 1 2 2 2 NIST Center for Neutron Research, Gaithersgurg, MD 20899, United States, University of Technology, Beijing, 100022, China Beijing Magnetic refrigeration, a clean and energy saving technology, offers a solid-state alternative to the traditional gas-compression-based cooling, and will eliminate the harmful refrigerant gases and reduce energy requirement. Mn2-xFexP1-yGey is a good candidate material used for room temperature magnetic refrigeration. It has a first order ferromagnetic transition with large entropy change in a wide and adjustable temperature range. To understand the materials’ crystal and magnetic structures and their relationships with the magnetic and thermal properties is of central interest for materials scientists. We use the neutron powder diffraction technique to determine, in situ, the crystal and magnetic structures, combined with magnetic and thermal properties measurements, to obtain the information necessary for understanding the relevant structural details and their relation to the physical properties at different temperatures and/or under magnetic fields. Our research results have demonstrated that changes of magnetization and the entropy are in proportion to the changes of the ferromagnetic phase fraction when decreasing the temperature or increasing an applied magnetic field. The determination of the crystallite size indicates that small crystallite sizes inhibit the paramagnetic to ferromagnetic transformation, suggesting that it is possible to achieve 100% transformation by increasing the crystallite size, and, therefore, obtain the maximum entropy change. We found that the compositional homogeneity is one of the main issues for the system performing under a low magnetic field, because that the Curie temperature Tc is very sensitive to the Mn and Ge contents and their distributions and, therefore, the chemical inhomogeneities are responsible the existence of an interval of Tc in which the two phases coexist.. By improving the chemical homogeneity we have made samples with as low as 1.2 tesla magnetic field requirements to achieve more than 80% ferromagnetic transformation. We expect that it is possible to improve the system so that the magnetocaloric effect will to exceed 100 J/kg-K under a magnetic field as low as less of 1 tesla. 07.23.7 Experimental and theoretical studies of the Magnetocaloric Effect (MCE) in the Mn5xFexSi3 series Michael Gottschlich , Olivier Gourdon , Michael Ohl , Joerg Persson , Thomas Brueckel 1 2 1,2 1,2 1,2 2 2 Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States, Research Center Juelich, Juelich, Nordrhein-Westfalen, Germany The magnetocaloric effect (MCE) based on entropy changes of magnetic materials in an applied magnetic field, holds the potential of applications for refrigeration without moving mechanical parts. Therefore it has recently attracted the attention of many scientific research groups. Although MCE was discovered a long time ago (1881) by Warburg in iron, we are still investigating new usable materials low cost and chemically stable and safe. Recently, after the characterization concerning magnetization measurements and refinements on x-ray data, neutron measurements on polycristalline samples Mn5-xFexSi3 have been collected on the HB2A neutron powder diffractometer at HFIR under various fields. These preliminary measurements emphasize a unique atomic distribution as well as a ferromagnetic ordering along the c axis which is in some extends “forced” by the field in the vicinity of the magnetic transition. Such effect is certainly directly linked to the MCE measured. Preliminary theoretical calculations which support our findings will also be presented. 07.23.8 Structure and Thermoelectric Properties of Selected Alkaline-Earth Cobalt Oxides Winnie Wong-Ng , Guangyao Liu , Tana Luo , Joshua Martin , Yonggao Yan , Evan 3 1 4 Thomas , Qing Huang , James Kaduk National Institute of Standards and Technology, Gaithersburg, MD, United States, University 3 of Maryland, College Park, MD, United States, Air Force Research Laboratory, Wright 4 Patterson, OH, United States, Poly Crystallography, Inc., Naperville, IL, United States In recent years, thermoelectric research has attracted considerable attention partly because of the green house gas emission problem and the ever-increasing gas price problem. There is a desperate need for efficient energy conversion materials and environmentally friendly technologies over the next twenty years. For energy conversion applications using waste heat, oxide materials of high temperature stability are potential candidates. This paper discusses our phase equilibria/structural/property studies of selected series of cobaltites, including those in the SrO-CaO-CoOx and CaO-ZnO-CoOx systems. 1 2 1 1 2 1 1 07.23.9 Crystallographic and theoretical studies of a family of new arsenides ACd4As3 (A = Na, K, and Rb) Hua He, Svilen Bobev University of Delaware, Newark, DE, United States In recent years, binary and ternary d-block metal pnictides have attracted much attention from the solid-state community. This intense research interest is due to the discoveries of superconductivity and high figure of merit for thermoelectrics among such compounds. In this presentation, we discuss the new arsenides, ACd4As3 (A = Na, K, and Rb), which have been synthesized by reactions of the elements at high temperature; and their crystal structures have been established by single-crystal X-ray diffraction. Despite of the large differences among the radii of the alkali metals, the title compounds are isostructural and crystallize with a new structure type in the rhombohedral space group R 3 m (No. 166) with the cell parameter c increasing dramatically on going from Na to Rb. The crystal structures can be rationalized + – as A cations and [Cd4As3] layers, which are made up of corner- and edge-shared CdAs4 tetrahedra. The potential application as thermoelectric material of the title compounds is discussed in the context of their electronic structure calculated by the density-functional method, as well as their close structural relationship with the layered alkaline-earth arsenides and antimonides EZn2Pn2 and ECd2Pn2 (E = Ca, Sr, Ba; CaAl2Si2 type structure). AW.03 From Paper Tape Input to Forensic Crystallography. Forty years of Small Molecule Computing A.L. Spek Utrecht University, Utrecht, Netherlands The lecture will largely follow the historical development of the program package PLATON. This program is probably best known and most used as part of the IUCr CheckCIF facility. Recently, it was instrumental in uncovering a large scale fraud with 'invented' structures that were published in Acta Cryst. E. PLATON has a history of 30 years. Development started as a second attempt to automate various procedures as part of our National Single Crystal Structure Service. The earlier attempt was based on software written in the ALGOL60 language that became obsolete with the introduction of a Control Data/Fortran mainframe in Utrecht. PLATON started off as a geometry calculations and molecular graphics program for local use. Over time, algorithms for the detection of solvent accessible voids in a structure and missed symmetry were added. This attracted the attention of Syd Hall, at that time Section Editor of Acta Cryst. C. Both tests were included as part of the development of an early version of a project to automate structure validation of structures to be published in Acta Cryst. C. Over time, more than 400 various tests have been implemented including warnings for missed twinning, problems with the reflection data or difference density map etc. One of the most powerful tests turned out to be the Hirshfeld Rigid Bond Test. PLATON also includes a large number of tools such as space group determination from systematic absences, the SQUEEZE algorithm to handle disordered solvent in the refinement and a version of the 'Charge Flipping' algorithm as an alternative tool to solve crystal structures. PLATON, in its native LINUX incarnation, also includes a tool named 'SYSTEM S'. The latter aims at solving, completing, refining and validation a crystal structure automatically. For that, it makes use of excellent external software such as SHELXL, SHELXS, SIR and DIRDIF. 01.04.1 Using Raman Microscopy to Map Reaction Pathways in Crystals in Real Time Paul Carey Case Western Reserve University, Cleveland, United States By combining the methods of Raman microscopy and Raman difference spectroscopy populations of reaction intermediates can be tracked in single crystals. Reactions are started using soak-in conditions at room temperature and the Raman data as a function of time provide both structural and kinetic data. These can be used to inform the crystallographer of the optimal conditions for flash freezing and trapping target intermediates for X-ray data collection. At the same time the X-ray structures provide reference points that are invaluable for interpreting the Raman spectra. Two examples will be given: the complex branched reaction pathway traversed by clinical drugs reacting with beta-lactamase drugs in antibiotic therapy (1); following the first step in RNA synthesis in the active site of an RNA polymerase (2). Advantages and limitations of the approach will be given. I am indebted to my collaborators listed in references (1) and (2). (1) P. S. Padayatti, A. Sheri, M. A. Totir, M. S. Helfand, M. P. Carey, V. E. Anderson, P. R. Carey, C. R. Bethel, R. A. Bonomo, J. D. Buynak and F. van den Akker. J. Amer. Chem. Soc. 128, 13235-13242 (2006). (2) M. L. Gleghorn, Y. Chen, E. K. Davydova, R. Basu, L. B. Rothman-Denes, P. R. Carey and K. S. Murakami, submitted for publication (2010). 01.04.2 Structural evidence for the staging of active site configurations that control diiron monooxygenase reactivity Brian Fox, Lucas Bailey, Justin Acheson University of Wisconsin, Madison, WI, United States Diiron monooxygenases are multi-protein complexes that catalyze the oxidation of hydrocarbons including methane, butane, toluene, ethene, and other compounds. Because of this powerful reactivity, microbes readily use these chemicals as the sole source of carbon and energy. Toluene-4 monooxygenase is a representative example from this enzyme family. It is composed of an electron transfer chain between an NADH oxidoreductase and a Riesketype [2Fe-2S] protein, a large hydroxylase protein containing the diiron center active site, and a small protein called the effector protein. Numerous studies have shown the effector protein is required for catalysis, but until recently, the structural bases of these observations were not known. High-resolution crystal structures of the stoichiometric complex of effector protein and hydroxylase have begun to give new insight into the role of protein-protein interaction in controlling the active site configuration, the reactions with substrates, and the formation of intermediates along the reaction pathway. Key findings from this work will be presented. This work supported by NSF MCB 0316232 and MCB 0843239. 01.04.3 Structural and mechanistic basis for hypoxia inducible factor hydroxylation by the oxygen sensing prolyl hydroxylases Rasheduzzaman Chowdhury, Michael A McDonough, Christopher J Schofield Chemistry Research Laboratory, University of Oxford, Oxford, OX1 3TA, United Kingdom Oxygen dependent prolyl-4-hydroxylation of the -subunit of hypoxia inducible transcription factor (HIF ) plays an essential role in the hypoxic response. Hydroxylation of prolines in the N- or C-terminal oxygen dependent degradation domains (NODD or CODD) increases the affinity of HIF to the von Hippel-Lindau protein (pVHL) by ~1000 fold so signaling for HIF degradation. With limiting oxygen, HIF hydroxylation slows, it dimerizes with HIF and activates the transcription of a gene array. Prolyl-4-hydroxylation also stabilizes the triple helix structure of collagen, the most abundant human protein. Both the collagen and the HIF prolyl hydroxylases (PHDs) are Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases. We determined crystal structures of PHD2 in complex with CODD. With biochemical analyses, the results demonstrate that catalysis involves a mobile region of PHD2 that encloses the hydroxylation site and stabilizes the PHD2.Fe(II).2OG complex. When bound to PHD2 the non-hydroxylated proline-residue adopts the C4-endo conformation. Evidence is provided that 4R-hydroxylation enables a stereoelectronic effect that changes the proline conformation to the C4-exo state, as observed when hydroxylated HIF is bound to pVHL and in collagen. The results rationalize selective recognition of NODD/CODD by three PHD isoforms and the effects of clinically observed mutations on PHD2 catalysis; they will be of use in the design of new types of PHD inhibitor aimed at treating anemia and ischemic disease. h h h h h h 01.04.4 Consequences of Exposure of Ferric Myoglobin Nitrite Crystals to Synchrotron X-ray Radiation George Richter Addo , Jun Yi , Allen Orville 1 1 1 2 2 University of Oklahoma, Norman, OK, United States, NSLS-BNL, Upton, NY, United States The nitrite anion is ubiquitous in the environment and is an important component of the global nitrogen cycle. The conversion of nitrite to nitric oxide (NO) has normally been associated primarily with the nitrite reductase (NiR) enzymes in the bacterial denitrification pathway. Recently, there has been a resurgence of interest in nitrite reduction by heme enzymes, due in a large part to literature reports that the mammalian proteins myoglobin (Mb) and hemoglobin (Hb) covert nitrite to NO in an apparent NiR reaction. We reported the X-ray crystal structure of the nitrite derivative of ferric horse heart Mb at 1.2 Å resolution and showed that the nitrite ligand was bound to the heme Fe via the nitrito O-binding mode. We extended the work to the nitrite derivative of ferric human Hb, and showed that the O-binding mode of nitrite was also extant in this Hb(ONO) complex (crystal structure at 1.8 Å resolution). Importantly, however, nitrite reduction by Mb (and Hb) occurs via the ferrous derivative and not the ferric derivative. Despite several attempts using different preparation routes, we were unable to prepare crystals of the ferrous Mb-nitrite complex derivative for crystal structural determination. We were intrigued by the new capabilities of the X26-c beamline at the National Synchrotron Light Source that allowed for correlated X-ray diffraction and UV-vis spectroscopy experiments. We thus pursued the goal of attempting to generate the ferrous Mb-nitrite complex from X-ray induced photoreduction of the ferric precursor. The results of this work will be presented and discussed. 01.04.5 xrs`kknfq`oghb? `mc? rhmfkd? bqxrs`k? rodbsq`k? `m`kxrhr? ne? sgd? odqnwhc`rd? edqqxk hmsdqldch`sd Tzanko Doukov , Yergalem Meharenna , Huiying Li , S. Michael Soltis , Thomas Poulos 1 1 2 2 1 2 m msm po m lz |{wv m s mr to ~mn rmto m m l rq p~mn t lz m mtm s m s mr mlz yxwv s m lz wpo m s mr l}l mlz tlz mzt u tm mtms rq pomn mlk ml k wm s mr l}l ~m n m q l}lz ji SSRL, Menlo Park, CA, United States, UCI, Irvine, CA, United States 2 The ferryl (Fe(IV)O) intermediate is important in many heme enzymes and thus the precise nature of the Fe(IV)-O bond is critical in understanding enzymatic mechanisms. The 1.40 Å crystal structure of cytochrome c peroxidase Compound I has been solved as a function of xray dose while monitoring the visible spectrum at BL9-2 at SSRL. Data were collected with an open air Helium cryostat at 65 K, the lowest safe temperature before nitrogen solidifies. Ninety six crystals were mounted by the SAM robot and were screened or data collected locally or remotely from Irvine, CA. Data from 25 crystals were collected, from which the best 19 were used in the experiment. For each crystal, data collections were carried out in 15 o separate runs. Run 1 consisted of 5 of data, representing the first 0.035 MGy of x-ray o exposure. Then the same 5 scanning angle were recollected 12 more times giving runs 2 o through 13 with increased x-ray dose. In run 14 a full 120 of data were collected in order to o fully reduce the crystal followed by run 15 which again repeated the same 5 representing the highest x-ray dose. The same 15-run data collection protocol was adopted for similarly sized crystals and the scanning angles were chosen to optimize the completeness of the data. o Each composite data set was assembled by merging 5 of data with identical run numbers from 19 crystals. Making the composite dataset in such manner ensures measuring the same reflections in every separate run and allows their monitoring as a function of time/dose. In addition the Fo-Fo difference maps are less noisy and more reliable. A total of 15 structures at 1.40 Å resolution were refined providing a picture of the structural changes associated with increasing x-ray dose. ? ? ‹? ‹‒ ‹ ‒ ‒› › ? ? „? « ? ›? ‒· ? ›? ›? ‒·? ‒‒ ? ? • ? ? ·‒ • ·‒ › • fi › ? · ? ? ? ›? ‒·? ? ? ‒„› ·‒ › ? · ?› ? ? ä? ‹ ? ‒„? ›? ‒·? ? ? ›?‚ ·‒ • › ? ‹? • ? • › ‒fi ? ? ä? ‹ ? fi ?fi ‒· › „ 01.05.1 Conformational Flexibility and Catalysis in a Phosphohexomutase: Crystallography, Kinetics, and NMR Lesa Beamer , Steven Van Doren , Cristina Furdui 1 1 1 2 2 Univeristy of Missouri, Columbia, MO, United States, Sciences, Winston-Salem, NC, United States Wake Forest University Health A critical role for protein dynamics and conformational flexibility in catalysis has been long suspected and often proposed, but difficult to demonstrate directly. However, powerful new methodologies, particularly NMR, can now greatly enhance the static “snapshots” typically provided by crystal structures. One system currently being studied by the complementary techniques of X-ray crystallography and NMR is the enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM), from the human pathogen P. aeruginosa. PMM/PGM has been well characterized in our laboratory by X-ray crystallography, kinetic studies, and site-directed mutagenesis. Ten crystal structures of the enzyme and various enzyme-ligand complexes have shown that the enzyme changes from an open to closed conformational state upon ligand binding. Recent studies characterizing the effects of mutants in a hinge region at a domain-domain interface show that increased conformational freedom is unfavorable for catalysis due to entropic factors. These results highlight the importance of conformational flexibility of the polypeptide backbone in catalytic efficiency. To further explore the role of protein dynamics and conformational change in enzyme mechanism, PMM/PGM is presently being investigated by NMR. A major accomplishment in this effort is successful completion of the backbone assignments of this 50 kD protein using triple resonance methods. Additional characterization of apo and ligandbound protein dynamics is underway via relaxation dispersion methods. A synthesis of the insights gained from this multi-disciplinary approach will be presented, with an emphasis on understanding the multi-step catalytic reaction of PMM/PGM. 01.05.2 A master switching motif with multiple effects on differential conformational stability 2+ and Mg -assisted catalysis suggests a general mechanism for transducing enzymes. Violetta Weinreb, Li Li, Brian Kuhlman, Charles Carter University of North Carolina at Chapel Hill, Chapel Hill, NC, United States B. stearothermophilus Tryptophanyl-tRNA synthetase (TrpRS) uses a sequence of different conformational states to catalyze tryptophan activation. Like other Class I aminoacyl-tRNA 2+ synthetases it also requires one Mg ion for optimal catalysis. The metal favors catalysis by 2+ 6.5 kcal/mol. However, catalytic assist by Mg occurs if, and only if, it interacts with the protein. We are trying to identify the metal-protein interactions that produce this catalytic 2+ effect. Physical interactions between Mg and TrpRS are mediated indirectly via active-site lysines K111, K192 and K195. Mutations of these lysines showed that they all stabilize the 2+ transition state. However, their interactions with the Mg have the opposite effects and significantly reduce their catalytic effects. This suggests that the balance of catalytically 2+ productive interactions between TrpRS and the Mg ion must arise from outside the active site. We identified a set of core residues we call the D1 switch because they move during the catalytic conformational transition. The D1 switch lies at the corner of the N-terminal ß-α -ß crossover opposite the HIGH sequence and KMSKS loop, forming links to I4 in the N-terminal ß strand and I140 in the Trp binding site from three of its side chains F26, Y33 and F37. These residues are prime candidates for mediating synergistic, catalytically-critical coupling to 2+ the Mg ion at the active site. The Rosetta Design program suggested mutations I4V, F26L, Y33F and F37I to ‘‘hyperstabilize’’ the PreTS along the structural reaction profile. Using 2+ 2+ multimutant thermodynamic cycles together with substitution of Mn for Mg and [ATP]dependent Michaelis-Menten kinetics we used these mutants to demonstrate long-range 2+ 2+ synergistic coupling between the D1 switch and the Mg ion. Thus, protein-Mg interactions within the active site oppose catalysis, while synergistic long-range interactions to the metal 2+ drive catalysis indirectly, by changing an inactive Mg coordination into one that can stabilize 2+ the transition state. In this way transition-state stabilization by Mg occurs if, and only if, conformational changes reposition it. This description may apply to a large number of NTPase 2+ enzymes that transduce chemical free energy by using control of Mg coordination to link catalyzed hydrolysis of their purine triphosphate substrates to conformational changes for cellular work and signaling. Supported by NIGMS 78227. 01.05.3 Structure of biosynthesis HydA ∆EFG , a key intermediate in [FeFe]-hydrogenase H-cluster David Mulder, Eric Boyd, Ranjana Sarma, Rachel Lange, James Endrizzi, Joan Broderick, John Peters Montana State University, Bozeman, MT, United States The [FeFe]-hydrogenase (HydA) contains a complex FeS cluster active site, termed the Hcluster, which exists as a [4Fe-4S] subcluster linked by a cysteine thiolate to a modified 2Fe subcluster with unique non protein ligands. Although the 2Fe subcluster is thought to be synthesized by the activities of the hydrogenase maturation enzymes HydE, HydF, and HydG, the precise mechanism by which it is assembled remains unclear. Here, we report the Δ EFG structure of HydA (HydA expressed in a genetic background devoid of the active site Hcluster biosynthetic genes hydE, hydF and hydG) determined to 1.97 Å resolution. The structure reveals the presence of a [4Fe-4S] cluster and an open channel for the insertion of the 2Fe subcluster. This indicates that H-cluster synthesis occurs in a stepwise manner, first with synthesis and insertion of the [4Fe–4S] subcluster by generalized host-cell machinery and then with synthesis and insertion of the 2Fe subcluster by specialized HydE, HydF, and HydG maturation machinery. 2Fe subcluster insertion presumably occurs via a cationically charged channel that collapses upon insertion through conformational changes in two conserved loop regions. Loop region conservation in organisms containing the 2Fe subcluster biosynthetic genes coupled with evolutionary analysis of HydA together indicate a bacterial origin for HydA that postdates the emergence of eukarya. By establishing parallels to FeMo-cofactor biosynthesis in nitrogenase maturation, general unifying themes from complex FeS cluster biosynthesis are revealed. 01.05.4 Mitochondrial and Cytosolic Phenylalanyl-tRNA Synthetases Catalyze Incorporation of ROS-damaged Amino Acid into Eukaryotic Proteins. Liron Klipcan , Nina Moor , Naama Kessler , Mark Safro 1 1 2 1 1 Weizmann Institute of Science, Rehovot, Israel, Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russian Federation The accumulation of proteins damaged by reactive oxygen species (ROS), conventionally regarded as having pathological potentials, is associated with age-related diseases such as Alzheimer's, atherosclerosis, and cataractogenesis. Exposure of the aromatic amino acid phenylalanine to ROS-generating systems produces multiple isomers of tyrosine: m-tyrosine (m-Tyr), o-tyrosine (o-Tyr), and the standard p-tyrosine (Tyr). Previously it was demonstrated that exogenously supplied, oxidized amino acids could be incorporated into bacterial and eukaryotic proteins. It is, therefore, likely that in many cases, in vivo-damaged amino acids are available for de novo synthesis of proteins. Although the involvement of aminoacyl-tRNA synthetases in this process has been hypothesized, the specific pathway by which ROSdamaged amino acids are incorporated into proteins remains unclear. We provide an evidence that mitochondrial and cytoplasmic phenylalanyl-tRNA synthetases in tandem catalyze direct attachment of m-Tyr to tRNA-Phe, thereby opening the way for delivery of the misacylated tRNA to the ribosome and incorporation of ROS-damaged amino acid into eukaryotic proteins. Crystal structures of human mitochondrial, human cytoplasmic and bacterial PheRSs complexed with m-Tyr and other ROS-damaged and medically important amino acids will be discussed. 2 01.05.5 Applying structure-based drug design to the moonlighting enzyme dihydrolipoamide dehydrogenase to target detrimental oxidative stress Donald Berkholz, Rachael Vaubel, Christina Andrist, Grazia Isaya, James Thompson Mayo Clinic College of Medicine, Rochester, MN, United States Numerous mitochondria-related diseases including Friedreich ataxia cause neurodegeneration and lack effective treatments. Evidence suggests changes in oligomerization of the abundant mitochondrial enzyme dihydrolipoamide dehydrogenase (DLD) result in a significant yet unappreciated source of disease symptoms including mitochondrial iron imbalance and oxidative stress. Dimeric DLD exhibits dehydrogenase activity that is an essential component of large complexes involved in energy metabolism. Critically, it can dissociate into monomers with proteolytic and diaphorase activities not seen in the physiological dimer that degrade vital mitochondrial proteins in vitro and increase oxidative stress in vitro and in vivo. These detrimental activities oppose DLD’s primary activity, with changes in its oligomeric state regulating whether DLD promotes oxidative metabolism or oxidative damage. We hypothesized that stabilizing dimeric DLD would decrease proteolytic and diaphorase activities of monomeric DLD. To test this hypothesis, we are using structure-based drug design to create high-affinity compounds from fragment-sized molecules with a combination of crystallographic and computational approaches. In vitro screening of a chemically diverse fragment library discovered 16 chemically similar compounds that bound to and stabilized DLD – 4 of which bound with micromolar affinity – amounting to a 4% hit rate. Analysis of the crystallographic dimer suggested favorable binding pockets within the dimer interface. Intriguingly, docking studies of substrate-bound enzyme suggest that all of these compounds bind to the same site within the interface pocket, which is being confirmed crystallographically. This provides the basis for further drug design to target mitochondrial diseases involving iron imbalance or oxidative stress. 01.05.6 Structure-guided therapeutic targeting of the essential protein farnesyltransferase from the AIDS-associated pathogen Cryptococcus neoformans Michael A. Hast , Connie B. Nichols , J. Andrew Alspaugh , Lorena S. Beese 1 2 1 2 2 1 Duke University Medical Center, Department of Biochemistry, Durham, NC, United States, Duke University Medical Center, Department of Medicine, Durham, NC, United States Cryptococcus neoformans is a human fungal pathogen that causes life-threatening respiratory and neurological infections in immunocompromised individuals, including transplant recipients and HIV patients. An ortholog of protein farnesyltransferase (FTase) that is essential for viability has been identified as a potential drug target in C. neoformans. FTase catalyzes a critical post-translational lipid modification of over 60 important signal transduction proteins in the eukaryotic cell. We present a series of crystal structures of the essential C.neoformans FTase (CnFTase) in complex with substrates and inhibitors and identify dominant structural determinants of ligand selection. We show that previously identified FTase inhibitors exhibit fungicidal activity against C. neoformans. Furthermore, we show that the most potent fungicidal FTI causes mislocalization of Ras1 in the organism, suggesting inhibition of prenylation of this important signaling molecule. Our combined structural and functional studies provide a foundation for the design of a new generation of antifungal FTase inhibitors (FTIs). This work was supported by NIH grants GM052382 to L.S.B. and AI050128 to J.A.A. 01.05.7 Error-free DNA replication opposite an oxidative DNA lesion by Human Y family polymerase iota Kevin Kirouac, Hong Ling University of Western Ontario, London, On, Canada Human Y family DNA polymerase iota (polι ) is one of a few DNA polymerases that incorporate the correct cytosine nucleotide with high specificity opposite 8-oxo-guanine; the most abundant oxidative DNA lesion in cells. The structural basis of preferential cytosine incorporation opposite 8-oxo-guanine by a eukaryotic DNA polymerase is currently unknown. We present four crystal structures of polι in complex with DNA containing an 8-oxo-guanine lesion, paired with correct dCTP or incorrect dATP, dGTP, or dTTP nucleotides. A narrow active site restricts the 8-oxo-guanine in a syn conformation, which favours incoming dCTP due to hydrogen bonding potential, base stacking interaction and optimal conformer orientation. We demonstrate the importance of the finger domain residue Gln59 in template 8-oxo-guanine orientation and polι enzymatic activity using site directed mutagenesis. Lastly, we propose a model for how polι may protect cells against oxidative DNA damaged by reversing replication induced mutagenesis via a Base Excision Repair pathway. 02.04.1 Crystal Structures Viewed as Dynamical Systems Carroll Johnson , Michael Burnett , Bryan Chakoimakos 1 1 1 2 Chemical Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Bldg 4500S, 2 Oak Ridge, Tennessee 37981-6197, United States, Neutron Scattering Sciences Division, Oak Ridge National Laboratory P.O. Box 2008, Bldg 7962, Oak Ridge, Tennessee 378316393, United States The excellent 800 page book [1] describes ergodic theory, topological dynamics, and theory of smooth dynamical systems in great theoretical detail, but the applications are also mainly theoretical. However, most of what they cover is directly applicable to crystal structures, but that fact is seldom mentioned. Our massive databases of space group symmetry, unit cell parameters, chemical contents, atom ID, positional anisotropic thermal parameters may provide reality tests for some of that theory. All we need to do is provide a few good examples and in the process we might learn things that could expand our own horizons. Below are some areas of crystallography where these theories apply. Flows, vector fields (of vector valued functions) and ordinary differential equations are basic operations in dynamical systems theory. In crystallography, these translate to a thermal motion flow [2] along a sequence of bonded thermal ellipsoids in an ORTEP drawing, and is determined by the Radon-Nikodym derivative ratio (a cocycle [3]) for each ellipsoid pair. The flow is from smaller to larger equiprobability ellipsoids to form a unidirectional network. The calculations are made directly from the atoms' thermal and positional parameters, perhaps using a future version of the ORTEP program. Also, dynamical flow theory gives the possibility of calculating rotation torsions about bonds, an unsolved problem in crystallography, using a cylindrical filter flow network derived from the above network. [1] A. Katok, B. Hasselblatt (1995) Introduction to Modern Theory of Dynamical Systems, Cambridge. [2] T. Izer (1991) Theories of Intramolecular Vibrational Energy Transfer; Physics Reports 199, no.3, 73-146, North-Holland. [3] V. Kaimanovich, K. Schmidt (2001) Ergodicity of Cocycles. 1: General Theory; unpublished preprintThis research is supported in part by UT Battelle. LLC under Contract No. DE-AC05-00OR22725 for the U.S. Department of Energy, Office of Science. 02.04.2 Substituent and Solvation Effects on N-H---O Hydrogen Bonds. Ronald See, Justin Hileman Indiana University of PA, Indiana, PA, United States A database and computational study was done to determine the factors which effect hydrogen bond strength in N-H---O hydrogen bonds. The importance of hydrogen bonding in general has been well established, but N-H---O hydrogen bonds remain an understudied system. These interactions dictate the form of the a-helix and the b-sheet, are crucial for base pairing in DNA, and play a crucial role in the differential binding of O2 in the active site of myoglobin. However, studies that detail the factors causing variation in the energy of N-H---O hydrogen bonds are not available. Using the imidazole---OCX2 (where X = CH3, NH2, H, F and others) system, N-H---O hydrogen bonds were studied computationally (B3LYP/6-31G**) to quantify the effects of substituents and solvation. These hydrogen bonds varied in gas-phase energies from 34-17 kJ/mol, and solvents decreased the attractive interaction based on a function of the dielectric constant of the solvent. Database studies were also carried out on N-H---O hydrogen bonding species, focusing on the effect of substituent groups. The results will be interpreted in the context of I.D. Brown's Bond Valence Model of hydrogen bonds. 02.04.3 A List of Organic Kryptoracemates. Carolyn Brock , László Fábián 1 1 2 2 University of Kentucky, Lexington, KY, United States, Pfizer Institute for Pharmaceutical Materials Science, Cambridge Crystallographic Data Centre, Cambridge, United Kingdom A list of 181 organic kryptoracemates has been compiled.** This class of crystallographic oddities is made up of racemic compounds (i.e., pairs of resolvable enantiomers) that happen to crystallize in Sohnke space groups (i.e., groups that include only proper symmetry operations); the two enantiomers are crystallographically independent. Most (151) of the 181 structures could have crystallized as ordered structures in non-Sohnke groups; the remaining 30 do not fully meet this criterion but would have been classified as kryptoracemates by previous authors. Examples were found and checked with the aid of available software for searching the Cambridge Structural Database, for generating and comparing InChI (IUPAC International Chemical Identifier) strings, and for validating crystal structures. The pairs of enantiomers in the true kryptoracemates usually have very similar conformations; often the match is nearperfect. There is a pseudosymmetric relationship of the enantiomers in about 60% of the kryptoracemate structures but the deviations from inversion or glide symmetry are usually quite easy to spot. Kryptoracemates were found to account for 0.1% of all organic structures containing either a racemic compound, a meso molecule, or some other achiral molecule. The centroid of a pair of enantiomers is more likely (99.9% vs. 99% probability) to be located on an inversion center than is the centroid of a potentially centrosymmetric molecule, probably because the overall shape and size of the van der Waals surface of an enantiomer pair is more variable than is the corresponding surface of a single molecule. *Current address for L. Fábián: Department of Chemistry, University College Cork, Cork, Ireland’ * Fábián , L & Brock, C. P. (2010). Acta Cryst. B66, 94–103 02.04.5 The 3.4 Å structure of the 122 kDa Mtr4 protein: refinement and revelation of a novel arch domain Sean Johnson , Ryan Jackson , Bradley Hintze , Alejandra Klauer , Ambro van Hoof 1 2 1 1 1 2 2 Utah State University, Logan, Utah, United States, University of Texas Health Science Center-Houston, Houston, Texas, United States The RNA helicase Mtr4 performs a critical role in RNA processing and degradation as an activator of the nuclear exosome. The molecular basis for this vital function is not understood and detailed analysis is significantly limited by the lack of structural data. Here we present a 3.4 Å crystal structure of the 122 kDa Mtr4 protein from S. cerevisiae. The structure reveals a novel arch-like domain that is specific to Mtr4 and Ski2 (the cytosolic homolog of Mtr4). In vivo and in vitro analyses demonstrate that the Mtr4 arch domain is required for proper 5.8 S rRNA processing, and suggest that the arch functions independently of canonical helicase activity. Additionally, extensive conservation along the face of the putative RNA exit site highlights a potential interface with the exosome. These studies provide a molecular framework for understanding fundamental aspects of helicase function in exosome activation, and more broadly define the molecular architecture of Ski2-like helicases. Because of the limited resolution of the Mtr4 diffraction data, extensive use of secondary structure restraints was required to complete refinement. To aid in definition of these restraints, interactive python-based tools ("ResDe" - Restraint Definer) were developed to rapidly define and edit distance restraints using the PyMol graphical interface. These tools alleviate the daunting task of manually editing an extensive text file when a large number of restraints are needed. We believe this tool will be of general interest to the crystallographic community. 02.04.6 Nanovolume Optimization of Protein Crystal Growth Using the Microcapillary Protein Crystallization System Cory Gerdts , Glenn Stahl , Peter Nollert , Alberto Napuli , Wes Van Voorhis , Peter 4,5 1,4 1,4 Myler , Bart Staker , Lance Stewart Emerald BioStructures, Inc., Bainbridge Island, WA, United States, Emerald BioSystems, 3 Inc., Bainbridge Island, WA, United States, University of Washington, Seattle, WA, United 4 States, Seattle Structural Genomics Center for Infectious Disease, Seattle, WA, United 5 States, Seattle Biomedical Research Institute, Seattle, WA, United States The Microcapillary Protein Crystallization System (MPCS) is a microfluidic, plug-based crystallization technology that generates diffraction-ready protein crystals in nanoliter volumes. Using proteins from the Seattle Structural Genomics Center for Infectious Disease (SSGCID), we sought to determine the rate of crystallization success using MPCS nanovolume microbatch methods, relative to the more traditional microliter volume vapor diffusion methods. SSGCID proteins underwent random sparse matrix crystallization screening by vapor diffusion methods and crystallization hit conditions were applied in a chemical gradient optimization using the MPCS. 120 different protein/precipitant combinations were optimized using the MPCS technology, with a 75% crystallization success rate. Our results also demonstrate recapitulated crystallization success for 93% of the 29 proteins tested. Moreover, the resulting crystals produced high quality X-ray diffraction data leading to 6 novel protein structure determinations from crystals harvested directly from the MPCS CrystalCards. These results suggest that the MPCS can be used to achieve chemical gradient based optimization of vapor diffusion crystallization hits with high probability of success using ~50-fold less protein than would be used in vapor diffusion based optimization experiments. The MPCS technology has now been encapsulated within a new automated instrument called the MPCS Plug Maker, winner of the 2010 Lab Automation New Product Award. 1 2 1,2 1 1,2 3,4 3,4 This work was supported in part by the NIGMS-NCRR co-sponsored PSI-2 Specialized Center Grant U54 GM074961 and by federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN272200700055C. 02.04.7 Solvent and Temperature effects upon the resulting Crystal Structure James Fettinger Univ. of California, Davis, CA, United States During the past few decades low temperature data collections have become the preferred method of choice for many desirable reasons; however there are structures that exhibit undesirable changes when the data collection temperature is set too low. These effects are frequently due to the temperature itself and/or the solvent of choice being incorporated into the lattice itself resulting in an unexpectedly large Z' and/or twinning. A series of structures will be described that exhibit these effects. 04.01.1 Combined X-Ray Scattering (SAXS) and Crystallography to Accurately Characterize Dynamic DNA Repair Complexes in Solution Robert Rambo , Greg Hura , Susan Tsutakawa , Michal Hammel , John Tainer 1 2 2 2 2 2 1,2 The Scripps Research Institute, La Jolla, CA, United States, aLawrence Berkeley National Laboratory, Berkeley, CA, United States Protein, DNA, and RNA shapes, as well as their detailed structural chemistry, encode key information about connections needed to define the “interactomes” critical to biological outcomes in cell biology. We are developing SAXS combined with crystallography as a premiere tool for defining macromolecular conformations and connections at the proteomic 1-4 scale . Crystallography supplies unparalleled structural detail for mechanistic analyses; however, it is restricted to describing conformations of macromolecules within crystal lattices. In principle, SAXS can provide reliable complementary data on small and large macromolecules. Our results on dynamic DNA repair protein and DNA complexes show that SAXS has great potential to provide accurate shapes, conformations, and assembly states in 5-7 solution and inform biological functions in fundamental ways . 04.01.2 A Conformational Switch in the Scaffolding Protein NHERF1 Controls Autoinhibition and Complex Formation Zimei Bu 1 1,2 2 City College of New York, New York, NY, United States, Fox Chase Cancer Center, Philadelphia, PA, United States Scaffolding proteins are molecular switches that control diverse signaling events. A particularly important example is the scaffolding protein NHERF1, which assembles and regulates the localization and intracellular trafficking of a number of important membrane proteins. At its N-terminus, NHERF1 begins with two modular protein-protein interaction domains-PDZ1 and PDZ2-and ends with a C-terminal (CT) domain. The CT domain binds to ezrin, which, in turn, interacts with cytosekeletal actin. Previously we have shown that ezrin binding to NHERF1 increases the binding capabilities of both PDZ domains. Our solution small angle neutron scattering and NMR experiments reveal the autoregulated intramolecular domain-domain interactions, as well as much longer range conformational changes in NHERF1 upon activation by ezrin binding. The results provide a structural explanation, at both mesoscopic scales and atomic resolution, of the allosteric control of NHERF1 by ezrin as it assembles protein complexes. We propose that this long-range allosteric regulation of NHERF1 by ezrin enables the membrane-cytoskeleton to assemble protein complexes that control cross-talk and regulate the strength and duration of signaling. 04.01.3 Crystallography without Crystals: Breaking the Crystallization Paradigm Dilano Saldin , Hin-Cheuck Poon , Henry Chapman , John Spence 1 1 1 2 3 2 University of Wisconsin-Milwaukee, Milwaukee, WI, United States, Center for Free Electron 3 Laser Science, Hamburg, Germany, Arizona State University, Tempe, AZ, United States The extreme brilliance of the x-ray free electron laser (XFEL) has provoked speculation about 1 the possibility of protein structure determination from single molecules . However, even in the proposed diffract and destroy experiment, the number of scattered photons per detector pixel 2 from a typical protein is estimated to be a number much less than unity . Methods have been 2,3,4 proposed for structure determination even under such circumstances . We propose a method for structure determination of uncrystallized proteins, by directly recovering the diffraction pattern of a single molecule from that of multiple randomly positioned and randomly oriented ones through their correlated scattering, and subsequent iterative phasing of this 5 diffraction pattern to recover the molecular electron density . We suggest a possible 6 application to structure determinations of membrane proteins in situ . Possible applications for the proposed single-molecule XFEL experiments will also be discussed, as well as ideas for 3,7 extracting time-resolved information . References [1] R. Neutze, et al., Nature 406, 752 (2000). [2] R. Fung et al., Nature Physics 5, 64 (2009). [3] D. K. Saldin et al., J. Phys: Condens. Matter 21, 134014 (2009). [4] N.-T. D. Loh and V. Elser, Phys. Rev. E 80, 026705 (2009). [5] D. K. Saldin et al., New J. Phys., in press [6] D. K. Saldin et al., submitted to Phys. Rev. B. [7] J. C. H. Spence et al., Abstracts of the Meeting of the Microscopical Society of America (2010). 04.01.4 Stucture of a bacterial ribonuclease P holoenzyme in complex with tRNA Nicholas Reiter , Amy Osterman , Alfredo Torres-Larios , Kerren Swinger , Tao Pan , 1 Alfonso Mondragón 1 1 1 1 1 1,2 Northwestern, Evanston, IL, United States, University of Chicago, Chicago, IL, United States 2 Maturation of transfer RNA (tRNA) requires processing at both its 3’ and 5’ ends. Ribonuclease (RNase) P is the ribozyme responsible for 5’ -end tRNA processing and is found in all three domains of life. We report the 3.85 Å resolution crystal structure of Thermotoga Phe maritima RNase P holoenzyme in complex with tRNA . The entire 154 kDa complex, consisting of a large catalytic RNA (P RNA), a small protein cofactor, and mature tRNA, is revealed in the structure. The structure shows the presence of tertiary RNA-RNA and RNAprotein interactions that mediate substrate recognition and catalysis. Specific contacts at the RNase P/tRNA interface explain the structural basis for substrate recognition. The structure identifies the active site location and shows that it is composed of phosphate backbone moieties, a universally conserved nucleotide, and metal ions. Additional experiments position the leader sequence and show the interactions with the protein and the RNA component. Overall, the structure is consistent with existing biochemical and biophysical data. The active site structure and conserved RNase P/tRNA contacts suggest a universal mechanism of catalysis by RNase P. 04.01.5 Structural Studies of the Complement-Like Innate Immune Response in anopheles gambiae. presented. Richard Baxter1, Stefanie Steinert2, Yoga Chelliah1, Gloria Volohonsky2, Elena Levashina2, Johann Deisenhofer1 University of Texas Southwestern Medical Center, Dallas, Texas, United States Minor Outlying Islands, 2Université de Strasbourg, Strasbourg, France Structural studies of the complement-like innate immune response in Anopheles gambiaeMalaria, the world's most devastating parasitic disease, is cause by apicomplexan parasites of the genus Plasmodium. The major vector for malaria in Africa is the mosquito Anopheles gambiae. Significant progress has been made in the past decade in demonstrating that A. gambiae possesses a robust innate immune response to infection by Plasmodium parasites that may be a potential source of novel vector control strategies. The complement-like protein thioestercontaining protein 1 (TEP1) labels P. berghei ookinetes for lytic destruction in the basal lamina of the midgut epithelium. The three dimensional structure of TEP1 is homologous to human complement factor C3. Further studies however, demonstrate that the mechanism of TEP1 activation is distinct from vertebrate complement factors, involving a pair of leucine-rich repeat proteins, LRIM1 and APL1. Recent results of crystallographic and solution x-ray scattering studies regarding LRIM1 and APL1 shall be 1 04.01.6 Morphological and Structural Changes of Lignocellulosic Biomass during Dilute Acid Pretreatment using SANS Sai Venkatesh Pingali , Volker Urban , William Heller , Hugh O'Neill , Marcus Foston , Dean 1 2 1 Myles , Arthur Ragauskas , Barbara Evans 1 1 1 1 1 2 Oak Ridge National Laboratory, Oak Ridge, TN, United States, Technology, Atlanta, GA, United States ? ¦› ›› ‹? ¡ ‒ 2 Georgia Institute of \·\ ‹‒›r £‹¡¦ mr ¡? £ ? ¡? ‒ ‹
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¡ ¡‹ \ t‹ ⁄ ‒¢› ‹· ›\ „ « › ¡¦ ¦ £\ ‒‹ ‹? ?¡ ⁄ ¦ ›¡ ‹· \ ?‒\ ¡¡ ·‒ › ?¢ ä «‹ \£ ‒¡ ¡? · 04.01.7 Structure of the Natural Killer (NK) cell activating receptor NKp30 and identification of its ligand binding site by utilization of a NKp30 blocking antibody and specific NKp30 peptides which map the protein surface M. Gordon Joyce , Marco Colonna , Peter D. Sun 1 2 1 2 1 NIAID/NIH, Rockville, MD, United States, Washington University School of Medicine, St. Louis, MO, United States NK cells are a group of innate immune cells which are critical for the detection and destruction of virally infected or cancerous cells on a daily basis in humans. These lymphocytes are activated without any prior immune priming and elicit their effects using a group of activating molecules (NKp30, NKp46 and NKp44). NKp30 is known to be the dominant receptor responsible for the destruction of a number of tumor cell types. The precise mechanism of how NKp30 interacts with cancerous cells initiating NK cell killing is an intriguing question and to date, a number of NKp30 interacting molecules have been proposed. These molecules are quite diverse ranging from Heparan sulphate to a Plasmodium falciparum molecule and most recently a B7 Immunoglobulin-like homolog protein. However, the precise mechanism of how NKp30 recognizes these molecules and initiates NK cell killing is not known. To address this question we have determined the crystal structure of human NKp30. Multiple protein constructs were expressed to find a protein construct which could be crystallized. Crystals diffracted to 1.85 angstrom and although the closest known structure has a seq id of 22%; utilizing Balbes and EPMR, the structure was solved by Molecular Replacement. NKp30 has an I-type Ig-like structure, with a large dimer interface (previously unknown) and it is structurally quite different from both NKp44 and NKp46. The closest homologue to NKp30 is Programmed Death Ligand-1 and a structure of this protein in complex with its ligand, Programmed Death-1 is known. Comparing our structure NKp30 with this complex indicates that NKp30 ligand binding may utilize one face of the protein, specifically sheets C, F and G. Cell killing assays using NK cells and cancer cells allowed us to identify an antibody which strongly blocks NKp30 activation and prevents cancer cell killing. We also designed a group of biotinylated peptides (each 15 aa in size) which map the entire surface of NKp30. Binding studies with the NKp30 peptides and blocking antibody allowed us to identify Sheet F as a critical portion for NKp30 activation. A NKp30 polyclonal antibody and a scrambled peptide were used as controls. Together, the structure determination of NKp30, comparison of NKp30 to its homologues and the identification of a specific NKp30 region critical for NK cell killing is the first step in understanding how NKp30 binds diverse ligands resulting in the destruction of cancerous cells. 04.02.1 DNA-directed colloidal crystal formation: assembly and reorganization Byeongdu Lee , Robert Macfarlane , Haley Hill , Soenke Seifert , Chad Mirkin Argonne National Laboratory, Argonne, IL, United States, Evanston, IL, United States 1 2 1 2 2 1 2 Northwestern University, While it was shown more than a decade ago that DNA oligonucleotides can be attached to Au nanoparticles to direct the formation of larger assemblies, the conceptually simple yet powerful idea that functionalized nanoparticles might serve as basic building blocks that can be rationally assembled through programmable base-pairing interactions into highly ordered macroscopic materials remains poorly developed, and the approach has mainly resulted in polymerization having no long range order, or periodicity between particles within the assembled material. Recently, it is demonstrated that DNA can be used to control the crystallization of nanoparticle–oligonucleotide conjugates to the extent that different DNA sequences guide the assembly of the same type of inorganic nanoparticle into different 1,2 crystalline states . Several factors that found critical to govern the process will be discussed. Crystallization mechanism studied by in-situ isothermal and nonisothermal SAXS experiment will be also presented to show that the crystals grow via a three step process: an initial “random binding” phase resulting in disordered DNA-AuNP aggregates, followed by localized reorganization and subsequent growth of crystalline domain size, where the resulting crystals are well-ordered at all subsequent stages of growth. 04.02.2 In situ SAXS studies of silver nanoparticles in synthetic lung fluid Andrew Allen , Vincent Hackley , Robert MacCuspie , Jan Ilavsky 1 1 1 1 2 2 Materials Science and Engineering Lab., NIST, Gaithersburg, MD, United States, Advanced Photon Source, Argonne Natl. Lab., Argonne, IL, United States Silver nanoparticles (AgNPs) have emerged as the most commonly identified nanoscale material in consumer products, principally because of their broad-spectrum biocidal properties. It remains unclear if nanoscale silver presents a new form of silver, or is simply a new vector for solubilized Ag ions. Consequently, research on the environmental, health, and safety (EHS) impact and risk of AgNPs has gained substantial momentum in recent years. Within this context, the dispersion stabilization of AgNPs in synthetic lung fluid has been studied to interrogate the effects on colloidal stability of the principal constituents in the fluid. The colloidal stability of 20 nm citrate-AgNPs dispersed in the presence of each constituent of the synthetic lung fluid (individually, the complete fluid, and without additives) was observed during the titration of an increasing sodium chloride concentration into the solution. In situ small-angle X-ray scattering (SAXS) measurements using a capillary flow cell were combined with other complementary measurement techniques (dynamic light scattering, ultravioletvisible absorption spectroscopy, and atomic force microscopy). It was observed that AgNPs continue to adsorb bovine serum albumin (BSA) protein from the synthetic lung fluid solution as the sodium chloride concentration increases, until a maximum BSA coating is achieved prior to reaching the physiological sodium chloride concentration of 154 mmol/L. BSA was determined to be the constituent of the synthetic lung fluid required to provide colloidal stability at high salt loadings, though phospholipid constituents also exert a subtle effect. Since AgNPs are a distinctly different class of nanoparticles from the carbon nanotubes and titania nanoparticles initially reported to be dispersible using this fluid, the work demonstrates the broad applicability of synthetic lung fluid in providing stable dispersions for engineered nanoparticles. Colloidal stability is inherently entangled with the surface functionalization of nanoparticles. Since the surface that a nanoparticle presents to a living cell will impact its biological fate and toxicity profile, understanding and controlling the nanoparticle surface in dispersion protocols is a key element of correctly interpreting data in nano-EHS studies. This AgNP study demonstrates how SAXS can play a critical role in understanding the EHS consequences of nanoparticle interactions with soft matter and biological systems. 04.02.3 New opportunities for Anomalous Small-Angle X-Ray Scattering to characterize Charged Soft Matter Systems Michael Sztucki, Emanuela Di Cola, Theyencheri Narayanan European Synchrotron Radiation Facility, Grenoble, France Better understanding of charged soft matter systems is of direct relevance to many areas of biological sciences. In general, electrostatic forces are more difficult to handle in modeling and simulation owing to their long-range character. Examples include charge stabilized colloids, polyectrolytes, proteins, surfactant micelles, membranes, etc. One of the key parameters in the complete understanding of such systems is the spatial distribution of counterions. In this contest, quantitative Anomalous Small-Angle X-ray Scattering (ASAXS) offers a unique method for the structural characterization of charged systems. The spatial distribution of counterions can be deduced with high precision by tuning the energy in the vicinity of the absorption edge of the counterions. This information is not readily accessible by conventional scattering techniques as the contributions of the counterions and the macroions superimpose and thus cannot be distinguished. This presentation will give an overview of recent results from different charged systems: e.g. flexible hydrophobic polyelectrolytes in semi-dilute regime with rubidium as counterions (Kedge, 15.2keV) and micellar surfactant systems studied near the bromine K-edge (13.474keV). In the case of polyelectrolytes, the ASAXS effect is rather weak. Nevertheless, the normalized intensities can be decomposed into three components: the energy independent normal SAXS, a cross-term involving the amplitudes of normal SAXS and the resonant scattering of the counterions and the elusive resonant scattering term due to counterions. This latter term allows determining directly the spatial distribution of counterions. On the other hand, the strong ASAXS effect in case of the micellar systems allows direct modeling of the charge distribution. The low concentration of anomalous species as well as the stability and radiation sensitivity of soft matter systems pose high demands on the instrumental setup for ASAXS. This contribution will also review the experimental requirements and recent technical advances in ASAXS instrumentation at the high brilliance SAXS beamline (ID2), ESRF. 04.02.4 Scattering and Cryo-TEM studies of oleic acid based emulsions for investigating selfassembly during human digestion Heiner Santner , Stefan Salentinig , Otto Glatter 1 2 1 2 2 Anton Paar GmbH, Graz, Austria, Department of Chemistry, University of Graz, Graz, Austria This contribution presents self-assembly structures in biological relevant emulsified oleic acid - monoolein (OA-MO) mixtures at different pH values. Small angle x-ray scattering (SAXS), performed with the SAXSess system, as well as Cryo-TEM and dynamic light scattering (DLS) were used to investigate structures and follow structure transition. The solubilization of OA in MO based cubosomes decreases the interfacial curvature of the liquid crystalline phase to more negative values. Structure transitions from bicontinuous cubosomes, to hexosomes, emulsified Fd3m and EME occur with increasing OA concentration. Similar effects were recently reported for the solubilization of tetradecane in monolinolein based emulsions [1]. pH variation between 2 and 8 in a OA-MO system shows that the internal particle structure strongly depends on the pH of the aqueous phase. At high enough OA concentration, transformations from structure less emulsions to emulsified microemulsion (EME), emulsified Fd3m, hexosomes, bicontinuous cubosomes and vesicles can be observed as a function of pH. Interestingly, the liquid crystalline structure to vesicle transition always occurs at intestinal pH values. The hydrodynamic radius of the particles decreases from around 120nm for internally structured particles to around 60nm for vesicles [2]. All transitions with pH are reversible. An apparent pKa for OA in MO is evaluated from the change of structure with pH. This value is within in the physiological pH range of the intestine (between pH 5.5 and 7.5). For pure OA a higher pKa value between 8 and 8.5 was found [3]. [1] A. Yaghmur, et al., Langmuir 21, 569 (2005). [2] S. Salentinig, et al., J. of Coll. and Interf. Sci. 326, 211 (2008). [3] D.P. Cistola, et al., Biochemistry, 27, 1881 (1988). 04.02.5 Evaluation of the Microstructure of Semicrystalline Solid Dispersions Qing Zhu, Lynne Taylor, Michael Harris Purdue University, West Lafayette, United States As a result of an increase in the number of emerging therapies with dissolution limited bioavailability, formulation strategies such as solid dispersions[1] that enhance the rate of solubilization are of interest.[2-5] In this study, the microstructure of solid dispersions prepared with polyethylene glycol (PEG) and several model compounds with different physicochemical properties were evaluated using a variety of experimental techniques. Solid dispersions were prepared by fusion and evaluated using small angle X-ray scattering (SAXS), powder X-ray diffraction (PXRD) etc. SAXS results indicated that, aceclofenac and chlorpropamide solid dispersions favored the interlamellar aggregation of the drug in the PEG matrix. Optical microscopy did not show any evidence of interspherulitic accumulation for any of the model compounds. Haloperidol was highly crystalline in the dispersions, whereas evidence of amorphous material was found for the other model compounds. Results indicated that both the crystallization tendency of the drug and its solubility in amorphous regions of PEG played important roles in determining the aggregation mode and size range of the drug within the dispersion. References 1. Sekiguchi, K.; Obi, N. Chem. Pharm. Bull. 1961, 9, 866-872. 2. Chiou, W. L.; Riegelman, S. J. Pharm. Sci. 1971, 60, 1281-1302. 3. Serajuddin, A. T. M. J. Pharm. Sci. 1999, 88, 1058-1066. 4. Leuner, C.; Dressman, J. Eur. J. Pharm. Biopharm. 2000, 50, 47-60. 5. Craig, D. Q. M. Int. J. Pharm. 2002, 231, 131-144. 04.02.6 Review of Application of Ultra-Small-Angle X-ray Scattering (USAXS) to Polymers and Colloids Jan Ilavsky , Fan Zhang , Gabrielle Long , Pete Jemian 1 1 2 1 1 2 Argonne National Laboratory, Argonne, IL, United States, NIST, Gaithersburg, MD, United States Ultra-small-angle X-ray scattering (USAXS) is capable of probing, in one single measurement, structural inhomogeneities in the size range of 1 to 1000 nm. Recent developments of X-ray sources and optics make USAXS increasingly relevant to polymer and colloids research. In this review, we examine the current unique capabilities of USAXS and how these are matched to the needs of these fields. We will present selected examples of USAXS applications in areas of polymer nanocomposites, polymer gels and solutions, polymer blends, polymer micelles and microemulsions, and colloidal sciences. As case example, we will review published study of a unique colloidal stabilization mechanism, known as 1 nanoparticle “haloing”. This mechanism has been predicted theoretically and inferred experimentally in microsphere-nanoparticle mixtures that possess high charge and size asymmetry. The term “halo” implies the existence of a non-zero separation distance between the highly charged nanoparticles and the negligibly charged microspheres that they surround. Direct characterization of this system by means other than USAXS is challenging due to large size difference between the two components of the system - approximately micron sized spheres and ~50 nanometer particles forming the “halo”. However, using the USAXS, it was possible to, quantitatively, characterize not only the separation distance of the “halo”, but also number of particles in the “halo” and ratio of particles in the “halo” and surrounding suspension. In conclusion, we predict more USAXS studies on polymeric and colloidal systems, especially those with large-scale structure or hierarchical microstructures. 1 F. Zhang, G.G. Long, P.R. Jemian, J. Ilavsky, V.T. Milam, and J.A. Lewis, Langmuir, 2008, 24 (13), p. 6504-6508 Acknowledgement: Use of the Advanced Photon Source at Argonne National Laboratory was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. 04.02.7 In Situ Studies by Small-Angle X-ray Scattering of Kerogens under High Pressure CO2 Randall Winans , Darren Locke , Soenke Seifert , Tony Clemens , Joseph Calo 1 2 1 1 1 2 3 Argonne National Laboratory, Argonne, IL, United States, CRL Energy Limited, Lower Hutt, 3 New Zealand, Brown University, Providence, RI, United States «? ‹? ‚ ‒„? ‹ ‒ £ £ ? ?⁄ ? ?· ? ? · ‹ › ? ⁄‹ ??‒·? ¢? › £ ‹ ·‒ › ‹? ›‒ ? ? « ? ⁄? ?? ? ‚ ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? wL‒\„ M? ? s⁄ ? «¡ ⁄› ? \ ›•¡ ? ⁄¡? ‒¡\ L «¡? ¡ ¡‒« ‹\ ›‹? ›¢? fi›‒› „? ¦⁄\‹£¡ ? ·¡? ›? ¦›\ •¡ ‹£? \‹ ? ¡ •¡ ‹£M? ? s⁄¡? \ \? ·££¡ ¡ ? ⁄\ ? ? « £⁄ ? ¡? fi› ¡? ›? ¡ ¡‒« ‹¡ \ ›‒fi ›‹?\‹ ?fi›‒¡?¢ ‹£? ‹? ⁄¡?¦›\ ? \«fi ¡ ?fl·\‹ \ ¡ „M?b\ ¦· \ ¡ ? ¦\ ¡‒ ‹£? \ \?¢‒›« \‒£¡? «› ¡¦· \‒? «› ¡ ? ›¢? \? ¤¡‒›£¡‹? \‹ ? \? «› ¡ ? • ⁄? bnQ? fl·\ \ ¡ „? ¡ ¦‒ ¡ ? •⁄\ ? › ¡‒ ¡ ? ‹? ⁄¡? ¡‚fi¡‒ «¡‹ M? s⁄¡? ¢ £·‒¡? ¡ ›•? ⁄›• ? ⁄¡? ¦⁄\‹£¡ ? › ¡‒ ¡ ‹ \ „? \ ? 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Specifically, the application of the method to branched polyolefin systems will be discussed. 3 4 The approach is useful in estimating the short-chain and long-chain branch contents in these polymers. Results from this approach are compared and related to data obtained from 4 common techniques such as NMR and rheological measurements. Additionally, the method provides a unique measure of the average long-chain branch length and the number of inner segments in polyolefins. Inner segments reflect the degree of hyperbranching (branch-onbranch) in the polymer systems. To quantify the topology in polyolefin systems, the scaling method is applied to small-angle 3-4 neutron scattering (SANS) data from dilute solutions of polymer in deuterated good solvent. Characteristic mass-fractal dimensions describing the topology (tortuosity and connectivity) of branched polyolefins can be obtained. Further, quantities such as mole-fraction long-chain branch content (Φ br), number of long-chain branches per chain (nbr), average branch length (zbr) and number of inner segments per chain (ni) are estimated, the former two quantities 4 being unique to this approach. Such extensive topological information can be useful in better understanding the effects of various catalyst systems and processing conditions on the molecular structure of polyolefin resins. The scaling method can also be adapted to systems other than polyolefins. For example, biomolecules, nano-aggregates cyclic and star polymers. 1. Beaucage, G., Determination of branch fraction and minimum dimension of massfractal aggregates. Physical Review E 2004, 70 (3). 2. Kulkarni, A. S.; Beaucage, G., Quantification of branching in disordered materials. Journal of Polymer Science Part B-Polymer Physics 2006, 44 (10), 1395-1405. 3. Ramachandran, R.; Beaucage, G.; Kulkarni, A. S.; McFaddin, D.; Merrick-Mack, J.; Galiatsatos, V., Persistence Length of Short-Chain Branched Polyethylene. Macromolecules 2008, 41 (24), 9802-9806. 4. Ramachandran, R.; Beaucage, G.; Kulkarni, A. S.; McFaddin, D.; Merrick-Mack, J.; Galiatsatos, V., Branch Content of Metallocene Polyethylene. Macromolecules 2009, 42 1, 2 07.24.1 Phase transitions in perovskites - further uses of symmetry Christopher J. Howard University of Newcastle, Callaghan, Australia In collaborative work with H.T. Stokes and others, the author has made extensive use of computer program ISOTROPY to establish hierarchies of possible structures based on various proposed distortions. Such hierarchies and their application (to the elucidation structures in SrZrO3, for example) were reviewed five years ago [1]. The exploration of complex distortions using ISODISPLACE is proving useful in more recent work [2]. The typical experimental study follows the evolution with temperature or pressure of lattice parameters (these are particularly precise if measured using HRPD at the ISIS neutron facility) and internal coordinates. It is often advantageous to construct symmetry-adapted strains from the lattice parameters, and symmetry-adapted (normal mode) coordinates from the internal coordinates. These symmetry-adapted quantities transform according to different irreps of the parent space group. Certain of these quantities represent the order parameter(s) for the transitions, and the connections between, say, the strains and order parameters can be checked against the predictions of Landau theory. Illustrative examples to be described may include further analysis of data from SrZrO3, results from a complex perovskite in the (Ca,Sr)TiO3 solid solution, and the onset of Jahn-Teller distortion monitored by examination of the relevant mode amplitude. The influence of strain on phase transitions is nicely illustrated through recent measurements of the 'plateau effect' on the system (Pr,La)AlO3 [3]. Low levels of the La dopant have no effect on the transition temperature (strain field around La dopant atoms do not overlap), but doping to around 2% La starts to reduce this temperature. This result leads to an estimate of the scale of the strain fields around impurity atoms in perovskites. [1] C J Howard & H T Stokes, Acta Cryst. A61, 93-111 (2005) [2] C J Howard & M A Carpenter. Acta Cryst. B66, 40-50 (2010) [3] M A Carpenter, R E A McKnight, C J Howard, Q Zhou, B J Kennedy & K S Knight, Phys. Rev. B 80, 214101 (2009). 07.24.2 Phonons, phase transitions, and symmetry-mode analysis: examples from perovskites and perovskite-related materials. Ian Swainson National Research Council of Canada, Chalk River, ON, Canada Applying symmetry analysis to phonon modes can be done in a simple manner: one approach is by inspection of a simple structure-type in direct-space, looking at the basic mechanics of the structure. Frequently, a few candidate distortions can be revealed, which can then be mapped onto a modulation vector in k-space. Symmetry programs, such as ISOTROPY, can then be interrogated to find the basis functions of irreducible representations that correspond to these phonon modes. I will give an example of the layered, A2BX4, organic-inorganic perovskite-related structures. Specifically, I will discuss the propylammonium tetrachlorometallates in which there is a complex series of transitions to tilt structures, and to both commensurately and incommensurately modulated structures, in which the layers are buckled. Using such methods it is possible to show that the inorganic sublattice solely determines the possible distortions in these structures, while the final choice is made by the energetics of the overall system. The same codes can be very useful in the analysis of real phonon spectra: an example will be shown of unusual zone boundary scattering from the relaxor perovskite, PMN, Pb(Mg1/3Nb2/3)O3. The relevant phonon branches can be assigned, based on compatibility, observations of the lifting of degeneracy, and by comparing the structure factor calculations from the basis functions of candidate irreducible representations to measured intensity variations. From this, one can identify incipient zone boundary soft modes, and identify the nature of the fluctuations. 07.24.3 Phase transitions in framework materials: insights from diffraction, NMR and theory John Evans Durham University, Durham, United Kingdom Phase transitions are ubiquitous in functional materials and often directly related to the emergence of specific properties. Examples range from paraelectric to ferroelectric transitions in perovskites, through structural phase transitions in cuprate and pnictide superconductors, to volume-reducing phase transitions in negative thermal expansion materials. In this contribution I'll discuss two examples where understanding phase transitions and their structural consequences is crucial for understanding the material's properties, and will show how insight from ISODISPLACE symmetry mode analysis provides insight.[1] The first study relates to displacive phase transitions in (MoO2)2P2O7 where combining information from 31 powder diffraction, multi-dimensional solid state P NMR and DFT calculations is crucial in unravelling the structural complexity.[2] In the second example I'll discuss the phase transitions and structural/magnetic properties of a new family of transition metal oxychalcogenides related to the pnictide superconductors.[3] [1] e.g. Stokes, H.T., Campbell, B.J., Hatch, D.M., http://stokes.byu.edu/isodisplace.html. [2] Lister, S.E., A. Soleilhavoup, R.L. Withers, P. Hodgkinson, and J.S.O. Evans, Structures and Phase Transitions in (MoO2)2P2O7. Inorganic Chemistry, 2010. 49(5): p. 2290-2301. [3] McCabe, E.E., Free, D.G., Evans, J.S.O., unpublished results. 07.24.4 The fundamental role of secondary modes in perovskite phase transitions Ross Angel , Di Wang , Yonggang Yu , Michael Carpenter 1 2 1 1 1 2 Virginia Tech, Blacksburg, VA, United States, University of Cambridge, Cambridge, United Kingdom The description of perovskite structures and the phase transitions between them in terms of the tilts of essentially rigid octahedra is long established, and has provided a consistent framework within which to analyse perovskite structures that do not involve electronic effects such as Jahn-Teller distortions or ferroelectric displacements. The formal description of these tilts in terms of two 3-dimensional order parameters associated with the M- and R- points of the Brillouin zone of the cubic parent phase has allowed the character of the transitions to be constrained through Landau theory, the detailed coupling between the order parameters and the spontaneous strain to be determined, and the resulting evolution of the elastic tensor coefficients to be predicted. However, if the octahedra were truly rigid as assumed in this analysis, then volume reduction under high pressure could only be achieved by increasing octahedral tilts, and thus phase transitions to lower-symmetry structures. However, in 3:3 perovskites such as YAlO3, LaGaO3 or LaAlO3, pressure results in decreased tilts and transitions to structures with higher symmetry. We have used DFT calculations to determine the structural evolution of orthorhombic YAlO3 over a pressure range (and with a precision) not available to experimental measurement. The structure undergoes a Pnma to Imma phase transition at ~140 GPa at 0K, a transition normally attributed solely to the tilts associated with the M-point becoming zero, and the amplitude of the corresponding M3+ mode becoming zero. However, this does not fully describe the structural evolution of the Pnma phase, and in particular it does not describe the simultaneous compression and reduction in distortion of the AlO6 octahedra. Analysis with the Isodisplace software shows that, in addition, the amplitudes of the X5+and R5+ symmetry-adapted modes evolve significantly in the Pnma phase, while the M2+ mode remains essentially zero. As expected from the crystal-chemical argument outlined above, our analysis therefore shows that these additional modes must be considered in any complete analysis of the physics of perovskite phase transitions. 07.24.5 Phase transition mechanisms in clinopyroxenes under non-ambient conditions Matteo Alvaro , Fabrizio Nestola , Fernando Cámara , Chiara M. Domeneghetti , Vittorio 2 1 Tazzoli , Ross J. Angel 1 1 3 4 2 Virginia Tech, Blacksburg, VA, United States, Universita' di Pavia, Pavia, Italy, Universita' 4 di Padova, Padova, Italy, C.N.R. - Istituto di Geoscienze e Georisorse, Pavia, Italy 2 3 Clinopyroxenes are chain silicate minerals, in which there are two chains, made up by cornersharing SiO4 - tetrahedra running parallel to the c – axis. The two chains are crosslinked by 2+ 3+ 2+ M1 (containing Mg, Fe , Fe , and Al) and M2 (containing Ca, Fe , Mg and Na ) polyhedra. There are three polymorphs (HT-C2/c, P21/c, HP-C2/c) stable at different pressure and temperature conditions. At ambient conditions the space group is normally P21/c, which transforms to HT-C2/c and HP-C2/c at high-temperature and high-pressure conditions respectively. The HP and HT C2/c structures have the same Wyckoff positions as one another but different geometries. Single crystal structure determinations under non-ambient conditions clearly show that the most significant changes affecting the whole structure geometry at the transition from P to C are those involving the kinking angle (angle between shared oxygens of the same tetrahedral chain), together with the volume of the M2 polyhedron. In particular the kinking angle plays a crucial role in the entire structural evolution as well as the phase transition mechanisms. As the two HP and HT polymorphs differ in their topology the transition mechanism is also expected to be different. By choosing a specific chemical composition we have been able to study the transition to both the HP and HT-C2/c polymorphs. At ambient conditions in the P21/c phase, both the tetrahedral chains are extremely kinked. Far from the transition point, with increasing P the structure becomes more “compressed”, as expected: the two distinct chains become more kinked with a decrease of volume of the M polyhedra. By contrast, at high-T the structure starts “expanding”; the chains extend and the M2 polyhedra expand. At the high-P transition there is an abrupt change of the rotation of the A chain and the two tetrahedral chains become equivalent (with a kinking angle that is smaller than those of the P21/c structure), and the M2 polyhedral volume undergoes a sudden decrease as expected for the first-order character of the transition. By contrast, the high-T transition is continuous, and within the P21/c phase there is a progressive change in rotation of both A and B chains until they become equal at the transition temperature. 07.24.6 Triple Structural Transition below Room Temperature in the Antifilarial Drug Diethylcarbamazine Citrate Javier Ellena , Cecilia C. P. da Silva , Felipe T. Martins Martins , Sara B. Honorato , Núbia 3 1 Boechat Boechat , Alejandro P. Ayala 1 2 1 1 1 2 Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, São Paulo, Brazil, Departamento de Física, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil, 3 Fundação Oswaldo Cruz - FioCruz, Instituto de Tecnologia em Fármacos–FarManguinhos, Rio de Janeiro, Rio de Janeiro, Brazil A very unusual triple structural transition pattern below room temperature was observed for the antifilarial drug diethylcarbamazine citrate. Besides the first thermal, crystallographic and vibrational investigations of this first-line drug used in clinical treatment for lymphatic filariasis, a noteworthy behavior with three structural transformations in function of temperature was demonstrated by differential scanning calorimetry, Raman spectroscopy and single-crystal Xray diffractometry. Our X-ray data on single crystals allow for a complete featuring and understanding of all transitions, since the four structures associated with the three solid-solid phase transformations were accurately determined. Two of three structural transitions show an order-disorder mechanism and temperature hysteresis with exothermic peaks at 224 K (T1’) and 213 K (T2’) upon cooling and endothermic ones at 248 K (T1) and 226 K (T2) upon heating. The other transition occurs at 108 K (T3) and it is temperature-rate sensitive. Molecular displacements onto the (010) plane and conformational changes of the diethylcarbamazine backbone as a consequence of the C—H•••N hydrogen bonding formation/cleavage between drug molecules explains the mechanism of the transitions at T1’/T2. However, such changes are observed only on alternate columns of the drug intercalated by citrate chains, which leads to a doubling of the lattice period along the a axis of the 235 K structure with respect to the 150 K and 293 K structures. At T2’/T1, these structural alterations are completed in the crystal. At T3, there is a rotation on the axis of the N—C bond between the carbamoyl moiety and an ethyl group of one crystallographically independent diethylcarbamazine molecule besides molecular shifts and other conformational alterations. The impact of this study is based on the fascinating finding in which the versatile capability of structural adaptation dependent on the thermal history was observed for a relatively simple organic salt, diethylcarbamazine citrate. T-261 Diluted kagomé antiferromagnet in a single-crystal of segnitite studied by neutron diffraction Huibo Cao Cao , Bryan C. Chakoumakos Chakoumakos , Brian C. Sales Sales , Stuart J. 3 Mills Mills 1 1 1 2 Neutron Scattering Science Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, 2 Oak Ridge, TN, United States, Materials Science and Technology Division, Oak Ridge 3 National Laboratory, Oak Ridge, TN 37831, Oak Ridge, TN, United States, University of British Columbia, Department of Earth and Ocean Sciences, Vancouver, Canada, Vancouver, BC, Canada Iron kagomé lattices in the alunite-jarosite family provide a model kagomé Heisenberg antiferromagnet, which is a highly geometrically frustrated system [1-2]. Segnitite, PbFe3(AsO4)2(OH)5(H2O), belongs to the alunite supergroup [3], and naturally formed single crystals offer a chance to perform neutron studies on this highly frustrated system. Single crystal neutron diffraction was undertaken on the HB-3A four-circle diffractometer at HFIR of ORNL. A 5% aluminum dilution in the iron kagomé layers was determined by both single crystal X-ray and neutron diffraction. SQUID magnetometer identifies an antiferromagnetic transition at T = 50 K, which lies well in the range of the antiferromagentic iron kagomé family [1]. But in the specific heat measurement, we found that its entropy of transition is only 8% of the total entropy, which also is much less than for pure iron kagomé lattices [1]. Preliminary neutron diffraction was carried out to search for the (1 1 3/2) reflection and other reflections with a propagation vector k = (0 0 3/2), where q-scans with a counting time of up to 60 s did not show any antiferromagnetic signal. Further study will be undertaken to fully develop a new magnetic model in this diluted kagomé antiferromagnet. This research is supported by UT Battelle, LLC under Contract No. DE-AC05-00OR22725 for the U.S. Department of Energy, Office of Science. [1] D. Grohol et al. Nature Mater. 4, 323 (2005), Phys. Rev. B 67, 064401 (2003). [2] J. Frunzke et al. J. Mater. Chem. 11, 179-185 (2001). [3] S. J. Mills et al. Eur. J. Mineral. 21, 1073-1080 (2009). 07.25.1 VECTOR APPROACH IN TWO-DIMENSIONAL X-RAY DIFFRACTION Bob He Bruker AXS, Madison, WI, United States Two-dimensional diffraction pattern contains information in a large solid angle. In the two2 dimensional X-ray diffraction (XRD ) geometry, the 2D image can be described by the diffraction intensity distribution in both 2θ and directions. Unit diffraction vector is used in the data analysis of the 2D diffraction pattern. The unit diffraction vector for all the pixels in the 2D pattern can be calculated in the laboratory coordinates. The data analysis requires the unit diffraction vector expressed in the sample coordinates, which can be obtained by vector transformation. The unit vector expression can be used for many applications. For stress {hkl } ( , , , ) ½ analysis, the fundamental equation is given by scalar product of the strain tensor ¾ ¾ ij with the unit vector where {h1 , h2 , h3} : ij hi h j {hkl } ( , , , ) is the measured strain from 2D pattern. For texture analysis, the pole figure angles ( , ) are given by pole mapping equations: The diffraction unit vector is also used in polarization correction, absorption correction and effective volume calculation for crystal size evaluation by -profile analysis. Ref: Bob He, Two-dimensional X-ray Diffraction, John Wiley & Sons, 2009 À sin 1 h3 ¿ cos 1 h12 2 h2 , cos 1 h1 h12 2 h2 0 0 if h2 if h2 0 0 07.25.2 Symmetry detection via symmetry-mode analysis Branton J. Campbell , Sean Kerman , John S. O. Evans , Francesca Perselli , Harold T. 1 Stokes Dept. of Physics & Astronomy, Brigham Young University, Provo, UT, United States, Dept. of Chemistry, University of Durham, Durham, United Kingdom For any crystal structure that can be viewed as a low-symmetry distortion of some highersymmetry parent structure, one can represent the details of the distorted structure in terms of symmetry-adapted distortion modes of the parent structure rather than the traditional list of atomic xyz coordinates. While both descriptions are entirely equivalent, and share the same number of structural degrees of freedom, the symmetry-motivated basis has the advantage that only a handful of the available degrees of freedom tend to be active (i.e. have nonnegligible values). Once these active modes are identified, even the Rietveld refinement of a highly complex distortion can become simple. Consider a subtle low-symmetry distortion of an otherwise high-symmetry parent structure that gives rise to a large supercell and a low point symmetry. If the peak splittings are small and the superlattice intensities weak, structural complexity can increase dramatically without a comparable increase in the information content of an experimental powder-diffraction pattern. In such a case, we will demonstrate that symmetry-mode analysis can reliably determine the real space-group symmetry and simplify the refinement of the distorted phase. 1 2 1 1 2 2 07.25.3 Synchrotron powder X-ray diffraction studies of the alkali metal phenolates to probe Kolbe-Schmitt reaction mechanism and intermediates Matthew Suchomel , Hyunsoo Park , Maren Pink 1 1 2 2 2 Argonne National Laboratory, Argonne, IL, United States, Indiana University, Bloomington, IN, United States Time-resolved powder X-ray diffraction is a powerful tool to investigate the reaction mechanisms, phase transformations and crystallization of solid-state processes. It can provide valuable insights into dynamic processes in extended structures such as zeolites, metal-organic frameworks and other porous crystalline solids to aid the rational design of new materials. In the current study, in-situ investigations of the carbon insertion mechanism in the KolbeSchmitt reaction were performed using powder XRD methods. This important reaction is widely used in industry for the? · ? ? K? ¡ ¡‒ ? \‹ ? fi⁄\‒«\¦¡· ¦\ Z ⁄›•¡ ¡‒? ⁄¡?¡‚\¦ ?«¡¦⁄\‹ «?›¢? ⁄¡?j› ¡Lr¦⁄« ?‒¡\¦ ›‹? ? ? ·‹¦ ¡\‒M??s›?⁄¡ fi?¢·‒ ⁄¡‒ ¡‹ ¢„? ⁄¡? ‒¡\¦ ›‹? ¡ \ ? \‹ ? ‹ ¡‒«¡ \ ¡ K time-resolved powder XRD measurements were performed on synchrotron powder diffraction beamlines at the Advanced Photon Source (1-BM and 11-BM). Time-resolved powder XRD techniques (1-BM) were used to observe the reaction between alkali metal phenolate and CO2 gas. The experiments were performed as a function of time, temperatures and CO2 pressures in order to probe the effects of reaction parameters on the reaction products. Crystalline reaction intermediates isolated during the insitu experiments were further characterized in detail via high-resolution powder XRD (11-BM). ÇÎÇÆÂ É ÍÆÈÌ ËÊÇÁ Éà ÈÃÇÆÅ ÄÃÂÁ In this presentation, we will discuss the possible reaction pathways of the solid-state KolbeSchmitt reaction and propose new crystal structures of the crystalline intermediate phases. The work will also highlight the potential of performing rapid in-situ powder X-ray diffraction measurements at high-intensity synchrotron sources like the Advanced Photon Source (APS) and identify resources available for similar experiments by potential users within the crystallography community. 07.25.4 Pressure-induced switching in magnetic framework materials. Gregory Halder, Karena Chapman, Peter Chupas, John Schlueter Argonne National Laboratory, Argonne, IL, United States The design and characterization of molecular materials with targeted functionalities, such as magnetism and/or nanoporosity, is part of a major international push aimed at developing systems with technologically important applications (e.g., molecular sensing and storage). As such, the accurate elucidation of their often complex structure-function relationships presents a crucial step in their advancement. For molecular magnetism, these approaches are commonly focused on variations of temperature and/or magnetic field, while comparatively little attention has been given to how these materials behave as a function of pressure. Here, we report magneto-structural investigations of magnetic molecular materials using synchrotron-based structural studies (powder diffraction and pair distribution function) and magnetic susceptibility measurements at high pressures. These studies have revealed a range of interesting phenomena such as orbital reorientations, spin crossover, phase transitions, and extreme compressibility. 07.25.5 Extending the Power of PowSnB Hongliang Xu , Charles M. Weeks , Robert H. 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K? ¢¡ ÑÕØÝ ÜÙÝÝÙõ ÑÐÔÝçÓÐàÙÜ âÔáÐÝÕ ÐÝ éÙÝâÙÓÓÐâ ûúùÿüþýùüûúùø÷ ¤£ ¢¡ ù ¤£ ù¤£ ÙÝçÝÔÝ àÑì ØâÜÕÙàÙë ¢¡ ß 07.25.6 Ab initio study of the structure of porous metal-organic frameworks and covalentorganic frameworks from powder X-ray diffraction using charge flipping Fernando Uribe-Romo, Felipe Gandara, Michael O'Keeffe, Omar Yaghi Center for Reticular Chemistry at the California NanoSystems Institute, Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, California, United States Metal-organic frameworks (MOFs) and covalent-organic frameworks (COFs) are crystalline extended materials that are constructed from rigid organic linkers connected either by inorganic metal clusters (MOFs) or completely organic building blocks (COFs), respectively. In the pursuit of the synthesis of functional porous crystalline materials, the preparation of single crystals for a complete structural assignment can be challenging; thus it is common that the only available source of structural information must be obtained from microcrystalline powder samples. Here we describe a series of MOFs and COFs that have been structurally characterized by analysis of their powder X-ray diffraction (PXRD) patterns. Electron density maps of the synthesized materials were calculated ab initio (no chemical or symmetry information) using the charge-flipping algorithm from laboratory PXRD data. Atomic resolution data (dmin = 1.3 Å) enabled the solution of the crystal structure of several MOFs whereas lowresolution data (dmin = 2.0 Å) yielded the assignment of the pore system (cavities) of COFs. For the MOF systems, we identified the atomic positions of the metal and organic units as well as the presence of guest molecules within the pore cavities. Despite the intrinsic low quality of the diffraction of COFs, we identified regions of electron density corresponding to the framework and volumes with very low electron density attributed to the void space of the pores. Indeed this pore system is in agreement with the proposed structural. 07.26.1 Practical Methods of Modeling Disordered Solvent Molecules Using SHELXTL and PLATON. Charles Campana , Bruce Noll , Holger Ott 1 1 1 2 2 Bruker AXS Inc., Madison, Wisconsin, United States, Germany Bruker AXS GmbH, Karlsruhe, Some of the most difficult problems in obtaining chemically reasonable and crystallographically accurate structures involve the use of appropriate techniques to model and account for disordered solvent molecules and counter ions within the crystal lattice. Through careful application of some of the advanced features of the SHELXTL and PLATON software packages, we have been able to significantly improve the quality of the final crystallographic results obtained for a variety of chemically important systems, often with low resolution data. We will present several examples to illustrate some these methods. 07.26.2 Solvent: The Necessary Evil Tom Emge Rutgers University, Piscataway, NJ, United States This brief survey reports on the deleterious effects of highly disordered solvent in a variety of crystal structures. The improvements of derived results and R indices based upon different schemes for modeling the disordered solvent are detailed. Structures analyzed include those with lattices containing large pores, such as MOFs and cavitands, and those containing disordered solvents or counterions of high molecular weight, such as bromoform, triphenyltritellurium and trisphenyltelluridomercurate. 07.26.3 Crystallographic Determination of Metallic Oxide Fullerenes: The Interplay Between Crystallographic and Computational Results Brandon Mercado , Christine Beavers , Steven Stevenson , Joseph Poblet , Marilyn 1 1 Olmstead , Alan Balch University of California - Davis, Davis, CA, United States, University of Southern Mississippi, 3 Hattiesburg, MS, United States, Universitat Rovira i Virgili, Tarragona, Spain, United States, 4 Advanced Light Source, Lawrence Berkeley National Lab, Berkeley, CA, United States Fullerenes have a variety of potential applications that require a better understanding of the interior environment of empty cage and endohedral metallofullerenes (EMFs). Crystallographic data of EMFs offer definitive answers to questions concerning fullerene cage symmetry, inter/intramolecular interactions. X-ray structure determination of EMF single crystals can present numerous problems due to issues such as large thermal motion and small crystal size. Our lab’s approach to solving these problems has included the application of engineered cocrystal systems, synchrotron radiation, and advanced refinement techniques. Computational optimizations of fullerene structures can also provide answers concerning the chemical nature of a fullerene, molecular orbital energy diagrams, and electrostatic potential maps. These results may or may not corroborate crystallographic data. Several new EMF structures will be presented here, specifically metallic oxide fullerenes: Sc4( 3-O)n@C80 (n = 2, 3 see Figure 1) and Sc2( 2-O)@C82. Special consideration will be Figure1. Sc4( 3-O)3@C80; the tetrahedral array of four scandium atoms with three of given to the comparison of crystallographic and the faces capped with oxygen atoms is the largest moiety to be encapsulated in an computational analyses. fullerene to date. 1 4 2 3 1 2 ð ð ¥ 07.26.4 Solution and Refinement with the cctbx and smtbx Luc Bourhis, Oleg Dolomanov, Richard Gildea, Judith Howard, Horst Puschmann Durham University, Durham, United Kingdom The Computational Crystallography Toolbox (cctbx) opened a new era in crystallographic computing by providing a free, open and comprehensive implementation of the fundamentals of crystallography (symmetries, Fourier, scattering, etc). As the foundation of the macromolecular suite PHENIX, it has a certain connotation which is undeserved since the algorithms and data structures it features are correct for any crystal structure. Thus we endeavoured to use it for small molecule work, as part of the EPSRC grant "Age Concern". This lead to the creation of a companion library, the Small Molecule Toolbox (smtbx). It shares the same philosophy as the cctbx: it is designed to make the writing of short scripts easy as well as to make it possible to build or to integrate it into large programs. One example of the latter is the program Olex 2, also developed under the same EPSRC grant, through which the practising crystallographer is given access to most smtbx features. It provides tools covering the whole workflow of small molecule work: e.g. charge flipping and map symmetry search for the solution stage, full matrix refinement for the refinement stage and solvent disorder modelling similar to the SQUEEZE procedure in PLATON. In this talk, we will give an overview of the capabilities of the smtbx/cctbx for small molecule work, focusing on those key computational details which have been the treasures of the classic programs CRYSTALS or SHELX. 07.26.5 A New Solvent Masking Procedure Richard Gildea, Luc Bourhis, Oleg Dolomanov, Judith Howard, Horst Puschmann Durham University, Durham, United Kingdom Disordered solvent molecules or counterions are most commonly modelled with two or more overlapping fragments, often requiring the extensive use of restraints and/or constraints to keep the model chemically reasonable. When appropriate, a somewhat more elegant alternative may be to model atoms as continuously disordered along some special figure, such as a line, a ring or the surface of a sphere, as featured by the program CRYSTALS [1]. However, there are often cases where extended disorder, unknown solvent composition, or incompatibility of the symmetry of the solvent molecule with its site, is such that neither approach is appropriate. Van der Sluis and Spek [2] suggested a method whereby the contribution to the calculated structure factors of the disordered solvent area is calculated via a Fourier transform of that area. This solvent contribution can then either be added to that calculated from the ordered part of the structure, or alternatively subtracted from the observed data before further cycles of refinement. This method has been made widely popular by the SQUEEZE routine available through the program PLATON [3]. We present a new implementation of the above method based on the cctbx (Computational Crystallographic Toolbox) [4], and available through the software Olex2 [5]. Our implementation is compatible with our own smtbx-based refinement program [6], and other refinement programs accepting a hkl file as input. 1. L. Schröder, D. J. Watkin, A. Cousson, R. I. Cooper and W. Paulus, J. Appl. Cryst. (2004). 37, 545-550. 2. P. van der Sluis and A. L. Spek, Acta Cryst. (1990). A46, 194-201. 3. A.L.Spek, J. Appl. Cryst. (2003). 36, 7-13. 4. http://cctbx.sourceforge.net 5. O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard and H. Puschmann, J. Appl. Cryst. (2009). 42, 339-341. 6. L. J. Bourhis, R. J. Gildea, O. V. Dolomanov, J. A. K. Howard and H. Puschmann, IUCr Comp. Comm. Newsletter (2009). 10, 18-31. 07.26.6 Truth is a Meadow – Solvent Molecules in Crystal Structures Peter Mueller MIT, Cambridge, MA, United States “Truth is”, according to German author Stefan Heym, “a meadow where everyone picks the flower he likes best”. Science is the search for knowledge (lat. scientia: knowledge), not truth; yet scientists strive to get as close to The Truth as possible, and the three-dimensional molecular models afforded by modern crystallographic methods certainly look “true”, however flawed they may be. On the example of a particularly challenging solvent disorder this presentation will offer a few flowers from the aforementioned meadow for the audience to examine. 07.26.7 Host-Guest Hydrogen Bonding in Clathrate Hydrates Konstantin Udachin, John Ripmeester National Research Council, Ottawa, Canada Clathrate hydrates with low melting points (often below –20 C) are difficult subjects for single crystal data collection. A high level of guest molecule disorder inside the high symmetry cages causes difficulty for structure determination of such crystals as well. Recent advances in single crystal X-ray diffraction have allowed this technique to be used as a valuable tool for the analysis of hydrate structure and composition. With detailed analysis of guest and water molecules disorder, not only the guest positions are clearly defined, but also it becomes possible to find interactions between guest and water molecules. For the first time, single crystal x-ray crystallography is used to detect the presence of guest host hydrogen bonding in structure I, II and structure H clathrate hydrates. Clathrates studied are the tert-butylamine (tBA) sII clathrate with H2S and Xe help gases, the pinacolone + H2S binary sH clathrate, 1,3-Dioxolane hydrate, chlorine, and bromine hydrates. X-ray structural analysis shows that the tBA nitrogen atom has a distance of 2.64 Å from the closest large cage oxygen atom. This water molecule is pulled inwards toward the tBA guest (cage center) and the structure of the large cage is substantially distorted in comparison to the ideal cage structure. The pinacolone oxygen atom is determined to have a distance of 2.96 Å from the closest large cage oxygen atom. This distance is compatible with pinacolone – water hydrogen bonding. ¦