SIBYLS - A SAXS and protein crystallography beamline at the ALS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trame, Christine; MacDowell, Alastair A.; Celestre, Richard S.

2003-08-22

The new Structurally Integrated BiologY for Life Sciences (SIBYLS) beamline at the Advanced Light Source will be dedicated to Macromolecular Crystallography (PX) and Small Angle X-ray Scattering (SAXS). SAXS will provide structural information of macromolecules in solutions and will complement high resolution PX studies on the same systems but in a crystalline state. The x-ray source is one of the 5 Tesla superbend dipoles recently installed at the ALS that allows for a hard x-ray program to be developed on the relatively low energy Advanced Light Source (ALS) ring (1.9 GeV). The beamline is equipped with fast interchangeable monochromator elements,more » consisting of either a pair of single Si(111) crystals for crystallography, or a pair of multilayers for the SAXS mode data collection (E/{Delta}E {approx} 1/110). Flux rates with Si(111) crystals for PX are measured as 2 x 10{sup 11} hv/sec/400 mA through a 100 {micro}m pinhole at 12.4 KeV. For SAXS the flux is up to 3 x 10{sup 13} photons/sec at 10 KeV with all apertures open when using the multilayer monochromator elements. The performance characteristics of this unique beamline will be described.« less

The accurate assessment of small-angle X-ray scattering data

DOE PAGES

Grant, Thomas D.; Luft, Joseph R.; Carter, Lester G.; ...

2015-01-23

Small-angle X-ray scattering (SAXS) has grown in popularity in recent times with the advent of bright synchrotron X-ray sources, powerful computational resources and algorithms enabling the calculation of increasingly complex models. However, the lack of standardized data-quality metrics presents difficulties for the growing user community in accurately assessing the quality of experimental SAXS data. Here, a series of metrics to quantitatively describe SAXS data in an objective manner using statistical evaluations are defined. These metrics are applied to identify the effects of radiation damage, concentration dependence and interparticle interactions on SAXS data from a set of 27 previously described targetsmore » for which high-resolution structures have been determined via X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. Studies show that these metrics are sufficient to characterize SAXS data quality on a small sample set with statistical rigor and sensitivity similar to or better than manual analysis. The development of data-quality analysis strategies such as these initial efforts is needed to enable the accurate and unbiased assessment of SAXS data quality.« less

Structure of the Full-length VEGFR-1 Extracellular Domain in Complex with VEGF-A.

PubMed

Markovic-Mueller, Sandra; Stuttfeld, Edward; Asthana, Mayanka; Weinert, Tobias; Bliven, Spencer; Goldie, Kenneth N; Kisko, Kaisa; Capitani, Guido; Ballmer-Hofer, Kurt

2017-02-07

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development upon activation of three receptor tyrosine kinases: VEGFR-1, -2, and -3. Partial structures of VEGFR/VEGF complexes based on single-particle electron microscopy, small-angle X-ray scattering, and X-ray crystallography revealed the location of VEGF binding and domain arrangement of individual receptor subdomains. Here, we describe the structure of the full-length VEGFR-1 extracellular domain in complex with VEGF-A at 4 Å resolution. We combined X-ray crystallography, single-particle electron microscopy, and molecular modeling for structure determination and validation. The structure reveals the molecular details of ligand-induced receptor dimerization, in particular of homotypic receptor interactions in immunoglobulin homology domains 4, 5, and 7. Functional analyses of ligand binding and receptor activation confirm the relevance of these homotypic contacts and identify them as potential therapeutic sites to allosterically inhibit VEGFR-1 activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Resolution of ab initio shapes determined from small-angle scattering.

PubMed

Tuukkanen, Anne T; Kleywegt, Gerard J; Svergun, Dmitri I

2016-11-01

Spatial resolution is an important characteristic of structural models, and the authors of structures determined by X-ray crystallography or electron cryo-microscopy always provide the resolution upon publication and deposition. Small-angle scattering of X-rays or neutrons (SAS) has recently become a mainstream structural method providing the overall three-dimensional structures of proteins, nucleic acids and complexes in solution. However, no quantitative resolution measure is available for SAS-derived models, which significantly hampers their validation and further use. Here, a method is derived for resolution assessment for ab initio shape reconstruction from scattering data. The inherent variability of the ab initio shapes is utilized and it is demonstrated how their average Fourier shell correlation function is related to the model resolution. The method is validated against simulated data for proteins with known high-resolution structures and its efficiency is demonstrated in applications to experimental data. It is proposed that henceforth the resolution be reported in publications and depositions of ab initio SAS models.

Resolution of ab initio shapes determined from small-angle scattering

PubMed Central

Tuukkanen, Anne T.; Kleywegt, Gerard J.; Svergun, Dmitri I.

2016-01-01

Spatial resolution is an important characteristic of structural models, and the authors of structures determined by X-ray crystallography or electron cryo-microscopy always provide the resolution upon publication and deposition. Small-angle scattering of X-rays or neutrons (SAS) has recently become a mainstream structural method providing the overall three-dimensional structures of proteins, nucleic acids and complexes in solution. However, no quantitative resolution measure is available for SAS-derived models, which significantly hampers their validation and further use. Here, a method is derived for resolution assessment for ab initio shape reconstruction from scattering data. The inherent variability of the ab initio shapes is utilized and it is demonstrated how their average Fourier shell correlation function is related to the model resolution. The method is validated against simulated data for proteins with known high-resolution structures and its efficiency is demonstrated in applications to experimental data. It is proposed that henceforth the resolution be reported in publications and depositions of ab initio SAS models. PMID:27840683

Better Ceramics Through Chemistry IV. Materials Research Society Sumposium Proceedings. Volume 180

DTIC Science & Technology

1991-03-31

GELS 117 L.F. Nazar, D.G. Napier, D. Lapham, and E. Epperson SMALL ANGLE X - RAY SCATTERING STUDIES Or POLYMERIC ZIRCONIUM SPECIES IN AQUEOUS SOLUTION...recently obtained the first X - ray crystallographic data on bismuth alkoxides, Bi(OR) 3 (R = C(CH3) 3 and C6 H4 (CH 3 )2 -2,6) [8]. These data showed that...d8, ppm): 12.5 (O2CMe), 9.6 (OCMe3). The complex was identified by X - ray crystallography. 1 crystallizes in space group P21/n with a = 13.149(2) A, b

Conformational variability of the stationary phase survival protein E from Xylella fastidiosa revealed by X-ray crystallography, small-angle X-ray scattering studies, and normal mode analysis.

PubMed

Machado, Agnes Thiane Pereira; Fonseca, Emanuella Maria Barreto; Reis, Marcelo Augusto Dos; Saraiva, Antonio Marcos; Santos, Clelton Aparecido Dos; de Toledo, Marcelo Augusto Szymanski; Polikarpov, Igor; de Souza, Anete Pereira; Aparicio, Ricardo; Iulek, Jorge

2017-10-01

Xylella fastidiosa is a xylem-limited bacterium that infects a wide variety of plants. Stationary phase survival protein E is classified as a nucleotidase, which is expressed when bacterial cells are in the stationary growth phase and subjected to environmental stresses. Here, we report four refined X-ray structures of this protein from X. fastidiosa in four different crystal forms in the presence and/or absence of the substrate 3'-AMP. In all chains, the conserved loop verified in family members assumes a closed conformation in either condition. Therefore, the enzymatic mechanism for the target protein might be different of its homologs. Two crystal forms exhibit two monomers whereas the other two show four monomers in the asymmetric unit. While the biological unit has been characterized as a tetramer, differences of their sizes and symmetry are remarkable. Four conformers identified by Small-Angle X-ray Scattering (SAXS) in a ligand-free solution are related to the low frequency normal modes of the crystallographic structures associated with rigid body-like protomer arrangements responsible for the longitudinal and symmetric adjustments between tetramers. When the substrate is present in solution, only two conformers are selected. The most prominent conformer for each case is associated to a normal mode able to elongate the protein by moving apart two dimers. To our knowledge, this work was the first investigation based on the normal modes that analyzed the quaternary structure variability for an enzyme of the SurE family followed by crystallography and SAXS validation. The combined results raise new directions to study allosteric features of XfSurE protein. © 2017 Wiley Periodicals, Inc.

Accurate determination of segmented X-ray detector geometry

PubMed Central

Yefanov, Oleksandr; Mariani, Valerio; Gati, Cornelius; White, Thomas A.; Chapman, Henry N.; Barty, Anton

2015-01-01

Recent advances in X-ray detector technology have resulted in the introduction of segmented detectors composed of many small detector modules tiled together to cover a large detection area. Due to mechanical tolerances and the desire to be able to change the module layout to suit the needs of different experiments, the pixels on each module might not align perfectly on a regular grid. Several detectors are designed to permit detector sub-regions (or modules) to be moved relative to each other for different experiments. Accurate determination of the location of detector elements relative to the beam-sample interaction point is critical for many types of experiment, including X-ray crystallography, coherent diffractive imaging (CDI), small angle X-ray scattering (SAXS) and spectroscopy. For detectors with moveable modules, the relative positions of pixels are no longer fixed, necessitating the development of a simple procedure to calibrate detector geometry after reconfiguration. We describe a simple and robust method for determining the geometry of segmented X-ray detectors using measurements obtained by serial crystallography. By comparing the location of observed Bragg peaks to the spot locations predicted from the crystal indexing procedure, the position, rotation and distance of each module relative to the interaction region can be refined. We show that the refined detector geometry greatly improves the results of experiments. PMID:26561117

Serial femtosecond X-ray diffraction of enveloped virus microcrystals

DOE PAGES

Lawrence, Robert M.; Conrad, Chelsie E.; Zatsepin, Nadia A.; ...

2015-08-20

Serial femtosecond crystallography (SFX) using X-ray free-electron lasers has produced high-resolution, room temperature, time-resolved protein structures. We report preliminary SFX of Sindbis virus, an enveloped icosahedral RNA virus with ~700 Å diameter. Microcrystals delivered in viscous agarose medium diffracted to ~40 Å resolution. Small-angle diffuse X-ray scattering overlaid Bragg peaks and analysis suggests this results from molecular transforms of individual particles. Viral proteins undergo structural changes during entry and infection, which could, in principle, be studied with SFX. This is a pertinent step toward determining room temperature structures from virus microcrystals that may enable time-resolved studies of enveloped viruses.

How the ESRF helps industry and how they help the ESRF

PubMed Central

Malbet-Monaco, Stéphanie; Leonard, Gordon A.; Mitchell, Edward P.; Gordon, Elspeth J.

2013-01-01

The ESRF has worked with, and provided services for, the pharmaceutical industry since the construction of its first protein crystallography beamline in the mid-1990s. In more recent times, industrial clients have benefited from a portfolio of beamlines which offer a wide range of functionality and beam characteristics, including tunability, microfocus and micro-aperture. Included in this portfolio is a small-angle X-­ray scattering beamline dedicated to the study of biological molecules in solution. The high demands on throughput and efficiency made by the ESRF’s industrial clients have been a major driving force in the evolution of the ESRF’s macromolecular crystallography resources, which now include remote access, the automation of crystal screening and data collection, and a beamline database allowing sample tracking, experiment reporting and real-time at-a-distance monitoring of experiments. This paper describes the key features of the functionality put in place on the ESRF structural biology beamlines and outlines the major advantages of the interaction of the ESRF with the pharmaceutical industry. PMID:23793155

Analysis of RNA structure using small-angle X-ray scattering

PubMed Central

Cantara, William A.; Olson, Erik D.; Musier-Forsyth, Karin

2016-01-01

In addition to their role in correctly attaching specific amino acids to cognate tRNAs, aminoacyl-tRNA synthetases (aaRS) have been found to possess many alternative functions and often bind to and act on other nucleic acids. In contrast to the well-defined 3D structure of tRNA, the structures of many of the other RNAs recognized by aaRSs have not been solved. Despite advances in the use of X-ray crystallography (XRC), nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (cryo-EM) for structural characterization of biomolecules, significant challenges to solving RNA structures still exist. Recently, small-angle X-ray scattering (SAXS) has been increasingly employed to characterize the 3D structures of RNAs and RNA-protein complexes. SAXS is capable of providing low-resolution tertiary structure information under physiological conditions and with less intensive sample preparation and data analysis requirements than XRC, NMR and cryo-EM. In this article, we describe best practices involved in the process of RNA and RNA-protein sample preparation, SAXS data collection, data analysis, and structural model building. PMID:27777026

Biological Small Angle Scattering: Techniques, Strategies and Tips

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chaudhuri, Barnali; Muñoz, Inés G.; Urban, Volker S.

This book provides a clear, comprehensible and up-to-date description of how Small Angle Scattering (SAS) can help structural biology researchers. SAS is an efficient technique that offers structural information on how biological macromolecules behave in solution. SAS provides distinct and complementary data for integrative structural biology approaches in combination with other widely used probes, such as X-ray crystallography, Nuclear magnetic resonance, Mass spectrometry and Cryo-electron Microscopy. The development of brilliant synchrotron small-angle X-ray scattering (SAXS) beam lines has increased the number of researchers interested in solution scattering. SAS is especially useful for studying conformational changes in proteins, highly flexible proteins,more » and intrinsically disordered proteins. Small-angle neutron scattering (SANS) with neutron contrast variation is ideally suited for studying multi-component assemblies as well as membrane proteins that are stabilized in surfactant micelles or vesicles. SAS is also used for studying dynamic processes of protein fibrillation in amyloid diseases, and pharmaceutical drug delivery. The combination with size-exclusion chromatography further increases the range of SAS applications.The book is written by leading experts in solution SAS methodologies. The principles and theoretical background of various SAS techniques are included, along with practical aspects that range from sample preparation to data presentation for publication. Topics covered include techniques for improving data quality and analysis, as well as different scientific applications of SAS. With abundant illustrations and practical tips, we hope the clear explanations of the principles and the reviews on the latest progresses will serve as a guide through all aspects of biological solution SAS.The scope of this book is particularly relevant for structural biology researchers who are new to SAS. Advanced users of the technique will find it helpful for exploring the diversity of solution SAS methods and applications.« less

Micro-crystallography comes of age

PubMed Central

Smith, Janet L.; Fischetti, Robert F.; Yamamoto, Masaki

2012-01-01

The latest revolution in macromolecular crystallography was incited by the development of dedicated, user friendly, micro-crystallography beamlines. Brilliant X-ray beams of diameter 20 microns or less, now available at most synchrotron sources, enable structure determination from samples that previously were inaccessible. Relative to traditional crystallography, crystals with one or more small dimensions have diffraction patterns with vastly improved signal-to-noise when recorded with an appropriately matched beam size. Structures can be solved from isolated, well diffracting regions within inhomogeneous samples. This review summarizes the technological requirements and approaches to producing micro-beams and how they continue to change the practice of crystallography. PMID:23021872

Teaching with the Case Study Method to Promote Active Learning in a Small Molecule Crystallography Course for Chemistry Students

ERIC Educational Resources Information Center

Campbell, Michael G.; Powers, Tamara M.; Zheng, Shao-Liang

2016-01-01

Implementing the case study method in a practical X-ray crystallography course designed for graduate or upper-level undergraduate chemistry students is described. Compared with a traditional lecture format, assigning small groups of students to examine literature case studies encourages more active engagement with the course material and…

Accurate determination of segmented X-ray detector geometry

DOE PAGES

Yefanov, Oleksandr; Mariani, Valerio; Gati, Cornelius; ...

2015-10-22

Recent advances in X-ray detector technology have resulted in the introduction of segmented detectors composed of many small detector modules tiled together to cover a large detection area. Due to mechanical tolerances and the desire to be able to change the module layout to suit the needs of different experiments, the pixels on each module might not align perfectly on a regular grid. Several detectors are designed to permit detector sub-regions (or modules) to be moved relative to each other for different experiments. Accurate determination of the location of detector elements relative to the beam-sample interaction point is critical formore » many types of experiment, including X-ray crystallography, coherent diffractive imaging (CDI), small angle X-ray scattering (SAXS) and spectroscopy. For detectors with moveable modules, the relative positions of pixels are no longer fixed, necessitating the development of a simple procedure to calibrate detector geometry after reconfiguration. We describe a simple and robust method for determining the geometry of segmented X-ray detectors using measurements obtained by serial crystallography. By comparing the location of observed Bragg peaks to the spot locations predicted from the crystal indexing procedure, the position, rotation and distance of each module relative to the interaction region can be refined. Furthermore, we show that the refined detector geometry greatly improves the results of experiments.« less

Micro-crystallography comes of age.

PubMed

Smith, Janet L; Fischetti, Robert F; Yamamoto, Masaki

2012-10-01

The latest revolution in macromolecular crystallography was incited by the development of dedicated, user friendly, micro-crystallography beam lines. Brilliant X-ray beams of diameter 20 μm or less, now available at most synchrotron sources, enable structure determination from samples that previously were inaccessible. Relative to traditional crystallography, crystals with one or more small dimensions have diffraction patterns with vastly improved signal-to-noise when recorded with an appropriately matched beam size. Structures can be solved from isolated, well diffracting regions within inhomogeneous samples. This review summarizes the technological requirements and approaches to producing micro-beams and how they continue to change the practice of crystallography. Copyright © 2012 Elsevier Ltd. All rights reserved.

AceDRG: a stereochemical description generator for ligands

PubMed Central

Emsley, Paul; Gražulis, Saulius; Merkys, Andrius; Vaitkus, Antanas

2017-01-01

The program AceDRG is designed for the derivation of stereochemical information about small molecules. It uses local chemical and topological environment-based atom typing to derive and organize bond lengths and angles from a small-molecule database: the Crystallography Open Database (COD). Information about the hybridization states of atoms, whether they belong to small rings (up to seven-membered rings), ring aromaticity and nearest-neighbour information is encoded in the atom types. All atoms from the COD have been classified according to the generated atom types. All bonds and angles have also been classified according to the atom types and, in a certain sense, bond types. Derived data are tabulated in a machine-readable form that is freely available from CCP4. AceDRG can also generate stereochemical information, provided that the basic bonding pattern of a ligand is known. The basic bonding pattern is perceived from one of the computational chemistry file formats, including SMILES, mmCIF, SDF MOL and SYBYL MOL2 files. Using the bonding chemistry, atom types, and bond and angle tables generated from the COD, AceDRG derives the ‘ideal’ bond lengths, angles, plane groups, aromatic rings and chirality information, and writes them to an mmCIF file that can be used by the refinement program REFMAC5 and the model-building program Coot. Other refinement and model-building programs such as PHENIX and BUSTER can also use these files. AceDRG also generates one or more coordinate sets corresponding to the most favourable conformation(s) of a given ligand. AceDRG employs RDKit for chemistry perception and for initial conformation generation, as well as for the interpretation of SMILES strings, SDF MOL and SYBYL MOL2 files. PMID:28177307

Langmuir-Blodgett nanotemplates for protein crystallography.

PubMed

Pechkova, Eugenia; Nicolini, Claudio

2017-12-01

The new generation of synchrotrons and microfocused beamlines has enabled great progress in X-ray protein crystallography, resulting in new 3D atomic structures for proteins of high interest to the pharmaceutical industry and life sciences. It is, however, often still challenging to produce protein crystals of sufficient size and quality (order, intensity of diffraction, radiation stability). In this protocol, we provide instructions for performing the Langmuir-Blodgett (LB) nanotemplate method, a crystallization approach that can be used for any protein (including membrane proteins). We describe how to produce highly ordered 2D LB protein monolayers at the air-water interface and deposit them on glass slides. LB-film formation can be observed by surface-pressure measurements and Brewster angle microscopy (BAM), although its quality can be characterized by atomic force microscopy (AFM) and nanogravimetry. Such films are then used as a 2D template for triggering 3D protein crystal formation by hanging-drop vapor diffusion. The procedure for forming the 2D template takes a few minutes. Structural information about the protein reorganization in the LB film during the crystallization process on the nano level can be obtained using an in situ submicron GISAXS (grazing-incidence small-angle X-ray scattering) method. MicroGISAXS spectra, measured directly at the interface of the LB films and protein solution in real time, as described in this protocol, can be interpreted in terms of the buildup of layers, islands, or holes. In our experience, the obtained LB crystals take 1-10 d to prepare and they are more ordered and radiation stable as compared with those produced using other crystallization methods.

Developing advanced X-ray scattering methods combined with crystallography and computation.

PubMed

Perry, J Jefferson P; Tainer, John A

2013-03-01

The extensive use of small angle X-ray scattering (SAXS) over the last few years is rapidly providing new insights into protein interactions, complex formation and conformational states in solution. This SAXS methodology allows for detailed biophysical quantification of samples of interest. Initial analyses provide a judgment of sample quality, revealing the potential presence of aggregation, the overall extent of folding or disorder, the radius of gyration, maximum particle dimensions and oligomerization state. Structural characterizations include ab initio approaches from SAXS data alone, and when combined with previously determined crystal/NMR, atomistic modeling can further enhance structural solutions and assess validity. This combination can provide definitions of architectures, spatial organizations of protein domains within a complex, including those not determined by crystallography or NMR, as well as defining key conformational states of a protein interaction. SAXS is not generally constrained by macromolecule size, and the rapid collection of data in a 96-well plate format provides methods to screen sample conditions. This includes screening for co-factors, substrates, differing protein or nucleotide partners or small molecule inhibitors, to more fully characterize the variations within assembly states and key conformational changes. Such analyses may be useful for screening constructs and conditions to determine those most likely to promote crystal growth of a complex under study. Moreover, these high throughput structural determinations can be leveraged to define how polymorphisms affect assembly formations and activities. This is in addition to potentially providing architectural characterizations of complexes and interactions for systems biology-based research, and distinctions in assemblies and interactions in comparative genomics. Thus, SAXS combined with crystallography/NMR and computation provides a unique set of tools that should be considered as being part of one's repertoire of biophysical analyses, when conducting characterizations of protein and other macromolecular interactions. Copyright © 2013 Elsevier Inc. All rights reserved.

Crystallography Open Database – an open-access collection of crystal structures

PubMed Central

Gražulis, Saulius; Chateigner, Daniel; Downs, Robert T.; Yokochi, A. F. T.; Quirós, Miguel; Lutterotti, Luca; Manakova, Elena; Butkus, Justas; Moeck, Peter; Le Bail, Armel

2009-01-01

The Crystallography Open Database (COD), which is a project that aims to gather all available inorganic, metal–organic and small organic molecule structural data in one database, is described. The database adopts an open-access model. The COD currently contains ∼80 000 entries in crystallographic information file format, with nearly full coverage of the International Union of Crystallography publications, and is growing in size and quality. PMID:22477773

Crystal structure of Streptococcus pneumoniae pneumolysin provides key insights into early steps of pore formation

PubMed Central

Lawrence, Sara L.; Feil, Susanne C.; Morton, Craig J.; Farrand, Allison J.; Mulhern, Terrence D.; Gorman, Michael A.; Wade, Kristin R.; Tweten, Rodney K.; Parker, Michael W.

2015-01-01

Pore-forming proteins are weapons often used by bacterial pathogens to breach the membrane barrier of target cells. Despite their critical role in infection important structural aspects of the mechanism of how these proteins assemble into pores remain unknown. Streptococcus pneumoniae is the world’s leading cause of pneumonia, meningitis, bacteremia and otitis media. Pneumolysin (PLY) is a major virulence factor of S. pneumoniae and a target for both small molecule drug development and vaccines. PLY is a member of the cholesterol-dependent cytolysins (CDCs), a family of pore-forming toxins that form gigantic pores in cell membranes. Here we present the structure of PLY determined by X-ray crystallography and, in solution, by small-angle X-ray scattering. The crystal structure reveals PLY assembles as a linear oligomer that provides key structural insights into the poorly understood early monomer-monomer interactions of CDCs at the membrane surface. PMID:26403197

Improving small-angle X-ray scattering data for structural analyses of the RNA world

PubMed Central

Rambo, Robert P.; Tainer, John A.

2010-01-01

Defining the shape, conformation, or assembly state of an RNA in solution often requires multiple investigative tools ranging from nucleotide analog interference mapping to X-ray crystallography. A key addition to this toolbox is small-angle X-ray scattering (SAXS). SAXS provides direct structural information regarding the size, shape, and flexibility of the particle in solution and has proven powerful for analyses of RNA structures with minimal requirements for sample concentration and volumes. In principle, SAXS can provide reliable data on small and large RNA molecules. In practice, SAXS investigations of RNA samples can show inconsistencies that suggest limitations in the SAXS experimental analyses or problems with the samples. Here, we show through investigations on the SAM-I riboswitch, the Group I intron P4-P6 domain, 30S ribosomal subunit from Sulfolobus solfataricus (30S), brome mosaic virus tRNA-like structure (BMV TLS), Thermotoga maritima asd lysine riboswitch, the recombinant tRNAval, and yeast tRNAphe that many problems with SAXS experiments on RNA samples derive from heterogeneity of the folded RNA. Furthermore, we propose and test a general approach to reducing these sample limitations for accurate SAXS analyses of RNA. Together our method and results show that SAXS with synchrotron radiation has great potential to provide accurate RNA shapes, conformations, and assembly states in solution that inform RNA biological functions in fundamental ways. PMID:20106957

From lows to highs: using low-resolution models to phase X-ray data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stuart, David I.; Diamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus, Didcot; Abrescia, Nicola G. A., E-mail: nabrescia@cicbiogune.es

2013-11-01

An unusual example of how virus structure determination pushes the limits of the molecular replacement method is presented. The study of virus structures has contributed to methodological advances in structural biology that are generally applicable (molecular replacement and noncrystallographic symmetry are just two of the best known examples). Moreover, structural virology has been instrumental in forging the more general concept of exploiting phase information derived from multiple structural techniques. This hybridization of structural methods, primarily electron microscopy (EM) and X-ray crystallography, but also small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy, is central to integrative structural biology. Here,more » the interplay of X-ray crystallography and EM is illustrated through the example of the structural determination of the marine lipid-containing bacteriophage PM2. Molecular replacement starting from an ∼13 Å cryo-EM reconstruction, followed by cycling density averaging, phase extension and solvent flattening, gave the X-ray structure of the intact virus at 7 Å resolution This in turn served as a bridge to phase, to 2.5 Å resolution, data from twinned crystals of the major coat protein (P2), ultimately yielding a quasi-atomic model of the particle, which provided significant insights into virus evolution and viral membrane biogenesis.« less

Protein Crystallography in Vaccine Research and Development.

PubMed

Malito, Enrico; Carfi, Andrea; Bottomley, Matthew J

2015-06-09

The use of protein X-ray crystallography for structure-based design of small-molecule drugs is well-documented and includes several notable success stories. However, it is less well-known that structural biology has emerged as a major tool for the design of novel vaccine antigens. Here, we review the important contributions that protein crystallography has made so far to vaccine research and development. We discuss several examples of the crystallographic characterization of vaccine antigen structures, alone or in complexes with ligands or receptors. We cover the critical role of high-resolution epitope mapping by reviewing structures of complexes between antigens and their cognate neutralizing, or protective, antibody fragments. Most importantly, we provide recent examples where structural insights obtained via protein crystallography have been used to design novel optimized vaccine antigens. This review aims to illustrate the value of protein crystallography in the emerging discipline of structural vaccinology and its impact on the rational design of vaccines.

Protein Crystallography in Vaccine Research and Development

PubMed Central

Malito, Enrico; Carfi, Andrea; Bottomley, Matthew J.

2015-01-01

The use of protein X-ray crystallography for structure-based design of small-molecule drugs is well-documented and includes several notable success stories. However, it is less well-known that structural biology has emerged as a major tool for the design of novel vaccine antigens. Here, we review the important contributions that protein crystallography has made so far to vaccine research and development. We discuss several examples of the crystallographic characterization of vaccine antigen structures, alone or in complexes with ligands or receptors. We cover the critical role of high-resolution epitope mapping by reviewing structures of complexes between antigens and their cognate neutralizing, or protective, antibody fragments. Most importantly, we provide recent examples where structural insights obtained via protein crystallography have been used to design novel optimized vaccine antigens. This review aims to illustrate the value of protein crystallography in the emerging discipline of structural vaccinology and its impact on the rational design of vaccines. PMID:26068237

Fixed target matrix for femtosecond time-resolved and in situ serial micro-crystallography

PubMed Central

Mueller, C.; Marx, A.; Epp, S. W.; Zhong, Y.; Kuo, A.; Balo, A. R.; Soman, J.; Schotte, F.; Lemke, H. T.; Owen, R. L.; Pai, E. F.; Pearson, A. R.; Olson, J. S.; Anfinrud, P. A.; Ernst, O. P.; Dwayne Miller, R. J.

2015-01-01

We present a crystallography chip enabling in situ room temperature crystallography at microfocus synchrotron beamlines and X-ray free-electron laser (X-FEL) sources. Compared to other in situ approaches, we observe extremely low background and high diffraction data quality. The chip design is robust and allows fast and efficient loading of thousands of small crystals. The ability to load a large number of protein crystals, at room temperature and with high efficiency, into prescribed positions enables high throughput automated serial crystallography with microfocus synchrotron beamlines. In addition, we demonstrate the application of this chip for femtosecond time-resolved serial crystallography at the Linac Coherent Light Source (LCLS, Menlo Park, California, USA). The chip concept enables multiple images to be acquired from each crystal, allowing differential detection of changes in diffraction intensities in order to obtain high signal-to-noise and fully exploit the time resolution capabilities of XFELs. PMID:26798825

Fixed target matrix for femtosecond time-resolved and in situ serial micro-crystallography.

PubMed

Mueller, C; Marx, A; Epp, S W; Zhong, Y; Kuo, A; Balo, A R; Soman, J; Schotte, F; Lemke, H T; Owen, R L; Pai, E F; Pearson, A R; Olson, J S; Anfinrud, P A; Ernst, O P; Dwayne Miller, R J

2015-09-01

We present a crystallography chip enabling in situ room temperature crystallography at microfocus synchrotron beamlines and X-ray free-electron laser (X-FEL) sources. Compared to other in situ approaches, we observe extremely low background and high diffraction data quality. The chip design is robust and allows fast and efficient loading of thousands of small crystals. The ability to load a large number of protein crystals, at room temperature and with high efficiency, into prescribed positions enables high throughput automated serial crystallography with microfocus synchrotron beamlines. In addition, we demonstrate the application of this chip for femtosecond time-resolved serial crystallography at the Linac Coherent Light Source (LCLS, Menlo Park, California, USA). The chip concept enables multiple images to be acquired from each crystal, allowing differential detection of changes in diffraction intensities in order to obtain high signal-to-noise and fully exploit the time resolution capabilities of XFELs.

Solution Structure of an Amyloid-Forming Protein During Photoinitiated Hexamer-Dodecamer Transitions Revealed Through Small-Angle Neutron Scattering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamill,A.; Wang, S.; Lee, Jr., C.

2007-01-01

Shape-reconstruction analysis applied to small angle neutron scattering (SANS) data is used to determine the in vitro conformations of {alpha}-chymotrypsin oligomers that form as a result of partial unfolding with a photoresponsive surfactant. In the presence of the photoactive surfactant under visible light, the native oligomers (dimers or compact hexamers) rearrange into expanded corkscrew-like hexamers. Converting the surfactant to the photopassive form with UV light illumination causes the hexamers to laterally aggregate and intertwine into dodecamers with elongated, twisted conformations containing cross-sectional dimensions similar to amyloid protofilaments. Secondary-structure measurements with FT-IR indicate that this photoinduced hexamer-to-dodecamer association occurs through intermolecularmore » {beta} sheets stabilized with hydrogen bonds, similar to amyloid formation. Traditional structural characterization techniques such as X-ray crystallography and NMR are not easily amenable to the study of these non-native protein conformations; however, SANS is ideally suited to the study of these associated intermediates, providing direct observation of the mechanism of oligomeric formation in an amyloid-forming protein. Combined with photoinitiated hexamer-to-dodecamer associations in the presence of the photoresponsive surfactant, this study could provide unique insight into the amyloidosis disease pathway, as well as novel disease treatment strategies.« less

Structural characterization of the RNA chaperone Hfq from the nitrogen-fixing bacterium Herbaspirillum seropedicae SmR1.

PubMed

Kadowaki, Marco Antonio Seiki; Iulek, Jorge; Barbosa, João Alexandre Ribeiro Gonçalves; Pedrosa, Fábio de Oliveira; de Souza, Emanuel Maltempi; Chubatsu, Leda Satie; Monteiro, Rose Adele; de Oliveira, Marco Aurélio Schüler; Steffens, Maria Berenice Reynaud

2012-02-01

The RNA chaperone Hfq is a homohexamer protein identified as an E. coli host factor involved in phage Qβ replication and it is an important posttranscriptional regulator of several types of RNA, affecting a plethora of bacterial functions. Although twenty Hfq crystal structures have already been reported in the Protein Data Bank (PDB), new insights into these protein structures can still be discussed. In this work, the structure of Hfq from the β-proteobacterium Herbaspirillum seropedicae, a diazotroph associated with economically important agricultural crops, was determined by X-ray crystallography and small-angle X-ray scattering (SAXS). Biochemical assays such as exclusion chromatography and RNA-binding by the electrophoretic shift assay (EMSA) confirmed that the purified protein is homogeneous and active. The crystal structure revealed a conserved Sm topology, composed of one N-terminal α-helix followed by five twisted β-strands, and a novel π-π stacking intra-subunit interaction of two histidine residues, absent in other Hfq proteins. Moreover, the calculated ab initio envelope based on small-angle X-ray scattering (SAXS) data agreed with the Hfq crystal structure, suggesting that the protein has the same folding structure in solution. Copyright © 2011 Elsevier B.V. All rights reserved.

Atomistic modelling of scattering data in the Collaborative Computational Project for Small Angle Scattering (CCP-SAS)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perkins, Stephen J.; Wright, David W.; Zhang, Hailiang

2016-10-14

The capabilities of current computer simulations provide a unique opportunity to model small-angle scattering (SAS) data at the atomistic level, and to include other structural constraints ranging from molecular and atomistic energetics to crystallography, electron microscopy and NMR. This extends the capabilities of solution scattering and provides deeper insights into the physics and chemistry of the systems studied. Realizing this potential, however, requires integrating the experimental data with a new generation of modelling software. To achieve this, the CCP-SAS collaboration (http://www.ccpsas.org/) is developing open-source, high-throughput and user-friendly software for the atomistic and coarse-grained molecular modelling of scattering data. Robust state-of-the-artmore » molecular simulation engines and molecular dynamics and Monte Carlo force fields provide constraints to the solution structure inferred from the small-angle scattering data, which incorporates the known physical chemistry of the system. The implementation of this software suite involves a tiered approach in whichGenAppprovides the deployment infrastructure for running applications on both standard and high-performance computing hardware, andSASSIEprovides a workflow framework into which modules can be plugged to prepare structures, carry out simulations, calculate theoretical scattering data and compare results with experimental data.GenAppproduces the accessible web-based front end termedSASSIE-web, andGenAppandSASSIEalso make community SAS codes available. Applications are illustrated by case studies: (i) inter-domain flexibility in two- to six-domain proteins as exemplified by HIV-1 Gag, MASP and ubiquitin; (ii) the hinge conformation in human IgG2 and IgA1 antibodies; (iii) the complex formed between a hexameric protein Hfq and mRNA; and (iv) synthetic `bottlebrush' polymers.« less

Report of the wwPDB Small-Angle Scattering Task Force: data requirements for biomolecular modeling and the PDB.

PubMed

Trewhella, Jill; Hendrickson, Wayne A; Kleywegt, Gerard J; Sali, Andrej; Sato, Mamoru; Schwede, Torsten; Svergun, Dmitri I; Tainer, John A; Westbrook, John; Berman, Helen M

2013-06-04

This report presents the conclusions of the July 12-13, 2012 meeting of the Small-Angle Scattering Task Force of the worldwide Protein Data Bank (wwPDB; Berman et al., 2003) at Rutgers University in New Brunswick, New Jersey. The task force includes experts in small-angle scattering (SAS), crystallography, data archiving, and molecular modeling who met to consider questions regarding the contributions of SAS to modern structural biology. Recognizing there is a rapidly growing community of structural biology researchers acquiring and interpreting SAS data in terms of increasingly sophisticated molecular models, the task force recommends that (1) a global repository is needed that holds standard format X-ray and neutron SAS data that is searchable and freely accessible for download; (2) a standard dictionary is required for definitions of terms for data collection and for managing the SAS data repository; (3) options should be provided for including in the repository SAS-derived shape and atomistic models based on rigid-body refinement against SAS data along with specific information regarding the uniqueness and uncertainty of the model, and the protocol used to obtain it; (4) criteria need to be agreed upon for assessment of the quality of deposited SAS data and the accuracy of SAS-derived models, and the extent to which a given model fits the SAS data; (5) with the increasing diversity of structural biology data and models being generated, archiving options for models derived from diverse data will be required; and (6) thought leaders from the various structural biology disciplines should jointly define what to archive in the PDB and what complementary archives might be needed, taking into account both scientific needs and funding. Copyright © 2013 Elsevier Ltd. All rights reserved.

Atomistic modelling of scattering data in the Collaborative Computational Project for Small Angle Scattering (CCP-SAS).

PubMed

Perkins, Stephen J; Wright, David W; Zhang, Hailiang; Brookes, Emre H; Chen, Jianhan; Irving, Thomas C; Krueger, Susan; Barlow, David J; Edler, Karen J; Scott, David J; Terrill, Nicholas J; King, Stephen M; Butler, Paul D; Curtis, Joseph E

2016-12-01

The capabilities of current computer simulations provide a unique opportunity to model small-angle scattering (SAS) data at the atomistic level, and to include other structural constraints ranging from molecular and atomistic energetics to crystallography, electron microscopy and NMR. This extends the capabilities of solution scattering and provides deeper insights into the physics and chemistry of the systems studied. Realizing this potential, however, requires integrating the experimental data with a new generation of modelling software. To achieve this, the CCP-SAS collaboration (http://www.ccpsas.org/) is developing open-source, high-throughput and user-friendly software for the atomistic and coarse-grained molecular modelling of scattering data. Robust state-of-the-art molecular simulation engines and molecular dynamics and Monte Carlo force fields provide constraints to the solution structure inferred from the small-angle scattering data, which incorporates the known physical chemistry of the system. The implementation of this software suite involves a tiered approach in which GenApp provides the deployment infrastructure for running applications on both standard and high-performance computing hardware, and SASSIE provides a workflow framework into which modules can be plugged to prepare structures, carry out simulations, calculate theoretical scattering data and compare results with experimental data. GenApp produces the accessible web-based front end termed SASSIE-web , and GenApp and SASSIE also make community SAS codes available. Applications are illustrated by case studies: (i) inter-domain flexibility in two- to six-domain proteins as exemplified by HIV-1 Gag, MASP and ubiquitin; (ii) the hinge conformation in human IgG2 and IgA1 antibodies; (iii) the complex formed between a hexameric protein Hfq and mRNA; and (iv) synthetic 'bottlebrush' polymers.

X-ray Crystallography Facility

NASA Technical Reports Server (NTRS)

2000-01-01

Edward Snell, a National Research Council research fellow at NASA's Marshall Space Flight Center (MSFC), prepares a protein crystal for analysis by x-ray crystallography as part of NASA's structural biology program. The small, individual crystals are bombarded with x-rays to produce diffraction patterns, a map of the intensity of the x-rays as they reflect through the crystal.

Crystallographic Determination of Molecular Parameters for K2SiF6: A Physical Chemistry Laboratory Experiment.

ERIC Educational Resources Information Center

Loehlin, James H.; Norton, Alexandra P.

1988-01-01

Describes a crystallography experiment using both diffraction-angle and diffraction-intensity information to determine the lattice constant and a lattice independent molecular parameter, while still employing standard X-ray powder diffraction techniques. Details the method, experimental details, and analysis for this activity. (CW)

Crystallography and Molecular Arrangement of Polymorphic Monolayer J-Aggregates of a Cyanine Dye: Multiangle Polarized Light Fluorescence Optical Microscopy Study.

PubMed

Prokhorov, Valery V; Pozin, Sergey I; Perelygina, Olga M; Mal'tsev, Eugene I

2018-04-24

The molecular orientation in monolayer J-aggregates of 3,3-di(γ-sulfopropyl)-5,5-dichlorotiamonomethinecyanine dye has been precisely estimated using improved linear polarization measurements in the fluorescence microscope in which a multiangle set of polarization data is obtained using sample rotation. The estimated molecular orientation supplemented with the previously established crystallographic constraints based on the analysis of the well-developed two-dimensional J-aggregate shapes unambiguously indicate the staircase type of molecular arrangement for striplike J-aggregates with the staircases oriented along strips. The molecular transition dipoles are inclined at an angle of ∼25° to the strip direction, whereas the characteristic strip vertex angle ∼45° is formed by the [100] and [1-10] directions of the monoclinic unit cell. Measurements of the geometry of partially unwound tubes and their polarization properties support the model of tube formation by close-packed helical winding of flexible monolayer strips. In the tubes, the long molecular axes are oriented at a small angle in the range of 5-15° to the normal to the tube axis providing low bending energy. At a nanoscale, high-resolution atomic force microscopy imaging of J-aggregate monolayers reveals a complex quasi-one-dimensional organization.

Crystallography Open Database (COD): an open-access collection of crystal structures and platform for world-wide collaboration

PubMed Central

Gražulis, Saulius; Daškevič, Adriana; Merkys, Andrius; Chateigner, Daniel; Lutterotti, Luca; Quirós, Miguel; Serebryanaya, Nadezhda R.; Moeck, Peter; Downs, Robert T.; Le Bail, Armel

2012-01-01

Using an open-access distribution model, the Crystallography Open Database (COD, http://www.crystallography.net) collects all known ‘small molecule / small to medium sized unit cell’ crystal structures and makes them available freely on the Internet. As of today, the COD has aggregated ∼150 000 structures, offering basic search capabilities and the possibility to download the whole database, or parts thereof using a variety of standard open communication protocols. A newly developed website provides capabilities for all registered users to deposit published and so far unpublished structures as personal communications or pre-publication depositions. Such a setup enables extension of the COD database by many users simultaneously. This increases the possibilities for growth of the COD database, and is the first step towards establishing a world wide Internet-based collaborative platform dedicated to the collection and curation of structural knowledge. PMID:22070882

Fixed target matrix for femtosecond time-resolved and in situ serial micro-crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mueller, C.; Marx, A.; Epp, S. W.

We present a crystallography chip enabling in situ room temperature crystallography at microfocus synchrotron beamlines and X-ray free-electron laser (X-FEL) sources. Compared to other in situ approaches, we observe extremely low background and high diffraction data quality. The chip design is robust and allows fast and efficient loading of thousands of small crystals. The ability to load a large number of protein crystals, at room temperature and with high efficiency, into prescribed positions enables high throughput automated serial crystallography with microfocus synchrotron beamlines. In addition, we demonstrate the application of this chip for femtosecond time-resolved serial crystallography at the Linacmore » Coherent Light Source (LCLS, Menlo Park, California, USA). As a result, the chip concept enables multiple images to be acquired from each crystal, allowing differential detection of changes in diffraction intensities in order to obtain high signal-to-noise and fully exploit the time resolution capabilities of XFELs.« less

Fixed target matrix for femtosecond time-resolved and in situ serial micro-crystallography

DOE PAGES

Mueller, C.; Marx, A.; Epp, S. W.; ...

2015-08-18

We present a crystallography chip enabling in situ room temperature crystallography at microfocus synchrotron beamlines and X-ray free-electron laser (X-FEL) sources. Compared to other in situ approaches, we observe extremely low background and high diffraction data quality. The chip design is robust and allows fast and efficient loading of thousands of small crystals. The ability to load a large number of protein crystals, at room temperature and with high efficiency, into prescribed positions enables high throughput automated serial crystallography with microfocus synchrotron beamlines. In addition, we demonstrate the application of this chip for femtosecond time-resolved serial crystallography at the Linacmore » Coherent Light Source (LCLS, Menlo Park, California, USA). As a result, the chip concept enables multiple images to be acquired from each crystal, allowing differential detection of changes in diffraction intensities in order to obtain high signal-to-noise and fully exploit the time resolution capabilities of XFELs.« less

FINAL REPORT: DOE CONTRACT NUMBER FG0205ER64026 Biological Neutron Scattering: A Collaboration with the Oak Ridge Center for Structural Molecular Biology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trewhella, Jill

2011-01-12

The overarching goal of this project was to promote applications of small-angle scattering in structural molecular biology by providing model examples of cutting edge applications that demonstrate the unique capabilities and potential of the DOE national user facilities at Oak Ridge, especially the newly commissioned BioSANS. The approach taken was three-fold: (1) to engage in high impact collaborative research projects that would benefit from small-angle neutron scattering to both demonstrate the power of the technique while expanding the potential user community; (2) to provide access to scattering facilities established at the University of Utah to as broad a set ofmore » researchers as possible to increase the expertise in small-angle scattering generally; and (3) to develop new methods and tools for small-angle scattering. To these ends, three major research collaborations were pursued that resulted in a significant body of published work where neutron scattering and contrast variation played a major role. These major collaborations involved studies of protein complexes involved in (1) bacterial transcription regulation and adaptive response (a DOE/BER priority area); (2) regulation of cardiac muscle; and (3) neuronal disorders. In addition, to broaden the impact of the project, smaller collaborative efforts were supported that used either small-angle X-ray or neutron scattering. Finally, the DOE supported facilities at the University of Utah were made available to researchers on a service basis and a number of independent groups took advantage of this opportunity. In all of this work, there was an emphasis on the training of students and post docs in scattering techniques, and a set of publications (a book chapter, a review, and an encyclopedia article) were produced to guide the non-specialist potential user of scattering techniques in successful applications of the techniques. We also developed a suite of user friendly web-based computational tools currently being accessed world-wide by researchers as an aid in neutron scattering data interpretation. In all, these collaborative projects and resulted in 29 original refereed journal articles published between 2005 and 2010 and engaged groups from at least 14 Universities (10 US, 4 international) and 3 National Laboratories (2 US, 1 international). An important final initiative from this project was to begin a process for international community agreement on a set of standards for the publication of biomolecular small-angle scattering data. This initiative is being championed with the International Union of Crystallography and has engaged a number of Journal Editors and is a very important step in the maturing of this now burgeoning field.« less

Validation and extraction of molecular-geometry information from small-molecule databases.

PubMed

Long, Fei; Nicholls, Robert A; Emsley, Paul; Graǽulis, Saulius; Merkys, Andrius; Vaitkus, Antanas; Murshudov, Garib N

2017-02-01

A freely available small-molecule structure database, the Crystallography Open Database (COD), is used for the extraction of molecular-geometry information on small-molecule compounds. The results are used for the generation of new ligand descriptions, which are subsequently used by macromolecular model-building and structure-refinement software. To increase the reliability of the derived data, and therefore the new ligand descriptions, the entries from this database were subjected to very strict validation. The selection criteria made sure that the crystal structures used to derive atom types, bond and angle classes are of sufficiently high quality. Any suspicious entries at a crystal or molecular level were removed from further consideration. The selection criteria included (i) the resolution of the data used for refinement (entries solved at 0.84 Å resolution or higher) and (ii) the structure-solution method (structures must be from a single-crystal experiment and all atoms of generated molecules must have full occupancies), as well as basic sanity checks such as (iii) consistency between the valences and the number of connections between atoms, (iv) acceptable bond-length deviations from the expected values and (v) detection of atomic collisions. The derived atom types and bond classes were then validated using high-order moment-based statistical techniques. The results of the statistical analyses were fed back to fine-tune the atom typing. The developed procedure was repeated four times, resulting in fine-grained atom typing, bond and angle classes. The procedure will be repeated in the future as and when new entries are deposited in the COD. The whole procedure can also be applied to any source of small-molecule structures, including the Cambridge Structural Database and the ZINC database.

The linkage between ribosomal crystallography, metal ions, heteropolytungstates and functional flexibility

PubMed Central

Bashan, Anat; Yonath, Ada

2009-01-01

Crystallography of ribosomes, the universal cell nucleoprotein assemblies facilitating the translation of the genetic-code into proteins, met with severe problems owing to their large size, complex structure, inherent flexibility and high conformational variability. For the case of the small ribosomal subunit, which caused extreme difficulties, post crystallization treatment by minute amounts of a heteropolytungstate cluster allowed structure determination at atomic resolution. This cluster played a dual role in ribosomal crystallography: providing anomalous phasing power and dramatically increased the resolution, by stabilization of a selected functional conformation. Thus, four out of the fourteen clusters that bind to each of the crystallized small subunits are attached to a specific ribosomal protein in a fashion that may control a significant component of the subunit internal flexibility, by “gluing” symmetrical related subunits. Here we highlight basic issues in the relationship between metal ions and macromolecules and present common traits controlling in the interactions between polymetalates and various macromolecules, which may be extended towards the exploitation of polymetalates for therapeutical treatment. PMID:19915655

First Results from a Microfocus X-Ray System for Macromolecular Crystallography

NASA Technical Reports Server (NTRS)

Gubarev, Mikhail; Ciszak, Ewa; Ponomarev, Igor; Gibson, Walter; Joy, Marshall

1999-01-01

The design and performance of a 40 Watt laboratory crystallography system optimized for the structure determination of small protein crystals are described. This system combines a microfocus x-ray generator (40 microns FWHM spot size at a power level of 40 Watts) and a short focal length (F = 2.6 mm) polycapillary collimating optic, and produces a small diameter quasi-parallel x-ray beam. Measurements of x-ray flux, divergence and spectral purity of the resulting x-ray beam are presented. The x-ray flux in a 250 microns diameter aperture produced by the microfocus system is 14.7 times higher .than that from a 3.15 kW rotating anode generator equipped with graphite monochromator. Crystallography data taken with the microfocus system are presented, and indicate that the divergence and spectral purity of the x-ray are sufficient to refine the diffraction data using a standard crystallographic software. Significant additional improvements in flux and beam divergence are possible, and plans for achieving these coals are discussed.

EIGER detector: application in macromolecular crystallography.

PubMed

Casanas, Arnau; Warshamanage, Rangana; Finke, Aaron D; Panepucci, Ezequiel; Olieric, Vincent; Nöll, Anne; Tampé, Robert; Brandstetter, Stefan; Förster, Andreas; Mueller, Marcus; Schulze-Briese, Clemens; Bunk, Oliver; Wang, Meitian

2016-09-01

The development of single-photon-counting detectors, such as the PILATUS, has been a major recent breakthrough in macromolecular crystallography, enabling noise-free detection and novel data-acquisition modes. The new EIGER detector features a pixel size of 75 × 75 µm, frame rates of up to 3000 Hz and a dead time as low as 3.8 µs. An EIGER 1M and EIGER 16M were tested on Swiss Light Source beamlines X10SA and X06SA for their application in macromolecular crystallography. The combination of fast frame rates and a very short dead time allows high-quality data acquisition in a shorter time. The ultrafine ϕ-slicing data-collection method is introduced and validated and its application in finding the optimal rotation angle, a suitable rotation speed and a sufficient X-ray dose are presented. An improvement of the data quality up to slicing at one tenth of the mosaicity has been observed, which is much finer than expected based on previous findings. The influence of key data-collection parameters on data quality is discussed.

In vivo crystallography at X-ray free-electron lasers: the next generation of structural biology?

PubMed

Gallat, François-Xavier; Matsugaki, Naohiro; Coussens, Nathan P; Yagi, Koichiro J; Boudes, Marion; Higashi, Tetsuya; Tsuji, Daisuke; Tatano, Yutaka; Suzuki, Mamoru; Mizohata, Eiichi; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Park, Jaehyun; Song, Changyong; Hatsui, Takaki; Yabashi, Makina; Nango, Eriko; Itoh, Kohji; Coulibaly, Fasséli; Tobe, Stephen; Ramaswamy, S; Stay, Barbara; Iwata, So; Chavas, Leonard M G

2014-07-17

The serendipitous discovery of the spontaneous growth of protein crystals inside cells has opened the field of crystallography to chemically unmodified samples directly available from their natural environment. On the one hand, through in vivo crystallography, protocols for protein crystal preparation can be highly simplified, although the technique suffers from difficulties in sampling, particularly in the extraction of the crystals from the cells partly due to their small sizes. On the other hand, the extremely intense X-ray pulses emerging from X-ray free-electron laser (XFEL) sources, along with the appearance of serial femtosecond crystallography (SFX) is a milestone for radiation damage-free protein structural studies but requires micrometre-size crystals. The combination of SFX with in vivo crystallography has the potential to boost the applicability of these techniques, eventually bringing the field to the point where in vitro sample manipulations will no longer be required, and direct imaging of the crystals from within the cells will be achievable. To fully appreciate the diverse aspects of sample characterization, handling and analysis, SFX experiments at the Japanese SPring-8 angstrom compact free-electron laser were scheduled on various types of in vivo grown crystals. The first experiments have demonstrated the feasibility of the approach and suggest that future in vivo crystallography applications at XFELs will be another alternative to nano-crystallography. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

Structural characterization of Staphylococcus aureus biotin protein ligase and interaction partners: an antibiotic target.

PubMed

Pendini, Nicole R; Yap, Min Y; Traore, D A K; Polyak, Steven W; Cowieson, Nathan P; Abell, Andrew; Booker, Grant W; Wallace, John C; Wilce, Jacqueline A; Wilce, Matthew C J

2013-06-01

The essential metabolic enzyme biotin protein ligase (BPL) is a potential target for the development of new antibiotics required to combat drug-resistant pathogens. Staphylococcus aureus BPL (SaBPL) is a bifunctional protein, possessing both biotin ligase and transcription repressor activities. This positions BPL as a key regulator of several important metabolic pathways. Here, we report the structural analysis of both holo- and apo-forms of SaBPL using X-ray crystallography. We also present small-angle X-ray scattering data of SaBPL in complex with its biotin-carboxyl carrier protein substrate as well as the SaBPL:DNA complex that underlies repression. This has revealed the molecular basis of ligand (biotinyl-5'-AMP) binding and conformational changes associated with catalysis and repressor function. These data provide new information to better understand the bifunctional activities of SaBPL and to inform future strategies for antibiotic discovery. © 2013 The Protein Society.

Structural characterization of Staphylococcus aureus biotin protein ligase and interaction partners: An antibiotic target

PubMed Central

Pendini, Nicole R; Yap, Min Y; Polyak, Steven W; Cowieson, Nathan P; Abell, Andrew; Booker, Grant W; Wallace, John C; Wilce, Jacqueline A; Wilce, Matthew C J

2013-01-01

The essential metabolic enzyme biotin protein ligase (BPL) is a potential target for the development of new antibiotics required to combat drug-resistant pathogens. Staphylococcus aureus BPL (SaBPL) is a bifunctional protein, possessing both biotin ligase and transcription repressor activities. This positions BPL as a key regulator of several important metabolic pathways. Here, we report the structural analysis of both holo- and apo-forms of SaBPL using X-ray crystallography. We also present small-angle X-ray scattering data of SaBPL in complex with its biotin-carboxyl carrier protein substrate as well as the SaBPL:DNA complex that underlies repression. This has revealed the molecular basis of ligand (biotinyl-5′-AMP) binding and conformational changes associated with catalysis and repressor function. These data provide new information to better understand the bifunctional activities of SaBPL and to inform future strategies for antibiotic discovery. PMID:23559560

The structure and dynamics in solution of Cu(I) pseudoazurin from Paracoccus pantotrophus.

PubMed Central

Thompson, G. S.; Leung, Y. C.; Ferguson, S. J.; Radford, S. E.; Redfield, C.

2000-01-01

The solution structure and backbone dynamics of Cu(I) pseudoazurin, a 123 amino acid electron transfer protein from Paracoccus pantotrophus, have been determined using NMR methods. The structure was calculated to high precision, with a backbone RMS deviation for secondary structure elements of 0.35+/-0.06 A, using 1,498 distance and 55 torsion angle constraints. The protein has a double-wound Greek-key fold with two alpha-helices toward its C-terminus, similar to that of its oxidized counterpart determined by X-ray crystallography. Comparison of the Cu(I) solution structure with the X-ray structure of the Cu(II) protein shows only small differences in the positions of some of the secondary structure elements. Order parameters S2, measured for amide nitrogens, indicate that the backbone of the protein is rigid on the picosecond to nanosecond timescale. PMID:10850794

Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sierra, Raymond G.; Gati, Cornelius; Laksmono, Hartawan

We describe a concentric-flow electrokinetic injector for efficiently delivering microcrystals for serial femtosecond X-ray crystallography analysis that enables studies of challenging biological systems in their unadulterated mother liquor. We used the injector to analyze microcrystals of Geobacillus stearothermophilus thermolysin (2.2-Å structure), Thermosynechococcus elongatus photosystem II (<3-Å diffraction) and Thermus thermophilus small ribosomal subunit bound to the antibiotic paromomycin at ambient temperature (3.4-Å structure).

Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II.

PubMed

Sierra, Raymond G; Gati, Cornelius; Laksmono, Hartawan; Dao, E Han; Gul, Sheraz; Fuller, Franklin; Kern, Jan; Chatterjee, Ruchira; Ibrahim, Mohamed; Brewster, Aaron S; Young, Iris D; Michels-Clark, Tara; Aquila, Andrew; Liang, Mengning; Hunter, Mark S; Koglin, Jason E; Boutet, Sébastien; Junco, Elia A; Hayes, Brandon; Bogan, Michael J; Hampton, Christina Y; Puglisi, Elisabetta V; Sauter, Nicholas K; Stan, Claudiu A; Zouni, Athina; Yano, Junko; Yachandra, Vittal K; Soltis, S Michael; Puglisi, Joseph D; DeMirci, Hasan

2016-01-01

We describe a concentric-flow electrokinetic injector for efficiently delivering microcrystals for serial femtosecond X-ray crystallography analysis that enables studies of challenging biological systems in their unadulterated mother liquor. We used the injector to analyze microcrystals of Geobacillus stearothermophilus thermolysin (2.2-Å structure), Thermosynechococcus elongatus photosystem II (<3-Å diffraction) and Thermus thermophilus small ribosomal subunit bound to the antibiotic paromomycin at ambient temperature (3.4-Å structure).

Programming new geometry restraints: Parallelity of atomic groups

DOE PAGES

Sobolev, Oleg V.; Afonine, Pavel V.; Adams, Paul D.; ...

2015-08-01

Improvements in structural biology methods, in particular crystallography and cryo-electron microscopy, have created an increased demand for the refinement of atomic models against low-resolution experimental data. One way to compensate for the lack of high-resolution experimental data is to use a priori information about model geometry that can be utilized in refinement in the form of stereochemical restraints or constraints. Here, the definition and calculation of the restraints that can be imposed on planar atomic groups, in particular the angle between such groups, are described. Detailed derivations of the restraint targets and their gradients are provided so that they canmore » be readily implemented in other contexts. Practical implementations of the restraints, and of associated data structures, in the Computational Crystallography Toolbox( cctbx) are presented.« less

Synthesis, X-ray crystallography, and computational analysis of 1-azafenestranes.

PubMed

Denmark, Scott E; Montgomery, Justin I; Kramps, Laurenz A

2006-09-06

The tandem [4+2]/[3+2] cycloaddition of nitroalkenes has been employed in the synthesis of 1-azafenestranes, molecules of theoretical interest because of planarizing distortion of their central carbon atoms. The synthesis of c,c,c,c-[5.5.5.5]-1-azafenestrane was completed in good yield from a substituted nitrocyclopentene, and its borane adduct was analyzed through X-ray crystallography, which showed a moderate distortion from ideal tetrahedral geometry. The syntheses of two members of the [4.5.5.5] family of 1-azafenestranes are also reported, including one with a trans fusion at a bicyclic ring junction which brings about considerable planarization of one of the central angles (16.8 degrees deviation from tetrahedral geometry). While investigating the [4.5.5.5]-1-azafenestranes, a novel dyotropic rearrangement that converts nitroso acetals into tetracyclic aminals was discovered. Through conformational analysis, a means to prevent this molecular reorganization was formulated and realized experimentally with the use of a bulky vinyl ether in the key [4+2] cycloaddition reaction. Finally, DFT calculations on relative strain energy for the 1-azafenestranes, as well as their predicted central angles, are disclosed.

Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sierra, Raymond G.; Gati, Cornelius; Laksmono, Hartawan

In this paper, we describe a concentric-flow electrokinetic injector for efficiently delivering microcrystals for serial femtosecond X-ray crystallography analysis that enables studies of challenging biological systems in their unadulterated mother liquor. Finally, we used the injector to analyze microcrystals of Geobacillus stearothermophilus thermolysin (2.2-Å structure), Thermosynechococcus elongatus photosystem II (<3-Å diffraction) and Thermus thermophilus small ribosomal subunit bound to the antibiotic paromomycin at ambient temperature (3.4-Å structure).

Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II

DOE PAGES

Sierra, Raymond G.; Gati, Cornelius; Laksmono, Hartawan; ...

2015-11-30

In this paper, we describe a concentric-flow electrokinetic injector for efficiently delivering microcrystals for serial femtosecond X-ray crystallography analysis that enables studies of challenging biological systems in their unadulterated mother liquor. Finally, we used the injector to analyze microcrystals of Geobacillus stearothermophilus thermolysin (2.2-Å structure), Thermosynechococcus elongatus photosystem II (<3-Å diffraction) and Thermus thermophilus small ribosomal subunit bound to the antibiotic paromomycin at ambient temperature (3.4-Å structure).

Dislocations and charge density distributions of {gamma} phase in Ti47.5Al2.5V deformed at room temperature and 400 {degree}C

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, J.; Gao, Y.; Miao, Y.

The observations on quantity and configuration of dislocations by TEM conventional diffraction contrast method as well as the determinations of the electron charge density distributions by the quantitative electron crystallography method in Ti47.5Al2.5V deformed at 400 C and room temperature (R.T.) have been carried out. The metallic bonding between Al-Al or Ti-Ti atom pair along {l_angle}110] and Ti-Ti along {l_angle}112] direction is strengthened; while the metallic bonding between Ti-Al atom pair both along {l_angle}101] and {l_angle}121] direction is weakened at 400 C. The quantities of a/2{l_angle}110], a/2{l_angle}112] and dissociated a{l_angle}101] (a[101]{yields}a/2[1{bar 1}0] + a/3[112] + SISF + a/6[112]) dislocations aremore » increased at 400 C, compared with that at R. T.. The a/2 {l_angle}121] super dislocations have not been seen both at 400 C and R.T.« less

A split-beam probe-pump-probe scheme for femtosecond time resolved protein X-ray crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

van Thor, Jasper J.; Madsen, Anders

In order to exploit the femtosecond pulse duration of X-ray Free-Electron Lasers (XFEL) operating in the hard X-ray regime for ultrafast time-resolved protein crystallography experiments, critical parameters that determine the crystallographic signal-to-noise (I/σI) must be addressed. For single-crystal studies under low absorbed dose conditions, it has been shown that the intrinsic pulse intensity stability as well as mode structure and jitter of this structure, significantly affect the crystallographic signal-to-noise. Here, geometrical parameters are theoretically explored for a three-beam scheme: X-ray probe, optical pump, X-ray probe (or “probe-pump-probe”) which will allow experimental determination of the photo-induced structure factor amplitude differences, ΔF,more » in a ratiometric manner, thereby internally referencing the intensity noise of the XFEL source. In addition to a non-collinear split-beam geometry which separates un-pumped and pumped diffraction patterns on an area detector, applying an additional convergence angle to both beams by focusing leads to integration over mosaic blocks in the case of well-ordered stationary protein crystals. Ray-tracing X-ray diffraction simulations are performed for an example using photoactive yellow protein crystals in order to explore the geometrical design parameters which would be needed. The specifications for an X-ray split and delay instrument that implements both an offset angle and focused beams are discussed, for implementation of a probe-pump-probe scheme at the European XFEL. We discuss possible extension of single crystal studies to serial femtosecond crystallography, particularly in view of the expected X-ray damage and ablation due to the first probe pulse.« less

A split-beam probe-pump-probe scheme for femtosecond time resolved protein X-ray crystallography

DOE PAGES

van Thor, Jasper J.; Madsen, Anders

2015-01-01

In order to exploit the femtosecond pulse duration of X-ray Free-Electron Lasers (XFEL) operating in the hard X-ray regime for ultrafast time-resolved protein crystallography experiments, critical parameters that determine the crystallographic signal-to-noise (I/σI) must be addressed. For single-crystal studies under low absorbed dose conditions, it has been shown that the intrinsic pulse intensity stability as well as mode structure and jitter of this structure, significantly affect the crystallographic signal-to-noise. Here, geometrical parameters are theoretically explored for a three-beam scheme: X-ray probe, optical pump, X-ray probe (or “probe-pump-probe”) which will allow experimental determination of the photo-induced structure factor amplitude differences, ΔF,more » in a ratiometric manner, thereby internally referencing the intensity noise of the XFEL source. In addition to a non-collinear split-beam geometry which separates un-pumped and pumped diffraction patterns on an area detector, applying an additional convergence angle to both beams by focusing leads to integration over mosaic blocks in the case of well-ordered stationary protein crystals. Ray-tracing X-ray diffraction simulations are performed for an example using photoactive yellow protein crystals in order to explore the geometrical design parameters which would be needed. The specifications for an X-ray split and delay instrument that implements both an offset angle and focused beams are discussed, for implementation of a probe-pump-probe scheme at the European XFEL. We discuss possible extension of single crystal studies to serial femtosecond crystallography, particularly in view of the expected X-ray damage and ablation due to the first probe pulse.« less

A split-beam probe-pump-probe scheme for femtosecond time resolved protein X-ray crystallography

PubMed Central

van Thor, Jasper J.; Madsen, Anders

2015-01-01

In order to exploit the femtosecond pulse duration of X-ray Free-Electron Lasers (XFEL) operating in the hard X-ray regime for ultrafast time-resolved protein crystallography experiments, critical parameters that determine the crystallographic signal-to-noise (I/σI) must be addressed. For single-crystal studies under low absorbed dose conditions, it has been shown that the intrinsic pulse intensity stability as well as mode structure and jitter of this structure, significantly affect the crystallographic signal-to-noise. Here, geometrical parameters are theoretically explored for a three-beam scheme: X-ray probe, optical pump, X-ray probe (or “probe-pump-probe”) which will allow experimental determination of the photo-induced structure factor amplitude differences, ΔF, in a ratiometric manner, thereby internally referencing the intensity noise of the XFEL source. In addition to a non-collinear split-beam geometry which separates un-pumped and pumped diffraction patterns on an area detector, applying an additional convergence angle to both beams by focusing leads to integration over mosaic blocks in the case of well-ordered stationary protein crystals. Ray-tracing X-ray diffraction simulations are performed for an example using photoactive yellow protein crystals in order to explore the geometrical design parameters which would be needed. The specifications for an X-ray split and delay instrument that implements both an offset angle and focused beams are discussed, for implementation of a probe-pump-probe scheme at the European XFEL. We discuss possible extension of single crystal studies to serial femtosecond crystallography, particularly in view of the expected X-ray damage and ablation due to the first probe pulse. PMID:26798786

"XANSONS for COD": a new small BOINC project in crystallography

NASA Astrophysics Data System (ADS)

Neverov, Vladislav S.; Khrapov, Nikolay P.

2018-04-01

"XANSONS for COD" (http://xansons4cod.com) is a new BOINC project aimed at creating the open-access database of simulated x-ray and neutron powder diffraction patterns for nanocrystalline phase of materials from the collection of the Crystallography Open Database (COD). The project uses original open-source software XaNSoNS to simulate diffraction patterns on CPU and GPU. This paper describes the scientific problem this project solves, the project's internal structure, its operation principles and organization of the final database.

Magic Angle Spinning NMR of Viruses

PubMed Central

Quinn, Caitlin; Lu, Manman; Suiter, Christopher L.; Hou, Guangjin; Zhang, Huilan; Polenova, Tatyana

2015-01-01

Viruses, relatively simple pathogens, are able to replicate in many living organisms and to adapt to various environments. Conventional atomic-resolution structural biology techniques, X-ray crystallography and solution NMR spectroscopy provided abundant information on the structures of individual proteins and nucleic acids comprising viruses; however, viral assemblies are not amenable to analysis by these techniques because of their large size, insolubility, and inherent lack of long-range order. In this article, we review the recent advances in magic angle spinning NMR spectroscopy that enabled atomic-resolution analysis of structure and dynamics of large viral systems and give examples of several exciting case studies. PMID:25919197

Electron crystallography of ultrathin 3D protein crystals: Atomic model with charges

PubMed Central

Yonekura, Koji; Kato, Kazuyuki; Ogasawara, Mitsuo; Tomita, Masahiro; Toyoshima, Chikashi

2015-01-01

Membrane proteins and macromolecular complexes often yield crystals too small or too thin for even the modern synchrotron X-ray beam. Electron crystallography could provide a powerful means for structure determination with such undersized crystals, as protein atoms diffract electrons four to five orders of magnitude more strongly than they do X-rays. Furthermore, as electron crystallography yields Coulomb potential maps rather than electron density maps, it could provide a unique method to visualize the charged states of amino acid residues and metals. Here we describe an attempt to develop a methodology for electron crystallography of ultrathin (only a few layers thick) 3D protein crystals and present the Coulomb potential maps at 3.4-Å and 3.2-Å resolution, respectively, obtained from Ca2+-ATPase and catalase crystals. These maps demonstrate that it is indeed possible to build atomic models from such crystals and even to determine the charged states of amino acid residues in the Ca2+-binding sites of Ca2+-ATPase and that of the iron atom in the heme in catalase. PMID:25730881

Electron crystallography of ultrathin 3D protein crystals: atomic model with charges.

PubMed

Yonekura, Koji; Kato, Kazuyuki; Ogasawara, Mitsuo; Tomita, Masahiro; Toyoshima, Chikashi

2015-03-17

Membrane proteins and macromolecular complexes often yield crystals too small or too thin for even the modern synchrotron X-ray beam. Electron crystallography could provide a powerful means for structure determination with such undersized crystals, as protein atoms diffract electrons four to five orders of magnitude more strongly than they do X-rays. Furthermore, as electron crystallography yields Coulomb potential maps rather than electron density maps, it could provide a unique method to visualize the charged states of amino acid residues and metals. Here we describe an attempt to develop a methodology for electron crystallography of ultrathin (only a few layers thick) 3D protein crystals and present the Coulomb potential maps at 3.4-Å and 3.2-Å resolution, respectively, obtained from Ca(2+)-ATPase and catalase crystals. These maps demonstrate that it is indeed possible to build atomic models from such crystals and even to determine the charged states of amino acid residues in the Ca(2+)-binding sites of Ca(2+)-ATPase and that of the iron atom in the heme in catalase.

X-ray evaluation of SEM technique for determining the crystallography of echinoid skeletons.

PubMed

Dillaman, R M; Hart, H V

1981-01-01

Coronal plates of the sea urchin Strongylocentrotus purpuratus have a microstructure typified by smooth textured trabeculae. When plates were decorated with calcite crystals each rhombohedron had the same orientation regardless of its location on the plate. When the sample was oriented so that the three edges of the rhombohedron formed equal 120 degrees angles and the three crystal faces appeared to form equal angles with the plane of the photograph, the c-axis of the plate paralleled the electron beam and the three a-axes were 30 degrees counterclockwise from the edges. These a-axes were then related to a plate edge recorded in a low magnification micrograph. Directions of the a-axes of each plate were also measured using a back-reflection Laue x-ray diffraction camera. A comparison of a-axes measured by the two techniques showed an average difference of 3 degrees, indicating that decorated crystal grew in crystallographic continuity with the plate. Assuming this relationship remains constant, the decoration technique appears to be an accurate and efficient method for evaluating the crystallography of echinoid skeletal units. Analysis of a polar plot of a-axes for 11 plates indicated that the a-axes were not randomly oriented; however, definitive relationships must await more extensive investigations.

Lipidic cubic phase serial millisecond crystallography using synchrotron radiation

PubMed Central

Nogly, Przemyslaw; James, Daniel; Wang, Dingjie; White, Thomas A.; Zatsepin, Nadia; Shilova, Anastasya; Nelson, Garrett; Liu, Haiguang; Johansson, Linda; Heymann, Michael; Jaeger, Kathrin; Metz, Markus; Wickstrand, Cecilia; Wu, Wenting; Båth, Petra; Berntsen, Peter; Oberthuer, Dominik; Panneels, Valerie; Cherezov, Vadim; Chapman, Henry; Schertler, Gebhard; Neutze, Richard; Spence, John; Moraes, Isabel; Burghammer, Manfred; Standfuss, Joerg; Weierstall, Uwe

2015-01-01

Lipidic cubic phases (LCPs) have emerged as successful matrixes for the crystallization of membrane proteins. Moreover, the viscous LCP also provides a highly effective delivery medium for serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs). Here, the adaptation of this technology to perform serial millisecond crystallography (SMX) at more widely available synchrotron microfocus beamlines is described. Compared with conventional microcrystallography, LCP-SMX eliminates the need for difficult handling of individual crystals and allows for data collection at room temperature. The technology is demonstrated by solving a structure of the light-driven proton-pump bacteriorhodopsin (bR) at a resolution of 2.4 Å. The room-temperature structure of bR is very similar to previous cryogenic structures but shows small yet distinct differences in the retinal ligand and proton-transfer pathway. PMID:25866654

Suite of three protein crystallography beamlines with single superconducting bend magnet as the source.

PubMed

MacDowell, Alastair A; Celestre, Rich S; Howells, Malcolm; McKinney, Wayne; Krupnick, James; Cambie, Daniella; Domning, Edward E; Duarte, Robert M; Kelez, Nicholas; Plate, David W; Cork, Carl W; Earnest, Thomas N; Dickert, Jeffery; Meigs, George; Ralston, Corie; Holton, James M; Alber, Tom; Berger, James M; Agard, David A; Padmore, Howard A

2004-11-01

At the Advanced Light Source, three protein crystallography beamlines have been built that use as a source one of the three 6 T single-pole superconducting bending magnets (superbends) that were recently installed in the ring. The use of such single-pole superconducting bend magnets enables the development of a hard X-ray program on a relatively low-energy 1.9 GeV ring without taking up insertion-device straight sections. The source is of relatively low power but, owing to the small electron beam emittance, it has high brightness. X-ray optics are required to preserve the brightness and to match the illumination requirements for protein crystallography. This was achieved by means of a collimating premirror bent to a plane parabola, a double-crystal monochromator followed by a toroidal mirror that focuses in the horizontal direction with a 2:1 demagnification. This optical arrangement partially balances aberrations from the collimating and toroidal mirrors such that a tight focused spot size is achieved. The optical properties of the beamline are an excellent match to those required by the small protein crystals that are typically measured. The design and performance of these new beamlines are described.

A new small-angle X-ray scattering set-up on the crystallography beamline I711 at MAX-lab.

PubMed

Knaapila, M; Svensson, C; Barauskas, J; Zackrisson, M; Nielsen, S S; Toft, K N; Vestergaard, B; Arleth, L; Olsson, U; Pedersen, J S; Cerenius, Y

2009-07-01

A small-angle X-ray scattering (SAXS) set-up has recently been developed at beamline I711 at the MAX II storage ring in Lund (Sweden). An overview of the required modifications is presented here together with a number of application examples. The accessible q range in a SAXS experiment is 0.009-0.3 A(-1) for the standard set-up but depends on the sample-to-detector distance, detector offset, beamstop size and wavelength. The SAXS camera has been designed to have a low background and has three collinear slit sets for collimating the incident beam. The standard beam size is about 0.37 mm x 0.37 mm (full width at half-maximum) at the sample position, with a flux of 4 x 10(10) photons s(-1) and lambda = 1.1 A. The vacuum is of the order of 0.05 mbar in the unbroken beam path from the first slits until the exit window in front of the detector. A large sample chamber with a number of lead-throughs allows different sample environments to be mounted. This station is used for measurements on weakly scattering proteins in solutions and also for colloids, polymers and other nanoscale structures. A special application supported by the beamline is the effort to establish a micro-fluidic sample environment for structural analysis of samples that are only available in limited quantities. Overall, this work demonstrates how a cost-effective SAXS station can be constructed on a multipurpose beamline.

Defining NADH-Driven Allostery Regulating Apoptosis-Inducing Factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brosey, Chris A.; Ho, Chris; Long, Winnie Z.

Apoptosis-inducing factor (AIF) is critical for mitochondrial respiratory complex biogenesis and for mediating necroptotic parthanatos; these functions are seemingly regulated by enigmatic allosteric switching driven by NADH charge-transfer complex (CTC) formation. In this paper, we define molecular pathways linking AIF's active site to allosteric switching regions by characterizing dimer-permissive mutants using small-angle X-ray scattering (SAXS) and crystallography and by probing AIF-CTC communication networks using molecular dynamics simulations. Collective results identify two pathways propagating allostery from the CTC active site: (1) active-site H454 links to S480 of AIF's central β-strand to modulate a hydrophobic border at the dimerization interface, and (2)more » an interaction network links AIF's FAD cofactor, central β-strand, and Cβ-clasp whereby R529 reorientation initiates C-loop release during CTC formation. Finally, this knowledge of AIF allostery and its flavoswitch mechanism provides a foundation for biologically understanding and biomedically controlling its participation in mitochondrial homeostasis and cell death.« less

Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism.

PubMed

Liberato, Marcelo V; Silveira, Rodrigo L; Prates, Érica T; de Araujo, Evandro A; Pellegrini, Vanessa O A; Camilo, Cesar M; Kadowaki, Marco A; Neto, Mario de O; Popov, Alexander; Skaf, Munir S; Polikarpov, Igor

2016-04-01

Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.

Mechanistic Understanding of Tungsten Oxide In-Plane Nanostructure Growth via Sequential Infiltration Synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Jae Jin; Suh, Hyo Seon; Zhou, Chun

Tungsten oxide (WO3-x) nanostructures with hexagonal in-plane arrangements were fabricated by sequential infiltration synthesis (SIS), using the selective interaction of gas phase precursors with functional groups in one domain of a block copolymer (BCP) self-assembled template. Such structures are highly desirable for various practical applications and as model systems for fundamental studies. The nanostructures were characterized by cross-sectional scanning electron microscopy, grazing-incidence small/wide-angle X-ray scattering (GISAXS/GIWAXS), and X-ray absorption near edge structure (XANES) measurements at each stage during the SIS process and subsequent thermal treatments, to provide a comprehensive picture of their evolution in morphology, crystallography and electronic structure. Inmore » particular, we discuss the critical role of SIS Al2O3 seeds toward modifying the chemical affinity and free volume in a polymer for subsequent infiltration of gas phase precursors. The insights into SIS growth obtained from this study are valuable to the design and fabrication of a wide range of targeted nanostructures.« less

Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism

NASA Astrophysics Data System (ADS)

Liberato, Marcelo V.; Silveira, Rodrigo L.; Prates, Érica T.; de Araujo, Evandro A.; Pellegrini, Vanessa O. A.; Camilo, Cesar M.; Kadowaki, Marco A.; Neto, Mario De O.; Popov, Alexander; Skaf, Munir S.; Polikarpov, Igor

2016-04-01

Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.

Defining NADH-Driven Allostery Regulating Apoptosis-Inducing Factor

DOE PAGES

Brosey, Chris A.; Ho, Chris; Long, Winnie Z.; ...

2016-11-03

Apoptosis-inducing factor (AIF) is critical for mitochondrial respiratory complex biogenesis and for mediating necroptotic parthanatos; these functions are seemingly regulated by enigmatic allosteric switching driven by NADH charge-transfer complex (CTC) formation. In this paper, we define molecular pathways linking AIF's active site to allosteric switching regions by characterizing dimer-permissive mutants using small-angle X-ray scattering (SAXS) and crystallography and by probing AIF-CTC communication networks using molecular dynamics simulations. Collective results identify two pathways propagating allostery from the CTC active site: (1) active-site H454 links to S480 of AIF's central β-strand to modulate a hydrophobic border at the dimerization interface, and (2)more » an interaction network links AIF's FAD cofactor, central β-strand, and Cβ-clasp whereby R529 reorientation initiates C-loop release during CTC formation. Finally, this knowledge of AIF allostery and its flavoswitch mechanism provides a foundation for biologically understanding and biomedically controlling its participation in mitochondrial homeostasis and cell death.« less

Structure-Function Perturbation and Dissociation of Tetrameric Urate Oxidase by High Hydrostatic Pressure

PubMed Central

Girard, Eric; Marchal, Stéphane; Perez, Javier; Finet, Stéphanie; Kahn, Richard; Fourme, Roger; Marassio, Guillaume; Dhaussy, Anne-Claire; Prangé, Thierry; Giffard, Marion; Dulin, Fabienne; Bonneté, Françoise; Lange, Reinhard; Abraini, Jacques H.; Mezouar, Mohamed; Colloc'h, Nathalie

2010-01-01

Abstract Structure-function relationships in the tetrameric enzyme urate oxidase were investigated using pressure perturbation. As the active sites are located at the interfaces between monomers, enzyme activity is directly related to the integrity of the tetramer. The effect of hydrostatic pressure on the enzyme was investigated by x-ray crystallography, small-angle x-ray scattering, and fluorescence spectroscopy. Enzymatic activity was also measured under pressure and after decompression. A global model, consistent with all measurements, discloses structural and functional details of the pressure-induced dissociation of the tetramer. Before dissociating, the pressurized protein adopts a conformational substate characterized by an expansion of its substrate binding pocket at the expense of a large neighboring hydrophobic cavity. This substate should be adopted by the enzyme during its catalytic mechanism, where the active site has to accommodate larger intermediates and product. The approach, combining several high-pressure techniques, offers a new (to our knowledge) means of exploring structural and functional properties of transient states relevant to protein mechanisms. PMID:20483346

Full-length model of the human galectin-4 and insights into dynamics of inter-domain communication

NASA Astrophysics Data System (ADS)

Rustiguel, Joane K.; Soares, Ricardo O. S.; Meisburger, Steve P.; Davis, Katherine M.; Malzbender, Kristina L.; Ando, Nozomi; Dias-Baruffi, Marcelo; Nonato, Maria Cristina

2016-09-01

Galectins are proteins involved in diverse cellular contexts due to their capacity to decipher and respond to the information encoded by β-galactoside sugars. In particular, human galectin-4, normally expressed in the healthy gastrointestinal tract, displays differential expression in cancerous tissues and is considered a potential drug target for liver and lung cancer. Galectin-4 is a tandem-repeat galectin characterized by two carbohydrate recognition domains connected by a linker-peptide. Despite their relevance to cell function and pathogenesis, structural characterization of full-length tandem-repeat galectins has remained elusive. Here, we investigate galectin-4 using X-ray crystallography, small- and wide-angle X-ray scattering, molecular modelling, molecular dynamics simulations, and differential scanning fluorimetry assays and describe for the first time a structural model for human galectin-4. Our results provide insight into the structural role of the linker-peptide and shed light on the dynamic characteristics of the mechanism of carbohydrate recognition among tandem-repeat galectins.

A Consensus Model of Human Apolipoprotein A-I in its Monomeric and Lipid-free State

PubMed Central

Melchior, John T.; Walker, Ryan G.; Cooke, Allison L.; Morris, Jamie; Castleberry, Mark; Thompson, Thomas B.; Jones, Martin K.; Song, Hyun D.; Rye, Kerry-Anne; Oda, Mike N.; Sorci-Thomas, Mary G.; Thomas, Michael J.; Heinecke, Jay W.; Mei, Xiaohu; Atkinson, David; Segrest, Jere P.; Lund-Katz, Sissel; Phillips, Michael C.; Davidson, W. Sean

2017-01-01

Apolipoprotein (apo)A-I is an organizing scaffold protein that is critical to high density lipoprotein (HDL) structure and metabolism, likely mediating many of its cardioprotective properties. However, HDL biogenesis is poorly understood as lipid-free apoA-I has been notoriously resistant to high resolution structural study. Published models from low resolution techniques share certain features but vary considerably in shape and secondary structure. To tackle this central issue in lipoprotein biology, we assembled an unprecedented team of lipoprotein structural biologists and set out to build a consensus model of monomeric lipid-free human apoA-I. Combining novel and published cross-link constraints, small angle X-ray scattering (SAXS), hydrogen-deuterium exchange (H-DX) and crystallography data, we propose a time averaged model consistent with much of the experimental data published over the last 40 years. The model provides a long sought platform for understanding and testing details of HDL biogenesis, structure and function. PMID:29131142

Neutron Nucleic Acid Crystallography.

PubMed

Chatake, Toshiyuki

2016-01-01

The hydration shells surrounding nucleic acids and hydrogen-bonding networks involving water molecules and nucleic acids are essential interactions for the structural stability and function of nucleic acids. Water molecules in the hydration shells influence various conformations of DNA and RNA by specific hydrogen-bonding networks, which often contribute to the chemical reactivity and molecular recognition of nucleic acids. However, X-ray crystallography could not provide a complete description of structural information with respect to hydrogen bonds. Indeed, X-ray crystallography is a powerful tool for determining the locations of water molecules, i.e., the location of the oxygen atom of H2O; however, it is very difficult to determine the orientation of the water molecules, i.e., the orientation of the two hydrogen atoms of H2O, because X-ray scattering from the hydrogen atom is very small.Neutron crystallography is a specialized tool for determining the positions of hydrogen atoms. Neutrons are not diffracted by electrons, but are diffracted by atomic nuclei; accordingly, neutron scattering lengths of hydrogen and its isotopes are comparable to those of non-hydrogen atoms. Therefore, neutron crystallography can determine both of the locations and orientations of water molecules. This chapter describes the current status of neutron nucleic acid crystallographic research as well as the basic principles of neutron diffraction experiments performed on nucleic acid crystals: materials, crystallization, diffraction experiments, and structure determination.

New developments in crystallography: exploring its technology, methods and scope in the molecular biosciences.

PubMed

Helliwell, John R

2017-08-31

Since the Protein Data Bank (PDB) was founded in 1971, there are now over 120,000 depositions, the majority of which are from X-ray crystallography and 90% of those made use of synchrotron beamlines. At the Cambridge Structure Database (CSD), founded in 1965, there are more than 800,000 'small molecule' crystal structure depositions and a very large number of those are relevant in the biosciences as ligands or cofactors. The technology for crystal structure analysis is still developing rapidly both at synchrotrons and in home labs. Determination of the details of the hydrogen atoms in biological macromolecules is well served using neutrons as probe. Large multi-macromolecular complexes cause major challenges to crystallization; electrons as probes offer unique advantages here. Methods developments naturally accompany technology change, mainly incremental but some, such as the tuneability, intensity and collimation of synchrotron radiation, have effected radical changes in capability of biological crystallography. In the past few years, the X-ray laser has taken X-ray crystallography measurement times into the femtosecond range. In terms of applications many new discoveries have been made in the molecular biosciences. The scope of crystallographic techniques is indeed very wide. As examples, new insights into chemical catalysis of enzymes and relating ligand bound structures to thermodynamics have been gained but predictive power is seen as not yet achieved. Metal complexes are also an emerging theme for biomedicine applications. Our studies of coloration of live and cooked lobsters proved to be an unexpected favourite with the public and schoolchildren. More generally, public understanding of the biosciences and crystallography's role within the field have been greatly enhanced by the United Nations International Year of Crystallography coordinated by the International Union of Crystallography. This topical review describes each of these areas along with illustrative results to document the scope of each methodology. © 2017 The Author(s).

Diffraction-based study of fatigue crack initiation and propagation in aerospace aluminum alloys

NASA Astrophysics Data System (ADS)

Gupta, Vipul K.

The crack initiation sites and microstructure-sensitive growth of small fatigue cracks are experimentally characterized in two precipitation-hardened aluminum alloys, 7075-T651 and 7050-T7451, stressed in ambient temperature moist-air (warm-humid) and -50°C dry N2 (cold-dry) environmental conditions. Backscattered electron imaging (BSE) and energy dispersive spectroscopy (EDS) of the fracture surfaces showed that Fe-Cu rich constituent particle clusters are the most common initiation sites within both alloys stressed in either environment. The crack growth within each alloy, on average, was observed to be slowed in the cold-dry environment than in the warm-humid environment, but only at longer crack lengths. Although no overwhelming effects of grain boundaries and grain orientations on small-crack growth were observed, crack growth data showed local fluctuations within individual grains. These observations are understood as crack propagation through the underlying substructure at the crack surface and frequent interaction with low/high-angle grain and subgrain boundaries, during cyclic loading, and, are further attributed to periodic changes in crack propagation path and multiple occurrences of crack-branching observed in the current study. SEM-based stereology in combination with electron backscattered diffraction (EBSD) established fatigue crack surface crystallography within the region from ˜1 to 50 mum of crack initiating particle clusters. Fatigue crack facets were parallel to a wide variety of crystallographic planes, with pole orientations distributed broadly across the irreducible stereographic triangle between the {001} and {101}-poles within both warm-humid and cold-dry environments. The results indicate environmentally affected fatigue cracking in both cases, given the similarity between the observed morphology and crystallography with that of a variety of aerospace aluminum alloys cracked in the presence of moist-air. There was no evidence of crystallographic {111} slip-plane cracking typical of the Stage I crack growth mode observed in single crystals and high purity polycrystals of face centered cubic metals, and which has presently been assumed for the present materials within fatigue crack initiation models. Rather, the facets tend to have near-Mode I spatial orientation, which is another indicator of the importance of environmentally affected fatigue damage. The results provide a physical basis to develop microstructurally-based next generation multi-stage fatigue (MSF) models that should include a new crack decohesion criteria based upon environmental fatigue cracking mechanisms. EBSD study of small-cracks in alloy 7050-T7451, stressed in warm-humid environment, showed that crack-path orientation changes and crack-branching occurred at both low/high-angle grain and subgrain boundaries. Single surface trace analysis suggests that the crack-path differs substantially from crystallographic slip-planes. EBSD-based observations of small-crack propagation through subgrain structure, either formed by cyclic plastic strain accumulation or pre-existing (typical of unrecrystallized grain structure in the present materials), suggest that subgrain structure plays a crucial role in small fatigue crack propagation. As mentioned earlier, local fluctuations in small-crack growth rates appear to be caused by frequent interaction with subgrain boundaries, and multiple occurrences of crack-branching and crack-path orientation changes at low/high-angle grain and subgrain boundaries. The aforementioned deviation from low-index {001}/{101}-planes and the occurrence of high-index cracking planes observed by EBSD/Stereology, in this study and others, are interpreted as trans-subgranular decohesion or inter-subgranular cracking, due to trapped hydrogen. In summary, the results provide a firmer experimental foundation for, and clearer understanding of, the mechanisms of environmental fatigue cracking of aluminum alloys, especially the role of inter-subgranular cracking, which had previously been advanced based upon fracture surface observations alone.

Sample mounts for microcrystal crystallography

NASA Technical Reports Server (NTRS)

Thorne, Robert E. (Inventor); Kmetko, Jan (Inventor); Stum, Zachary (Inventor); O'Neill, Kevin (Inventor)

2007-01-01

Sample mounts (10) for mounting microcrystals of biological macromolecules for X-ray crystallography are prepared by using patterned thin polyimide films (12) that have curvature imparted thereto, for example, by being attached to a curved outer surface of a small metal rod (16). The patterned film (12) preferably includes a tapered tip end (24) for holding a crystal. Preferably, a small sample aperture is disposed in the film for reception of the crystal. A second, larger aperture can also be provided that is connected to the sample aperture by a drainage channel, allowing removal of excess liquid and easier manipulation in viscous solutions. The curvature imparted to the film (12) increases the film's rigidity and allows a convenient scoop-like action for retrieving crystals. The polyimide contributes minimally to background and absorption, and can be treated to obtain desired hydrophobicity or hydrophilicity.

Sample mounts for microcrystal crystallography

NASA Technical Reports Server (NTRS)

O'Neill, Kevin (Inventor); Kmetko, Jan (Inventor); Thorne, Robert E. (Inventor); Stum, Zachary (Inventor)

2009-01-01

Sample mounts (10) for mounting microcrystals of biological macromolecules for X-ray crystallography are prepared by using patterned thin polyimide films (12) that have curvature imparted thereto, for example, by being attached to a curved outer surface of a small metal rod (16). The patterned film (12) preferably includes a tip end (24) for holding a crystal. Preferably, a small sample aperture is disposed in the film for reception of the crystal. A second, larger aperture can also be provided that is connected to the sample aperture by a drainage channel, allowing removal of excess liquid and easier manipulation in viscous solutions. The curvature imparted to the film (12) increases the film's rigidity and allows a convenient scoop-like action for retrieving crystals. The polyimide contributes minimally to background and absorption, and can be treated to obtain desired hydrophobicity or hydrophilicity.

Remote Control by Inter-Enzyme Allostery: A Novel Paradigm for Regulation of the Shikimate Pathway.

PubMed

Munack, Steffi; Roderer, Kathrin; Ökvist, Mats; Kamarauskaite, Jurate; Sasso, Severin; van Eerde, André; Kast, Peter; Krengel, Ute

2016-03-27

DAHP synthase and chorismate mutase catalyze key steps in the shikimate biosynthetic pathway en route to aromatic amino acids. In Mycobacterium tuberculosis, chorismate mutase (MtCM; Rv0948c), located at the branch point toward phenylalanine and tyrosine, has poor activity on its own. However, it is efficiently activated by the first enzyme of the pathway, DAHP synthase (MtDS; Rv2178c), through formation of a non-covalent MtCM-MtDS complex. Here, we show how MtDS serves as an allosteric platform for feedback regulation of both enzymes, using X-ray crystallography, small-angle X-ray scattering, size-exclusion chromatography, and multi-angle light scattering. Crystal structures of the fully inhibited MtDS and the allosterically down-regulated MtCM-MtDS complex, solved at 2.8 and 2.7Å, respectively, reveal how effector binding at the internal MtDS subunit interfaces regulates the activity of MtDS and MtCM. While binding of all three metabolic end products to MtDS shuts down the entire pathway, the binding of phenylalanine jointly with tyrosine releases MtCM from the MtCM-MtDS complex, hence suppressing MtCM activation by 'inter-enzyme allostery'. This elegant regulatory principle, invoking a transient allosteric enzyme interaction, seems to be driven by dynamics and is likely a general strategy used by nature. Copyright © 2016 Elsevier Ltd. All rights reserved.

Biological Activity Predictions and Hydrogen Bonding Analysis in Quinolines

NASA Astrophysics Data System (ADS)

Gupta, Palvi; Kamni

The paper has been designed to make a comprehensive review of a particular series of organic molecular assembly in the form of compendium. An overview of general description of fifteen quinoline derivatives has been given. The biological activity spectra of quinoline derivatives have been correlated on structure activity relationships base which provides the different Pa (possibility of activity) and Pi (possibility of inactivity) values. Expositions of the role of intermolecular interactions in the identified derivatives have been discussed with the standard distance and angle cut-off criteria criteria as proposed by Desiraju and Steiner (1999) in an International monogram on crystallography. Distance-angle scatter plots for intermolecular interactions are presented for a better understanding of the packing interactions which exist in quinoline derivatives.

Single-crystal X-ray diffraction and NMR crystallography of a 1:1 cocrystal of dithianon and pyrimethanil.

PubMed

Pöppler, Ann Christin; Corlett, Emily K; Pearce, Harriet; Seymour, Mark P; Reid, Matthew; Montgomery, Mark G; Brown, Steven P

2017-03-01

A single-crystal X-ray diffraction structure of a 1:1 cocrystal of two fungicides, namely dithianon (DI) and pyrimethanil (PM), is reported [systematic name: 5,10-dioxo-5H,10H-naphtho[2,3-b][1,4]dithiine-2,3-dicarbonitrile-4,6-dimethyl-N-phenylpyrimidin-2-amine (1/1), C 14 H 4 N 2 O 2 S 2 ·C 12 H 13 N 2 ]. Following an NMR crystallography approach, experimental solid-state magic angle spinning (MAS) NMR spectra are presented together with GIPAW (gauge-including projector augmented wave) calculations of NMR chemical shieldings. Specifically, experimental 1 H and 13 C chemical shifts are determined from two-dimensional 1 H- 13 C MAS NMR correlation spectra recorded with short and longer contact times so as to probe one-bond C-H connectivities and longer-range C...H proximities, whereas H...H proximities are identified in a 1 H double-quantum (DQ) MAS NMR spectrum. The performing of separate GIPAW calculations for the full periodic crystal structure and for isolated molecules allows the determination of the change in chemical shift upon going from an isolated molecule to the full crystal structure. For the 1 H NMR chemical shifts, changes of 3.6 and 2.0 ppm correspond to intermolecular N-H...O and C-H...O hydrogen bonding, while changes of -2.7 and -1.5 ppm are due to ring current effects associated with C-H...π interactions. Even though there is a close intermolecular S...O distance of 3.10 Å, it is of note that the molecule-to-crystal chemical shifts for the involved sulfur or oxygen nuclei are small.

Combination of X-ray crystallography, SAXS and DEER to obtain the structure of the FnIII-3, 4 domains of integrin α6β4

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alonso-García, Noelia; García-Rubio, Inés; Academia General Militar, Carretera de Huesca s/n, 50090 Zaragoza

The structure of the FnIII-3, 4 region of integrin β4 was solved using a hybrid approach that combines crystallographic structures, SAXS, DEER and molecular modelling. The structure helps in understanding how integrin β4 might bind to other hemidesmosomal proteins and mediate signalling. Integrin α6β4 is a major component of hemidesmosomes that mediate the stable anchorage of epithelial cells to the underlying basement membrane. Integrin α6β4 has also been implicated in cell proliferation and migration and in carcinoma progression. The third and fourth fibronectin type III domains (FnIII-3, 4) of integrin β4 mediate binding to the hemidesmosomal proteins BPAG1e and BPAG2,more » and participate in signalling. Here, it is demonstrated that X-ray crystallography, small-angle X-ray scattering and double electron–electron resonance (DEER) complement each other to solve the structure of the FnIII-3, 4 region. The crystal structures of the individual FnIII-3 and FnIII-4 domains were solved and the relative arrangement of the FnIII domains was elucidated by combining DEER with site-directed spin labelling. Multiple structures of the interdomain linker were modelled by Monte Carlo methods complying with DEER constraints, and the final structures were selected against experimental scattering data. FnIII-3, 4 has a compact and cambered flat structure with an evolutionary conserved surface that is likely to correspond to a protein-interaction site. Finally, this hybrid method is of general application for the study of other macromolecules and complexes.« less

X-ray scattering data and structural genomics

NASA Astrophysics Data System (ADS)

Doniach, Sebastian

2003-03-01

High throughput structural genomics has the ambitious goal of determining the structure of all, or a very large number of protein folds using the high-resolution techniques of protein crystallography and NMR. However, the program is facing significant bottlenecks in reaching this goal, which include problems of protein expression and crystallization. In this talk, some preliminary results on how the low-resolution technique of small-angle X-ray solution scattering (SAXS) can help ameliorate some of these bottlenecks will be presented. One of the most significant bottlenecks arises from the difficulty of crystallizing integral membrane proteins, where only a handful of structures are available compared to thousands of structures for soluble proteins. By 3-dimensional reconstruction from SAXS data, the size and shape of detergent-solubilized integral membrane proteins can be characterized. This information can then be used to classify membrane proteins which constitute some 25% of all genomes. SAXS may also be used to study the dependence of interparticle interference scattering on solvent conditions so that regions of the protein solution phase diagram which favor crystallization can be elucidated. As a further application, SAXS may be used to provide physical constraints on computational methods for protein structure prediction based on primary sequence information. This in turn can help in identifying structural homologs of a given protein, which can then give clues to its function. D. Walther, F. Cohen and S. Doniach. "Reconstruction of low resolution three-dimensional density maps from one-dimensional small angle x-ray scattering data for biomolecules." J. Appl. Cryst. 33(2):350-363 (2000). Protein structure prediction constrained by solution X-ray scattering data and structural homology identification Zheng WJ, Doniach S JOURNAL OF MOLECULAR BIOLOGY , v. 316(#1) pp. 173-187 FEB 8, 2002

Structural insights into the interaction of IL-33 with its receptors.

PubMed

Liu, Xi; Hammel, Michal; He, Yanfeng; Tainer, John A; Jeng, U-Ser; Zhang, Linqi; Wang, Shuying; Wang, Xinquan

2013-09-10

Interleukin (IL)-33 is an important member of the IL-1 family that has pleiotropic activities in innate and adaptive immune responses in host defense and disease. It signals through its ligand-binding primary receptor ST2 and IL-1 receptor accessory protein (IL-1RAcP), both of which are members of the IL-1 receptor family. To clarify the interaction of IL-33 with its receptors, we determined the crystal structure of IL-33 in complex with the ectodomain of ST2 at a resolution of 3.27 Å. Coupled with structure-based mutagenesis and binding assay, the structural results define the molecular mechanism by which ST2 specifically recognizes IL-33. Structural comparison with other ligand-receptor complexes in the IL-1 family indicates that surface-charge complementarity is critical in determining ligand-binding specificity of IL-1 primary receptors. Combined crystallography and small-angle X-ray-scattering studies reveal that ST2 possesses hinge flexibility between the D3 domain and D1D2 module, whereas IL-1RAcP exhibits a rigid conformation in the unbound state in solution. The molecular flexibility of ST2 provides structural insights into domain-level conformational change of IL-1 primary receptors upon ligand binding, and the rigidity of IL-1RAcP explains its inability to bind ligands directly. The solution architecture of IL-33-ST2-IL-1RAcP complex from small-angle X-ray-scattering analysis resembles IL-1β-IL-1RII-IL-1RAcP and IL-1β-IL-1RI-IL-1RAcP crystal structures. The collective results confer IL-33 structure-function relationships, supporting and extending a general model for ligand-receptor assembly and activation in the IL-1 family.

Residue-Specific Side-Chain Polymorphisms via Particle Belief Propagation.

PubMed

Ghoraie, Laleh Soltan; Burkowski, Forbes; Li, Shuai Cheng; Zhu, Mu

2014-01-01

Protein side chains populate diverse conformational ensembles in crystals. Despite much evidence that there is widespread conformational polymorphism in protein side chains, most of the X-ray crystallography data are modeled by single conformations in the Protein Data Bank. The ability to extract or to predict these conformational polymorphisms is of crucial importance, as it facilitates deeper understanding of protein dynamics and functionality. In this paper, we describe a computational strategy capable of predicting side-chain polymorphisms. Our approach extends a particular class of algorithms for side-chain prediction by modeling the side-chain dihedral angles more appropriately as continuous rather than discrete variables. Employing a new inferential technique known as particle belief propagation, we predict residue-specific distributions that encode information about side-chain polymorphisms. Our predicted polymorphisms are in relatively close agreement with results from a state-of-the-art approach based on X-ray crystallography data, which characterizes the conformational polymorphisms of side chains using electron density information, and has successfully discovered previously unmodeled conformations.

Structural study of piracetam polymorphs and cocrystals: crystallography redetermination and quantum mechanics calculations.

PubMed

Tilborg, Anaëlle; Jacquemin, Denis; Norberg, Bernadette; Perpète, Eric; Michaux, Catherine; Wouters, Johan

2011-12-01

Pharmaceutical compounds are mostly developed as solid dosage forms containing a single-crystal form. It means that the selection of a particular crystal state for a given molecule is an important step for further clinical outlooks. In this context, piracetam, a pharmaceutical molecule known since the sixties for its nootropic properties, is considered in the present work. This molecule is analyzed using several experimental and theoretical approaches. First, the conformational space of the molecule has been systematically explored by performing a quantum mechanics scan of the two most relevant dihedral angles of the lateral chain. The predicted stable conformations have been compared to all the reported experimental geometries retrieved from the Cambridge Structural Database (CSD) covering polymorphs and cocrystals structures. In parallel, different batches of powders have been recrystallized. Under specific conditions, single crystals of polymorph (III) of piracetam have been obtained, an outcome confirmed by crystallographic analysis. © 2011 International Union of Crystallography. Printed in Singapore – all rights reserved.

Crystallography of some lunar plagioclases

USGS Publications Warehouse

Stewart, D.B.; Appleman, D.E.; Huebner, J.S.; Clark, J.R.

1970-01-01

Crystals of calcic bytownite from type B rocks have space group U with c ??? 14 angstroms. Bytownite crystals from type A rocks are more sodic and have space group C1, c ??? 7 angstroms. Cell parameters of eight bulk feldspar separates from crystalline rocks indicate that the range of angle gamma is about 23 times the standard error of measurement, and its value might be useful for estimation of composition. Cell parameters of seven ilmenites are close to those of pure FeTiO3.

Fast fluorescence techniques for crystallography beamlines

PubMed Central

Stepanov, Sergey; Hilgart, Mark; Yoder, Derek W.; Makarov, Oleg; Becker, Michael; Sanishvili, Ruslan; Ogata, Craig M.; Venugopalan, Nagarajan; Aragão, David; Caffrey, Martin; Smith, Janet L.; Fischetti, Robert F.

2011-01-01

This paper reports on several developments of X-ray fluorescence techniques for macromolecular crystallography recently implemented at the National Institute of General Medical Sciences and National Cancer Institute beamlines at the Advanced Photon Source. These include (i) three-band on-the-fly energy scanning around absorption edges with adaptive positioning of the fine-step band calculated from a coarse pass; (ii) on-the-fly X-ray fluorescence rastering over rectangular domains for locating small and invisible crystals with a shuttle-scanning option for increased speed; (iii) fluorescence rastering over user-specified multi-segmented polygons; and (iv) automatic signal optimization for reduced radiation damage of samples. PMID:21808424

Direct methods in protein crystallography.

PubMed

Karle, J

1989-11-01

It is pointed out that the 'direct methods' of phase determination for small-structure crystallography do not have immediate applicability to macromolecular structures. The term 'direct methods in macromolecular crystallography' is suggested to categorize a spectrum of approaches to macromolecular structure determination in which the analyses are characterized by the use of two-phase and higher-order-phase invariants. The evaluation of the invariants is generally obtained by the use of heavy-atom techniques. The results of a number of the more recent algebraic and probabilistic studies involving isomorphous replacement and anomalous dispersion thus become valid subjects for discussion here. These studies are described and suggestions are also presented concerning future applicability. Additional discussion concerns the special techniques of filtering, the use of non-crystallographic symmetry, some features of maximum entropy and attempts to apply phase-determining formulas to the refinement of macromolecular structure. It is noted that, in addition to the continuing remarkable progress in macromolecular crystallography based on the traditional applications of isomorphous replacement and anomalous dispersion, recent valuable advances have been made in the application of non-crystallographic symmetry, in particular, to virus structures and in applications of filtering. Good progress has also been reported in the application of exact linear algebra to multiple-wavelength anomalous-dispersion investigations of structures containing anomalous scatterers of only moderate scattering power.

An expanded allosteric network in PTP1B by multitemperature crystallography, fragment screening, and covalent tethering.

PubMed

Keedy, Daniel A; Hill, Zachary B; Biel, Justin T; Kang, Emily; Rettenmaier, T Justin; Brandao-Neto, Jose; Pearce, Nicholas M; von Delft, Frank; Wells, James A; Fraser, James S

2018-06-07

Allostery is an inherent feature of proteins, but it remains challenging to reveal the mechanisms by which allosteric signals propagate. A clearer understanding of this intrinsic circuitry would afford new opportunities to modulate protein function. Here we have identified allosteric sites in protein tyrosine phosphatase 1B (PTP1B) by combining multiple-temperature X-ray crystallography experiments and structure determination from hundreds of individual small-molecule fragment soaks. New modeling approaches reveal 'hidden' low-occupancy conformational states for protein and ligands. Our results converge on allosteric sites that are conformationally coupled to the active-site WPD loop and are hotspots for fragment binding. Targeting one of these sites with covalently tethered molecules or mutations allosterically inhibits enzyme activity. Overall, this work demonstrates how the ensemble nature of macromolecular structure, revealed here by multitemperature crystallography, can elucidate allosteric mechanisms and open new doors for long-range control of protein function. © 2018, Keedy et al.

X-Ray Crystallography as a Tool to Determine Three-Dimensional Structures of Commercial Enzymes Subjected to Treatment in Pressurized Fluids.

PubMed

Feiten, Mirian Cristina; Di Luccio, Marco; Santos, Karine F; de Oliveira, Débora; Oliveira, J Vladimir

2017-06-01

The study of enzyme function often involves a multi-disciplinary approach. Several techniques are documented in the literature towards determining secondary and tertiary structures of enzymes, and X-ray crystallography is the most explored technique for obtaining three-dimensional structures of proteins. Knowledge of three-dimensional structures is essential to understand reaction mechanisms at the atomic level. Additionally, structures can be used to modulate or improve functional activity of enzymes by the production of small molecules that act as substrates/cofactors or by engineering selected mutants with enhanced biological activity. This paper presentes a short overview on how to streamline sample preparation for crystallographic studies of treated enzymes. We additionally revise recent developments on the effects of pressurized fluid treatment on activity and stability of commercial enzymes. Future directions and perspectives on the the role of crystallography as a tool to access the molecular mechanisms underlying enzymatic activity modulation upon treatment in pressurized fluids are also addressed.

X-rays in the Cryo-EM Era: Structural Biology’s Dynamic Future

PubMed Central

Shoemaker, Susannah C.; Ando, Nozomi

2018-01-01

Over the past several years, single-particle cryo-electron microscopy (cryo-EM) has emerged as a leading method for elucidating macromolecular structures at near-atomic resolution, rivaling even the established technique of X-ray crystallography. Cryo-EM is now able to probe proteins as small as hemoglobin (64 kDa), while avoiding the crystallization bottleneck entirely. The remarkable success of cryo-EM has called into question the continuing relevance of X-ray methods, particularly crystallography. To say that the future of structural biology is either cryo-EM or crystallography, however, would be misguided. Crystallography remains better suited to yield precise atomic coordinates of macromolecules under a few hundred kDa in size, while the ability to probe larger, potentially more disordered assemblies is a distinct advantage of cryo-EM. Likewise, crystallography is better equipped to provide high-resolution dynamic information as a function of time, temperature, pressure, and other perturbations, whereas cryo-EM offers increasing insight into conformational and energy landscapes, particularly as algorithms to deconvolute conformational heterogeneity become more advanced. Ultimately, the future of both techniques depends on how their individual strengths are utilized to tackle questions on the frontiers of structural biology. Structure determination is just one piece of a much larger puzzle: a central challenge of modern structural biology is to relate structural information to biological function. In this perspective, we share insight from several leaders in the field and examine the unique and complementary ways in which X-ray methods and cryo-EM can shape the future of structural biology. PMID:29227642

The C-terminal region of translesion synthesis DNA polymerase η is partially unstructured and has high conformational flexibility

PubMed Central

Powers, Kyle T; Washington, M Todd

2018-01-01

Abstract Eukaryotic DNA polymerase η catalyzes translesion synthesis of thymine dimers and 8-oxoguanines. It is comprised of a polymerase domain and a C-terminal region, both of which are required for its biological function. The C-terminal region mediates interactions with proliferating cell nuclear antigen (PCNA) and other translesion synthesis proteins such as Rev1. This region contains a ubiquitin-binding/zinc-binding (UBZ) motif and a PCNA-interacting protein (PIP) motif. Currently little structural information is available for this region of polymerase η. Using a combination of approaches—including genetic complementation assays, X-ray crystallography, Langevin dynamics simulations, and small-angle X-ray scattering—we show that the C-terminal region is partially unstructured and has high conformational flexibility. This implies that the C-terminal region acts as a flexible tether linking the polymerase domain to PCNA thereby increasing its local concentration. Such tethering would facilitate the sampling of translesion synthesis polymerases to ensure that the most appropriate one is selected to bypass the lesion. PMID:29385534

Nucleotide-induced asymmetry within ATPase activator ring drives σ54-RNAP interaction and ATP hydrolysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sysoeva, Tatyana A.; Chowdhury, Saikat; Guo, Liang

2013-12-10

It is largely unknown how the typical homomeric ring geometry of ATPases associated with various cellular activities enables them to perform mechanical work. Small-angle solution X-ray scattering, crystallography, and electron microscopy (EM) reconstructions revealed that partial ATP occupancy caused the heptameric closed ring of the bacterial enhancer-binding protein (bEBP) NtrC1 to rearrange into a hexameric split ring of striking asymmetry. The highly conserved and functionally crucial GAFTGA loops responsible for interacting with σ54–RNA polymerase formed a spiral staircase. We propose that splitting of the ensemble directs ATP hydrolysis within the oligomer, and the ring's asymmetry guides interaction between ATPase andmore » the complex of σ54 and promoter DNA. Similarity between the structure of the transcriptional activator NtrC1 and those of distantly related helicases Rho and E1 reveals a general mechanism in homomeric ATPases whereby complex allostery within the ring geometry forms asymmetric functional states that allow these biological motors to exert directional forces on their target macromolecules.« less

Structural Aspects of N-Glycosylations and the C-terminal Region in Human Glypican-1*

PubMed Central

Awad, Wael; Adamczyk, Barbara; Örnros, Jessica; Karlsson, Niclas G.; Mani, Katrin; Logan, Derek T.

2015-01-01

Glypicans are multifunctional cell surface proteoglycans involved in several important cellular signaling pathways. Glypican-1 (Gpc1) is the predominant heparan sulfate proteoglycan in the developing and adult human brain. The two N-linked glycans and the C-terminal domain that attach the core protein to the cell membrane are not resolved in the Gpc1 crystal structure. Therefore, we have studied Gpc1 using crystallography, small angle x-ray scattering, and chromatographic approaches to elucidate the composition, structure, and function of the N-glycans and the C terminus and also the topology of Gpc1 with respect to the membrane. The C terminus is shown to be highly flexible in solution, but it orients the core protein transverse to the membrane, directing a surface evolutionarily conserved in Gpc1 orthologs toward the membrane, where it may interact with signaling molecules and/or membrane receptors on the cell surface, or even the enzymes involved in heparan sulfate substitution in the Golgi apparatus. Furthermore, the N-glycans are shown to extend the protein stability and lifetime by protection against proteolysis and aggregation. PMID:26203194

Broadly neutralizing epitopes in the Plasmodium vivax vaccine candidate Duffy Binding Protein

DOE PAGES

Chen, Edwin; Salinas, Nichole D.; Huang, Yining; ...

2016-05-18

Plasmodium vivax Duffy Binding Protein (PvDBP) is the most promising vaccine candidate for P. vivax malaria. The polymorphic nature of PvDBP induces strain-specific immune responses, however, and the epitopes of broadly neutralizing antibodies are unknown. These features hamper the rational design of potent DBP-based vaccines and necessitate the identification of globally conserved epitopes. Using X-ray crystallography, small-angle X-ray scattering, hydrogen-deuterium exchange mass spectrometry, and mutational mapping, we have defined epitopes for three inhibitory mAbs (mAbs 2D10, 2H2, and 2C6) and one noninhibitory mAb (3D10) that engage DBP. These studies expand the currently known inhibitory epitope repertoire by establishing protective motifsmore » in subdomain three outside the receptor-binding and dimerization residues of DBP, and introduce globally conserved protective targets. All of the epitopes are highly conserved among DBP alleles. In conclusion, the identification of broadly conserved epitopes of inhibitory antibodies provides critical motifs that should be retained in the next generation of potent vaccines for P. vivax malaria.« less

Broadly neutralizing epitopes in the Plasmodium vivax vaccine candidate Duffy Binding Protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Edwin; Salinas, Nichole D.; Huang, Yining

Plasmodium vivax Duffy Binding Protein (PvDBP) is the most promising vaccine candidate for P. vivax malaria. The polymorphic nature of PvDBP induces strain-specific immune responses, however, and the epitopes of broadly neutralizing antibodies are unknown. These features hamper the rational design of potent DBP-based vaccines and necessitate the identification of globally conserved epitopes. Using X-ray crystallography, small-angle X-ray scattering, hydrogen-deuterium exchange mass spectrometry, and mutational mapping, we have defined epitopes for three inhibitory mAbs (mAbs 2D10, 2H2, and 2C6) and one noninhibitory mAb (3D10) that engage DBP. These studies expand the currently known inhibitory epitope repertoire by establishing protective motifsmore » in subdomain three outside the receptor-binding and dimerization residues of DBP, and introduce globally conserved protective targets. All of the epitopes are highly conserved among DBP alleles. In conclusion, the identification of broadly conserved epitopes of inhibitory antibodies provides critical motifs that should be retained in the next generation of potent vaccines for P. vivax malaria.« less

Periodic magnetic field as a polarized and focusing thermal neutron spectrometer and monochromator.

PubMed

Cremer, J T; Williams, D L; Fuller, M J; Gary, C K; Piestrup, M A; Pantell, R H; Feinstein, J; Flocchini, R G; Boussoufi, M; Egbert, H P; Kloh, M D; Walker, R B

2010-01-01

A novel periodic magnetic field (PMF) optic is shown to act as a prism, lens, and polarizer for neutrons and particles with a magnetic dipole moment. The PMF has a two-dimensional field in the axial direction of neutron propagation. The PMF alternating magnetic field polarity provides strong gradients that cause separation of neutrons by wavelength axially and by spin state transversely. The spin-up neutrons exit the PMF with their magnetic spins aligned parallel to the PMF magnetic field, and are deflected upward and line focus at a fixed vertical height, proportional to the PMF period, at a downstream focal distance that increases with neutron energy. The PMF has no attenuation by absorption or scatter, as with material prisms or crystal monochromators. Embodiments of the PMF include neutron spectrometer or monochromator, and applications include neutron small angle scattering, crystallography, residual stress analysis, cross section measurements, and reflectometry. Presented are theory, experimental results, computer simulation, applications of the PMF, and comparison of its performance to Stern-Gerlach gradient devices and compound material and magnetic refractive prisms.

Periodic magnetic field as a polarized and focusing thermal neutron spectrometer and monochromator

PubMed Central

Cremer, J. T.; Williams, D. L.; Fuller, M. J.; Gary, C. K.; Piestrup, M. A.; Pantell, R. H.; Feinstein, J.; Flocchini, R. G.; Boussoufi, M.; Egbert, H. P.; Kloh, M. D.; Walker, R. B.

2010-01-01

A novel periodic magnetic field (PMF) optic is shown to act as a prism, lens, and polarizer for neutrons and particles with a magnetic dipole moment. The PMF has a two-dimensional field in the axial direction of neutron propagation. The PMF alternating magnetic field polarity provides strong gradients that cause separation of neutrons by wavelength axially and by spin state transversely. The spin-up neutrons exit the PMF with their magnetic spins aligned parallel to the PMF magnetic field, and are deflected upward and line focus at a fixed vertical height, proportional to the PMF period, at a downstream focal distance that increases with neutron energy. The PMF has no attenuation by absorption or scatter, as with material prisms or crystal monochromators. Embodiments of the PMF include neutron spectrometer or monochromator, and applications include neutron small angle scattering, crystallography, residual stress analysis, cross section measurements, and reflectometry. Presented are theory, experimental results, computer simulation, applications of the PMF, and comparison of its performance to Stern–Gerlach gradient devices and compound material and magnetic refractive prisms. PMID:20113108

Three-dimensional Structures of Carotenoids by X-ray Crystallography

NASA Astrophysics Data System (ADS)

Helliwell, Madeleine

The number of crystal structures of carotenoid molecules and carotenoid derivatives deposited in the Cambridge Crystallographic Data Centre [1] is still relatively small, but has increased compared with the previous survey [2]. The list is summarized in Table 1.

Semiautomated model building for RNA crystallography using a directed rotameric approach.

PubMed

Keating, Kevin S; Pyle, Anna Marie

2010-05-04

Structured RNA molecules play essential roles in a variety of cellular processes; however, crystallographic studies of such RNA molecules present a large number of challenges. One notable complication arises from the low resolutions typical of RNA crystallography, which results in electron density maps that are imprecise and difficult to interpret. This problem is exacerbated by the lack of computational tools for RNA modeling, as many of the techniques commonly used in protein crystallography have no equivalents for RNA structure. This leads to difficulty and errors in the model building process, particularly in modeling of the RNA backbone, which is highly error prone due to the large number of variable torsion angles per nucleotide. To address this, we have developed a method for accurately building the RNA backbone into maps of intermediate or low resolution. This method is semiautomated, as it requires a crystallographer to first locate phosphates and bases in the electron density map. After this initial trace of the molecule, however, an accurate backbone structure can be built without further user intervention. To accomplish this, backbone conformers are first predicted using RNA pseudotorsions and the base-phosphate perpendicular distance. Detailed backbone coordinates are then calculated to conform both to the predicted conformer and to the previously located phosphates and bases. This technique is shown to produce accurate backbone structure even when starting from imprecise phosphate and base coordinates. A program implementing this methodology is currently available, and a plugin for the Coot model building program is under development.

Sub-atomic resolution X-ray crystallography and neutron crystallography: promise, challenges and potential.

PubMed

Blakeley, Matthew P; Hasnain, Samar S; Antonyuk, Svetlana V

2015-07-01

The International Year of Crystallography saw the number of macromolecular structures deposited in the Protein Data Bank cross the 100000 mark, with more than 90000 of these provided by X-ray crystallography. The number of X-ray structures determined to sub-atomic resolution (i.e. ≤1 Å) has passed 600 and this is likely to continue to grow rapidly with diffraction-limited synchrotron radiation sources such as MAX-IV (Sweden) and Sirius (Brazil) under construction. A dozen X-ray structures have been deposited to ultra-high resolution (i.e. ≤0.7 Å), for which precise electron density can be exploited to obtain charge density and provide information on the bonding character of catalytic or electron transfer sites. Although the development of neutron macromolecular crystallography over the years has been far less pronounced, and its application much less widespread, the availability of new and improved instrumentation, combined with dedicated deuteration facilities, are beginning to transform the field. Of the 83 macromolecular structures deposited with neutron diffraction data, more than half (49/83, 59%) were released since 2010. Sub-mm(3) crystals are now regularly being used for data collection, structures have been determined to atomic resolution for a few small proteins, and much larger unit-cell systems (cell edges >100 Å) are being successfully studied. While some details relating to H-atom positions are tractable with X-ray crystallography at sub-atomic resolution, the mobility of certain H atoms precludes them from being located. In addition, highly polarized H atoms and protons (H(+)) remain invisible with X-rays. Moreover, the majority of X-ray structures are determined from cryo-cooled crystals at 100 K, and, although radiation damage can be strongly controlled, especially since the advent of shutterless fast detectors, and by using limited doses and crystal translation at micro-focus beams, radiation damage can still take place. Neutron crystallography therefore remains the only approach where diffraction data can be collected at room temperature without radiation damage issues and the only approach to locate mobile or highly polarized H atoms and protons. Here a review of the current status of sub-atomic X-ray and neutron macromolecular crystallography is given and future prospects for combined approaches are outlined. New results from two metalloproteins, copper nitrite reductase and cytochrome c', are also included, which illustrate the type of information that can be obtained from sub-atomic-resolution (∼0.8 Å) X-ray structures, while also highlighting the need for complementary neutron studies that can provide details of H atoms not provided by X-ray crystallography.

Solvent minimization induces preferential orientation and crystal clustering in serial micro-crystallography on micro-meshes, in situ plates and on a movable crystal conveyor belt

DOE Office of Scientific and Technical Information (OSTI.GOV)

Soares, Alexei S.; Mullen, Jeffrey D.; Parekh, Ruchi M.

X-ray diffraction data were obtained at the National Synchrotron Light Source from insulin and lysozyme crystals that were densely deposited on three types of surfaces suitable for serial micro-crystallography: MiTeGen MicroMeshes™, Greiner Bio-One Ltdin situmicro-plates, and a moving kapton crystal conveyor belt that is used to deliver crystals directly into the X-ray beam. 6° wedges of data were taken from ~100 crystals mounted on each material, and these individual data sets were merged to form nine complete data sets (six from insulin crystals and three from lysozyme crystals). Insulin crystals have a parallelepiped habit with an extended flat face thatmore » preferentially aligned with the mounting surfaces, impacting the data collection strategy and the design of the serial crystallography apparatus. Lysozyme crystals had a cuboidal habit and showed no preferential orientation. Preferential orientation occluded regions of reciprocal space when the X-ray beam was incident normal to the data-collection medium surface, requiring a second pass of data collection with the apparatus inclined away from the orthogonal. In addition, crystals measuring less than 20 µm were observed to clump together into clusters of crystals. Clustering required that the X-ray beam be adjusted to match the crystal size to prevent overlapping diffraction patterns. No additional problems were encountered with the serial crystallography strategy of combining small randomly oriented wedges of data from a large number of specimens. Lastly, high-quality data able to support a realistic molecular replacement solution were readily obtained from both crystal types using all three serial crystallography strategies.« less

Solvent minimization induces preferential orientation and crystal clustering in serial micro-crystallography on micro-meshes, in situ plates and on a movable crystal conveyor belt.

PubMed

Soares, Alexei S; Mullen, Jeffrey D; Parekh, Ruchi M; McCarthy, Grace S; Roessler, Christian G; Jackimowicz, Rick; Skinner, John M; Orville, Allen M; Allaire, Marc; Sweet, Robert M

2014-11-01

X-ray diffraction data were obtained at the National Synchrotron Light Source from insulin and lysozyme crystals that were densely deposited on three types of surfaces suitable for serial micro-crystallography: MiTeGen MicroMeshes™, Greiner Bio-One Ltd in situ micro-plates, and a moving kapton crystal conveyor belt that is used to deliver crystals directly into the X-ray beam. 6° wedges of data were taken from ∼100 crystals mounted on each material, and these individual data sets were merged to form nine complete data sets (six from insulin crystals and three from lysozyme crystals). Insulin crystals have a parallelepiped habit with an extended flat face that preferentially aligned with the mounting surfaces, impacting the data collection strategy and the design of the serial crystallography apparatus. Lysozyme crystals had a cuboidal habit and showed no preferential orientation. Preferential orientation occluded regions of reciprocal space when the X-ray beam was incident normal to the data-collection medium surface, requiring a second pass of data collection with the apparatus inclined away from the orthogonal. In addition, crystals measuring less than 20 µm were observed to clump together into clusters of crystals. Clustering required that the X-ray beam be adjusted to match the crystal size to prevent overlapping diffraction patterns. No additional problems were encountered with the serial crystallography strategy of combining small randomly oriented wedges of data from a large number of specimens. High-quality data able to support a realistic molecular replacement solution were readily obtained from both crystal types using all three serial crystallography strategies.

Solvent minimization induces preferential orientation and crystal clustering in serial micro-crystallography on micro-meshes, in situ plates and on a movable crystal conveyor belt

DOE PAGES

Soares, Alexei S.; Mullen, Jeffrey D.; Parekh, Ruchi M.; ...

2014-10-09

X-ray diffraction data were obtained at the National Synchrotron Light Source from insulin and lysozyme crystals that were densely deposited on three types of surfaces suitable for serial micro-crystallography: MiTeGen MicroMeshes™, Greiner Bio-One Ltdin situmicro-plates, and a moving kapton crystal conveyor belt that is used to deliver crystals directly into the X-ray beam. 6° wedges of data were taken from ~100 crystals mounted on each material, and these individual data sets were merged to form nine complete data sets (six from insulin crystals and three from lysozyme crystals). Insulin crystals have a parallelepiped habit with an extended flat face thatmore » preferentially aligned with the mounting surfaces, impacting the data collection strategy and the design of the serial crystallography apparatus. Lysozyme crystals had a cuboidal habit and showed no preferential orientation. Preferential orientation occluded regions of reciprocal space when the X-ray beam was incident normal to the data-collection medium surface, requiring a second pass of data collection with the apparatus inclined away from the orthogonal. In addition, crystals measuring less than 20 µm were observed to clump together into clusters of crystals. Clustering required that the X-ray beam be adjusted to match the crystal size to prevent overlapping diffraction patterns. No additional problems were encountered with the serial crystallography strategy of combining small randomly oriented wedges of data from a large number of specimens. Lastly, high-quality data able to support a realistic molecular replacement solution were readily obtained from both crystal types using all three serial crystallography strategies.« less

Solvent minimization induces preferential orientation and crystal clustering in serial micro-crystallography on micro-meshes, in situ plates and on a movable crystal conveyor belt

PubMed Central

Soares, Alexei S.; Mullen, Jeffrey D.; Parekh, Ruchi M.; McCarthy, Grace S.; Roessler, Christian G.; Jackimowicz, Rick; Skinner, John M.; Orville, Allen M.; Allaire, Marc; Sweet, Robert M.

2014-01-01

X-ray diffraction data were obtained at the National Synchrotron Light Source from insulin and lysozyme crystals that were densely deposited on three types of surfaces suitable for serial micro-crystallography: MiTeGen MicroMeshes™, Greiner Bio-One Ltd in situ micro-plates, and a moving kapton crystal conveyor belt that is used to deliver crystals directly into the X-ray beam. 6° wedges of data were taken from ∼100 crystals mounted on each material, and these individual data sets were merged to form nine complete data sets (six from insulin crystals and three from lysozyme crystals). Insulin crystals have a parallelepiped habit with an extended flat face that preferentially aligned with the mounting surfaces, impacting the data collection strategy and the design of the serial crystallography apparatus. Lysozyme crystals had a cuboidal habit and showed no preferential orientation. Preferential orientation occluded regions of reciprocal space when the X-ray beam was incident normal to the data-collection medium surface, requiring a second pass of data collection with the apparatus inclined away from the orthogonal. In addition, crystals measuring less than 20 µm were observed to clump together into clusters of crystals. Clustering required that the X-ray beam be adjusted to match the crystal size to prevent overlapping diffraction patterns. No additional problems were encountered with the serial crystallography strategy of combining small randomly oriented wedges of data from a large number of specimens. High-quality data able to support a realistic molecular replacement solution were readily obtained from both crystal types using all three serial crystallography strategies. PMID:25343789

The use of a mini-κ goniometer head in macromolecular crystallography diffraction experiments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brockhauser, Sandor; UJF–EMBL–CNRS UMI 3265, 6 Rue Jules Horowitz, 38043 Grenoble; Ravelli, Raimond B. G.

2013-07-01

Hardware and software solutions for MX data-collection strategies using the EMBL/ESRF miniaturized multi-axis goniometer head are presented. Most macromolecular crystallography (MX) diffraction experiments at synchrotrons use a single-axis goniometer. This markedly contrasts with small-molecule crystallography, in which the majority of the diffraction data are collected using multi-axis goniometers. A novel miniaturized κ-goniometer head, the MK3, has been developed to allow macromolecular crystals to be aligned. It is available on the majority of the structural biology beamlines at the ESRF, as well as elsewhere. In addition, the Strategy for the Alignment of Crystals (STAC) software package has been developed to facilitatemore » the use of the MK3 and other similar devices. Use of the MK3 and STAC is streamlined by their incorporation into online analysis tools such as EDNA. The current use of STAC and MK3 on the MX beamlines at the ESRF is discussed. It is shown that the alignment of macromolecular crystals can result in improved diffraction data quality compared with data obtained from randomly aligned crystals.« less

Application of two-dimensional crystallography and image processing to atomic resolution Z-contrast images.

PubMed

Morgan, David G; Ramasse, Quentin M; Browning, Nigel D

2009-06-01

Zone axis images recorded using high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM or Z-contrast imaging) reveal the atomic structure with a resolution that is defined by the probe size of the microscope. In most cases, the full images contain many sub-images of the crystal unit cell and/or interface structure. Thanks to the repetitive nature of these images, it is possible to apply standard image processing techniques that have been developed for the electron crystallography of biological macromolecules and have been used widely in other fields of electron microscopy for both organic and inorganic materials. These methods can be used to enhance the signal-to-noise present in the original images, to remove distortions in the images that arise from either the instrumentation or the specimen itself and to quantify properties of the material in ways that are difficult without such data processing. In this paper, we describe briefly the theory behind these image processing techniques and demonstrate them for aberration-corrected, high-resolution HAADF-STEM images of Si(46) clathrates developed for hydrogen storage.

Thermally induced lamellar reorganization and thickening in spherical poly (L-lactic acid) crystalsome

NASA Astrophysics Data System (ADS)

Staub, Mark; Qi, Hao; Li, Christopher

Understanding fundamental aspects of spherical crystals is important for a variety of applications such as encapsulation and drug delivery. The curved nature of these crystals gives rise to differences in key crystallographic concepts such as grain boundaries and defect formation when compared to flat crystals. This curved crystallography is difficult to study experimentally, especially at the nanoscale. Our group has recently shown how an oil in water miniemulsion can be used to direct the crystallization of poly (L-lactic acid) (PLLA) at a curved liquid/liquid interface. This produces nanosized, polymer single-crystal-like capsules termed crystalsomes with increased stability and mechanical properties compared with non-crystalline counterparts. This system will serve as our model for examining spherical crystallography. In this work, combined wide angle X-ray diffraction, Atomic force microscopy, and differential scanning calorimetry is employed to examine how the curved interface effects crystal thickening and reorganization compared to flat PLLA crystals. The influence of degree of curvature on these processes is also studied by examining crystalsomes with differing diameters.

Crystallography: past and present

NASA Astrophysics Data System (ADS)

Hodeau, J.-L.; Guinebretiere, R.

2007-12-01

In the 19th century, crystallography referred to the study of crystal shapes. Such studies by Haüy and Bravais allowed the establishment of important hypotheses such as (i) “les molécules intégrantes qui sont censées être les plus petits solides que l’on puisse extraire d’un minéral” [1], (ii) the definition of the crystal lattice and (iii) “le cristal est clivable parallèlement à deux ou trois formes cristallines” [2]. This morphological crystallography defined a crystal like “a chemically homogeneous solid, wholly or partly bounded by natural planes that intersect at predetermined angles” [3]. It described the main symmetry elements and operations, nomenclatures of different crystal forms and also the theory of twinning. A breakthrough appeared in 1912 with the use of X-rays by M. von Laue and W.H. and W.L. Bragg. This experimental development allowed the determination of the atomic content of each unit cell constituting the crystal and defined a crystal as “any solid in which an atomic pattern is repeated periodically in three dimensions, that is, any solid that “diffracts” an incident X-ray beam” [3]. Mathematical tools like the Patterson methods, the direct methods, were developed. The way for solving crystalline structure was opened first for simple compounds and at that time crystallography was associated mainly with perfect crystals. In the fifties, crystallographers already had most apparatus and fundamental methods at their disposal; however, we had to wait for the development of computers to see the full use of these tools. Furthermore the development of new sources of neutrons, electrons and synchrotron X-rays allowed the studies of complex compounds like large macromolecules in biology. Nowadays, one of the new frontiers for crystallographers is to relate the crystal structure to its physical-chemical-biological properties, this means that an accurate structural determination is needed to focus on a selective part of the structure (chemical order, anisotropy, charge transfer, magnetic order) versus an external parameter like temperature, pressure, magnetic or electric field. Modern crystallography is also extended to the study of very small crystals, powders, ill-ordered or non-crystallized materials. Thus presently, crystallography is concerned with any solid that “scatters” an incident beam. Nevertheless, as quoted by A. Guinier, “the problems facing crystallographers have only changed, ... new ones have appeared which require reflection and imagination, ... and which in turn may still bring much joy to all those who like crystallography” [4]. Such developments open up crystallography to modern materials like artificial ones and nanostructures with low- and/or multi-scaled-periodicities and/or extremely small “crystal size” and to materials of the “real world”, with mixtures of phases and/or amorphous contribution and/or defects, a common characteristic of ancient materials analysed in patrimonial research. In our contribution we will show by selected examples that these improvements were allowed (i) by the use of powerful sources, apparatus and detectors which allow micro-diffraction, in-situ diffraction, spectroscopy, resonant scattering, inelastic scattering, coherent scattering, (ii) by the development of methods like diffraction anomalous fine structure (DAFS), pair distribution function (PDF), simulated annealing, single object reconstruction, (iii) by combination of scattering and spectroscopy and by combination of scattering and microscopy. Such combination of different approaches is very efficient and, as said by H. Curien at the IUCr Bordeaux Congress in 1990, “in crystallography, there is a constant alternation between the crystal space and its associated reciprocal space, ... the alternation between experiment and model building is another feature of crystallography activity ..., the crystallographer relies both on his computer and on his diffractometer ..., all these dualistic complementarities are responsible for the fact that crystallographers tend to waver between the description of atomic structures and the explanation of macroscopic properties of solid matter” [5].

Synthesis of N-(β-D-glycuronopyranosyl)alkanamides and 1-(β-D-glycuronopyranosyl)-4-phenyl-[1,2,3]-triazoles as N-glycoprotein linkage region analogs: examination of the effect of C5 substituent on the N-glycosidic torsion (ΦN) based on X-ray crystallography.

PubMed

Mathiselvam, Manoharan; Loganathan, Duraikkannu; Varghese, Babu

2013-10-18

The torsion angle around the N-glycoprotein linkage region (GlcNAc-Asn) is an important factor for presenting sugar on the cell surface which is crucial for many biological processes. Earlier studies using model and analogs showed that this important torsion angle is greatly influenced by substitutions in the sugar part. In the present work, uronic acid alkanamides and triazole derivatives have been designed and synthesized as newer analogs of N-glycoprotein linkage region to understand the influence of the carboxylic group on linkage region torsion as well as on molecular packing. Crystal structure of N-(β-D-galacturonopyranosyl)acetamide is solved with the space group of P22121. Comparison of the torsion angle and molecular packing of this compound with N-(β-D-galactopyranosyl)acetamide showed that changing the C6-hydoxymethyl group to the carboxylic acid group has minimum influence on the N-glycosidic torsion angle, ΦN and significant influence on the molecular packing. Copyright © 2013 Elsevier Ltd. All rights reserved.

Fifteen years of the Protein Crystallography Station: the coming of age of macromolecular neutron crystallography.

PubMed

Chen, Julian C-H; Unkefer, Clifford J

2017-01-01

The Protein Crystallography Station (PCS), located at the Los Alamos Neutron Scattering Center (LANSCE), was the first macromolecular crystallography beamline to be built at a spallation neutron source. Following testing and commissioning, the PCS user program was funded by the Biology and Environmental Research program of the Department of Energy Office of Science (DOE-OBER) for 13 years (2002-2014). The PCS remained the only dedicated macromolecular neutron crystallography station in North America until the construction and commissioning of the MaNDi and IMAGINE instruments at Oak Ridge National Laboratory, which started in 2012. The instrument produced a number of research and technical outcomes that have contributed to the field, clearly demonstrating the power of neutron crystallo-graphy in helping scientists to understand enzyme reaction mechanisms, hydrogen bonding and visualization of H-atom positions, which are critical to nearly all chemical reactions. During this period, neutron crystallography became a technique that increasingly gained traction, and became more integrated into macromolecular crystallography through software developments led by investigators at the PCS. This review highlights the contributions of the PCS to macromolecular neutron crystallography, and gives an overview of the history of neutron crystallography and the development of macromolecular neutron crystallography from the 1960s to the 1990s and onwards through the 2000s.

Single-crystal X-ray diffraction and NMR crystallography of a 1:1 cocrystal of di­thia­non and pyrimethanil

PubMed Central

Pöppler, Ann-Christin; Corlett, Emily K.; Pearce, Harriet; Seymour, Mark P.; Reid, Matthew; Montgomery, Mark G.

2017-01-01

A single-crystal X-ray diffraction structure of a 1:1 cocrystal of two fungicides, namely di­thia­non (DI) and pyrimethanil (PM), is reported [systematic name: 5,10-dioxo-5H,10H-naphtho­[2,3-b][1,4]dithiine-2,3-dicarbo­nitrile–4,6-dimethyl-N-phenyl­pyrimidin-2-amine (1/1), C14H4N2O2S2·C12H13N2]. Following an NMR crystallography approach, experimental solid-state magic angle spinning (MAS) NMR spectra are presented together with GIPAW (gauge-including projector augmented wave) calculations of NMR chemical shieldings. Specifically, experimental 1H and 13C chemical shifts are determined from two-dimensional 1H–13C MAS NMR correlation spectra recorded with short and longer contact times so as to probe one-bond C—H connectivities and longer-range C⋯H proximities, whereas H⋯H proximities are identified in a 1H double-quantum (DQ) MAS NMR spectrum. The performing of separate GIPAW calculations for the full periodic crystal structure and for isolated mol­ecules allows the determination of the change in chemical shift upon going from an isolated mol­ecule to the full crystal structure. For the 1H NMR chemical shifts, changes of 3.6 and 2.0 ppm correspond to inter­molecular N—H⋯O and C—H⋯O hydrogen bonding, while changes of −2.7 and −1.5 ppm are due to ring current effects associated with C—H⋯π inter­actions. Even though there is a close inter­molecular S⋯O distance of 3.10 Å, it is of note that the mol­ecule-to-crystal chemical shifts for the involved sulfur or oxygen nuclei are small. PMID:28257008

Protein crystal structure from non-oriented, single-axis sparse X-ray data

DOE PAGES

Wierman, Jennifer L.; Lan, Ti-Yen; Tate, Mark W.; ...

2016-01-01

X-ray free-electron lasers (XFELs) have inspired the development of serial femtosecond crystallography (SFX) as a method to solve the structure of proteins. SFX datasets are collected from a sequence of protein microcrystals injected across ultrashort X-ray pulses. The idea behind SFX is that diffraction from the intense, ultrashort X-ray pulses leaves the crystal before the crystal is obliterated by the effects of the X-ray pulse. The success of SFX at XFELs has catalyzed interest in analogous experiments at synchrotron-radiation (SR) sources, where data are collected from many small crystals and the ultrashort pulses are replaced by exposure times that aremore » kept short enough to avoid significant crystal damage. The diffraction signal from each short exposure is so `sparse' in recorded photons that the process of recording the crystal intensity is itself a reconstruction problem. Using theEMCalgorithm, a successful reconstruction is demonstrated here in a sparsity regime where there are no Bragg peaks that conventionally would serve to determine the orientation of the crystal in each exposure. In this proof-of-principle experiment, a hen egg-white lysozyme (HEWL) crystal rotating about a single axis was illuminated by an X-ray beam from an X-ray generator to simulate the diffraction patterns of microcrystals from synchrotron radiation. Millions of these sparse frames, typically containing only ~200 photons per frame, were recorded using a fast-framing detector. It is shown that reconstruction of three-dimensional diffraction intensity is possible using theEMCalgorithm, even with these extremely sparse frames and without knowledge of the rotation angle. Further, the reconstructed intensity can be phased and refined to solve the protein structure using traditional crystallographic software. In conclusion, this suggests that synchrotron-based serial crystallography of micrometre-sized crystals can be practical with the aid of theEMCalgorithm even in cases where the data are sparse.« less

Protein crystal structure from non-oriented, single-axis sparse X-ray data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wierman, Jennifer L.; Lan, Ti-Yen; Tate, Mark W.

X-ray free-electron lasers (XFELs) have inspired the development of serial femtosecond crystallography (SFX) as a method to solve the structure of proteins. SFX datasets are collected from a sequence of protein microcrystals injected across ultrashort X-ray pulses. The idea behind SFX is that diffraction from the intense, ultrashort X-ray pulses leaves the crystal before the crystal is obliterated by the effects of the X-ray pulse. The success of SFX at XFELs has catalyzed interest in analogous experiments at synchrotron-radiation (SR) sources, where data are collected from many small crystals and the ultrashort pulses are replaced by exposure times that aremore » kept short enough to avoid significant crystal damage. The diffraction signal from each short exposure is so `sparse' in recorded photons that the process of recording the crystal intensity is itself a reconstruction problem. Using theEMCalgorithm, a successful reconstruction is demonstrated here in a sparsity regime where there are no Bragg peaks that conventionally would serve to determine the orientation of the crystal in each exposure. In this proof-of-principle experiment, a hen egg-white lysozyme (HEWL) crystal rotating about a single axis was illuminated by an X-ray beam from an X-ray generator to simulate the diffraction patterns of microcrystals from synchrotron radiation. Millions of these sparse frames, typically containing only ~200 photons per frame, were recorded using a fast-framing detector. It is shown that reconstruction of three-dimensional diffraction intensity is possible using theEMCalgorithm, even with these extremely sparse frames and without knowledge of the rotation angle. Further, the reconstructed intensity can be phased and refined to solve the protein structure using traditional crystallographic software. In conclusion, this suggests that synchrotron-based serial crystallography of micrometre-sized crystals can be practical with the aid of theEMCalgorithm even in cases where the data are sparse.« less

Fifteen years of the Protein Crystallography Station: The coming of age of macromolecular neutron crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Julian C.-H.; Unkefer, Clifford Jay

The Protein Crystallography Station (PCS), located at the Los Alamos Neutron Scattering Center (LANSCE), was the first macromolecular crystallography beamline to be built at a spallation neutron source. Following testing and commissioning, the PCS user program was funded by the Biology and Environmental Research program of the Department of Energy Office of Science (DOE-OBER) for 13 years (2002–2014). The PCS remained the only dedicated macromolecular neutron crystallography station in North America until the construction and commissioning of the MaNDi and IMAGINE instruments at Oak Ridge National Laboratory, which started in 2012. The instrument produced a number of research and technicalmore » outcomes that have contributed to the field, clearly demonstrating the power of neutron crystallography in helping scientists to understand enzyme reaction mechanisms, hydrogen bonding and visualization of H-atom positions, which are critical to nearly all chemical reactions. During this period, neutron crystallography became a technique that increasingly gained traction, and became more integrated into macromolecular crystallography through software developments led by investigators at the PCS. As a result, this review highlights the contributions of the PCS to macromolecular neutron crystallography, and gives an overview of the history of neutron crystallography and the development of macromolecular neutron crystallography from the 1960s to the 1990s and onwards through the 2000s.« less

Fifteen years of the Protein Crystallography Station: The coming of age of macromolecular neutron crystallography

DOE PAGES

Chen, Julian C.-H.; Unkefer, Clifford Jay

2017-01-01

The Protein Crystallography Station (PCS), located at the Los Alamos Neutron Scattering Center (LANSCE), was the first macromolecular crystallography beamline to be built at a spallation neutron source. Following testing and commissioning, the PCS user program was funded by the Biology and Environmental Research program of the Department of Energy Office of Science (DOE-OBER) for 13 years (2002–2014). The PCS remained the only dedicated macromolecular neutron crystallography station in North America until the construction and commissioning of the MaNDi and IMAGINE instruments at Oak Ridge National Laboratory, which started in 2012. The instrument produced a number of research and technicalmore » outcomes that have contributed to the field, clearly demonstrating the power of neutron crystallography in helping scientists to understand enzyme reaction mechanisms, hydrogen bonding and visualization of H-atom positions, which are critical to nearly all chemical reactions. During this period, neutron crystallography became a technique that increasingly gained traction, and became more integrated into macromolecular crystallography through software developments led by investigators at the PCS. As a result, this review highlights the contributions of the PCS to macromolecular neutron crystallography, and gives an overview of the history of neutron crystallography and the development of macromolecular neutron crystallography from the 1960s to the 1990s and onwards through the 2000s.« less

Understanding pre-mRNA splicing through crystallography.

PubMed

Espinosa, Sara; Zhang, Lingdi; Li, Xueni; Zhao, Rui

2017-08-01

Crystallography is a powerful tool to determine the atomic structures of proteins and RNAs. X-ray crystallography has been used to determine the structure of many splicing related proteins and RNAs, making major contributions to our understanding of the molecular mechanism and regulation of pre-mRNA splicing. Compared to other structural methods, crystallography has its own advantage in the high-resolution structural information it can provide and the unique biological questions it can answer. In addition, two new crystallographic methods - the serial femtosecond crystallography and 3D electron crystallography - were developed to overcome some of the limitations of traditional X-ray crystallography and broaden the range of biological problems that crystallography can solve. This review discusses the theoretical basis, instrument requirements, troubleshooting, and exciting potential of these crystallographic methods to further our understanding of pre-mRNA splicing, a critical event in gene expression of all eukaryotes. Copyright © 2017 Elsevier Inc. All rights reserved.

Microfocus/Polycapillary-Optic Crystallographic X-Ray System

NASA Technical Reports Server (NTRS)

Joy, Marshall; Gubarev, Mikhail; Ciszak, Ewa

2005-01-01

A system that generates an intense, nearly collimated, nearly monochromatic, small-diameter x-ray beam has been developed for use in macromolecular crystallography. A conventional x-ray system for macromolecular crystallography includes a rotating-anode x-ray source, which is massive (.500 kg), large (approximately 2 by 2 by 1 m), and power-hungry (between 2 and 18 kW). In contrast, the present system generates a beam of the required brightness from a microfocus source, which is small and light enough to be mounted on a laboratory bench, and operates at a power level of only tens of watts. The figure schematically depicts the system as configured for observing x-ray diffraction from a macromolecular crystal. In addition to the microfocus x-ray source, the system includes a polycapillary optic . a monolithic block (typically a bundle of fused glass tubes) that contains thousands of straight or gently curved capillary channels, along which x-rays propagate with multiple reflections. This particular polycapillary optic is configured to act as a collimator; the x-ray beam that emerges from its output face consists of quasi-parallel subbeams with a small angular divergence and a diameter comparable to the size of a crystal to be studied. The gap between the microfocus x-ray source and the input face of the polycapillary optic is chosen consistently with the focal length of the polycapillary optic and the need to maximize the solid angle subtended by the optic in order to maximize the collimated x-ray flux. The spectrum from the source contains a significant component of Cu K (photon energy is 8.08 keV) radiation. The beam is monochromatized (for Cu K ) by a nickel filter 10 m thick. In a test, this system was operated at a power of 40 W (current of 897 A at an accelerating potential of 45 kV), with an anode x-ray spot size of 41+/-2 microns. Also tested, in order to provide a standard for comparison, was a commercial rotating-anode x-ray crystallographic system with a pyrolytic graphite monochromator and a 250-micron pinhole collimator, operating at a power of 3.15 kW (current of 70 mA at an accelerating potential of 45 kV). The flux of collimated Cu K radiation in this system was found to be approximately 16 times that in the rotatinganode system. Data on x-ray diffraction from crystals of tetragonal form of lysozyme (protein) in this system were found to be of high quality and to be reducible by use of standard crystallographic software.

Energy optimization of a regular macromolecular crystallography beamline for ultra-high-resolution crystallography

DOE PAGES

Rosenbaum, Gerd; Ginell, Stephan L.; Chen, Julian C.-H.

2015-01-01

In this study, a practical method for operating existing undulator synchrotron beamlines at photon energies considerably higher than their standard operating range is described and applied at beamline 19-ID of the Structural Biology Center at the Advanced Photon Source enabling operation at 30 keV. Adjustments to the undulator spectrum were critical to enhance the 30 keV flux while reducing the lower- and higher-energy harmonic contamination. A Pd-coated mirror and Al attenuators acted as effective low- and high-bandpass filters. The resulting flux at 30 keV, although significantly lower than with X-ray optics designed and optimized for this energy, allowed for accuratemore » data collection on crystals of the small protein crambin to 0.38 Å resolution.« less

Large-volume protein crystal growth for neutron macromolecular crystallography.

PubMed

Ng, Joseph D; Baird, James K; Coates, Leighton; Garcia-Ruiz, Juan M; Hodge, Teresa A; Huang, Sijay

2015-04-01

Neutron macromolecular crystallography (NMC) is the prevailing method for the accurate determination of the positions of H atoms in macromolecules. As neutron sources are becoming more available to general users, finding means to optimize the growth of protein crystals to sizes suitable for NMC is extremely important. Historically, much has been learned about growing crystals for X-ray diffraction. However, owing to new-generation synchrotron X-ray facilities and sensitive detectors, protein crystal sizes as small as in the nano-range have become adequate for structure determination, lessening the necessity to grow large crystals. Here, some of the approaches, techniques and considerations for the growth of crystals to significant dimensions that are now relevant to NMC are revisited. These include experimental strategies utilizing solubility diagrams, ripening effects, classical crystallization techniques, microgravity and theoretical considerations.

Effect of impurities and post-experimental purification in SAD phasing with serial femtosecond crystallography data.

PubMed

Zhang, Tao; Gu, Yuanxin; Fan, Haifu

2016-06-01

In serial crystallography (SX) with either an X-ray free-electron laser (XFEL) or synchrotron radiation as the light source, huge numbers of micrometre-sized crystals are used in diffraction data collection. For a SAD experiment using a derivative with introduced heavy atoms, it is difficult to completely exclude crystals of the native protein from the sample. In this paper, simulations were performed to study how the inclusion of native crystals in the derivative sample could affect the result of SAD phasing and how the post-experimental purification proposed by Zhang et al. [(2015), Acta Cryst. D71, 2513-2518] could be used to remove the impurities. A gadolinium derivative of lysozyme and the corresponding native protein were used in the test. Serial femtosecond crystallography (SFX) diffraction snapshots were generated by CrystFEL. SHELXC/D, Phaser, DM, ARP/wARP and REFMAC were used for automatic structure solution. It is shown that a small amount of impurities (snapshots from native crystals) in the set of derivative snapshots can strongly affect the SAD phasing results. On the other hand, post-experimental purification can efficiently remove the impurities, leading to results similar to those from a pure sample.

Canadian macromolecular crystallography facility: a suite of fully automated beamlines.

PubMed

Grochulski, Pawel; Fodje, Michel; Labiuk, Shaunivan; Gorin, James; Janzen, Kathryn; Berg, Russ

2012-06-01

The Canadian light source is a 2.9 GeV national synchrotron radiation facility located on the University of Saskatchewan campus in Saskatoon. The small-gap in-vacuum undulator illuminated beamline, 08ID-1, together with the bending magnet beamline, 08B1-1, constitute the Canadian Macromolecular Crystallography Facility (CMCF). The CMCF provides service to more than 50 Principal Investigators in Canada and the United States. Up to 25% of the beam time is devoted to commercial users and the general user program is guaranteed up to 55% of the useful beam time through a peer-review process. CMCF staff provides "Mail-In" crystallography service to users with the highest scored proposals. Both beamlines are equipped with very robust end-stations including on-axis visualization systems, Rayonix 300 CCD series detectors and Stanford-type robotic sample auto-mounters. MxDC, an in-house developed beamline control system, is integrated with a data processing module, AutoProcess, allowing full automation of data collection and data processing with minimal human intervention. Sample management and remote monitoring of experiments is enabled through interaction with a Laboratory Information Management System developed at the facility.

Microgravity

NASA Image and Video Library

2000-04-20

Edward Snell, a National Research Council research fellow at NASA's Marshall Space Flight Center (MSFC), prepares a protein crystal for analysis by x-ray crystallography as part of NASA's structural biology program. The small, individual crystals are bombarded with x-rays to produce diffraction patterns, a map of the intensity of the x-rays as they reflect through the crystal.

Three Dimensional Characterization of Tin Crystallography and Cu6Sn5 Intermetallics in Solder Joints by Multiscale Tomography

NASA Astrophysics Data System (ADS)

Kirubanandham, A.; Lujan-Regalado, I.; Vallabhaneni, R.; Chawla, N.

2016-11-01

Decreasing pitch size in electronic packaging has resulted in a drastic decrease in solder volumes. The Sn grain crystallography and fraction of intermetallic compounds (IMCs) in small-scale solder joints evolve much differently at the smaller length scales. A cross-sectional study limits the morphological analysis of microstructural features to two dimensions. This study utilizes serial sectioning technique in conjunction with electron backscatter diffraction to investigate the crystallographic orientation of both Sn grains and Cu6Sn5 IMCs in Cu/Pure Sn/Cu solder joints in three dimensional (3D). Quantification of grain aspect ratio is affected by local cooling rate differences within the solder volume. Backscatter electron imaging and focused ion beam serial sectioning enabled the visualization of morphology of both nanosized Cu6Sn5 IMCs and the hollow hexagonal morphology type Cu6Sn5 IMCs in 3D. Quantification and visualization of microstructural features in 3D thus enable us to better understand the microstructure and deformation mechanics within these small scale solder joints.

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography.

PubMed

Gicquel, Yannig; Schubert, Robin; Kapis, Svetlana; Bourenkov, Gleb; Schneider, Thomas; Perbandt, Markus; Betzel, Christian; Chapman, Henry N; Heymann, Michael

2018-04-24

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 µm grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography.

PubMed

Ishchenko, Andrii; Cherezov, Vadim; Liu, Wei

2016-09-20

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural information, typically obtained by crystallography; however MPs are difficult to crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization methods, which typically yield well-diffracting, but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. The development of new-generation X-ray free-electron laser (XFEL) sources that produce extremely bright femtosecond pulses has enabled room temperature data collection from microcrystals with no or negligible radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, which represent a notoriously difficult target for structure determination. In the LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of MP microcrystals to the intersection of the injector stream with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can substantially improve the diffraction resolution when only sub-10 µm crystals are available, or when the use of smaller crystals at room temperature can overcome various problems associated with larger cryocooled crystals, such as accumulation of defects, high mosaicity and cryocooling artifacts. Future advancements in X-ray sources and detector technologies should make serial crystallography highly attractive and practicable for implementation not only at XFELs, but also at more accessible synchrotron beamlines. Here we present detailed visual protocols for the preparation, characterization and delivery of microcrystals in LCP for serial crystallography experiments. These protocols include methods for conducting crystallization experiments in syringes, detecting and characterizing the crystal samples, optimizing crystal density, loading microcrystal laden LCP into the injector device and delivering the sample to the beam for data collection.

Molecular structures of carotenoids as predicted by MNDO-AM1 molecular orbital calculations

NASA Astrophysics Data System (ADS)

Hashimoto, Hideki; Yoda, Takeshi; Kobayashi, Takayoshi; Young, Andrew J.

2002-02-01

Semi-empirical molecular orbital calculations using AM1 Hamiltonian (MNDO-AM1 method) were performed for a number of biologically important carotenoid molecules, namely all- trans-β-carotene, all- trans-zeaxanthin, and all- trans-violaxanthin (found in higher plants and algae) together with all- trans-canthaxanthin, all- trans-astaxanthin, and all- trans-tunaxanthin in order to predict their stable structures. The molecular structures of all- trans-β-carotene, all- trans-canthaxanthin, and all- trans-astaxanthin predicted based on molecular orbital calculations were compared with those determined by X-ray crystallography. Predicted bond lengths, bond angles, and dihedral angles showed an excellent agreement with those determined experimentally, a fact that validated the present theoretical calculations. Comparison of the bond lengths, bond angles and dihedral angles of the most stable conformer among all the carotenoid molecules showed that the displacements are localized around the substituent groups and hence around the cyclohexene rings. The most stable conformers of all- trans-zeaxanthin and all- trans-violaxanthin gave rise to a torsion angle around the C6-C7 bond to be ±48.7 and -84.8°, respectively. This difference is a key factor in relation to the biological function of these two carotenoids in plants and algae (the xanthophyll cycle). Further analyses by calculating the atomic charges and using enpartment calculations (division of bond energies between component atoms) were performed to ascribe the cause of the different observed torsion angles.

Fifteen years of the Protein Crystallography Station: the coming of age of macromolecular neutron crystallography

PubMed Central

Chen, Julian C.-H.

2017-01-01

The Protein Crystallography Station (PCS), located at the Los Alamos Neutron Scattering Center (LANSCE), was the first macromolecular crystallography beamline to be built at a spallation neutron source. Following testing and commissioning, the PCS user program was funded by the Biology and Environmental Research program of the Department of Energy Office of Science (DOE-OBER) for 13 years (2002–2014). The PCS remained the only dedicated macromolecular neutron crystallography station in North America until the construction and commissioning of the MaNDi and IMAGINE instruments at Oak Ridge National Laboratory, which started in 2012. The instrument produced a number of research and technical outcomes that have contributed to the field, clearly demonstrating the power of neutron crystallo­graphy in helping scientists to understand enzyme reaction mechanisms, hydrogen bonding and visualization of H-atom positions, which are critical to nearly all chemical reactions. During this period, neutron crystallography became a technique that increasingly gained traction, and became more integrated into macromolecular crystallography through software developments led by investigators at the PCS. This review highlights the contributions of the PCS to macromolecular neutron crystallography, and gives an overview of the history of neutron crystallography and the development of macromolecular neutron crystallography from the 1960s to the 1990s and onwards through the 2000s. PMID:28250943

⁵¹V NMR Crystallography of Vanadium Chloroperoxidase and Its Directed Evolution P395D/L241V/T343A Mutant: Protonation Environments of the Active Site.

PubMed

Gupta, Rupal; Hou, Guangjin; Renirie, Rokus; Wever, Ron; Polenova, Tatyana

2015-04-29

Vanadium-dependent haloperoxidases (VHPOs) perform two-electron oxidation of halides using hydrogen peroxide. Their mechanism, including the factors determining the substrate specificity and the pH-dependence of the catalytic rates, is poorly understood. The vanadate cofactor in the active site of VHPOs contains "spectroscopically silent" V(V), which does not change oxidation state during the reaction. We employed an NMR crystallography approach based on (51)V magic angle spinning NMR spectroscopy and Density Functional Theory, to gain insights into the structure and coordination environment of the cofactor in the resting state of vanadium-dependent chloroperoxidases (VCPO). The cofactor environments in the wild-type VCPO and its P395D/L241V/T343A mutant exhibiting 5-100-fold improved catalytic activity are examined at various pH values. Optimal sensitivity attained due to the fast MAS probe technologies enabled the assignment of the location and number of protons on the vanadate as a function of pH. The vanadate cofactor changes its protonation from quadruply protonated at pH 6.3 to triply protonated at pH 7.3 to doubly protonated at pH 8.3. In contrast, in the mutant, the vanadate protonation is the same at pH 5.0 and 8.3, and the cofactor is doubly protonated. This methodology to identify the distinct protonation environments of the cofactor, which are also pH-dependent, could help explain the different reactivities of the wild-type and mutant VCPO and their pH-dependence. This study demonstrates that (51)V-based NMR crystallography can be used to derive the detailed coordination environments of vanadium centers in large biological molecules.

Crystallography of decahedral and icosahedral particles. II - High symmetry orientations

NASA Technical Reports Server (NTRS)

Yang, C. Y.; Yacaman, M. J.; Heinemann, K.

1979-01-01

Based on the exact crystal structure of decahedral and icosahedral particles, high energy electron diffraction patterns and image profiles have been derived for various high symmetry orientations of the particles with respect to the incident beam. These results form a basis for the identification of small metal particle structures with advanced methods of transmission electron microscopy.

Structure of catalase determined by MicroED

PubMed Central

Nannenga, Brent L; Shi, Dan; Hattne, Johan; Reyes, Francis E; Gonen, Tamir

2014-01-01

MicroED is a recently developed method that uses electron diffraction for structure determination from very small three-dimensional crystals of biological material. Previously we used a series of still diffraction patterns to determine the structure of lysozyme at 2.9 Å resolution with MicroED (Shi et al., 2013). Here we present the structure of bovine liver catalase determined from a single crystal at 3.2 Å resolution by MicroED. The data were collected by continuous rotation of the sample under constant exposure and were processed and refined using standard programs for X-ray crystallography. The ability of MicroED to determine the structure of bovine liver catalase, a protein that has long resisted atomic analysis by traditional electron crystallography, demonstrates the potential of this method for structure determination. DOI: http://dx.doi.org/10.7554/eLife.03600.001 PMID:25303172

Large-volume protein crystal growth for neutron macromolecular crystallography

DOE PAGES

Ng, Joseph D.; Baird, James K.; Coates, Leighton; ...

2015-03-30

Neutron macromolecular crystallography (NMC) is the prevailing method for the accurate determination of the positions of H atoms in macromolecules. As neutron sources are becoming more available to general users, finding means to optimize the growth of protein crystals to sizes suitable for NMC is extremely important. Historically, much has been learned about growing crystals for X-ray diffraction. However, owing to new-generation synchrotron X-ray facilities and sensitive detectors, protein crystal sizes as small as in the nano-range have become adequate for structure determination, lessening the necessity to grow large crystals. Here, some of the approaches, techniques and considerations for themore » growth of crystals to significant dimensions that are now relevant to NMC are revisited. We report that these include experimental strategies utilizing solubility diagrams, ripening effects, classical crystallization techniques, microgravity and theoretical considerations.« less

Large-volume protein crystal growth for neutron macromolecular crystallography

PubMed Central

Ng, Joseph D.; Baird, James K.; Coates, Leighton; Garcia-Ruiz, Juan M.; Hodge, Teresa A.; Huang, Sijay

2015-01-01

Neutron macromolecular crystallography (NMC) is the prevailing method for the accurate determination of the positions of H atoms in macromolecules. As neutron sources are becoming more available to general users, finding means to optimize the growth of protein crystals to sizes suitable for NMC is extremely important. Historically, much has been learned about growing crystals for X-ray diffraction. However, owing to new-generation synchrotron X-ray facilities and sensitive detectors, protein crystal sizes as small as in the nano-range have become adequate for structure determination, lessening the necessity to grow large crystals. Here, some of the approaches, techniques and considerations for the growth of crystals to significant dimensions that are now relevant to NMC are revisited. These include experimental strategies utilizing solubility diagrams, ripening effects, classical crystallization techniques, microgravity and theoretical considerations. PMID:25849493

Large-volume protein crystal growth for neutron macromolecular crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ng, Joseph D.; Baird, James K.; Coates, Leighton

Neutron macromolecular crystallography (NMC) is the prevailing method for the accurate determination of the positions of H atoms in macromolecules. As neutron sources are becoming more available to general users, finding means to optimize the growth of protein crystals to sizes suitable for NMC is extremely important. Historically, much has been learned about growing crystals for X-ray diffraction. However, owing to new-generation synchrotron X-ray facilities and sensitive detectors, protein crystal sizes as small as in the nano-range have become adequate for structure determination, lessening the necessity to grow large crystals. Here, some of the approaches, techniques and considerations for themore » growth of crystals to significant dimensions that are now relevant to NMC are revisited. We report that these include experimental strategies utilizing solubility diagrams, ripening effects, classical crystallization techniques, microgravity and theoretical considerations.« less

Room-temperature serial crystallography using a kinetically optimized microfluidic device for protein crystallization and on-chip X-ray diffraction

PubMed Central

Heymann, Michael; Opthalage, Achini; Wierman, Jennifer L.; Akella, Sathish; Szebenyi, Doletha M. E.; Gruner, Sol M.; Fraden, Seth

2014-01-01

An emulsion-based serial crystallographic technology has been developed, in which nanolitre-sized droplets of protein solution are encapsulated in oil and stabilized by surfactant. Once the first crystal in a drop is nucleated, the small volume generates a negative feedback mechanism that lowers the supersaturation. This mechanism is exploited to produce one crystal per drop. Diffraction data are measured, one crystal at a time, from a series of room-temperature crystals stored on an X-ray semi-transparent microfluidic chip, and a 93% complete data set is obtained by merging single diffraction frames taken from different unoriented crystals. As proof of concept, the structure of glucose isomerase was solved to 2.1 Å, demonstrating the feasibility of high-throughput serial X-ray crystallography using synchrotron radiation. PMID:25295176

Electron crystallography and aquaporins.

PubMed

Schenk, Andreas D; Hite, Richard K; Engel, Andreas; Fujiyoshi, Yoshinori; Walz, Thomas

2010-01-01

Electron crystallography of two-dimensional (2D) crystals can provide information on the structure of membrane proteins at near-atomic resolution. Originally developed and used to determine the structure of bacteriorhodopsin (bR), electron crystallography has recently been applied to elucidate the structure of aquaporins (AQPs), a family of membrane proteins that form pores mostly for water but also other solutes. While electron crystallography has made major contributions to our understanding of the structure and function of AQPs, structural studies on AQPs, in turn, have fostered a number of technical developments in electron crystallography. In this contribution, we summarize the insights electron crystallography has provided into the biology of AQPs, and describe technical advancements in electron crystallography that were driven by structural studies on AQP 2D crystals. In addition, we discuss some of the lessons that were learned from electron crystallographic work on AQPs. Copyright © 2010 Elsevier Inc. All rights reserved.

X-ray Crystallography Facility

NASA Technical Reports Server (NTRS)

1999-01-01

University of Alabama engineer Lance Weiss briefs NASA astronaut Dr. Bornie Dunbar about the design and capabilities of the X-ray Crystallography Facility under development at the Center for Macromolecular Crystallography of the University of Alabama at Birmingham, AL, April 21, 1999. The X-ray Crystallography Facility is designed to speed the collection of protein structure information from crystals grown aboard the International Space Station. By measuring and mapping the protein crystal structure in space, researchers will avoid exposing the delicate crystals to the rigors of space travel and make important research data available to scientists much faster. The X-ray Crystallography facility is being designed and developed by the Center for Macromolecular Crystallography of the University of Alabama at Birmingham, a NASA Commercial Space Center.

X-ray Crystallography Facility

NASA Technical Reports Server (NTRS)

1999-01-01

University of Alabama engineer Stacey Giles briefs NASA astronaut Dr. Bornie Dunbar about the design and capabilities of the X-ray Crystallography Facility under development at the Center for Macromolecular Crystallography of the University of Alabama at Birmingham, AL, April 21, 1999. The X-ray Crystallography Facility is designed to speed the collection of protein structure information from crystals grown aboard the International Space Station. By measuring and mapping the protein crystal structure in space, researchers will avoid exposing the delicate crystals to the rigors of space travel and make important research data available to scientists much faster. The X-ray Crystallography facility is being designed and developed by the Center for Macromolecular Crystallography of the University of Alabama at Birmingham, a NASA Commercial Space Center.

A glimpse of structural biology through X-ray crystallography.

PubMed

Shi, Yigong

2014-11-20

Since determination of the myoglobin structure in 1957, X-ray crystallography, as the anchoring tool of structural biology, has played an instrumental role in deciphering the secrets of life. Knowledge gained through X-ray crystallography has fundamentally advanced our views on cellular processes and greatly facilitated development of modern medicine. In this brief narrative, I describe my personal understanding of the evolution of structural biology through X-ray crystallography-using as examples mechanistic understanding of protein kinases and integral membrane proteins-and comment on the impact of technological development and outlook of X-ray crystallography.

Fragment Screening and HIV Therapeutics

PubMed Central

Bauman, Joseph D.; Patel, Disha; Arnold, Eddy

2013-01-01

Fragment screening has proven to be a powerful alternative to traditional methods for drug discovery. Biophysical methods, such as X-ray crystallography, NMR spectroscopy, and surface plasmon resonance, are used to screen a diverse library of small molecule compounds. Although compounds identified via this approach have relatively weak affinity, they provide a good platform for lead development and are highly efficient binders with respect to their size. Fragment screening has been utilized for a wide-range of targets, including HIV-1 proteins. Here, we review the fragment screening studies targeting HIV-1 proteins using X-ray crystallography or surface plasmon resonance. These studies have successfully detected binding of novel fragments to either previously established or new sites on HIV-1 protease and reverse transcriptase. In addition, fragment screening against HIV-1 reverse transcriptase has been used as a tool to better understand the complex nature of ligand binding to a flexible target. PMID:21972022

In-situ data collection at the photon factory macromolecular crystallography beamlines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamada, Yusuke, E-mail: yusuke.yamada@kek.jp; Matsugaki, Naohiro; Kato, Ryuichi

Crystallization trial is one of the most important but time-consuming steps in macromolecular crystallography, and in-situ diffraction experiment has a capability to make researchers to proceed this step more efficiently. At the Photon Factory, a new tabletop diffractometer for in-situ diffraction experiments has been developed. It consists of XYZ translation stages with a plate handler, an on-axis viewing system and a plate rack with a capacity for ten crystallization plates. These components sit on a common plate and are able to be placed on the existing diffractometer table. The CCD detector with a large active area and a pixel arraymore » detector with a small active area are used for acquiring diffraction images from crystals. Dedicated control software and a user interface have also been developed. The new diffractometer has been operational for users and used for evaluation of crystallization screening since 2014.« less

Protein-ligand complex structure from serial femtosecond crystallography using soaked thermolysin microcrystals and comparison with structures from synchrotron radiation.

PubMed

Naitow, Hisashi; Matsuura, Yoshinori; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Hatsui, Takaki; Yabashi, Makina; Tanaka, Rie; Tanaka, Tomoyuki; Sugahara, Michihiro; Kobayashi, Jun; Nango, Eriko; Iwata, So; Kunishima, Naoki

2017-08-01

Serial femtosecond crystallography (SFX) with an X-ray free-electron laser is used for the structural determination of proteins from a large number of microcrystals at room temperature. To examine the feasibility of pharmaceutical applications of SFX, a ligand-soaking experiment using thermolysin microcrystals has been performed using SFX. The results were compared with those from a conventional experiment with synchrotron radiation (SR) at 100 K. A protein-ligand complex structure was successfully obtained from an SFX experiment using microcrystals soaked with a small-molecule ligand; both oil-based and water-based crystal carriers gave essentially the same results. In a comparison of the SFX and SR structures, clear differences were observed in the unit-cell parameters, in the alternate conformation of side chains, in the degree of water coordination and in the ligand-binding mode.

Using more than 801 296 small-molecule crystal structures to aid in protein structure refinement and analysis

PubMed Central

Cole, Jason C.

2017-01-01

The Cambridge Structural Database (CSD) is the worldwide resource for the dissemination of all published three-dimensional structures of small-molecule organic and metal–organic compounds. This paper briefly describes how this collection of crystal structures can be used en masse in the context of macromolecular crystallography. Examples highlight how the CSD and associated software aid protein–ligand complex validation, and show how the CSD could be further used in the generation of geometrical restraints for protein structure refinement. PMID:28291758

3D Microstructural Characterization of Uranium Oxide as a Surrogate Nuclear Fuel: Effect of Oxygen Stoichiometry on Grain Boundary Distributions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rudman, K.; Dickerson, P.; Byler, Darrin David

The initial microstructure of an oxide fuel can play a key role in its performance. At low burn-ups, the diffusion of fission products can depend strongly on grain size and grain boundary (GB) characteristics, which in turn depend on processing conditions and oxygen stoichiometry. Serial sectioning techniques using Focused Ion Beam were developed to obtain Electron Backscatter Diffraction (EBSD) data for depleted UO2 pellets that were processed to obtain 3 different oxygen stoichiometries. The EBSD data were used to create 3D microstructure reconstructions and to gather statistical information on the grain and GB crystallography, with emphasis on identifying the charactermore » (twist, tilt, mixed) for GBs that meet the Coincident Site Lattice (CSL) criterion as well as GBs with the most common misorientation angles. Data on dihedral angles at triple points were also collected. The results were compared across different samples to understand effects of oxygen content on microstructure evolution.« less

The Upgrade Programme for the Structural Biology beamlines at the European Synchrotron Radiation Facility - High throughput sample evaluation and automation

NASA Astrophysics Data System (ADS)

Theveneau, P.; Baker, R.; Barrett, R.; Beteva, A.; Bowler, M. W.; Carpentier, P.; Caserotto, H.; de Sanctis, D.; Dobias, F.; Flot, D.; Guijarro, M.; Giraud, T.; Lentini, M.; Leonard, G. A.; Mattenet, M.; McCarthy, A. A.; McSweeney, S. M.; Morawe, C.; Nanao, M.; Nurizzo, D.; Ohlsson, S.; Pernot, P.; Popov, A. N.; Round, A.; Royant, A.; Schmid, W.; Snigirev, A.; Surr, J.; Mueller-Dieckmann, C.

2013-03-01

Automation and advances in technology are the key elements in addressing the steadily increasing complexity of Macromolecular Crystallography (MX) experiments. Much of this complexity is due to the inter-and intra-crystal heterogeneity in diffraction quality often observed for crystals of multi-component macromolecular assemblies or membrane proteins. Such heterogeneity makes high-throughput sample evaluation an important and necessary tool for increasing the chances of a successful structure determination. The introduction at the ESRF of automatic sample changers in 2005 dramatically increased the number of samples that were tested for diffraction quality. This "first generation" of automation, coupled with advances in software aimed at optimising data collection strategies in MX, resulted in a three-fold increase in the number of crystal structures elucidated per year using data collected at the ESRF. In addition, sample evaluation can be further complemented using small angle scattering experiments on the newly constructed bioSAXS facility on BM29 and the micro-spectroscopy facility (ID29S). The construction of a second generation of automated facilities on the MASSIF (Massively Automated Sample Screening Integrated Facility) beam lines will build on these advances and should provide a paradigm shift in how MX experiments are carried out which will benefit the entire Structural Biology community.

Structure and crystallography of foliated and chalk shell microstructures of the oyster Magallana: the same materials grown under different conditions.

PubMed

Checa, Antonio G; Harper, Elizabeth M; González-Segura, Alicia

2018-05-14

Oyster shells are mainly composed of layers of foliated microstructure and lenses of chalk, a highly porous, apparently poorly organized and mechanically weak material. We performed a structural and crystallographic study of both materials, paying attention to the transitions between them. The morphology and crystallography of the laths comprising both microstructures are similar. The main differences were, in general, crystallographic orientation and texture. Whereas the foliated microstructure has a moderate sheet texture, with a defined 001 maximum, the chalk has a much weaker sheet texture, with a defined 011 maximum. This is striking because of the much more disorganized aspect of the chalk. We hypothesize that part of the unanticipated order is inherited from the foliated microstructure by means of, possibly, [Formula: see text] twinning. Growth line distribution suggests that during chalk formation, the mantle separates from the previous shell several times faster than for the foliated material. A shortage of structural material causes the chalk to become highly porous and allows crystals to reorient at a high angle to the mantle surface, with which they continue to keep contact. In conclusion, both materials are structurally similar and the differences in orientation and aspect simply result from differences in growth conditions.

Small angle neutron scattering study of nano sized microstructure in Fe-Cr ODS steels for gen IV in-core applications.

PubMed

Han, Young-Soo; Mao, Xiadong; Jang, Jinsung

2013-11-01

The nano-sized microstructures in Fe-Cr oxide dispersion strengthened steel for Gen IV in-core applications were studied using small angle neutron scattering. The oxide dispersion strengthened steel was manufactured through hot isostatic pressing with various chemical compositions and fabrication conditions. Small angle neutron scattering experiments were performed using a 40 m small angle neutron scattering instrument at HANARO. Nano sized microstructures, namely, yttrium oxides and Cr-oxides were quantitatively analyzed by small angle neutron scattering. The yttrium oxides and Cr-oxides were also observed by transmission electron microscopy. The microstructural analysis results from small angle neutron scattering were compared with those obtained by transmission electron microscopy. The effects of the chemical compositions and fabrication conditions on the microstructure were investigated in relation to the quantitative microstructural analysis results obtained by small angle neutron scattering. The volume fraction of Y-oxide increases after fabrication, and this result is considered to be due to the formation of non-stochiometric Y-Ti-oxides.

Chemical Crystallography at the Advanced Light Source

DOE Office of Scientific and Technical Information (OSTI.GOV)

McCormick, Laura; Giordano, Nico; Teat, Simon

Chemical crystallography at synchrotrons was pioneered at the Daresbury SRS station 9.8. The chemical crystallography beamlines at the Advanced Light Source seek to follow that example, with orders of magnitude more flux than a lab source, and various in situ experiments. This article thus attempts to answer why a chemist would require synchrotron X-rays, to describe the techniques available at the ALS chemical crystallography beamlines, and place the current facilities in a historical context.

Chemical Crystallography at the Advanced Light Source

DOE PAGES

McCormick, Laura; Giordano, Nico; Teat, Simon; ...

2017-12-18

Chemical crystallography at synchrotrons was pioneered at the Daresbury SRS station 9.8. The chemical crystallography beamlines at the Advanced Light Source seek to follow that example, with orders of magnitude more flux than a lab source, and various in situ experiments. This article thus attempts to answer why a chemist would require synchrotron X-rays, to describe the techniques available at the ALS chemical crystallography beamlines, and place the current facilities in a historical context.

Recent advances in racemic protein crystallography.

PubMed

Yan, Bingjia; Ye, Linzhi; Xu, Weiliang; Liu, Lei

2017-09-15

Solution of the three-dimensional structures of proteins is a critical step in deciphering the molecular mechanisms of their bioactivities. Among the many approaches for obtaining protein crystals, racemic protein crystallography has been developed as a unique method to solve the structures of an increasing number of proteins. Exploiting unnatural protein enantiomers in crystallization and resolution, racemic protein crystallography manifests two major advantages that are 1) to increase the success rate of protein crystallization, and 2) to obviate the phase problem in X-ray diffraction. The requirement of unnatural protein enantiomers in racemic protein crystallography necessitates chemical protein synthesis, which is hitherto accomplished through solid phase peptide synthesis and chemical ligation reactions. This review highlights the fundamental ideas of racemic protein crystallography and surveys the harvests in the field of racemic protein crystallography over the last five years from early 2012 to late 2016. Copyright © 2017. Published by Elsevier Ltd.

Protein Crystallography from the Perspective of Technology Developments

PubMed Central

Su, Xiao-Dong; Zhang, Heng; Terwilliger, Thomas C.; Liljas, Anders; Xiao, Junyu; Dong, Yuhui

2015-01-01

Early on, crystallography was a domain of mineralogy and mathematics and dealt mostly with symmetry properties and imaginary crystal lattices. This changed when Wilhelm Conrad Röntgen discovered X-rays in 1895, and in 1912 Max von Laue and his associates discovered X-ray irradiated salt crystals would produce diffraction patterns that could reveal the internal atomic periodicity of the crystals. In the same year the father-and-son team, Henry and Lawrence Bragg successfully solved the first crystal structure of sodium chloride and the era of modern crystallography began. Protein crystallography (PX) started some 20 years later with the pioneering work of British crystallographers. In the past 50-60 years, the achievements of modern crystallography and particularly those in protein crystallography have been due to breakthroughs in theoretical and technical advancements such as phasing and direct methods; to more powerful X-ray sources such as synchrotron radiation (SR); to more sensitive and efficient X-ray detectors; to ever faster computers and to improvements in software. The exponential development of protein crystallography has been accelerated by the invention and applications of recombinant DNA technology that can yield nearly any protein of interest in large amounts and with relative ease. Novel methods, informatics platforms, and technologies for automation and high-throughput have allowed the development of large-scale, high efficiency macromolecular crystallography efforts in the field of structural genomics (SG). Very recently, the X-ray free-electron laser (XFEL) sources and its applications in protein crystallography have shown great potential for revolutionizing the whole field again in the near future. PMID:25983389

Microgravity

NASA Image and Video Library

1999-04-21

University of Alabama engineer Stacey Giles briefs NASA astronaut Dr. Bornie Dunbar about the design and capabilities of the X-ray Crystallography Facility under development at the Center for Macromolecular Crystallography of the University of Alabama at Birmingham, AL, April 21, 1999. The X-ray Crystallography Facility is designed to speed the collection of protein structure information from crystals grown aboard the International Space Station. By measuring and mapping the protein crystal structure in space, researchers will avoid exposing the delicate crystals to the rigors of space travel and make important research data available to scientists much faster. The X-ray Crystallography facility is being designed and developed by the Center for Macromolecular Crystallography of the University of Alabama at Birmingham, a NASA Commercial Space Center.

Racemic DNA crystallography.

PubMed

Mandal, Pradeep K; Collie, Gavin W; Kauffmann, Brice; Huc, Ivan

2014-12-22

Racemates increase the chances of crystallization by allowing molecular contacts to be formed in a greater number of ways. With the advent of protein synthesis, the production of protein racemates and racemic-protein crystallography are now possible. Curiously, racemic DNA crystallography had not been investigated despite the commercial availability of L- and D-deoxyribo-oligonucleotides. Here, we report a study into racemic DNA crystallography showing the strong propensity of racemic DNA mixtures to form racemic crystals. We describe racemic crystal structures of various DNA sequences and folded conformations, including duplexes, quadruplexes, and a four-way junction, showing that the advantages of racemic crystallography should extend to DNA. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Advances in structural and functional analysis of membrane proteins by electron crystallography

PubMed Central

Wisedchaisri, Goragot; Reichow, Steve L.; Gonen, Tamir

2011-01-01

Summary Electron crystallography is a powerful technique for the study of membrane protein structure and function in the lipid environment. When well-ordered two-dimensional crystals are obtained the structure of both protein and lipid can be determined and lipid-protein interactions analyzed. Protons and ionic charges can be visualized by electron crystallography and the protein of interest can be captured for structural analysis in a variety of physiologically distinct states. This review highlights the strengths of electron crystallography and the momentum that is building up in automation and the development of high throughput tools and methods for structural and functional analysis of membrane proteins by electron crystallography. PMID:22000511

Advances in structural and functional analysis of membrane proteins by electron crystallography.

PubMed

Wisedchaisri, Goragot; Reichow, Steve L; Gonen, Tamir

2011-10-12

Electron crystallography is a powerful technique for the study of membrane protein structure and function in the lipid environment. When well-ordered two-dimensional crystals are obtained the structure of both protein and lipid can be determined and lipid-protein interactions analyzed. Protons and ionic charges can be visualized by electron crystallography and the protein of interest can be captured for structural analysis in a variety of physiologically distinct states. This review highlights the strengths of electron crystallography and the momentum that is building up in automation and the development of high throughput tools and methods for structural and functional analysis of membrane proteins by electron crystallography. Copyright © 2011 Elsevier Ltd. All rights reserved.

Microgravity

NASA Image and Video Library

1999-04-21

University of Alabama engineer Lance Weiss briefs NASA astronaut Dr. Bornie Dunbar about the design and capabilities of the X-ray Crystallography Facility under development at the Center for Macromolecular Crystallography of the University of Alabama at Birmingham, AL, April 21, 1999. The X-ray Crystallography Facility is designed to speed the collection of protein structure information from crystals grown aboard the International Space Station. By measuring and mapping the protein crystal structure in space, researchers will avoid exposing the delicate crystals to the rigors of space travel and make important research data available to scientists much faster. The X-ray Crystallography facility is being designed and developed by the Center for Macromolecular Crystallography of the University of Alabama at Birmingham, a NASA Commercial Space Center.

Non-linearity of the collagen triple helix in solution and implications for collagen function.

PubMed

Walker, Kenneth T; Nan, Ruodan; Wright, David W; Gor, Jayesh; Bishop, Anthony C; Makhatadze, George I; Brodsky, Barbara; Perkins, Stephen J

2017-06-16

Collagen adopts a characteristic supercoiled triple helical conformation which requires a repeating (Xaa-Yaa-Gly) n sequence. Despite the abundance of collagen, a combined experimental and atomistic modelling approach has not so far quantitated the degree of flexibility seen experimentally in the solution structures of collagen triple helices. To address this question, we report an experimental study on the flexibility of varying lengths of collagen triple helical peptides, composed of six, eight, ten and twelve repeats of the most stable Pro-Hyp-Gly (POG) units. In addition, one unblocked peptide, (POG) 10unblocked , was compared with the blocked (POG) 10 as a control for the significance of end effects. Complementary analytical ultracentrifugation and synchrotron small angle X-ray scattering data showed that the conformations of the longer triple helical peptides were not well explained by a linear structure derived from crystallography. To interpret these data, molecular dynamics simulations were used to generate 50 000 physically realistic collagen structures for each of the helices. These structures were fitted against their respective scattering data to reveal the best fitting structures from this large ensemble of possible helix structures. This curve fitting confirmed a small degree of non-linearity to exist in these best fit triple helices, with the degree of bending approximated as 4-17° from linearity. Our results open the way for further studies of other collagen triple helices with different sequences and stabilities in order to clarify the role of molecular rigidity and flexibility in collagen extracellular and immune function and disease. © 2017 The Author(s).

Crystallography of decahedral and icosahedral particles. I - Geometry of twinning

NASA Technical Reports Server (NTRS)

Yang, C. Y.

1979-01-01

The crystal structure of the tetrahedral twins in multiply-twinned particles with decahedral and icosahedral point group symmetries has been examined and correlated with the face-centered cubic structure. Details on the crystal structure as well as the geometrical relationships among twins in each particle are presented. These crystallographic facts serve as a basis for the interpretation of small particle images obtained with advanced methods of transmission electron microscopy.

76 FR 10067 - Notice Pursuant to the National Cooperative Research and Production Act of 1993-Industrial...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-02-23

... Production Act of 1993--Industrial Macromolecular Crystallography Association Correction In notice document... National Cooperative Research and Production Act of 1993--Industrial Nacromolecular Crystallography...--Industrial Macromolecular Crystallography Association''. 2. On the same page, in the second column, in the...

Neutron Scattering Conferences

Science.gov Websites

Crystallography 2013 Location Aarhus, Denmark Contact Poul Nissen Email pn@mb.au.dk URL http://www.bioxray.au.dk /conf/gcnb2012/ June 9-22, 2013 The Zürich School of Crystallography Location Zürich, Switzerland July 8-12, 2013 International Workshop: Powder & Electron Crystallography Location Patras, Greece

The Cambridge Structural Database in retrospect and prospect.

PubMed

Groom, Colin R; Allen, Frank H

2014-01-13

The Cambridge Crystallographic Data Centre (CCDC) was established in 1965 to record numerical, chemical and bibliographic data relating to published organic and metal-organic crystal structures. The Cambridge Structural Database (CSD) now stores data for nearly 700,000 structures and is a comprehensive and fully retrospective historical archive of small-molecule crystallography. Nearly 40,000 new structures are added each year. As X-ray crystallography celebrates its centenary as a subject, and the CCDC approaches its own 50th year, this article traces the origins of the CCDC as a publicly funded organization and its onward development into a self-financing charitable institution. Principally, however, we describe the growth of the CSD and its extensive associated software system, and summarize its impact and value as a basis for research in structural chemistry, materials science and the life sciences, including drug discovery and drug development. Finally, the article considers the CCDC's funding model in relation to open access and open data paradigms. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Indexing amyloid peptide diffraction from serial femtosecond crystallography: New algorithms for sparse patterns

DOE PAGES

Brewster, Aaron S.; Sawaya, Michael R.; Rodriguez, Jose; ...

2015-01-23

Still diffraction patterns from peptide nanocrystals with small unit cells are challenging to index using conventional methods owing to the limited number of spots and the lack of crystal orientation information for individual images. New indexing algorithms have been developed as part of the Computational Crystallography Toolbox( cctbx) to overcome these challenges. Accurate unit-cell information derived from an aggregate data set from thousands of diffraction patterns can be used to determine a crystal orientation matrix for individual images with as few as five reflections. These algorithms are potentially applicable not only to amyloid peptides but also to any set ofmore » diffraction patterns with sparse properties, such as low-resolution virus structures or high-throughput screening of still images captured by raster-scanning at synchrotron sources. As a proof of concept for this technique, successful integration of X-ray free-electron laser (XFEL) data to 2.5 Å resolution for the amyloid segment GNNQQNY from the Sup35 yeast prion is presented.« less

Determination of the Molecular Structures of Ferric Enterobactin and Ferric Enantioenterobactin Using Racemic Crystallography.

PubMed

Johnstone, Timothy C; Nolan, Elizabeth M

2017-10-25

Enterobactin is a secondary metabolite produced by Enterobacteriaceae for acquiring iron, an essential metal nutrient. The biosynthesis and utilization of enterobactin permits many Gram-negative bacteria to thrive in environments where low soluble iron concentrations would otherwise preclude survival. Despite extensive work carried out on this celebrated molecule since its discovery over 40 years ago, the ferric enterobactin complex has eluded crystallographic structural characterization. We report the successful growth of single crystals containing ferric enterobactin using racemic crystallization, a method that involves cocrystallization of a chiral molecule with its mirror image. The structures of ferric enterobactin and ferric enantioenterobactin obtained in this work provide a definitive assignment of the stereochemistry at the metal center and reveal secondary coordination sphere interactions. The structures were employed in computational investigations of the interactions of these complexes with two enterobactin-binding proteins, which illuminate the influence of metal-centered chirality on these interactions. This work highlights the utility of small-molecule racemic crystallography for obtaining elusive structures of coordination complexes.

Goniometer-based femtosecond crystallography with X-ray free electron lasers

DOE PAGES

Cohen, Aina E.; Soltis, S. Michael; González, Ana; ...

2014-10-31

The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. With smaller crystals, high-density grids were usedmore » to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β 2-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.« less

Current status and future prospects of an automated sample exchange system PAM for protein crystallography

NASA Astrophysics Data System (ADS)

Hiraki, M.; Yamada, Y.; Chavas, L. M. G.; Matsugaki, N.; Igarashi, N.; Wakatsuki, S.

2013-03-01

To achieve fully-automated and/or remote data collection in high-throughput X-ray experiments, the Structural Biology Research Centre at the Photon Factory (PF) has installed PF automated mounting system (PAM) for sample exchange robots at PF macromolecular crystallography beamlines BL-1A, BL-5A, BL-17A, AR-NW12A and AR-NE3A. We are upgrading the experimental systems, including the PAM for stable and efficient operation. To prevent human error in automated data collection, we installed a two-dimensional barcode reader for identification of the cassettes and sample pins. Because no liquid nitrogen pipeline in the PF experimental hutch is installed, the users commonly add liquid nitrogen using a small Dewar. To address this issue, an automated liquid nitrogen filling system that links a 100-liter tank to the robot Dewar has been installed on the PF macromolecular beamline. Here we describe this new implementation, as well as future prospects.

Goniometer-based femtosecond crystallography with X-ray free electron lasers

PubMed Central

Cohen, Aina E.; Soltis, S. Michael; González, Ana; Aguila, Laura; Alonso-Mori, Roberto; Barnes, Christopher O.; Baxter, Elizabeth L.; Brehmer, Winnie; Brewster, Aaron S.; Brunger, Axel T.; Calero, Guillermo; Chang, Joseph F.; Chollet, Matthieu; Ehrensberger, Paul; Eriksson, Thomas L.; Feng, Yiping; Hattne, Johan; Hedman, Britt; Hollenbeck, Michael; Holton, James M.; Keable, Stephen; Kobilka, Brian K.; Kovaleva, Elena G.; Kruse, Andrew C.; Lemke, Henrik T.; Lin, Guowu; Lyubimov, Artem Y.; Manglik, Aashish; Mathews, Irimpan I.; McPhillips, Scott E.; Nelson, Silke; Peters, John W.; Sauter, Nicholas K.; Smith, Clyde A.; Song, Jinhu; Stevenson, Hilary P.; Tsai, Yingssu; Uervirojnangkoorn, Monarin; Vinetsky, Vladimir; Wakatsuki, Soichi; Weis, William I.; Zadvornyy, Oleg A.; Zeldin, Oliver B.; Zhu, Diling; Hodgson, Keith O.

2014-01-01

The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. For smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β2-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources. PMID:25362050

Inhibition of Retinoblastoma Protein Inactivation

DTIC Science & Technology

2017-11-01

SUBJECT TERMS cell cycle, Retinoblastoma protein, E2F transcription factor, high throughput screen, drug discovery, x-ray crystallography 16. SECURITY...screening by x-ray crystallography . 2.0 KEYWORDS Retinoblastoma (Rb) pathway, E2F transcription factor, cancer, cell-cycle inhibition, activation...modulation, inhibition, high throughput screening, fragment-based screening, x-ray crystallography . 3.0 ACCOMPLISHMENTS Summary: We

X-Ray Crystallography: One Century of Nobel Prizes

ERIC Educational Resources Information Center

Galli, Simona

2014-01-01

In 2012, the United Nations General Assembly declared 2014 the International Year of Crystallography. Throughout the year 2014 and beyond, all the crystallographic associations and societies active all over the world are organizing events to attract the wider public toward crystallography and the numerous topics to which it is deeply interlinked.…

Fragment-based screening by protein crystallography: successes and pitfalls.

PubMed

Chilingaryan, Zorik; Yin, Zhou; Oakley, Aaron J

2012-10-08

Fragment-based drug discovery (FBDD) concerns the screening of low-molecular weight compounds against macromolecular targets of clinical relevance. These compounds act as starting points for the development of drugs. FBDD has evolved and grown in popularity over the past 15 years. In this paper, the rationale and technology behind the use of X-ray crystallography in fragment based screening (FBS) will be described, including fragment library design and use of synchrotron radiation and robotics for high-throughput X-ray data collection. Some recent uses of crystallography in FBS will be described in detail, including interrogation of the drug targets β-secretase, phenylethanolamine N-methyltransferase, phosphodiesterase 4A and Hsp90. These examples provide illustrations of projects where crystallography is straightforward or difficult, and where other screening methods can help overcome the limitations of crystallography necessitated by diffraction quality.

Fragment-Based Screening by Protein Crystallography: Successes and Pitfalls

PubMed Central

Chilingaryan, Zorik; Yin, Zhou; Oakley, Aaron J.

2012-01-01

Fragment-based drug discovery (FBDD) concerns the screening of low-molecular weight compounds against macromolecular targets of clinical relevance. These compounds act as starting points for the development of drugs. FBDD has evolved and grown in popularity over the past 15 years. In this paper, the rationale and technology behind the use of X-ray crystallography in fragment based screening (FBS) will be described, including fragment library design and use of synchrotron radiation and robotics for high-throughput X-ray data collection. Some recent uses of crystallography in FBS will be described in detail, including interrogation of the drug targets β-secretase, phenylethanolamine N-methyltransferase, phosphodiesterase 4A and Hsp90. These examples provide illustrations of projects where crystallography is straightforward or difficult, and where other screening methods can help overcome the limitations of crystallography necessitated by diffraction quality. PMID:23202926

On the state of crystallography at the dawn of the electron microscopy revolution.

PubMed

Higgins, Matthew K; Lea, Susan M

2017-10-01

While protein crystallography has, for many years, been the most used method for structural analysis of macromolecular complexes, remarkable recent advances in high-resolution electron cryo-microscopy led to suggestions that 'the revolution will not be crystallised'. Here we highlight the current success rate, speed and ease of modern crystallographic structure determination and some recent triumphs of both 'classical' crystallography and the use of X-ray free electron lasers. We also outline fundamental differences between structure determination using X-ray crystallography and electron microscopy. We suggest that crystallography will continue to co-exist with electron microscopy as part of an integrated array of methods, allowing structural biologists to focus on fundamental biological questions rather than being constrained by the methods available. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

A 1:2 crystalline complex of ApA:proflavine: a model for binding to single-stranded regions in RNA.

PubMed Central

Neidle, S; Taylor, G; Sanderson, M

1978-01-01

The structure of a 1"2 complex of adenylyl-(3',5')-adenosine phosphate and proflavine hemisulfate has been determined using the methods of x-ray crystallography. Since the ApA does not form a mini double helix, it may serve as a model for the interaction of planar molecules with single stranded nucleic acids. The dinucleotide adopts an extended conformation with the adenines in adjacent molecules forming base pairs. A most unusual feature of the molecule is that it does not obey the "rigid nucleotide" concept although none of the torsion angles occur in energetically unfavourable regions. This is most probably due to the strong interactions between the proflavine and the oligonucleotide. PMID:724521

Grazing-Incidence Neutron Optics based on Wolter Geometries

NASA Technical Reports Server (NTRS)

Gubarev, M. V.; Ramsey, B. D.; Mildner, D. F. R.

2008-01-01

The feasibility of grazing-incidence neutron imaging optics based on the Wolter geometries have been successfully demonstrated. Biological microscopy, neutron radiography, medical imaging, neutron crystallography and boron neutron capture therapy would benefit from high resolution focusing neutron optics. Two bounce optics can also be used to focus neutrons in SANS experiments. Here, the use of the optics would result in lower values of obtainable scattering angles. The high efficiency of the optics permits a decrease in the minimum scattering vector without lowering the neutron intensity on sample. In this application, a significant advantage of the reflective optics over refractive optics is that the focus is independent of wavelength, so that the technique can be applied to polychromatic beams at pulsed neutron sources.

Structure-function insights of membrane and soluble proteins revealed by electron crystallography.

PubMed

Dreaden, Tina M; Devarajan, Bharanidharan; Barry, Bridgette A; Schmidt-Krey, Ingeborg

2013-01-01

Electron crystallography is emerging as an important method in solving protein structures. While it has found extensive applications in the understanding of membrane protein structure and function at a wide range of resolutions, from revealing oligomeric arrangements to atomic models, electron crystallography has also provided invaluable information on the soluble α/β-tubulin which could not be obtained by any other method to date. Examples of critical insights from selected structures of membrane proteins as well as α/β-tubulin are described here, demonstrating the vast potential of electron crystallography that is first beginning to unfold.

Inhibition of Retinoblastoma Protein Inactivation

DTIC Science & Technology

2016-09-01

Retinoblastoma protein, E2F transcription factor, high throughput screen, drug discovery, x-ray crystallography 16. SECURITY CLASSIFICATION OF: 17...developed a method to perform fragment based screening by x-ray crystallography . 2.0 KEYWORDS Retinoblastoma (Rb) pathway, E2F transcription factor...cancer, cell-cycle inhibition, activation, modulation, inhibition, high throughput screening, fragment-based screening, x-ray crystallography

The Coherent Backscattering Opposition Effect: Measurements at Very Small Phase Angles

NASA Technical Reports Server (NTRS)

Nelson, R.; Hapke, B.; Smythe, W.; Horn, L.; Herrera, P.; Gharakanian, V.

1993-01-01

This oral presentation explains that measurements of the opposition surge (the nonlinear increase in reflectance seen in particulate materials when observed at small phase angles) are the first ever made using the JPL long-arm goniometer, which permits very small phase angle measuremnets to be made.

Multi-Conformation Monte Carlo: A Method for Introducing Flexibility in Efficient Simulations of Many-Protein Systems.

PubMed

Prytkova, Vera; Heyden, Matthias; Khago, Domarin; Freites, J Alfredo; Butts, Carter T; Martin, Rachel W; Tobias, Douglas J

2016-08-25

We present a novel multi-conformation Monte Carlo simulation method that enables the modeling of protein-protein interactions and aggregation in crowded protein solutions. This approach is relevant to a molecular-scale description of realistic biological environments, including the cytoplasm and the extracellular matrix, which are characterized by high concentrations of biomolecular solutes (e.g., 300-400 mg/mL for proteins and nucleic acids in the cytoplasm of Escherichia coli). Simulation of such environments necessitates the inclusion of a large number of protein molecules. Therefore, computationally inexpensive methods, such as rigid-body Brownian dynamics (BD) or Monte Carlo simulations, can be particularly useful. However, as we demonstrate herein, the rigid-body representation typically employed in simulations of many-protein systems gives rise to certain artifacts in protein-protein interactions. Our approach allows us to incorporate molecular flexibility in Monte Carlo simulations at low computational cost, thereby eliminating ambiguities arising from structure selection in rigid-body simulations. We benchmark and validate the methodology using simulations of hen egg white lysozyme in solution, a well-studied system for which extensive experimental data, including osmotic second virial coefficients, small-angle scattering structure factors, and multiple structures determined by X-ray and neutron crystallography and solution NMR, as well as rigid-body BD simulation results, are available for comparison.

ISPyB for BioSAXS, the gateway to user autonomy in solution scattering experiments.

PubMed

De Maria Antolinos, Alejandro; Pernot, Petra; Brennich, Martha E; Kieffer, Jérôme; Bowler, Matthew W; Delageniere, Solange; Ohlsson, Staffan; Malbet Monaco, Stephanie; Ashton, Alun; Franke, Daniel; Svergun, Dmitri; McSweeney, Sean; Gordon, Elspeth; Round, Adam

2015-01-01

Logging experiments with the laboratory-information management system ISPyB (Information System for Protein crystallography Beamlines) enhances the automation of small-angle X-ray scattering of biological macromolecules in solution (BioSAXS) experiments. The ISPyB interface provides immediate user-oriented online feedback and enables data cross-checking and downstream analysis. To optimize data quality and completeness, ISPyBB (ISPyB for BioSAXS) makes it simple for users to compare the results from new measurements with previous acquisitions from the same day or earlier experiments in order to maximize the ability to collect all data required in a single synchrotron visit. The graphical user interface (GUI) of ISPyBB has been designed to guide users in the preparation of an experiment. The input of sample information and the ability to outline the experimental aims in advance provides feedback on the number of measurements required, calculation of expected sample volumes and time needed to collect the data: all of this information aids the users to better prepare for their trip to the synchrotron. A prototype version of the ISPyBB database is now available at the European Synchrotron Radiation Facility (ESRF) beamline BM29 and is already greatly appreciated by academic users and industrial clients. It will soon be available at the PETRA III beamline P12 and the Diamond Light Source beamlines I22 and B21.

ISPyB for BioSAXS, the gateway to user autonomy in solution scattering experiments

PubMed Central

De Maria Antolinos, Alejandro; Pernot, Petra; Brennich, Martha E.; Kieffer, Jérôme; Bowler, Matthew W.; Delageniere, Solange; Ohlsson, Staffan; Malbet Monaco, Stephanie; Ashton, Alun; Franke, Daniel; Svergun, Dmitri; McSweeney, Sean; Gordon, Elspeth; Round, Adam

2015-01-01

Logging experiments with the laboratory-information management system ISPyB (Information System for Protein crystallography Beamlines) enhances the automation of small-angle X-ray scattering of biological macromolecules in solution (BioSAXS) experiments. The ISPyB interface provides immediate user-oriented online feedback and enables data cross-checking and downstream analysis. To optimize data quality and completeness, ISPyBB (ISPyB for BioSAXS) makes it simple for users to compare the results from new measurements with previous acquisitions from the same day or earlier experiments in order to maximize the ability to collect all data required in a single synchrotron visit. The graphical user interface (GUI) of ISPyBB has been designed to guide users in the preparation of an experiment. The input of sample information and the ability to outline the experimental aims in advance provides feedback on the number of measurements required, calculation of expected sample volumes and time needed to collect the data: all of this information aids the users to better prepare for their trip to the synchrotron. A prototype version of the ISPyBB database is now available at the European Synchrotron Radiation Facility (ESRF) beamline BM29 and is already greatly appreciated by academic users and industrial clients. It will soon be available at the PETRA III beamline P12 and the Diamond Light Source beamlines I22 and B21. PMID:25615862

Microstructure of Amorphous and Semi-Crystalline Polymers.

DTIC Science & Technology

1981-06-07

of these materials. Further, the occurrence of nodular structures is difficult to reconcile with the results of studies of small angle neutron ...scattering and small angle neutron scattering studies of the same materials. Based on the combined results of these studies , it is suggested that the nodular...relevance here were reviewed by Flory.’ In addition to these, the results of studies using small angle neutron scattering’ and wide angle X-ray scattering

The Structure of the Plakin Domain of Plectin Reveals an Extended Rod-like Shape*

PubMed Central

Carballido, Ana M.

2016-01-01

Plakins are large multi-domain proteins that interconnect cytoskeletal structures. Plectin is a prototypical plakin that tethers intermediate filaments to membrane-associated complexes. Most plakins contain a plakin domain formed by up to nine spectrin repeats (SR1–SR9) and an SH3 domain. The plakin domains of plectin and other plakins harbor binding sites for junctional proteins. We have combined x-ray crystallography with small angle x-ray scattering (SAXS) to elucidate the structure of the plakin domain of plectin, extending our previous analysis of the SR1 to SR5 region. Two crystal structures of the SR5-SR6 region allowed us to characterize its uniquely wide inter-repeat conformational variability. We also report the crystal structures of the SR7-SR8 region, refined to 1.8 Å, and the SR7–SR9 at lower resolution. The SR7–SR9 region, which is conserved in all other plakin domains, forms a rigid segment stabilized by uniquely extensive inter-repeat contacts mediated by unusually long helices in SR8 and SR9. Using SAXS we show that in solution the SR3–SR6 and SR7–SR9 regions are rod-like segments and that SR3–SR9 of plectin has an extended shape with a small central kink. Other plakins, such as bullous pemphigoid antigen 1 and microtubule and actin cross-linking factor 1, are likely to have similar extended plakin domains. In contrast, desmoplakin has a two-segment structure with a central flexible hinge. The continuous versus segmented structures of the plakin domains of plectin and desmoplakin give insight into how different plakins might respond to tension and transmit mechanical signals. PMID:27413182

X-ray crystallography over the past decade for novel drug discovery - where are we heading next?

PubMed

Zheng, Heping; Handing, Katarzyna B; Zimmerman, Matthew D; Shabalin, Ivan G; Almo, Steven C; Minor, Wladek

2015-01-01

Macromolecular X-ray crystallography has been the primary methodology for determining the three-dimensional structures of proteins, nucleic acids and viruses. Structural information has paved the way for structure-guided drug discovery and laid the foundations for structural bioinformatics. However, X-ray crystallography still has a few fundamental limitations, some of which may be overcome and complemented using emerging methods and technologies in other areas of structural biology. This review describes how structural knowledge gained from X-ray crystallography has been used to advance other biophysical methods for structure determination (and vice versa). This article also covers current practices for integrating data generated by other biochemical and biophysical methods with those obtained from X-ray crystallography. Finally, the authors articulate their vision about how a combination of structural and biochemical/biophysical methods may improve our understanding of biological processes and interactions. X-ray crystallography has been, and will continue to serve as, the central source of experimental structural biology data used in the discovery of new drugs. However, other structural biology techniques are useful not only to overcome the major limitation of X-ray crystallography, but also to provide complementary structural data that is useful in drug discovery. The use of recent advancements in biochemical, spectroscopy and bioinformatics methods may revolutionize drug discovery, albeit only when these data are combined and analyzed with effective data management systems. Accurate and complete data management is crucial for developing experimental procedures that are robust and reproducible.

Inhibition of 53BP1: Potential for Restoring Homologous Recombination In Ovarian Cancer Cells

DTIC Science & Technology

2017-08-01

crystallography ; NMR spectroscopy; Calorimetry 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE...ray crystallography ; NMR spectroscopy; Calorimetry 3. ACCOMPLISHMENTS: The PI is reminded that the recipient organization is required to obtain...originally planned. Each aim combines structural studies using X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy and functional

Advanced Communication for Wireless Sensor Networks

DTIC Science & Technology

2016-08-22

characteristic peaks of the PZT crystallography and the presence of non-desired secondary phases. Regarding the spinel thin film, a new sol gel process...particles with the desired crystallography were obtained. As shown in Figure 21 (left), the particle morphology and size can be seen using TEM...film. The crystallography of the films was characterized at different annealing temperatures in order to determine the crystallization kinetics. It

Introduction to electron crystallography.

PubMed

Kühlbrandt, Werner

2013-01-01

From the earliest work on regular arrays in negative stain, electron crystallography has contributed greatly to our understanding of the structure and function of biological macromolecules. The development of electron cryo-microscopy (cryo-EM) then lead to the first groundbreaking atomic models of the membrane proteins bacteriorhodopsin and light harvesting complex II within lipid bilayers. Key contributions towards cryo-EM and electron crystallography methods included specimen preparation and vitrification, liquid-helium cooling, data collection, and image processing. These methods are now applied almost routinely to both membrane and soluble proteins. Here we outline the advances and the breakthroughs that paved the way towards high-resolution structures by electron crystallography, both in terms of methods development and biological milestones.

Cleavage crystallography of liquid metal embrittled aluminum alloys

NASA Technical Reports Server (NTRS)

Reynolds, A. P.; Stoner, G. E.

1991-01-01

The crystallography of liquid metal-induced transgranular cleavage in six aluminum alloys having a variety of microstructures has been determined via Laue X-ray back reflection. The cleavage crystallography was independent of alloy microstructure, and the cleavage plane was 100-plane oriented in all cases. It was further determined that the cleavage crystallography was not influenced by alloy texture. Examination of the fracture surface indicated that there was not a unique direction of crack propagation. In addition, the existence of 100-plane cleavage on alloy 2024 fracture surfaces was inferred by comparison of secondary cleavage crack intersection geometry on the 2024 surfaces with the geometry of secondary cleavage crack intersections on the test alloys.

High-throughput Crystallography for Structural Genomics

PubMed Central

Joachimiak, Andrzej

2009-01-01

Protein X-ray crystallography recently celebrated its 50th anniversary. The structures of myoglobin and hemoglobin determined by Kendrew and Perutz provided the first glimpses into the complex protein architecture and chemistry. Since then, the field of structural molecular biology has experienced extraordinary progress and now over 53,000 proteins structures have been deposited into the Protein Data Bank. In the past decade many advances in macromolecular crystallography have been driven by world-wide structural genomics efforts. This was made possible because of third-generation synchrotron sources, structure phasing approaches using anomalous signal and cryo-crystallography. Complementary progress in molecular biology, proteomics, hardware and software for crystallographic data collection, structure determination and refinement, computer science, databases, robotics and automation improved and accelerated many processes. These advancements provide the robust foundation for structural molecular biology and assure strong contribution to science in the future. In this report we focus mainly on reviewing structural genomics high-throughput X-ray crystallography technologies and their impact. PMID:19765976

A Microbeam Small-Angle X-ray Scattering Study on Enamel Crystallites in Subsurface Lesion

NASA Astrophysics Data System (ADS)

Yagi, N.; Ohta, N.; Matsuo, T.; Tanaka, T.; Terada, Y.; Kamasaka, H.; Kometani, T.

2010-10-01

The early caries lesion in bovine tooth enamel was studied by two different X-ray diffraction systems at the SPring-8 third generation synchrotron radiation facility. Both allowed us simultaneous measurement of the small and large angle regions. The beam size was 6μm at BL40XU and 50μm at BL45XU. The small-angle scattering from voids in the hydroxyapatite crystallites and the wide-angle diffraction from the hydroxyapatite crystals were observed simultaneously. At BL40XU an X-ray image intensifier was used for the small-angle and a CMOS flatpanel detector for the large-angle region. At BL45XU, a large-area CCD detector was used to cover both regions. A linear microbeam scan at BL40XU showed a detailed distribution of voids and crystals and made it possible to examine the structural details in the lesion. The two-dimensional scan at BL45XU showed distribution of voids and crystals in a wider region in the enamel. The simultaneous small- and wide-angle measurement with a microbeam is a powerful tool to elucidate the mechanisms of demineralization and remineralization in the early caries lesion.

Asymmetry in serial femtosecond crystallography data.

PubMed

Sharma, Amit; Johansson, Linda; Dunevall, Elin; Wahlgren, Weixiao Y; Neutze, Richard; Katona, Gergely

2017-03-01

Serial crystallography is an increasingly important approach to protein crystallography that exploits both X-ray free-electron laser (XFEL) and synchrotron radiation. Serial crystallography recovers complete X-ray diffraction data by processing and merging diffraction images from thousands of randomly oriented non-uniform microcrystals, of which all observations are partial Bragg reflections. Random fluctuations in the XFEL pulse energy spectrum, variations in the size and shape of microcrystals, integrating over millions of weak partial observations and instabilities in the XFEL beam position lead to new types of experimental errors. The quality of Bragg intensity estimates deriving from serial crystallography is therefore contingent upon assumptions made while modeling these data. Here it is observed that serial femtosecond crystallography (SFX) Bragg reflections do not follow a unimodal Gaussian distribution and it is recommended that an idealized assumption of single Gaussian peak profiles be relaxed to incorporate apparent asymmetries when processing SFX data. The phenomenon is illustrated by re-analyzing data collected from microcrystals of the Blastochloris viridis photosynthetic reaction center and comparing these intensity observations with conventional synchrotron data. The results show that skewness in the SFX observations captures the essence of the Wilson plot and an empirical treatment is suggested that can help to separate the diffraction Bragg intensity from the background.

Crystallographic education in the 21st century

PubMed Central

Gražulis, Saulius; Sarjeant, Amy Alexis; Moeck, Peter; Stone-Sundberg, Jennifer; Snyder, Trevor J.; Kaminsky, Werner; Oliver, Allen G.; Stern, Charlotte L.; Dawe, Louise N.; Rychkov, Denis A.; Losev, Evgeniy A.; Boldyreva, Elena V.; Tanski, Joseph M.; Bernstein, Joel; Rabeh, Wael M.; Kantardjieff, Katherine A.

2015-01-01

There are many methods that can be used to incorporate concepts of crystallography into the learning experiences of students, whether they are in elementary school, at university or part of the public at large. It is not always critical that those who teach crystallography have immediate access to diffraction equipment to be able to introduce the concepts of symmetry, packing or molecular structure in an age- and audience-appropriate manner. Crystallography can be used as a tool for teaching general chemistry concepts as well as general research techniques without ever having a student determine a crystal structure. Thus, methods for younger students to perform crystal growth experiments of simple inorganic salts, organic compounds and even metals are presented. For settings where crystallographic instrumentation is accessible (proximally or remotely), students can be involved in all steps of the process, from crystal growth, to data collection, through structure solution and refinement, to final publication. Several approaches based on the presentations in the MS92 Microsymposium at the IUCr 23rd Congress and General Assembly are reported. The topics cover methods for introducing crystallography to undergraduate students as part of a core chemistry curriculum; a successful short-course workshop intended to bootstrap researchers who rely on crystallography for their work; and efforts to bring crystallography to secondary school children and non-science majors. In addition to these workshops, demonstrations and long-format courses, open-format crystallographic databases and three-dimensional printed models as tools that can be used to excite target audiences and inspire them to pursue a deeper understanding of crystallography are described. PMID:26664347

X-ray crystallography over the past decade for novel drug discovery – where are we heading next?

PubMed Central

Zheng, Heping; Handing, Katarzyna B; Zimmerman, Matthew D; Shabalin, Ivan G; Almo, Steven C; Minor, Wladek

2015-01-01

Introduction Macromolecular X-ray crystallography has been the primary methodology for determining the three-dimensional structures of proteins, nucleic acids and viruses. Structural information has paved the way for structure-guided drug discovery and laid the foundations for structural bioinformatics. However, X-ray crystallography still has a few fundamental limitations, some of which may be overcome and complemented using emerging methods and technologies in other areas of structural biology. Areas covered This review describes how structural knowledge gained from X-ray crystallography has been used to advance other biophysical methods for structure determination (and vice versa). This article also covers current practices for integrating data generated by other biochemical and biophysical methods with those obtained from X-ray crystallography. Finally, the authors articulate their vision about how a combination of structural and biochemical/biophysical methods may improve our understanding of biological processes and interactions. Expert opinion X-ray crystallography has been, and will continue to serve as, the central source of experimental structural biology data used in the discovery of new drugs. However, other structural biology techniques are useful not only to overcome the major limitation of X-ray crystallography, but also to provide complementary structural data that is useful in drug discovery. The use of recent advancements in biochemical, spectroscopy and bioinformatics methods may revolutionize drug discovery, albeit only when these data are combined and analyzed with effective data management systems. Accurate and complete data management is crucial for developing experimental procedures that are robust and reproducible. PMID:26177814

Monte Carlo calculation of large and small-angle electron scattering in air

NASA Astrophysics Data System (ADS)

Cohen, B. I.; Higginson, D. P.; Eng, C. D.; Farmer, W. A.; Friedman, A.; Grote, D. P.; Larson, D. J.

2017-11-01

A Monte Carlo method for angle scattering of electrons in air that accommodates the small-angle multiple scattering and larger-angle single scattering limits is introduced. The algorithm is designed for use in a particle-in-cell simulation of electron transport and electromagnetic wave effects in air. The method is illustrated in example calculations.

Human HDAC isoform selectivity achieved via exploitation of the acetate release channel with structurally unique small molecule inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whitehead, Lewis; Dobler, Markus R.; Radetich, Branko

2013-11-20

Herein we report the discovery of a family of novel yet simple, amino-acid derived class I HDAC inhibitors that demonstrate isoform selectivity via access to the internal acetate release channel. Isoform selectivity criteria is discussed on the basis of X-ray crystallography and molecular modeling of these novel inhibitors bound to HDAC8, potentially revealing insights into the mechanism of enzymatic function through novel structural features revealed at the atomic level.

Microgravity and Macromolecular Crystallography

NASA Technical Reports Server (NTRS)

Kundrot, Craig E.; Judge, Russell A.; Pusey, Marc L.; Snell, Edward H.; Rose, M. Franklin (Technical Monitor)

2000-01-01

Macromolecular crystal growth has been seen as an ideal experiment to make use of the reduced acceleration environment provided by an orbiting spacecraft. The experiments are small, simply operated and have a high potential scientific and economic impact. In this review we examine the theoretical reasons why microgravity should be a beneficial environment for crystal growth and survey the history of experiments on the Space Shuttle Orbiter, on unmanned spacecraft, and on the Mir space station. Finally we outline the direction for optimizing the future use of orbiting platforms.

Ab initio structure determination of nanocrystals of organic pharmaceutical compounds by electron diffraction at room temperature using a Timepix quantum area direct electron detector

DOE Office of Scientific and Technical Information (OSTI.GOV)

Genderen, E. van; Clabbers, M. T. B.; Center for Cellular Imaging and NanoAnalytics

A specialized quantum area detector for electron diffraction studies makes it possible to solve the structure of small organic compound nanocrystals in non-cryo conditions by direct methods. Until recently, structure determination by transmission electron microscopy of beam-sensitive three-dimensional nanocrystals required electron diffraction tomography data collection at liquid-nitrogen temperature, in order to reduce radiation damage. Here it is shown that the novel Timepix detector combines a high dynamic range with a very high signal-to-noise ratio and single-electron sensitivity, enabling ab initio phasing of beam-sensitive organic compounds. Low-dose electron diffraction data (∼0.013 e{sup −} Å{sup −2} s{sup −1}) were collected at roommore » temperature with the rotation method. It was ascertained that the data were of sufficient quality for structure solution using direct methods using software developed for X-ray crystallography (XDS, SHELX) and for electron crystallography (ADT3D/PETS, SIR2014)« less

NMR crystallography of α-poly(L-lactide).

PubMed

Pawlak, Tomasz; Jaworska, Magdalena; Potrzebowski, Marek J

2013-03-07

A complementary approach that combines NMR measurements, analysis of X-ray and neutron powder diffraction data and advanced quantum mechanical calculations was employed to study the α-polymorph of L-polylactide. Such a strategy, which is known as NMR crystallography, to the best of our knowledge, is used here for the first time for the fine refinement of the crystal structure of a synthetic polymer. The GIPAW method was used to compute the NMR shielding parameters for the different models, which included the α-PLLA structure obtained by 2-dimensional wide-angle X-ray diffraction (WAXD) at -150 °C (model M1) and at 25 °C (model M2), neutron diffraction (WAND) measurements (model M3) and the fully optimized geometry of the PLLA chains in the unit cell with defined size (model M4). The influence of changes in the chain conformation on the (13)C σ(ii) NMR shielding parameters is shown. The correlation between the σ(ii) and δ(ii) values for the M1-M4 models revealed that the M4 model provided the best fit. Moreover, a comparison of the experimental (13)C NMR spectra with the spectra calculated using the M1-M4 models strongly supports the data for the M4 model. The GIPAW method, via verification using NMR measurements, was shown to be capable of the fine refinement of the crystal structures of polymers when coarse X-ray diffraction data for powdered samples are available.

Investigation of the structure of unilamellar dimyristoylphosphatidylcholine vesicles in aqueous sucrose solutions by small-angle neutron and X-ray scattering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kiselev, M. A., E-mail: elena@jinr.ru; Zemlyanaya, E. V.; Zhabitskaya, E. I.

2015-01-15

The structure of a polydispersed population of unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles in sucrose solutions has been investigated by small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS). Calculations within the model of separated form factors (SFF) show that the structure of the vesicle system depends strongly on the sucrose concentration.

Monte Carlo calculation of large and small-angle electron scattering in air

DOE PAGES

Cohen, B. I.; Higginson, D. P.; Eng, C. D.; ...

2017-08-12

A Monte Carlo method for angle scattering of electrons in air that accommodates the small-angle multiple scattering and larger-angle single scattering limits is introduced. In this work, the algorithm is designed for use in a particle-in-cell simulation of electron transport and electromagnetic wave effects in air. The method is illustrated in example calculations.

Crystallography and Interphase Boundary of Martensite and Bainite in Steels

NASA Astrophysics Data System (ADS)

Furuhara, Tadashi; Chiba, Tadachika; Kaneshita, Takeshi; Wu, Huidong; Miyamoto, Goro

2017-06-01

Grain refinements in lath martensite and bainite structures are crucial for strengthening and toughening of high-strength structural steels. Clearly, crystallography of transformation plays an important role in determining the "grain" sizes in these structures. In the present study, crystallography and intrinsic boundary structure of martensite and bainite are described. Furthermore, various extrinsic factors affecting variant selection and growth kinetics, such as elastic/plastic strain and alloying effects on interphase boundary migration, are discussed.

NIST Standard Reference Material 3600: Absolute Intensity Calibration Standard for Small-Angle X-ray Scattering

DOE PAGES

Allen, Andrew J.; Zhang, Fan; Kline, R. Joseph; ...

2017-03-07

The certification of a new standard reference material for small-angle scattering [NIST Standard Reference Material (SRM) 3600: Absolute Intensity Calibration Standard for Small-Angle X-ray Scattering (SAXS)], based on glassy carbon, is presented. Creation of this SRM relies on the intrinsic primary calibration capabilities of the ultra-small-angle X-ray scattering technique. This article describes how the intensity calibration has been achieved and validated in the certified Q range, Q = 0.008–0.25 Å –1, together with the purpose, use and availability of the SRM. The intensity calibration afforded by this robust and stable SRM should be applicable universally to all SAXS instruments thatmore » employ a transmission measurement geometry, working with a wide range of X-ray energies or wavelengths. As a result, the validation of the SRM SAXS intensity calibration using small-angle neutron scattering (SANS) is discussed, together with the prospects for including SANS in a future renewal certification.« less

NIST Standard Reference Material 3600: Absolute Intensity Calibration Standard for Small-Angle X-ray Scattering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Allen, Andrew J.; Zhang, Fan; Kline, R. Joseph

The certification of a new standard reference material for small-angle scattering [NIST Standard Reference Material (SRM) 3600: Absolute Intensity Calibration Standard for Small-Angle X-ray Scattering (SAXS)], based on glassy carbon, is presented. Creation of this SRM relies on the intrinsic primary calibration capabilities of the ultra-small-angle X-ray scattering technique. This article describes how the intensity calibration has been achieved and validated in the certified Q range, Q = 0.008–0.25 Å –1, together with the purpose, use and availability of the SRM. The intensity calibration afforded by this robust and stable SRM should be applicable universally to all SAXS instruments thatmore » employ a transmission measurement geometry, working with a wide range of X-ray energies or wavelengths. As a result, the validation of the SRM SAXS intensity calibration using small-angle neutron scattering (SANS) is discussed, together with the prospects for including SANS in a future renewal certification.« less

NIST Standard Reference Material 3600: Absolute Intensity Calibration Standard for Small-Angle X-ray Scattering.

PubMed

Allen, Andrew J; Zhang, Fan; Kline, R Joseph; Guthrie, William F; Ilavsky, Jan

2017-04-01

The certification of a new standard reference material for small-angle scattering [NIST Standard Reference Material (SRM) 3600: Absolute Intensity Calibration Standard for Small-Angle X-ray Scattering (SAXS)], based on glassy carbon, is presented. Creation of this SRM relies on the intrinsic primary calibration capabilities of the ultra-small-angle X-ray scattering technique. This article describes how the intensity calibration has been achieved and validated in the certified Q range, Q = 0.008-0.25 Å -1 , together with the purpose, use and availability of the SRM. The intensity calibration afforded by this robust and stable SRM should be applicable universally to all SAXS instruments that employ a transmission measurement geometry, working with a wide range of X-ray energies or wavelengths. The validation of the SRM SAXS intensity calibration using small-angle neutron scattering (SANS) is discussed, together with the prospects for including SANS in a future renewal certification.

Time-resolved structural studies with serial crystallography: A new light on retinal proteins

PubMed Central

Panneels, Valérie; Wu, Wenting; Tsai, Ching-Ju; Nogly, Przemek; Rheinberger, Jan; Jaeger, Kathrin; Cicchetti, Gregor; Gati, Cornelius; Kick, Leonhard M.; Sala, Leonardo; Capitani, Guido; Milne, Chris; Padeste, Celestino; Pedrini, Bill; Li, Xiao-Dan; Standfuss, Jörg; Abela, Rafael; Schertler, Gebhard

2015-01-01

Structural information of the different conformational states of the two prototypical light-sensitive membrane proteins, bacteriorhodopsin and rhodopsin, has been obtained in the past by X-ray cryo-crystallography and cryo-electron microscopy. However, these methods do not allow for the structure determination of most intermediate conformations. Recently, the potential of X-Ray Free Electron Lasers (X-FELs) for tracking the dynamics of light-triggered processes by pump-probe serial femtosecond crystallography has been demonstrated using 3D-micron-sized crystals. In addition, X-FELs provide new opportunities for protein 2D-crystal diffraction, which would allow to observe the course of conformational changes of membrane proteins in a close-to-physiological lipid bilayer environment. Here, we describe the strategies towards structural dynamic studies of retinal proteins at room temperature, using injector or fixed-target based serial femtosecond crystallography at X-FELs. Thanks to recent progress especially in sample delivery methods, serial crystallography is now also feasible at synchrotron X-ray sources, thus expanding the possibilities for time-resolved structure determination. PMID:26798817

An effective introduction to structural crystallography using 1D Gaussian atoms

NASA Astrophysics Data System (ADS)

Smith, Emily; Evans, Gwyndaf; Foadi, James

2017-11-01

The most important quantitative aspects of computational structural crystallography can be introduced in a satisfactory way using 1D truncated and periodic Gaussian functions to represent the atoms in a crystal lattice. This paper describes in detail and demonstrates 1D structural crystallography starting with the definition of such truncated Gaussians. The availability of the computer programme CRONE makes possible the repetition of the examples provided in the paper as well as the creation of new ones.

Bringing diffuse X-ray scattering into focus

DOE PAGES

Wall, Michael E.; Wolff, Alexander M.; Fraser, James S.

2018-02-16

X-ray crystallography is experiencing a renaissance as a method for probing the protein conformational ensemble. The inherent limitations of Bragg analysis, however, which only reveals the mean structure, have given way to a surge in interest in diffuse scattering, which is caused by structure variations. Diffuse scattering is present in all macromolecular crystallography experiments. Recent studies are shedding light on the origins of diffuse scattering in protein crystallography, and provide clues for leveraging diffuse scattering to model protein motions with atomic detail.

Bringing diffuse X-ray scattering into focus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wall, Michael E.; Wolff, Alexander M.; Fraser, James S.

X-ray crystallography is experiencing a renaissance as a method for probing the protein conformational ensemble. The inherent limitations of Bragg analysis, however, which only reveals the mean structure, have given way to a surge in interest in diffuse scattering, which is caused by structure variations. Diffuse scattering is present in all macromolecular crystallography experiments. Recent studies are shedding light on the origins of diffuse scattering in protein crystallography, and provide clues for leveraging diffuse scattering to model protein motions with atomic detail.

Pinhole-type two-dimensional ultra-small-angle X-ray scattering on the micrometer scale

PubMed Central

Kishimoto, Hiroyuki; Shinohara, Yuya; Suzuki, Yoshio; Takeuchi, Akihisa; Yagi, Naoto; Amemiya, Yoshiyuki

2014-01-01

A pinhole-type two-dimensional ultra-small-angle X-ray scattering set-up at a so-called medium-length beamline at SPring-8 is reported. A long sample-to-detector distance, 160.5 m, can be used at this beamline and a small-angle resolution of 0.25 µm−1 was thereby achieved at an X-ray energy of 8 keV. PMID:24365910

Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners

PubMed Central

Felicori, Liza; Jameson, Katie H.; Roblin, Pierre; Fogg, Mark J.; Garcia-Garcia, Transito; Ventroux, Magali; Cherrier, Mickaël V.; Bazin, Alexandre; Noirot, Philippe; Wilkinson, Anthony J.; Molina, Franck; Terradot, Laurent; Noirot-Gros, Marie-Françoise

2016-01-01

YabA negatively regulates initiation of DNA replication in low-GC Gram-positive bacteria. The protein exerts its control through interactions with the initiator protein DnaA and the sliding clamp DnaN. Here, we combined X-ray crystallography, X-ray scattering (SAXS), modeling and biophysical approaches, with in vivo experimental data to gain insight into YabA function. The crystal structure of the N-terminal domain (NTD) of YabA solved at 2.7 Å resolution reveals an extended α-helix that contributes to an intermolecular four-helix bundle. Homology modeling and biochemical analysis indicates that the C-terminal domain (CTD) of YabA is a small Zn-binding domain. Multi-angle light scattering and SAXS demonstrate that YabA is a tetramer in which the CTDs are independent and connected to the N-terminal four-helix bundle via flexible linkers. While YabA can simultaneously interact with both DnaA and DnaN, we found that an isolated CTD can bind to either DnaA or DnaN, individually. Site-directed mutagenesis and yeast-two hybrid assays identified DnaA and DnaN binding sites on the YabA CTD that partially overlap and point to a mutually exclusive mode of interaction. Our study defines YabA as a novel structural hub and explains how the protein tetramer uses independent CTDs to bind multiple partners to orchestrate replication initiation in the bacterial cell. PMID:26615189

Mechanistic Insights from Structural Analyses of Ran-GTPase-Driven Nuclear Export of Proteins and RNAs.

PubMed

Matsuura, Yoshiyuki

2016-05-22

Understanding how macromolecules are rapidly exchanged between the nucleus and the cytoplasm through nuclear pore complexes is a fundamental problem in biology. Exportins are Ran-GTPase-dependent nuclear transport factors that belong to the karyopherin-β family and mediate nuclear export of a plethora of proteins and RNAs, except for bulk mRNA nuclear export. Exportins bind cargo macromolecules in a Ran-GTP-dependent manner in the nucleus, forming exportin-cargo-Ran-GTP complexes (nuclear export complexes). Transient weak interactions between exportins and nucleoporins containing characteristic FG (phenylalanine-glycine) repeat motifs facilitate nuclear pore complex passage of nuclear export complexes. In the cytoplasm, nuclear export complexes are disassembled, thereby releasing the cargo. GTP hydrolysis by Ran promoted in the cytoplasm makes the disassembly reaction virtually irreversible and provides thermodynamic driving force for the overall export reaction. In the past decade, X-ray crystallography of some of the exportins in various functional states coupled with functional analyses, single-particle electron microscopy, molecular dynamics simulations, and small-angle solution X-ray scattering has provided rich insights into the mechanism of cargo binding and release and also begins to elucidate how exportins interact with the FG repeat motifs. The knowledge gained from structural analyses of nuclear export is being translated into development of clinically useful inhibitors of nuclear export to treat human diseases such as cancer and influenza. Copyright © 2015 Elsevier Ltd. All rights reserved.

NMR crystallography of campho[2,3-c]pyrazole (Z' = 6): combining high-resolution 1H-13C solid-state MAS NMR spectroscopy and GIPAW chemical-shift calculations.

PubMed

Webber, Amy L; Emsley, Lyndon; Claramunt, Rosa M; Brown, Steven P

2010-09-30

(1)H-(13)C two-dimensional magic-angle spinning (MAS) solid-state NMR correlation spectra, recorded with the MAS-J-HMQC experiment, are presented for campho[2,3-c]pyrazole. For each (13)C moiety, there are six resonances associated with the six distinct molecules in the asymmetric unit cell (Z' = 6). The one-bond C-H correlations observed in the 2D (1)H-(13)C MAS-J-HMQC spectra allow the experimental determination of the (1)H and (13)C chemical shifts associated with the separate CH, CH(2), and CH(3) groups. (1)H and (13)C chemical shifts calculated by using the GIPAW (Gauge Including Projector Augmented Waves) plane-wave pseudopotential approach are presented. Calculations for the whole unit cell (12 × 29 = 348 atoms, with geometry optimization of all atoms) allow the assignment of the experimental (1)H and (13)C chemical shifts to the six distinct molecules. The calculated chemical shifts for the full crystal structure are compared with those for isolated molecules as extracted from the geometry-optimized crystal structure. In this way, the effect of intermolecular interactions on the observed chemical shifts is quantified. In particular, the calculations are sufficiently precise to differentiate the small (<1 ppm) differences between the (1)H chemical shifts of the six resonances associated with each distinct CH or CH(2) moiety.

Crystal structures, spectroscopic and theoretical study of novel Schiff bases of 2-(methylthiomethyl)anilines.

PubMed

Olalekan, Temitope E; Adejoro, Isaiah A; VanBrecht, Bernardus; Watkins, Gareth M

2015-03-15

New Schiff bases derived from p-methoxysalicylaldehyde and 2-(methylthiomethyl)anilines (substituted with methyl, methoxy, nitro) were synthesized and characterized by elemental analyses, FT-IR, NMR, electronic spectra and quantum chemical calculations. X-ray crystallography of two compounds showed the solid structures are stabilized by intramolecular and intermolecular H-bonds. The effect of OH⋯N interaction between the phenolic hydrogen and imine nitrogen on the proton and carbon NMR shifts, and the role of CH⋯O and CH⋯S contacts are discussed. The bond lengths and angles, (1)H and (13)C NMR data, E(LUMO-HOMO), dipole moments and polarizability of the compounds were predicted by density functional theory, DFT (B3LYP/6-31G∗∗) method. The experimental geometric parameters and the NMR shifts were compared with the calculated values, which gave good correlations. The electronic effects of aryl ring substituents (methyl, methoxy and nitro) on the properties of the resulting compounds, such as the color, NMR shifts, electronic spectra and the calculated energy band gaps, dipole moments and polarizability are discussed. Increase in electron density shifted the phenolic proton resonance to lower fields. The methoxy-substituted compound has a small dipole moment and subsequent large polarizability value. Highest polarity was indicated by the nitro compound which also showed high polarizability due to its larger size. The energy gaps obtained from E(LUMO-HOMO) calculations suggest these compounds may have applications as organic semiconducting materials. Copyright © 2014 Elsevier B.V. All rights reserved.

Structural model of the p14/SF3b155 · branch duplex complex.

PubMed

Schellenberg, Matthew J; Dul, Erin L; MacMillan, Andrew M

2011-01-01

Human p14 (SF3b14), a component of the spliceosomal U2 snRNP, interacts directly with the pre-mRNA branch adenosine within the context of the bulged duplex formed between the pre-mRNA branch region and U2 snRNA. This association occurs early in spliceosome assembly and persists within the fully assembled spliceosome. Analysis of the crystal structure of a complex containing p14 and a peptide derived from p14-associated SF3b155 combined with the results of cross-linking studies has suggested that the branch nucleotide interacts with a pocket on a non-canonical RNA binding surface formed by the complex. Here we report a structural model of the p14 · bulged duplex interaction based on a combination of X-ray crystallography of an adenine p14/SF3b155 peptide complex, biochemical comparison of a panel of disulfide cross-linked protein-RNA complexes, and small-angle X-ray scattering (SAXS). These studies reveal specific recognition of the branch adenosine within the p14 pocket and establish the orientation of the bulged duplex RNA bound on the protein surface. The intimate association of one surface of the bulged duplex with the p14/SF3b155 peptide complex described by this model buries the branch nucleotide at the interface and suggests that p14 · duplex interaction must be disrupted before the first step of splicing.

Structural model of the p14/SF3b155·branch duplex complex

PubMed Central

Schellenberg, Matthew J.; Dul, Erin L.; MacMillan, Andrew M.

2011-01-01

Human p14 (SF3b14), a component of the spliceosomal U2 snRNP, interacts directly with the pre-mRNA branch adenosine within the context of the bulged duplex formed between the pre-mRNA branch region and U2 snRNA. This association occurs early in spliceosome assembly and persists within the fully assembled spliceosome. Analysis of the crystal structure of a complex containing p14 and a peptide derived from p14-associated SF3b155 combined with the results of cross-linking studies has suggested that the branch nucleotide interacts with a pocket on a non-canonical RNA binding surface formed by the complex. Here we report a structural model of the p14•bulged duplex interaction based on a combination of X-ray crystallography of an adenine p14/SF3b155 peptide complex, biochemical comparison of a panel of disulfide cross-linked protein–RNA complexes, and small-angle X-ray scattering (SAXS). These studies reveal specific recognition of the branch adenosine within the p14 pocket and establish the orientation of the bulged duplex RNA bound on the protein surface. The intimate association of one surface of the bulged duplex with the p14/SF3b155 peptide complex described by this model buries the branch nucleotide at the interface and suggests that p14•duplex interaction must be disrupted before the first step of splicing. PMID:21062891

[Structural Study in the Platform for Drug Discovery, Informatics, and Structural Life Science].

PubMed

Senda, Toshiya

2016-01-01

The Platform for Drug Discovery, Informatics, and Structural Life Science (PDIS), which has been launched since FY2012, is a national project in the field of structural biology. The PDIS consists of three cores - structural analysis, control, and informatics - and aims to support life science researchers who are not familiar with structural biology. The PDIS project is able to provide full-scale support for structural biology research. The support provided by the PDIS project includes protein purification with various expression systems, large scale protein crystallization, crystal structure determination, small angle scattering (SAXS), NMR, electron microscopy, bioinformatics, etc. In order to utilize these methods of support, PDIS users need to submit an application form to the one-stop service office. Submitted applications will be reviewed by three referees. It is strongly encouraged that PDIS users have sufficient discussion with researchers in the PDIS project before submitting the application. This discussion is very useful in the process of project design, particularly for beginners in structural biology. In addition to this user support, the PDIS project has conducted R&D, which includes the development of synchrotron beamlines. In the PDIS project, PF and SPring-8 have developed beamlines for micro-crystallography, high-throughput data collection, supramolecular assembly, and native single anomalous dispersion (SAD) phasing. The newly developed beamlines have been open to all users, and have accelerated structural biology research. Beamlines for SAXS have also been developed, which has dramatically increased bio-SAXS users.

An Optical Crystallography Instructional Package on Videocassettes.

ERIC Educational Resources Information Center

Birnie, Richard W.

1980-01-01

Describes a self-teaching instructional package on color videocassettes, supplemented with audio descriptions, prepared from original super-8mm cinephotomicrographs for use in optical crystallography courses. Production techniques are also reviewed. (Author/JN)

From Macrocrystals to Microcrystals: A Strategy for Membrane Protein Serial Crystallography.

PubMed

Dods, Robert; Båth, Petra; Arnlund, David; Beyerlein, Kenneth R; Nelson, Garrett; Liang, Mengling; Harimoorthy, Rajiv; Berntsen, Peter; Malmerberg, Erik; Johansson, Linda; Andersson, Rebecka; Bosman, Robert; Carbajo, Sergio; Claesson, Elin; Conrad, Chelsie E; Dahl, Peter; Hammarin, Greger; Hunter, Mark S; Li, Chufeng; Lisova, Stella; Milathianaki, Despina; Robinson, Joseph; Safari, Cecilia; Sharma, Amit; Williams, Garth; Wickstrand, Cecilia; Yefanov, Oleksandr; Davidsson, Jan; DePonte, Daniel P; Barty, Anton; Brändén, Gisela; Neutze, Richard

2017-09-05

Serial protein crystallography was developed at X-ray free-electron lasers (XFELs) and is now also being applied at storage ring facilities. Robust strategies for the growth and optimization of microcrystals are needed to advance the field. Here we illustrate a generic strategy for recovering high-density homogeneous samples of microcrystals starting from conditions known to yield large (macro) crystals of the photosynthetic reaction center of Blastochloris viridis (RC vir ). We first crushed these crystals prior to multiple rounds of microseeding. Each cycle of microseeding facilitated improvements in the RC vir serial femtosecond crystallography (SFX) structure from 3.3-Å to 2.4-Å resolution. This approach may allow known crystallization conditions for other proteins to be adapted to exploit novel scientific opportunities created by serial crystallography. Copyright © 2017 Elsevier Ltd. All rights reserved.

Raman crystallography of RNA.

PubMed

Gong, Bo; Chen, Jui-Hui; Yajima, Rieko; Chen, Yuanyuan; Chase, Elaine; Chadalavada, Durga M; Golden, Barbara L; Carey, Paul R; Bevilacqua, Philip C

2009-10-01

Raman crystallography is the application of Raman spectroscopy to single crystals. This technique has been applied to a variety of protein molecules where it has provided unique information about biopolymer folding, substrate binding, and catalysis. Here, we describe the application of Raman crystallography to functional RNA molecules. RNA represents unique opportunities and challenges for Raman crystallography. One issue that confounds studies of RNA is its tendency to adopt multiple non-functional folds. Raman crystallography has the advantage that it isolates a single state of the RNA within the crystal and can evaluate its fold, metal ion binding properties (ligand identity, stoichiometry, and affinity), proton binding properties (identity, stoichiometry, and affinity), and catalytic potential. In particular, base-specific stretches can be identified and then associated with the binding of metal ions and protons. Because measurements are carried out in the hanging drop at ambient, rather than cryo, conditions and because RNA crystals tend to be approximately 70% solvent, RNA dynamics and conformational changes become experimentally accessible. This review focuses on experimental setup and procedures, acquisition and interpretation of Raman data, and determination of physicochemical properties of the RNA. Raman crystallographic and solution biochemical experiments on the HDV RNA enzyme are summarized and found to be in excellent agreement. Remarkably, characterization of the crystalline state has proven to help rather than hinder functional characterization of functional RNA, most likely because the tendency of RNA to fold heterogeneously is limited in a crystalline environment. Future applications of Raman crystallography to RNA are briefly discussed.

Small-angle X-ray scattering (SAXS) studies of the structure of mesoporous silicas

NASA Astrophysics Data System (ADS)

Zienkiewicz-Strzałka, M.; Skibińska, M.; Pikus, S.

2017-11-01

Mesoporous ordered silica nanostructures show strong interaction with X-ray radiation in the range of small-angles. Small-angle X-ray scattering (SAXS) measurements based on the elastically scattered X-rays are important in analysis of condensed matter. In the case of mesoporous silica materials SAXS technique provides information on the distribution of electron density in the mesoporous material, in particular describing their structure and size of the unit cell as well as type of ordered structure and finally their parameters. The characterization of nanopowder materials, nanocomposites and porous materials by Small-Angle X-ray Scattering seems to be valuable and useful. In presented work, the SAXS investigation of structures from the group of mesoporous ordered silicates was performed. This work has an objective to prepare functional materials modified by noble metal ions and nanoparticles and using the small-angle X-ray scattering to illustrate their properties. We report the new procedure for describing mesoporous materials belonging to SBA-15 and MCM-41 family modified by platinum, palladium and silver nanoparticles, based on detailed analysis of characteristic peaks in the small-angle range of X-ray scattering. This procedure allows to obtained the most useful parameters for mesoporous materials characterization and their successfully compare with experimental measurements reducing the time and material consumption with good precision for particles and pores with a size below 10 nm.

Semiconductor Based Transverse Bragg Resonance (TBR) Optical Amplifiers and Lasers

DTIC Science & Technology

2007-02-14

modes with small modal angles experience zero or very low radiation loss. We call these modes small modal angle (SMA) modes. SMA modes include both...lossless effective index-guided modes and low loss leaky modes. They are almost parallel to the graing and do not radiate significantly. As the modal...angle increases, all the modes experience higher radiation loss. However, around the transverse resonance angle of 13.80, low loss modes exist. These

A brief history of macromolecular crystallography, illustrated by a family tree and its Nobel fruits.

PubMed

Jaskolski, Mariusz; Dauter, Zbigniew; Wlodawer, Alexander

2014-09-01

As a contribution to the celebration of the year 2014, declared by the United Nations to be 'The International Year of Crystallography', the FEBS Journal is dedicating this issue to papers showcasing the intimate union between macromolecular crystallography and structural biology, both in historical perspective and in current research. Instead of a formal editorial piece, by way of introduction, this review discusses the most important, often iconic, achievements of crystallographers that led to major advances in our understanding of the structure and function of biological macromolecules. We identified at least 42 scientists who received Nobel Prizes in Physics, Chemistry or Medicine for their contributions that included the use of X-rays or neutrons and crystallography, including 24 who made seminal discoveries in macromolecular sciences. Our spotlight is mostly, but not only, on the recipients of this most prestigious scientific honor, presented in approximately chronological order. As a summary of the review, we attempt to construct a genealogy tree of the principal lineages of protein crystallography, leading from the founding members to the present generation. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Entering an era of dynamic structural biology….

PubMed

Orville, Allen M

2018-05-31

A recent paper in BMC Biology presents a general method for mix-and-inject serial crystallography, to facilitate the visualization of enzyme intermediates via time-resolved serial femtosecond crystallography (tr-SFX). They apply their method to resolve in near atomic detail the cleavage and inactivation of the antibiotic ceftriaxone by a β-lactamase enzyme from Mycobacterium tuberculosis. Their work demonstrates the general applicability of time-resolved crystallography, from which dynamic structures, at atomic resolution, can be obtained.See research article: https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-018-0524-5 .

Macromolecular crystallography beamline X25 at the NSLS

PubMed Central

Héroux, Annie; Allaire, Marc; Buono, Richard; Cowan, Matthew L.; Dvorak, Joseph; Flaks, Leon; LaMarra, Steven; Myers, Stuart F.; Orville, Allen M.; Robinson, Howard H.; Roessler, Christian G.; Schneider, Dieter K.; Shea-McCarthy, Grace; Skinner, John M.; Skinner, Michael; Soares, Alexei S.; Sweet, Robert M.; Berman, Lonny E.

2014-01-01

Beamline X25 at the NSLS is one of the five beamlines dedicated to macromolecular crystallography operated by the Brookhaven National Laboratory Macromolecular Crystallography Research Resource group. This mini-gap insertion-device beamline has seen constant upgrades for the last seven years in order to achieve mini-beam capability down to 20 µm × 20 µm. All major components beginning with the radiation source, and continuing along the beamline and its experimental hutch, have changed to produce a state-of-the-art facility for the scientific community. PMID:24763654

Invariant Deformation Element Model Interpretation to the Crystallography of Diffusional Body-Centered-Cube to Face-Centered-Cube Phase Transformations

NASA Astrophysics Data System (ADS)

Liu, Hongwei; Liu, Jiangwen; Su, Guangcai; Li, Weizhou; Zeng, Jianmin; Hu, Zhiliu

2012-10-01

The crystallography of body-centered-cube to face-centered cube (bcc-to-fcc) diffusion phase transformations in a duplex stainless steel and a Cu-Zn alloy, including long axis, orientation relationship (OR), habit plane (HP), and dislocation spacing, is successfully interpreted with one-step rotation from the Bain lattice relationship by applying a simplified invariant line (IL) analysis. It is proposed that the dislocation slipping direction in the matrix plays an important role in controlling the crystallography of precipitation.

Thermal gradient crystals as tuneable monochromator for high energy X-rays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ruett, U.; Schulte-Schrepping, H.; Heuer, J.

2010-06-23

At the high energy synchrotron radiation beamline BW5 at DORIS III at DESY a new monochromator providing broad energy bandwidth and high reflectivity is in use. On a small 10x10x5 mm{sup 3} silicon crystal scattering at the (311) reflection a thermal gradient is applied, which tunes the scattered energy bandwidth. The (311) reflection strongly suppresses the higher harmonics allowing the use of an image plate detector for crystallography. The monochromator can be used at photon energies above 60 keV.

Applications of SHAPES screening in drug discovery.

PubMed

Lepre, Christopher A; Peng, Jeffrey; Fejzo, Jasna; Abdul-Manan, Norzehan; Pocas, Jennifer; Jacobs, Marc; Xie, Xiaoling; Moore, Jonathan M

2002-12-01

The SHAPES strategy combines nuclear magnetic resonance (NMR) screening of a library of small drug-like molecules with a variety of complementary methods, such as virtual screening, high throughput enzymatic assays, combinatorial chemistry, X-ray crystallography, and molecular modeling, in a directed search for new medicinal chemistry leads. In the past few years, the SHAPES strategy has found widespread utility in pharmaceutical research. To illustrate a variety of different implementations of the method, we will focus in this review on recent applications of the SHAPES strategy in several drug discovery programs at Vertex Pharmaceuticals.

The Protein Micro-Crystallography Beamlines for Targeted Protein Research Program

NASA Astrophysics Data System (ADS)

Hirata, Kunio; Yamamoto, Masaki; Matsugaki, Naohiro; Wakatsuki, Soichi

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 Proteins Research Program by MEXT of Japan. The program aims to reveal the structure and function of proteins that are difficult to solve but have great importance in both academic research and industrial application. At SPring-8, a new 1-micron beam beamline for protein micro-crystallography, RIKEN Targeted Proteins Beamline (BL32XU), is developed. At KEK-PF a new low energy micro-beam beamline, BL-1A, is dedicated for SAD micro-crystallography. The two beamlines will start operation in the end of 2010. The present status of the research and development for protein micro-crystallography will be presented.

Development of the field of structural physiology

PubMed Central

FUJIYOSHI, Yoshinori

2015-01-01

Electron crystallography is especially useful for studying the structure and function of membrane proteins — key molecules with important functions in neural and other cells. Electron crystallography is now an established technique for analyzing the structures of membrane proteins in lipid bilayers that closely simulate their natural biological environment. Utilizing cryo-electron microscopes with helium-cooled specimen stages that were developed through a personal motivation to understand the functions of neural systems from a structural point of view, the structures of membrane proteins can be analyzed at a higher than 3 Å resolution. This review covers four objectives. First, I introduce the new research field of structural physiology. Second, I recount some of the struggles involved in developing cryo-electron microscopes. Third, I review the structural and functional analyses of membrane proteins mainly by electron crystallography using cryo-electron microscopes. Finally, I discuss multifunctional channels named “adhennels” based on structures analyzed using electron and X-ray crystallography. PMID:26560835

Overview of electron crystallography of membrane proteins: crystallization and screening strategies using negative stain electron microscopy.

PubMed

Nannenga, Brent L; Iadanza, Matthew G; Vollmar, Breanna S; Gonen, Tamir

2013-01-01

Electron cryomicroscopy, or cryoEM, is an emerging technique for studying the three-dimensional structures of proteins and large macromolecular machines. Electron crystallography is a branch of cryoEM in which structures of proteins can be studied at resolutions that rival those achieved by X-ray crystallography. Electron crystallography employs two-dimensional crystals of a membrane protein embedded within a lipid bilayer. The key to a successful electron crystallographic experiment is the crystallization, or reconstitution, of the protein of interest. This unit describes ways in which protein can be expressed, purified, and reconstituted into well-ordered two-dimensional crystals. A protocol is also provided for negative stain electron microscopy as a tool for screening crystallization trials. When large and well-ordered crystals are obtained, the structures of both protein and its surrounding membrane can be determined to atomic resolution.

Progress in protein crystallography.

PubMed

Dauter, Zbigniew; Wlodawer, Alexander

2016-01-01

Macromolecular crystallography evolved enormously from the pioneering days, when structures were solved by "wizards" performing all complicated procedures almost by hand. In the current situation crystal structures of large systems can be often solved very effectively by various powerful automatic programs in days or hours, or even minutes. Such progress is to a large extent coupled to the advances in many other fields, such as genetic engineering, computer technology, availability of synchrotron beam lines and many other techniques, creating the highly interdisciplinary science of macromolecular crystallography. Due to this unprecedented success crystallography is often treated as one of the analytical methods and practiced by researchers interested in structures of macromolecules, but not highly competent in the procedures involved in the process of structure determination. One should therefore take into account that the contemporary, highly automatic systems can produce results almost without human intervention, but the resulting structures must be carefully checked and validated before their release into the public domain.

Crystallography taken to the extreme

NASA Astrophysics Data System (ADS)

Dubrovinskaia, Natalia; Dubrovinsky, Leonid

2018-06-01

This article is a brief autobiographical account of our life in science and the path that we took in performing the research for which we were awarded the Gregori Aminoff Prize in Crystallography 2017 by the Royal Swedish Academy of Sciences. We were invited to write it by the editor-in-chief of Physica Scripta, Suzy Lidström, who charged us with the task of contributing to a series of autobiographical articles published since 2014, the International Year of Crystallography, on the lives of the Aminoff Prize winners. As this series is intended to be of particular interest to young scientists, teachers and lecturers and those researching the history of science, we tried to adhere to this purpose while writing our story. It does not pretend to be a comprehensive review either of our own scientific results or, especially, of covering the complete history of the research field of high-pressure crystallography in which we are active.

IMAGINE: first neutron protein structure and new capabilities for neutron macromolecular crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Munshi, Parthapratim; Myles, Dean A A; Robertson, Lee

2013-01-01

We report the first high resolution neutron protein structure of perdeuterated rubredoxin from Pyrococcus furiosus (PfRd) determined using the new IMAGINE macromolecular neutron crystallography instrument at the Oak Ridge National Laboratory. Neutron diffraction data extending to 1.65 resolution were collected from a relatively small 0.7 mm3 PfRd crystal using 2.5 days (60 h) of beam time. The refined structure contains 371 out of 391, or 95%, of the deuterium atoms of the protein, and 58 solvent molecules. The IMAGINE instrument is designed to provide neutron data at or near atomic resolutions (1.5 ) from crystals with volume < 1.0 mm3more » and with unit cell edges < 100 . Beam line features include elliptical focusing mirrors that deliver 3x107 n s-1 cm-2 into a 3.5 x 2.0 mm2 focal spot at the sample position, and variable short and long wavelength cutoff optics that provide automated exchange between multiple wavelength configurations ( min=2.0 , 2.8 , 3.3 - max =3.0 , 4.0 , 4.5 , ~20 ). Notably, the crystal used to collect this PfRd data is 5-10 times smaller than has been previously reported.« less

Optical metasurfaces for high angle steering at visible wavelengths

DOE PAGES

Lin, Dianmin; Melli, Mauro; Poliakov, Evgeni; ...

2017-05-23

Metasurfaces have facilitated the replacement of conventional optical elements with ultrathin and planar photonic structures. Previous designs of metasurfaces were limited to small deflection angles and small ranges of the angle of incidence. Here, we have created two types of Si-based metasurfaces to steer visible light to a large deflection angle. These structures exhibit high diffraction efficiencies over a broad range of angles of incidence. We have demonstrated metasurfaces working both in transmission and reflection modes based on conventional thin film silicon processes that are suitable for the large-scale fabrication of high-performance devices.

The Small-Angle Neutron Scattering Data Analysis of the Phospholipid Transport Nanosystem Structure

NASA Astrophysics Data System (ADS)

Zemlyanaya, E. V.; Kiselev, M. A.; Zhabitskaya, E. I.; Aksenov, V. L.; Ipatova, O. M.; Ivankov, O. I.

2018-05-01

The small-angle neutron scattering technique (SANS) is employed for investigation of structure of the phospholipid transport nanosystem (PTNS) elaborated in the V.N.Orekhovich Institute of Biomedical Chemistry (Moscow, Russia). The SANS spectra have been measured at the YuMO small-angle spectrometer of IBR-2 reactor (Joint Institute of Nuclear Research, Dubna, Russia). Basic characteristics of polydispersed population of PTNS unilamellar vesicles (average radius of vesicles, polydispersity, thickness of membrane, etc.) have been determined in three cases of the PTNS concentrations in D2O: 5%, 10%, and 25%. Numerical analysis is based on the separated form factors method (SFF). The results are discussed in comparison with the results of analysis of the small-angle X-ray scattering spectra collected at the Kurchatov Synchrotron Radiation Source of the National Research Center “Kurchatov Institute” (Moscow, Russia).

Structural evolution of photocrosslinked silk fibroin and silk fibroin-based hybrid hydrogels: A small angle and ultra-small angle scattering investigation.

PubMed

Whittaker, Jasmin L; Balu, Rajkamal; Knott, Robert; de Campo, Liliana; Mata, Jitendra P; Rehm, Christine; Hill, Anita J; Dutta, Naba K; Roy Choudhury, Namita

2018-07-15

Regenerated Bombyx mori silk fibroin (RSF) is a widely recognized protein for biomedical applications; however, its hierarchical gel structure is poorly understood. In this paper, the hierarchical structure of photocrosslinked RSF and RSF-based hybrid hydrogel systems: (i) RSF/Rec1-resilin and (ii) RSF/poly(N-vinylcaprolactam (PVCL) is reported for the first time using small-angle scattering (SAS) techniques. The structure of RSF in dilute to concentrated solution to fabricated hydrogels were characterized using small angle X-ray scattering (SAXS), small angle neutron scattering (SANS) and ultra-small angle neutron scattering (USANS) techniques. The RSF hydrogel exhibited three distinctive structural characteristics: (i) a Porod region in the length scale of 2 to 3nm due to hydrophobic domains (containing β-sheets) which exhibits sharp interfaces with the amorphous matrix of the hydrogel and the solvent, (ii) a Guinier region in the length scale of 4 to 20nm due to hydrophilic domains (containing turns and random coil), and (iii) a Porod-like region in the length scale of few micrometers due to water pores/channels exhibiting fractal-like characteristics. Addition of Rec1-resilin or PVCL to RSF and subsequent crosslinking systematically increased the nanoscale size of hydrophobic and hydrophilic domains, whereas decreased the homogeneity of pore size distribution in the microscale. The presented results have implications on the fundamental understanding of the structure-property relationship of RSF-based hydrogels. Copyright © 2018. Published by Elsevier B.V.

Prediction of the noise from a propeller at angle of attack

NASA Technical Reports Server (NTRS)

Krejsa, Eugene A.

1990-01-01

An analysis is presented to predict the noise of a propeller at angle of attack. The analysis is an extension of that reported by Mani which predicted the change in noise due to angle of attack to both unsteady loading and to azimuthal variation of the radiation efficiency of steady noise sources. Mani's analysis, however, was limited to small angles of attack. The analysis reported herein removes this small angle limitation. Results from the analysis are compared with the data of Woodward for a single rotation propeller and a counter rotating propeller. The comparison shows that including the effect of angle of attack on the steady noise sources significantly improves the agreement with data. Including higher order effects of angle of attack, while changing the predicted noise at far forward and aft angles, has little effect near the propeller plane.

Irena : tool suite for modeling and analysis of small-angle scattering.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ilavsky, J.; Jemian, P.

2009-04-01

Irena, a tool suite for analysis of both X-ray and neutron small-angle scattering (SAS) data within the commercial Igor Pro application, brings together a comprehensive suite of tools useful for investigations in materials science, physics, chemistry, polymer science and other fields. In addition to Guinier and Porod fits, the suite combines a variety of advanced SAS data evaluation tools for the modeling of size distribution in the dilute limit using maximum entropy and other methods, dilute limit small-angle scattering from multiple non-interacting populations of scatterers, the pair-distance distribution function, a unified fit, the Debye-Bueche model, the reflectivity (X-ray and neutron)more » using Parratt's formalism, and small-angle diffraction. There are also a number of support tools, such as a data import/export tool supporting a broad sampling of common data formats, a data modification tool, a presentation-quality graphics tool optimized for small-angle scattering data, and a neutron and X-ray scattering contrast calculator. These tools are brought together into one suite with consistent interfaces and functionality. The suite allows robust automated note recording and saving of parameters during export.« less

FASEB Science Research Conference on Ion Channel Regulation

DTIC Science & Technology

2015-11-02

advanced single-molecule fluorescence and microscopy, and x-ray crystallography . This grant provided support for a conference that brought...ray crystallography . This grant provided support for a conference that brought together scientists from a wide variety of disciplines but with several

Remote Access to the PXRR Macromolecular Crystallography Facilities at the NSLS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Soares, A.S.; Schneider, D. K.; Skinner, J. M.

2008-09-01

The most recent surge of innovations that have simplified and streamlined the process of determining macromolecular structures by crystallography owes much to the efforts of the structural genomics community. However, this was only the last step in a long evolution that saw the metamorphosis of crystallography from an heroic effort that involved years of dedication and skill into a straightforward measurement that is occasionally almost trivial. Many of the steps in this remarkable odyssey involved reducing the physical labor that is demanded of experimenters in the field. Other steps reduced the technical expertise required for conducting those experiments.

Remote Access to the PXRR Macromolecular Crystallography Facilities at the NSLS

DOE Office of Scientific and Technical Information (OSTI.GOV)

A Soares; D Schneider; J Skinner

2011-12-31

The most recent surge of innovations that have simplified and streamlined the process of determining macromolecular structures by crystallography owes much to the efforts of the structural genomics community. However, this was only the last step in a long evolution that saw the metamorphosis of crystallography from an heroic effort that involved years of dedication and skill into a straightforward measurement that is occasionally almost trivial. Many of the steps in this remarkable odyssey involved reducing the physical labor that is demanded of experimenters in the field. Other steps reduced the technical expertise required for conducting those experiments.

A Structural Biology and Protein Engineering Approach to the Engineering Highly Proficient Catalytic Bioscavengers for In Vivo Detoxification of a Broad Spectrum of Nerve Agents

DTIC Science & Technology

2016-06-30

enzyme. 2 6. The N-terminus of subunit B of PTE-A53 was shown by X-ray crystallography to protrude into the active site of subunit A in a symmetry... crystallography unit. 8. The tagless C23-A203L variant was over-expressed and purified , and was tested for protection against VX intoxication by Prof. Franz...2.3 A, was collected ’in house’, at the WIS X-ray Crystallography Facility. The data collected are summarized in Table 10. Figure 15: The crystals

Native sulfur/chlorine SAD phasing for serial femtosecond crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakane, Takanori; Song, Changyong; POSTECH, Pohang 790-784

Sulfur SAD phasing facilitates the structure determination of diverse native proteins using femtosecond X-rays from free-electron lasers via serial femtosecond crystallography. Serial femtosecond crystallography (SFX) allows structures to be determined with minimal radiation damage. However, phasing native crystals in SFX is not very common. Here, the structure determination of native lysozyme from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of sulfur and chlorine at a wavelength of 1.77 Å is successfully demonstrated. This sulfur SAD method can be applied to a wide range of proteins, which will improve the determination of native crystal structures.

Crystallography Open Databases and Preservation: a World-wide Initiative

NASA Astrophysics Data System (ADS)

Chateigner, Daniel

In 2003, an international team of crystallographers proposed the Crystallography Open Database (COD), a fully-free collection of crystal structure data, in the aim of ensuring their preservation. With nearly 250000 entries, this database represents a large open set of data for crystallographers, academics and industrials, located at five different places world-wide, and included in Thomson-Reuters’ ISI. As a large step towards data preservation, raw data can now be uploaded along with «digested» structure files, and COD can be questioned by most of the crystallography-linked industrial software. The COD initiative work deserves several other open developments.

Fab Chaperone-Assisted RNA Crystallography (Fab CARC).

PubMed

Sherman, Eileen; Archer, Jennifer; Ye, Jing-Dong

2016-01-01

Recent discovery of structured RNAs such as ribozymes and riboswitches shows that there is still much to learn about the structure and function of RNAs. Knowledge learned can be employed in both biochemical research and clinical applications. X-ray crystallography gives unparalleled atomic-level structural detail from which functional inferences can be deduced. However, the difficulty in obtaining high-quality crystals and their phasing information make it a very challenging task. RNA crystallography is particularly arduous due to several factors such as RNA's paucity of surface chemical diversity, lability, repetitive anionic backbone, and flexibility, all of which are counterproductive to crystal packing. Here we describe Fab chaperone assisted RNA crystallography (CARC), a systematic technique to increase RNA crystallography success by facilitating crystal packing as well as expediting phase determination through molecular replacement of conserved Fab domains. Major steps described in this chapter include selection of a synthetic Fab library displayed on M13 phage against a structured RNA crystallization target, ELISA for initial choice of binding Fabs, Fab expression followed by protein A affinity then cation exchange chromatography purification, final choice of Fab by binding specificity and affinity as determined by a dot blot assay, and lastly gel filtration purification of a large quantity of chosen Fabs for crystallization.

Future directions of electron crystallography.

PubMed

Fujiyoshi, Yoshinori

2013-01-01

In biological science, there are still many interesting and fundamental yet difficult questions, such as those in neuroscience, remaining to be answered. Structural and functional studies of membrane proteins, which are key molecules of signal transduction in neural and other cells, are essential for understanding the molecular mechanisms of many fundamental biological processes. Technological and instrumental advancements of electron microscopy have facilitated comprehension of structural studies of biological components, such as membrane proteins. While X-ray crystallography has been the main method of structure analysis of proteins including membrane proteins, electron crystallography is now an established technique to analyze structures of membrane proteins in the lipid bilayer, which is close to their natural biological environment. By utilizing cryo-electron microscopes with helium-cooled specimen stages, structures of membrane proteins were analyzed at a resolution better than 3 Å. Such high-resolution structural analysis of membrane proteins by electron crystallography opens up the new research field of structural physiology. Considering the fact that the structures of integral membrane proteins in their native membrane environment without artifacts from crystal contacts are critical in understanding their physiological functions, electron crystallography will continue to be an important technology for structural analysis. In this chapter, I will present several examples to highlight important advantages and to suggest future directions of this technique.

Using X-Ray Crystallography to Simplify and Accelerate Biologics Drug Development.

PubMed

Brader, Mark L; Baker, Edward N; Dunn, Michael F; Laue, Thomas M; Carpenter, John F

2017-02-01

Every major biopharmaceutical company incorporates a protein crystallography unit that is central to its structure-based drug discovery efforts. Yet these capabilities are rarely leveraged toward the formal higher order structural characterization that is so challenging but integral to large-scale biologics manufacturing. Although the biotech industry laments the shortcomings of its favored biophysical techniques, x-ray crystallography is not even considered for drug development. Why not? We suggest that this is due, at least in part, to outdated thinking (for a recent industry-wide survey, see Gabrielson JP, Weiss IV WF. Technical decision-making with higher order structure data: starting a new dialogue. J Pharm Sci. 2015;104(4):1240-1245). We examine some myths surrounding protein crystallography and highlight the inherent properties of protein crystals (molecular identity, biochemical purity, conformational uniformity, and macromolecular crowding) as having practicable commonalities with today's patient-focused liquid drug products. In the new millennium, protein crystallography has become essentially a routine analytical test. Its application may aid the identification of better candidate molecules that are more amenable to high-concentration processing, formulation, and analysis thereby helping to make biologics drug development quicker, simpler, and cheaper. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Evaluation of solution stability for two-component polydisperse systems by small-angle scattering

NASA Astrophysics Data System (ADS)

Kryukova, A. E.; Konarev, P. V.; Volkov, V. V.

2017-12-01

The article is devoted to the modelling of small-angle scattering data using the program MIXTURE designed for the study of polydisperse multicomponent mixtures. In this work we present the results of solution stability studies for theoretical small-angle scattering data sets from two-component models. It was demonstrated that the addition of the noise to the data influences the stability range of the restored structural parameters. The recommendations for the optimal minimization schemes that permit to restore the volume size distributions for polydisperse systems are suggested.

Structural changes of TasA in biofilm formation of Bacillus subtilis.

PubMed

Diehl, Anne; Roske, Yvette; Ball, Linda; Chowdhury, Anup; Hiller, Matthias; Molière, Noel; Kramer, Regina; Stöppler, Daniel; Worth, Catherine L; Schlegel, Brigitte; Leidert, Martina; Cremer, Nils; Erdmann, Natalja; Lopez, Daniel; Stephanowitz, Heike; Krause, Eberhard; van Rossum, Barth-Jan; Schmieder, Peter; Heinemann, Udo; Turgay, Kürşad; Akbey, Ümit; Oschkinat, Hartmut

2018-03-27

Microorganisms form surface-attached communities, termed biofilms, which can serve as protection against host immune reactions or antibiotics. Bacillus subtilis biofilms contain TasA as major proteinaceous component in addition to exopolysaccharides. In stark contrast to the initially unfolded biofilm proteins of other bacteria, TasA is a soluble, stably folded monomer, whose structure we have determined by X-ray crystallography. Subsequently, we characterized in vitro different oligomeric forms of TasA by NMR, EM, X-ray diffraction, and analytical ultracentrifugation (AUC) experiments. However, by magic-angle spinning (MAS) NMR on live biofilms, a swift structural change toward only one of these forms, consisting of homogeneous and protease-resistant, β-sheet-rich fibrils, was observed in vivo. Thereby, we characterize a structural change from a globular state to a fibrillar form in a functional prokaryotic system on the molecular level. Copyright © 2018 the Author(s). Published by PNAS.

Structural changes of TasA in biofilm formation of Bacillus subtilis

PubMed Central

Diehl, Anne; Roske, Yvette; Ball, Linda; Chowdhury, Anup; Hiller, Matthias; Molière, Noel; Kramer, Regina; Stöppler, Daniel; Worth, Catherine L.; Schlegel, Brigitte; Leidert, Martina; Cremer, Nils; Erdmann, Natalja; Lopez, Daniel; Stephanowitz, Heike; Krause, Eberhard; Schmieder, Peter; Akbey, Ümit; Oschkinat, Hartmut

2018-01-01

Microorganisms form surface-attached communities, termed biofilms, which can serve as protection against host immune reactions or antibiotics. Bacillus subtilis biofilms contain TasA as major proteinaceous component in addition to exopolysaccharides. In stark contrast to the initially unfolded biofilm proteins of other bacteria, TasA is a soluble, stably folded monomer, whose structure we have determined by X-ray crystallography. Subsequently, we characterized in vitro different oligomeric forms of TasA by NMR, EM, X-ray diffraction, and analytical ultracentrifugation (AUC) experiments. However, by magic-angle spinning (MAS) NMR on live biofilms, a swift structural change toward only one of these forms, consisting of homogeneous and protease-resistant, β-sheet–rich fibrils, was observed in vivo. Thereby, we characterize a structural change from a globular state to a fibrillar form in a functional prokaryotic system on the molecular level. PMID:29531041

Mannobiose Binding Induces Changes in Hydrogen Bonding and Protonation States of Acidic Residues in Concanavalin A As Revealed by Neutron Crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gerlits, Oksana O.; Coates, Leighton; Woods, Robert J.

Plant lectins are carbohydrate-binding proteins with various biomedical applications. Concanavalin A (Con A) holds promise in treating cancerous tumors. To better understand the Con A carbohydrate binding specificity, we obtained a room-temperature neutron structure of this legume lectin in complex with a disaccharide Manα1–2Man, mannobiose. The neutron structure afforded direct visualization of the hydrogen bonding between the protein and ligand, showing that the ligand is able to alter both protonation states and interactions for residues located close to and distant from the binding site. An unprecedented low-barrier hydrogen bond was observed forming between the carboxylic side chains of Asp28 andmore » Glu8, with the D atom positioned equidistant from the oxygen atoms having an O···D···O angle of 101.5°.« less

Effects of macromolecular crowding on the structure of a protein complex: A small-angle scattering study of superoxide dismutase

DOE PAGES

Rajapaksha, Ajith; Stanley, Christopher B.; Todd, Brian A.

2015-02-17

Macromolecular crowding can alter the structure and function of biological macromolecules. We used small angle scattering (SAS) to measure the change in size of a protein complex, superoxide dismutase (SOD), induced by macromolecular crowding. Crowding was induced using 400 MW polyethylene glycol (PEG), triethylene glycol (TEG), methyl- -glucoside ( -MG) and trimethylamine N-oxide (TMAO). Parallel small angle neutron scattering (SANS) and small angle x-ray scattering (SAXS) allowed us to unambiguously attribute apparent changes in radius of gyration to changes in the structure of SOD. For a 40% PEG solution, we find that the volume of SOD was reduced by 9%.more » Considering the osmotic pressure due to PEG, this deformation corresponds to a highly compressible structure. SAXS done in the presence of TEG suggests that for further deformation beyond a 9% decrease in volume the resistance to deformation may increase dramatically.« less

Creation of superwetting surfaces with roughness structures.

PubMed

Garg, Varun; Qiao, Lei; Sarwate, Prasha; Luo, Cheng

2014-12-09

In this work, we explored the possibility of creating superwetting surfaces, which are defined here as those with apparent contact angles of <5°, using roughness structures for the purpose of eliminating the surface tension effect on a floating small plate, which is denser than the surrounding liquid. The roughness ratio is often thought to play a critical role in generating superwetting surfaces. However, we found that the top surface ratio had more influence on apparent contact angles. When this ratio was <0.013, the resulting apparent contact angle might be less than 5°, when the intrinsic contact angle was ≥40°. Accordingly, hybrid micro- and nanostructures, which had such a small ratio, were chosen to create the superwetting surfaces. These surfaces were subsequently applied to eliminate the surface tension effect on a small plate. As a result of this elimination, the small plate sank down to the bottom of the liquid.

Evaluation of eyes with relative pupillary block by indentation ultrasound biomicroscopy gonioscopy.

PubMed

Matsunaga, Koichi; Ito, Kunio; Esaki, Koji; Sugimoto, Kota; Sano, Toru; Miura, Katsuya; Sasoh, Mikio; Uji, Yukitaka

2004-03-01

To investigate changes in anterior chamber angle configuration with indentation ultrasound biomicroscopy gonioscopy of relative pupillary block (RPB). Cross-sectional study. This study included 26 eyes of 26 patients with RPB. We determined angle opening distance 500 and angle recess area using indentation ultrasound biomicroscopy gonioscopy and compared a small-sized standard eye cup with a new eye cup with an area for inducing pressure. Indentation ultrasound biomicroscopy images documented concavity of the iris in eyes with RPB. Both the new and the small standard eye cups widened the anterior chamber angle significantly (P <.0001) without causing corneal damage. Angle changes were significantly greater for the new eye cup design. Indentation ultrasound biomicroscopy gonioscopy is a useful technique for observation and diagnosis of RPB. Using a small standard or the newly designed eye cup, the procedure can be performed easily and without causing corneal damage.

Determination of corrections to flow direction measurements obtained with a wing-tip mounted sensor. M.S. Thesis

NASA Technical Reports Server (NTRS)

Moul, T. M.

1983-01-01

The nature of corrections for flow direction measurements obtained with a wing-tip mounted sensor was investigated. Corrections for the angle of attack and sideslip, measured by sensors mounted in front of each wing tip of a general aviation airplane, were determined. These flow corrections were obtained from both wind-tunnel and flight tests over a large angle-of-attack range. Both the angle-of-attack and angle-of-sideslip flow corrections were found to be substantial. The corrections were a function of the angle of attack and angle of sideslip. The effects of wing configuration changes, small changes in Reynolds number, and spinning rotation on the angle-of-attack flow correction were found to be small. The angle-of-attack flow correction determined from the static wind-tunnel tests agreed reasonably well with the correction determined from flight tests.

Prediction of the noise from a propeller at angle of attack

NASA Technical Reports Server (NTRS)

Krejsa, Eugene A.

1990-01-01

An analysis is presented to predict the noise of a propeller at angle of attack. The analysis is an extension of that reported by Mani (1990) which predicted the change in noise due to angle of attack due to both unsteady loading and to azimuthal variation of the radiation efficiency of steady noise sources. Mani's analysis, however, was limited to small angles of attack. The analysis reported herein removes this small angle limitation. Results from the analysis are compared with the data of Woodward (1987, 1988), for a single rotation propeller and for a counter rotating propeller. The comparison shows that including the effect of angle of attack on the steady noise sources significantly improves the agreement with data. Including higher order effects of angle of attack, while changing the predicted noise at far forward and aft angles, has little effect near the propeller plane.

Microgravity

NASA Image and Video Library

2004-04-15

The loss of productivity due to flu is staggering. Costs range as much as $20 billio a year. High mutation rates of the flu virus have hindered development of new drugs or vaccines. The secret lies in a small molecule which is attached to the host cell's surface. Each flu virus, no matter what strain, must remove this small molecule to escape the host cell to spread infection. Using data from space and earth grown crystals, researchers from the Center of Macromolecular Crystallography (CMC) are desining drugs to bind with this protein's active site. This lock and key fit reduces the spread of flu in the body by blocking its escape route. In collaboration with its corporate partner, the CMC has refined drug structure in preparation for clinical trials. Tested and approved relief is expected to reach drugstores by year 2004.

Serial crystallography captures enzyme catalysis in copper nitrite reductase at atomic resolution from one crystal.

PubMed

Horrell, Sam; Antonyuk, Svetlana V; Eady, Robert R; Hasnain, S Samar; Hough, Michael A; Strange, Richard W

2016-07-01

Relating individual protein crystal structures to an enzyme mechanism remains a major and challenging goal for structural biology. Serial crystallography using multiple crystals has recently been reported in both synchrotron-radiation and X-ray free-electron laser experiments. In this work, serial crystallography was used to obtain multiple structures serially from one crystal (MSOX) to study in crystallo enzyme catalysis. Rapid, shutterless X-ray detector technology on a synchrotron MX beamline was exploited to perform low-dose serial crystallography on a single copper nitrite reductase crystal, which survived long enough for 45 consecutive 100 K X-ray structures to be collected at 1.07-1.62 Å resolution, all sampled from the same crystal volume. This serial crystallography approach revealed the gradual conversion of the substrate bound at the catalytic type 2 Cu centre from nitrite to nitric oxide, following reduction of the type 1 Cu electron-transfer centre by X-ray-generated solvated electrons. Significant, well defined structural rearrangements in the active site are evident in the series as the enzyme moves through its catalytic cycle, namely nitrite reduction, which is a vital step in the global denitrification process. It is proposed that such a serial crystallography approach is widely applicable for studying any redox or electron-driven enzyme reactions from a single protein crystal. It can provide a 'catalytic reaction movie' highlighting the structural changes that occur during enzyme catalysis. The anticipated developments in the automation of data analysis and modelling are likely to allow seamless and near-real-time analysis of such data on-site at some of the powerful synchrotron crystallographic beamlines.

Axial coordination and conformational heterogeneity of nickel(II) tetraphenylprophyrin complexes with nitrogenous bases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jia, S.L.; Song, X.Z.; Ma, J.G.

1998-08-24

Axial ligation of nickel(II) 5,10,15,20-tetraphenylporphyrin (NiTPP) with pyrrolidine or piperidine has been investigated using X-ray crystallography, UV-visible spectroscopy, resonance Raman spectroscopy, and molecular mechanics (MM) calculations. Distinct v{sub 4} Raman lines are found for the 4-, 5-, and 6-coordinate species of NiTPP. The equilibrium constants for addition of the first and second pyrrolidine axial ligands are 1.1 and 3.8 M{sup {minus}1}, respectively. The differences in the calculated energies of the conformers having different ligand rotational angles are small so they may coexist in solution. Because of the similarity in macrocyclic structural parameters of these conformers and the free rotation ofmore » the axial ligands, narrow and symmetric v{sub 2} and v{sub 8} Raman lines are observed. Nonetheless, the normal-coordinate structural-decomposition analysis of the nonplanar distortions of the calculated structures and the crystal structure of the bis(piperidine) complex reveals a relationship between the orientations of axial ligand(s) and the macrocyclic distortions. For the 5-coordinate complex with the plane of the axial ligand bisecting the Ni-N{sub pyrrole} bonds, a primarily ruffled deformation results. With the ligand plane eclipsing the Ni-N{sub pyrrole} bonds, a mainly saddled deformation occurs. With the addition of the second axial ligand, the small doming of the 5-coordinate complexes disappears, and ruffling or saddling deformations change depending on the relative orientation of the two axial ligands. The crystal structure of the NiTPP bis(piperidine) complex shows a macrocycle distortion composed of wav(x) and wav(y) symmetric deformations, but no ruffling, saddling, or doming. The difference in the calculated and observed distortions results partly from the phenyl group orientation imposed by crystal packing forces. MM calculations predict three stable conformers (ruf, sad, and planar) for 4-coordinate NiTPP, and resonance Raman evidence for these conformers was given previously.« less

Molecular structure in the solid state by X-ray crystallography and SSNMR and in solution by NMR of two 1,4-diazepines

NASA Astrophysics Data System (ADS)

Nieto, Carla I.; Sanz, Dionisia; Claramunt, Rosa M.; Torralba, M. Carmen; Torres, M. Rosario; Alkorta, Ibon; Elguero, José

2018-03-01

The crystals of two 1,4-diazepines prepared from curcuminoid β-diketones and ethylenediamine were studied by X-ray crystallography and NMR. Their tautomerism, intramolecular hydrogen bonds and conformation were determined.

Modified sine bar device measures small angles with high accuracy

NASA Technical Reports Server (NTRS)

Thekaekara, M.

1968-01-01

Modified sine bar device measures small angles with enough accuracy to calibrate precision optical autocollimators. The sine bar is a massive bar of steel supported by two cylindrical rods at one end and one at the other.

Recent progress in structural biology: lessons from our research history.

PubMed

Nitta, Ryo; Imasaki, Tsuyoshi; Nitta, Eriko

2018-05-16

The recent 'resolution revolution' in structural analyses of cryo-electron microscopy (cryo-EM) has drastically changed the research strategy for structural biology. In addition to X-ray crystallography and nuclear magnetic resonance spectroscopy, cryo-EM has achieved the structural analysis of biological molecules at near-atomic resolution, resulting in the Nobel Prize in Chemistry 2017. The effect of this revolution has spread within the biology and medical science fields affecting everything from basic research to pharmaceutical development by visualizing atomic structure. As we have used cryo-EM as well as X-ray crystallography since 2000 to elucidate the molecular mechanisms of the fundamental phenomena in the cell, here we review our research history and summarize our findings. In the first half of the review, we describe the structural mechanisms of microtubule-based motility of molecular motor kinesin by using a joint cryo-EM and X-ray crystallography method. In the latter half, we summarize our structural studies on transcriptional regulation by X-ray crystallography of in vitro reconstitution of a multi-protein complex.

Where is crystallography going?

PubMed Central

Ashton, Alun W.; Sorensen, Thomas

2018-01-01

Macromolecular crystallography (MX) has been a motor for biology for over half a century and this continues apace. A series of revolutions, including the production of recombinant proteins and cryo-crystallography, have meant that MX has repeatedly reinvented itself to dramatically increase its reach. Over the last 30 years synchrotron radiation has nucleated a succession of advances, ranging from detectors to optics and automation. These advances, in turn, open up opportunities. For instance, a further order of magnitude could perhaps be gained in signal to noise for general synchrotron experiments. In addition, X-ray free-electron lasers offer to capture fragments of reciprocal space without radiation damage, and open up the subpicosecond regime of protein dynamics and activity. But electrons have recently stolen the limelight: so is X-ray crystallography in rude health, or will imaging methods, especially single-particle electron microscopy, render it obsolete for the most interesting biology, whilst electron diffraction enables structure determination from even the smallest crystals? We will lay out some information to help you decide. PMID:29533241

The room temperature crystal structure of a bacterial phytochrome determined by serial femtosecond crystallography

DOE PAGES

Edlund, Petra; Takala, Heikki; Claesson, Elin; ...

2016-10-19

Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the room temperature crystal structure of the chromophore-binding domains of the Deinococcus radiodurans phytochrome at 2.1 Å resolution. The structure was obtained by serial femtosecond X-ray crystallography from microcrystals at an X-ray free electron laser. We find overall good agreement compared to a crystal structure at 1.35 Å resolution derived frommore » conventional crystallography at cryogenic temperatures, which we also report here. The thioether linkage between chromophore and protein is subject to positional ambiguity at the synchrotron, but is fully resolved with SFX. As a result, the study paves the way for time-resolved structural investigations of the phytochrome photocycle with time-resolved SFX.« less

The room temperature crystal structure of a bacterial phytochrome determined by serial femtosecond crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Edlund, Petra; Takala, Heikki; Claesson, Elin

Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the room temperature crystal structure of the chromophore-binding domains of the Deinococcus radiodurans phytochrome at 2.1 Å resolution. The structure was obtained by serial femtosecond X-ray crystallography from microcrystals at an X-ray free electron laser. We find overall good agreement compared to a crystal structure at 1.35 Å resolution derived frommore » conventional crystallography at cryogenic temperatures, which we also report here. The thioether linkage between chromophore and protein is subject to positional ambiguity at the synchrotron, but is fully resolved with SFX. As a result, the study paves the way for time-resolved structural investigations of the phytochrome photocycle with time-resolved SFX.« less

Influence of SiC grain boundary character on fission product transport in irradiated TRISO fuel

NASA Astrophysics Data System (ADS)

Lillo, T. M.; van Rooyen, I. J.

2016-05-01

In this study, the fission product precipitates at silicon carbide grain boundaries from an irradiated TRISO particle were identified and correlated with the associated grain boundary characteristics. Precession electron diffraction in the transmission electron microscope provided the crystallographic information needed to identify grain boundary misorientation and boundary type (i.e., low angle, random high angle or coincident site lattice (CSL)-related). The silicon carbide layer was found to be composed mainly of twin boundaries and small fractions of random high angle and low angle grain boundaries. Most fission products were found at random, high-angle grain boundaries, with small fractions at low-angle and CSL-related grain boundaries. Palladium (Pd) was found at all types of grain boundaries while Pd-uranium and Pd-silver precipitates were only associated with CSL-related and random, high-angle grain boundaries. Precipitates containing only Ag were found only at random, high-angle grain boundaries, but not at low angle or CSL-related grain boundaries.

Synthesis and structure elucidation of a series of pyranochromene chalcones and flavanones using 1D and 2D NMR spectroscopy and X-ray crystallography.

PubMed

Pawar, Sunayna S; Koorbanally, Neil A

2014-06-01

A series of novel pyranochromene chalcones and corresponding flavanones were synthesized. This is the first report on the confirmation of the absolute configuration of chromene-based flavanones using X-ray crystallography. These compounds were characterized by 2D NMR spectroscopy, and their assignments are reported herein. The 3D structure of the chalcone 3b and flavanone 4g was determined by X-ray crystallography, and the structure of the flavanone was confirmed to be in the S configuration at C-2. Copyright © 2014 John Wiley & Sons, Ltd.

A Maltose-Binding Protein Fusion Construct Yields a Robust Crystallography Platform for MCL1

PubMed Central

Clifton, Matthew C.; Dranow, David M.; Leed, Alison; Fulroth, Ben; Fairman, James W.; Abendroth, Jan; Atkins, Kateri A.; Wallace, Ellen; Fan, Dazhong; Xu, Guoping; Ni, Z. J.; Daniels, Doug; Van Drie, John; Wei, Guo; Burgin, Alex B.; Golub, Todd R.; Hubbard, Brian K.; Serrano-Wu, Michael H.

2015-01-01

Crystallization of a maltose-binding protein MCL1 fusion has yielded a robust crystallography platform that generated the first apo MCL1 crystal structure, as well as five ligand-bound structures. The ability to obtain fragment-bound structures advances structure-based drug design efforts that, despite considerable effort, had previously been intractable by crystallography. In the ligand-independent crystal form we identify inhibitor binding modes not observed in earlier crystallographic systems. This MBP-MCL1 construct dramatically improves the structural understanding of well-validated MCL1 ligands, and will likely catalyze the structure-based optimization of high affinity MCL1 inhibitors. PMID:25909780

Which Strategy for a Protein Crystallization Project?

NASA Technical Reports Server (NTRS)

Kundrot, Craig E.

2003-01-01

The three-dimensional, atomic-resolution protein structures produced by X-ray crystallography over the past 50+ years have led to tremendous chemical understanding of fundamental biochemical processes. The pace of discovery in protein crystallography has increased greatly with advances in molecular biology, crystallization techniques, cryo-crystallography, area detectors, synchrotrons and computing. While the methods used to produce single, well-ordered crystals have also evolved over the years in response to increased understanding and advancing technology, crystallization strategies continue to be rooted in trial-and-error approaches. This review summarizes the current approaches in protein crystallization and surveys the first results to emerge from the structural genomics efforts.

Optimized multi-step NMR-crystallography approach for structural characterization of a stable quercetin solvate.

PubMed

Filip, Xenia; Miclaus, Maria; Martin, Flavia; Filip, Claudiu; Grosu, Ioana Georgeta

2017-05-10

Herein we report the preparation and solid state structural investigation of the 1,4-dioxane-quercetin solvate. NMR crystallography methods were employed for crystal structure determination of the solvate from microcrystalline powder. The stability of the compound relative to other reported quercetin solvates is discussed and found to be in perfect agreement with the hydrogen bonding networks/supra-molecular architectures formed in each case. It is also clearly shown that NMR crystallography represents an ideal analytical tool in such cases when hydrogen-bonding networks are required to be constrained at a high accuracy level. Copyright © 2017 Elsevier B.V. All rights reserved.

Influence of laser beam incidence angle on laser lap welding quality of galvanized steels

NASA Astrophysics Data System (ADS)

Mei, Lifang; Yan, Dongbing; Chen, Genyu; Wang, Zhenhui; Chen, Shuixuan

2017-11-01

Based on the characteristics of laser welded structural parts of auto bodies, the influence of variation in laser beam incidence angle on the lap welding performance of galvanized auto-body sheets was studied. Lap welding tests were carried out on the galvanized sheets for auto-body application at different laser beam incidence angles by using the optimal welding parameters obtained through orthogonal experiment. The effects of incidence angle variation on seam appearance, cross-sectional shape, joint mechanical properties and microstructure of weldments were analyzed. In addition, the main factors influencing the value of incidence angle were investigated. According to the results, the weld seams had a good appearance as well as a fine, and uniform microstructure when the laser beam incidence angle was smaller than the critical incidence angle, and thus they could withstand great tensile and shear loads. Moreover, all tensile-shear specimens were fractured in the base material zone. When the laser beam incidence angle was larger than the critical incidence angle, defects like shrinkage and collapse tended to emerge, thereby resulting in the deteriorated weldability of specimens. Meanwhile, factors like the type and thickness of sheet, weld width as well as inter-sheet gap all had a certain effect on the value of laser beam incidence angle. When the sheet thickness was small and the weld width was narrow, the laser beam incidence angle could be increased appropriately. At the same time, small changes in the inter-sheet gap could greatly impact the value of incidence angle. When the inter-sheet gap was small, the laser beam incidence angle should not be too large.

Examination of Cement Pastes Hydrated Phases, and Synthetic Products by X-Ray Diffraction

DTIC Science & Technology

1972-04-01

International Union o, Crystallography Commission on Crystallo- graphic Data, Powder Data, Journal of Applied Crystallography, Vol 4, pp 81-86, 1971 . 21...Papes, Research Series 1, Building Research Station, 1962. 25. l,, Dosch, Rbntgen-Ceinstrukturuntersuchu , luftemp’indlicher pulverprlparate, Zement- Kall

Tinker Toys, Crystallography, and the Introductory Mineralogy Course

ERIC Educational Resources Information Center

Buseck, Peter R.

1970-01-01

Describes the use of Tinker Toys to construct three dimensional models of crystals useful in illustrating many concepts of crystallography. Space lattices representing all of the Bravais types can be constructed. Also discusses the use of appropriate models to demonstrate the various symmetry operations. Bibliography. (LC)

Single-particle cryo-EM using alignment by classification (ABC): the structure of Lumbricus terrestris haemoglobin.

PubMed

Afanasyev, Pavel; Seer-Linnemayr, Charlotte; Ravelli, Raimond B G; Matadeen, Rishi; De Carlo, Sacha; Alewijnse, Bart; Portugal, Rodrigo V; Pannu, Navraj S; Schatz, Michael; van Heel, Marin

2017-09-01

Single-particle cryogenic electron microscopy (cryo-EM) can now yield near-atomic resolution structures of biological complexes. However, the reference-based alignment algorithms commonly used in cryo-EM suffer from reference bias, limiting their applicability (also known as the 'Einstein from random noise' problem). Low-dose cryo-EM therefore requires robust and objective approaches to reveal the structural information contained in the extremely noisy data, especially when dealing with small structures. A reference-free pipeline is presented for obtaining near-atomic resolution three-dimensional reconstructions from heterogeneous ('four-dimensional') cryo-EM data sets. The methodologies integrated in this pipeline include a posteriori camera correction, movie-based full-data-set contrast transfer function determination, movie-alignment algorithms, (Fourier-space) multivariate statistical data compression and unsupervised classification, 'random-startup' three-dimensional reconstructions, four-dimensional structural refinements and Fourier shell correlation criteria for evaluating anisotropic resolution. The procedures exclusively use information emerging from the data set itself, without external 'starting models'. Euler-angle assignments are performed by angular reconstitution rather than by the inherently slower projection-matching approaches. The comprehensive 'ABC-4D' pipeline is based on the two-dimensional reference-free 'alignment by classification' (ABC) approach, where similar images in similar orientations are grouped by unsupervised classification. Some fundamental differences between X-ray crystallography versus single-particle cryo-EM data collection and data processing are discussed. The structure of the giant haemoglobin from Lumbricus terrestris at a global resolution of ∼3.8 Å is presented as an example of the use of the ABC-4D procedure.

Simultaneous multiplexed materials characterization using a high-precision hard X-ray micro-slit array.

PubMed

Zhang, Fan; Allen, Andrew J; Levine, Lyle E; Mancini, Derrick C; Ilavsky, Jan

2015-05-01

The needs both for increased experimental throughput and for in operando characterization of functional materials under increasingly realistic experimental conditions have emerged as major challenges across the whole of crystallography. A novel measurement scheme that allows multiplexed simultaneous measurements from multiple nearby sample volumes is presented. This new approach enables better measurement statistics or direct probing of heterogeneous structure, dynamics or elemental composition. To illustrate, the submicrometer precision that optical lithography provides has been exploited to create a multiplexed form of ultra-small-angle scattering based X-ray photon correlation spectroscopy (USAXS-XPCS) using micro-slit arrays fabricated by photolithography. Multiplexed USAXS-XPCS is applied to follow the equilibrium dynamics of a simple colloidal suspension. While the dependence of the relaxation time on momentum transfer, and its relationship with the diffusion constant and the static structure factor, follow previous findings, this measurements-in-parallel approach reduces the statistical uncertainties of this photon-starved technique to below those associated with the instrument resolution. More importantly, we note the potential of the multiplexed scheme to elucidate the response of different components of a heterogeneous sample under identical experimental conditions in simultaneous measurements. In the context of the X-ray synchrotron community, this scheme is, in principle, applicable to all in-line synchrotron techniques. Indeed, it has the potential to open a new paradigm for in operando characterization of heterogeneous functional materials, a situation that will be even further enhanced by the ongoing development of multi-bend achromat storage ring designs as the next evolution of large-scale X-ray synchrotron facilities around the world.

Simultaneous multiplexed materials characterization using a high-precision hard X-ray micro-slit array

DOE PAGES

Zhang, Fan; Allen, Andrew J.; Levine, Lyle E.; ...

2015-01-01

Here, the needs both for increased experimental throughput and forin operandocharacterization of functional materials under increasingly realistic experimental conditions have emerged as major challenges across the whole of crystallography. A novel measurement scheme that allows multiplexed simultaneous measurements from multiple nearby sample volumes is presented. This new approach enables better measurement statistics or direct probing of heterogeneous structure, dynamics or elemental composition. To illustrate, the submicrometer precision that optical lithography provides has been exploited to create a multiplexed form of ultra-small-angle scattering based X-ray photon correlation spectroscopy (USAXS-XPCS) using micro-slit arrays fabricated by photolithography. Multiplexed USAXS-XPCS is applied to followmore » the equilibrium dynamics of a simple colloidal suspension. While the dependence of the relaxation time on momentum transfer, and its relationship with the diffusion constant and the static structure factor, follow previous findings, this measurements-in-parallel approach reduces the statistical uncertainties of this photon-starved technique to below those associated with the instrument resolution. More importantly, we note the potential of the multiplexed scheme to elucidate the response of different components of a heterogeneous sample underidenticalexperimental conditions in simultaneous measurements. Lastly, in the context of the X-ray synchrotron community, this scheme is, in principle, applicable to all in-line synchrotron techniques. Indeed, it has the potential to open a new paradigm for in operando characterization of heterogeneous functional materials, a situation that will be even further enhanced by the ongoing development of multi-bend achromat storage ring designs as the next evolution of large-scale X-ray synchrotron facilities around the world.« less

Distinct structural features of the peroxide response regulator from group A Streptococcus drive DNA binding.

PubMed

Lin, Chang Sheng-Huei; Chao, Shi-Yu; Hammel, Michal; Nix, Jay C; Tseng, Hsiao-Ling; Tsou, Chih-Cheng; Fei, Chun-Hsien; Chiou, Huo-Sheng; Jeng, U-Ser; Lin, Yee-Shin; Chuang, Woei-Jer; Wu, Jiunn-Jong; Wang, Shuying

2014-01-01

Group A streptococcus (GAS, Streptococcus pyogenes) is a strict human pathogen that causes severe, invasive diseases. GAS does not produce catalase, but has an ability to resist killing by reactive oxygen species (ROS) through novel mechanisms. The peroxide response regulator (PerR), a member of ferric uptake regulator (Fur) family, plays a key role for GAS to cope with oxidative stress by regulating the expression of multiple genes. Our previous studies have found that expression of an iron-binding protein, Dpr, is under the direct control of PerR. To elucidate the molecular interactions of PerR with its cognate promoter, we have carried out structural studies on PerR and PerR-DNA complex. By combining crystallography and small-angle X-ray scattering (SAXS), we confirmed that the determined PerR crystal structure reflects its conformation in solution. Through mutagenesis and biochemical analysis, we have identified DNA-binding residues suggesting that PerR binds to the dpr promoter at the per box through a winged-helix motif. Furthermore, we have performed SAXS analysis and resolved the molecular architecture of PerR-DNA complex, in which two 30 bp DNA fragments wrap around two PerR homodimers by interacting with the adjacent positively-charged winged-helix motifs. Overall, we provide structural insights into molecular recognition of DNA by PerR and define the hollow structural arrangement of PerR-30bpDNA complex, which displays a unique topology distinct from currently proposed DNA-binding models for Fur family regulators.

Strategies for the structural analysis of multi-protein complexes: lessons from the 3D-Repertoire project.

PubMed

Collinet, B; Friberg, A; Brooks, M A; van den Elzen, T; Henriot, V; Dziembowski, A; Graille, M; Durand, D; Leulliot, N; Saint André, C; Lazar, N; Sattler, M; Séraphin, B; van Tilbeurgh, H

2011-08-01

Structural studies of multi-protein complexes, whether by X-ray diffraction, scattering, NMR spectroscopy or electron microscopy, require stringent quality control of the component samples. The inability to produce 'keystone' subunits in a soluble and correctly folded form is a serious impediment to the reconstitution of the complexes. Co-expression of the components offers a valuable alternative to the expression of single proteins as a route to obtain sufficient amounts of the sample of interest. Even in cases where milligram-scale quantities of purified complex of interest become available, there is still no guarantee that good quality crystals can be obtained. At this step, protein engineering of one or more components of the complex is frequently required to improve solubility, yield or the ability to crystallize the sample. Subsequent characterization of these constructs may be performed by solution techniques such as Small Angle X-ray Scattering and Nuclear Magnetic Resonance to identify 'well behaved' complexes. Herein, we recount our experiences gained at protein production and complex assembly during the European 3D Repertoire project (3DR). The goal of this consortium was to obtain structural information on multi-protein complexes from yeast by combining crystallography, electron microscopy, NMR and in silico modeling methods. We present here representative set case studies of complexes that were produced and analyzed within the 3DR project. Our experience provides useful insight into strategies that are more generally applicable for structural analysis of protein complexes. Copyright © 2011 Elsevier Inc. All rights reserved.

Crystallographic relationship of YTaO4 particles with matrix in Ta-containing 12Cr ODS steel

NASA Astrophysics Data System (ADS)

Mao, Xiaodong; Kim, Tae Kyu; Kim, Sung Soo; Han, Young Soo; Oh, Kyu Hwan; Jang, Jinsung

2015-06-01

The crystallography of monoclinic YTaO4 particles and the atomic structure at the particle/ferrite matrix interface in Ta-containing 12Cr ODS steel have been examined by means of SAD and HRTEM. Three different peaks in size distribution of oxide particles were detected by SANS, with the peak positions at 1.5 nm, 9 nm, and 100 nm in size. The results show that many YTaO4 particles are semi-coherent with the matrix, and the habit plane determined in most cases is { 0 5 1 } O / /{ 0 1 1 } M . Orientation relationships of (0 5 1) O / /(1 bar 1 bar 0) M , [ 7 1 5 bar ] O / /[ 1 bar 1 1 ] M ; (1 2 1) O / /(1 1 0) M , [ 2 bar 1 0 ] O / /[ 0 0 1 ] M ; (0 5 1) O / /(0 1 1) M , [ 7 1 5 bar ] O / /[ 0 1 bar 1 ] M and (0 5 bar 1) O / /(1 bar 1 bar 0) M , [ 3 bar 1 5 ] O / /[ 1 bar 1 3 ] M were found. These orientation relationships provide a very small misorientation between the specific planes of YTaO4 particles and {1 1 0} close packed planes of ferrite. Fine particles of around 4 nm in size exhibited incoherent relationship with the misfit angle of around 10° with the matrix. Observation on particles ranging from 7 to 50 nm in size revealed that the crystallographic relationship is semi-coherent between oxide particles and the matrix.

A small-angle large-acceptance detection system for hadrons

NASA Astrophysics Data System (ADS)

Kalantar-Nayestanaki, N.; Bacelar, J. C. S.; Brandenburg, S.; Huisman, H.; Messchendorp, J. G.; Mul, F. A.; Schadmand, S.; van der Schaaf, K.; Schippers, J. M.; Volkerts, M.

2000-04-01

The performance of a segmented large-acceptance detector, capable of measuring particles at small forward angles, is presented. The Small-Angle Large-Acceptance Detector (SALAD), was built to handle very high rates of particles impinging on the detector. Particles down to a few MeV can be detected with it. The position of charged particles is measured by two Multi-Wire Proportional Chambers while scintillator blocks are used to measure the energy of the detected particle. A stack of thin scintillators placed behind the energy detectors allows for a hardware rejection (veto) of high-energy particles going through the scintillator blocks.

Combining EPR spectroscopy and X-ray crystallography to elucidate the structure and dynamics of conformationally constrained spin labels in T4 lysozyme single crystals.

PubMed

Consentius, Philipp; Gohlke, Ulrich; Loll, Bernhard; Alings, Claudia; Heinemann, Udo; Wahl, Markus C; Risse, Thomas

2017-08-09

Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling is used to investigate the structure and dynamics of conformationally constrained spin labels in T4 lysozyme single crystals. Within a single crystal, the oriented ensemble of spin bearing moieties results in a strong angle dependence of the EPR spectra. A quantitative description of the EPR spectra requires the determination of the unit cell orientation with respect to the sample tube and the orientation of the spin bearing moieties within the crystal lattice. Angle dependent EPR spectra were analyzed by line shape simulations using the stochastic Liouville equation approach developed by Freed and co-workers and an effective Hamiltonian approach. The gain in spectral information obtained from the EPR spectra of single crystalline samples taken at different frequencies, namely the X-band and Q-band, allows us to discriminate between motional models describing the spectra of isotropic solutions similarly well. In addition, it is shown that the angle dependent single crystal spectra allow us to identify two spin label rotamers with very similar side chain dynamics. These results demonstrate the utility of single crystal EPR spectroscopy in combination with spectral line shape simulation techniques to extract valuable dynamic information not readily available from the analysis of isotropic systems. In addition, it will be shown that the loss of electron density in high resolution diffraction experiments at room temperature does not allow us to conclude that there is significant structural disorder in the system.

A New Camera for Powder Diffraction of Macromolecular Crystallography at SPring-8

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miura, Keiko; Inoue, Katsuaki; Goto, Shunji

2004-05-12

A powder diffractometer of Guinier geometry was developed and tested on a beamline, BL40B2, at SPring-8. The long specimen-to-detector distance, 1,000 mm, is advantageous in recording diffraction from Bragg spacing of 20 nm or larger. The angular resolution, 0.012 degrees, was realized together with the focusing optics, the long specimen-to-detector distance and the small pixel size of Blue-type Imaging Plate detector. Such a high resolution makes the peak separation possible in the powder diffraction from microcrystals with large unit cell and low symmetry of biological macromolecules.

Present and future of membrane protein structure determination by electron crystallography.

PubMed

Ubarretxena-Belandia, Iban; Stokes, David L

2010-01-01

Membrane proteins are critical to cell physiology, playing roles in signaling, trafficking, transport, adhesion, and recognition. Despite their relative abundance in the proteome and their prevalence as targets of therapeutic drugs, structural information about membrane proteins is in short supply. This chapter describes the use of electron crystallography as a tool for determining membrane protein structures. Electron crystallography offers distinct advantages relative to the alternatives of X-ray crystallography and NMR spectroscopy. Namely, membrane proteins are placed in their native membranous environment, which is likely to favor a native conformation and allow changes in conformation in response to physiological ligands. Nevertheless, there are significant logistical challenges in finding appropriate conditions for inducing membrane proteins to form two-dimensional arrays within the membrane and in using electron cryo-microscopy to collect the data required for structure determination. A number of developments are described for high-throughput screening of crystallization trials and for automated imaging of crystals with the electron microscope. These tools are critical for exploring the necessary range of factors governing the crystallization process. There have also been recent software developments to facilitate the process of structure determination. However, further innovations in the algorithms used for processing images and electron diffraction are necessary to improve throughput and to make electron crystallography truly viable as a method for determining atomic structures of membrane proteins. Copyright © 2010 Elsevier Inc. All rights reserved.

Present and future of membrane protein structure determination by electron crystallography

PubMed Central

Ubarretxena-Belandia, Iban; Stokes, David L.

2011-01-01

Membrane proteins are critical to cell physiology, playing roles in signaling, trafficking, transport, adhesion, and recognition. Despite their relative abundance in the proteome and their prevalence as targets of therapeutic drugs, structural information about membrane proteins is in short supply. This review describes the use of electron crystallography as a tool for determining membrane protein structures. Electron crystallography offers distinct advantages relative to the alternatives of X-ray crystallography and NMR spectroscopy. Namely, membrane proteins are placed in their native membranous environment, which is likely to favor a native conformation and allow changes in conformation in response to physiological ligands. Nevertheless, there are significant logistical challenges in finding appropriate conditions for inducing membrane proteins to form two-dimensional arrays within the membrane and in using electron cryo-microscopy to collect the data required for structure determination. A number of developments are described for high-throughput screening of crystallization trials and for automated imaging of crystals with the electron microscope. These tools are critical for exploring the necessary range of factors governing the crystallization process. There have also been recent software developments to facilitate the process of structure determination. However, further innovations in the algorithms used for processing images and electron diffraction are necessary to improve throughput and to make electron crystallography truly viable as a method for determining atomic structures of membrane proteins. PMID:21115172

Design of an all-attitude flight control system to execute commanded bank angles and angles of attack

NASA Technical Reports Server (NTRS)

Burgin, G. H.; Eggleston, D. M.

1976-01-01

A flight control system for use in air-to-air combat simulation was designed. The input to the flight control system are commanded bank angle and angle of attack, the output are commands to the control surface actuators such that the commanded values will be achieved in near minimum time and sideslip is controlled to remain small. For the longitudinal direction, a conventional linear control system with gains scheduled as a function of dynamic pressure is employed. For the lateral direction, a novel control system, consisting of a linear portion for small bank angle errors and a bang-bang control system for large errors and error rates is employed.

The effect of small angle of attack on the laminar-turbulent transition in boundary layer on swept wing at Mach number M=2

NASA Astrophysics Data System (ADS)

Semionov, N. V.; Yermolaev, Yu. G.; Kosinov, A. D.; Semenov, A. N.; Smorodsky, B. V.; Yatskikh, A. A.

2017-10-01

The paper is devoted to an experimental and theoretical study of effect of small angle of attack on disturbances evolution and laminar-turbulent transition in a supersonic boundary layer on swept wing at Mach number M=2. The experiments are conducted at the low nose supersonic wind tunnel T-325 of ITAM. Model is a symmetrical wing with a 45° sweep angle, a 3 percent-thick circular-arc airfoil. The transition location is determined using a hot-wire anemometer. Confirmed monotonous growth of the transition Reynolds numbers with increasing of angle of attack from -2° to 2.5°. The experimental data on the influence of the angle of attack on the disturbances evolution in the supersonic boundary layer on the swept wing model are obtained. Calculations on the effect of small angles of attack on the development of perturbations are made in the framework of the linear theory of stability. A good qualitative correspondence of theoretical and experimental data are obtained.

Energetic Materials for Bio-Agent Destruction

DTIC Science & Technology

2017-03-01

Delocalization of electrons in 4 The structure of 5 was obtained by X-ray single crystal crystallography . The crystallographic data are summarized in...4 22.2 49.6 - - 8.36 5 18.8 41.7 - - 5.07 The structure of 5 was obtained by X-ray single crystal crystallography . The

Using Two-Dimensional Colloidal Crystals to Understand Crystallography

ERIC Educational Resources Information Center

Bosse, Stephanie A.; Loening, Nikolaus M.

2008-01-01

X-ray crystallography is an essential technique for modern chemistry and biochemistry, but it is infrequently encountered by undergraduate students owing to lack of access to equipment, the time-scale for generating diffraction-quality molecular crystals, and the level of mathematics involved in analyzing the resulting diffraction patterns.…

76 FR 6497 - Notice Pursuant to the National Cooperative Research and Production Act of 1993-Industrial...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-02-04

... DEPARTMENT OF JUSTICE Antitrust Division Notice Pursuant to the National Cooperative Research and Production Act of 1993--Industrial Nacromolecular Crystallography Association Notice is hereby given that, on..., 15 U.S.C. 4301 et seq. (``the Act''), Industrial Nacromolecular Crystallography Association (``INCA...

Novel Catalytic Mechanisms For The Chemical Reduction Of Carbon Dioxide To Energy-Dense Liquids

DTIC Science & Technology

2016-12-14

spectroscopy and X-ray crystallography . Synchrotron radiation techniques such as EXAFS and XANES are being used to characterize the structure and...Chemistry and Catalysis using Soft X-rays at LCLS” 23rd Congress and General Assembly of the International Union of Crystallography , Montreal (2014). A

A Filtering Method to Reveal Crystalline Patterns from Atom Probe Microscopy Desorption Maps

DTIC Science & Technology

2016-03-26

Gault, S.P. Ringer, J.M. Cairney, Atom probe crystallography : characterization of grain boundary orientation relationships in nanocrystalline...J.M. Cairney, Atom probe crystallography : atomic- scale 3-D orientation mapping, Scr. Mater. 66 (11) (2012) 907. L. Yao /MethodsX 3 (2016) 268–273 273

Models as an Aid to Courses in Crystallography and Mineralogy.

ERIC Educational Resources Information Center

Brady, K. T.

1983-01-01

Three models used in teaching crystallography/mineralogy at the University of Technology (Papua, New Guinea) are described. These include stereographic projection model, optical indicatrix models for Istropic/Anisotropic minerals, and model showing effect of anisotropic minerals under crossed polars. Photographs of the models are also included.…

Johann Deisenhofer, Crystallography, and Proteins

Science.gov Websites

research using X-ray crystallography to elucidate for the first time the three-dimensional structure of a large membrane-bound protein molecule. This structure helped explain the process of photosynthesis, by a protein structure determination that relied on complementary features of two different beam lines

A flavor symmetry model for bilarge leptonic mixing and the lepton masses

NASA Astrophysics Data System (ADS)

Ohlsson, Tommy; Seidl, Gerhart

2002-11-01

We present a model for leptonic mixing and the lepton masses based on flavor symmetries and higher-dimensional mass operators. The model predicts bilarge leptonic mixing (i.e., the mixing angles θ12 and θ23 are large and the mixing angle θ13 is small) and an inverted hierarchical neutrino mass spectrum. Furthermore, it approximately yields the experimental hierarchical mass spectrum of the charged leptons. The obtained values for the leptonic mixing parameters and the neutrino mass squared differences are all in agreement with atmospheric neutrino data, the Mikheyev-Smirnov-Wolfenstein large mixing angle solution of the solar neutrino problem, and consistent with the upper bound on the reactor mixing angle. Thus, we have a large, but not close to maximal, solar mixing angle θ12, a nearly maximal atmospheric mixing angle θ23, and a small reactor mixing angle θ13. In addition, the model predicts θ 12≃ {π}/{4}-θ 13.

Macromolecular diffractive imaging using imperfect crystals

PubMed Central

Ayyer, Kartik; Yefanov, Oleksandr; Oberthür, Dominik; Roy-Chowdhury, Shatabdi; Galli, Lorenzo; Mariani, Valerio; Basu, Shibom; Coe, Jesse; Conrad, Chelsie E.; Fromme, Raimund; Schaffer, Alexander; Dörner, Katerina; James, Daniel; Kupitz, Christopher; Metz, Markus; Nelson, Garrett; Lourdu Xavier, Paulraj; Beyerlein, Kenneth R.; Schmidt, Marius; Sarrou, Iosifina; Spence, John C. H.; Weierstall, Uwe; White, Thomas A.; Yang, Jay-How; Zhao, Yun; Liang, Mengning; Aquila, Andrew; Hunter, Mark S.; Robinson, Joseph S.; Koglin, Jason E.; Boutet, Sébastien; Fromme, Petra; Barty, Anton; Chapman, Henry N.

2016-01-01

The three-dimensional structures of macromolecules and their complexes are predominantly elucidated by X-ray protein crystallography. A major limitation is access to high-quality crystals, to ensure X-ray diffraction extends to sufficiently large scattering angles and hence yields sufficiently high-resolution information that the crystal structure can be solved. The observation that crystals with shrunken unit-cell volumes and tighter macromolecular packing often produce higher-resolution Bragg peaks1,2 hints that crystallographic resolution for some macromolecules may be limited not by their heterogeneity but rather by a deviation of strict positional ordering of the crystalline lattice. Such displacements of molecules from the ideal lattice give rise to a continuous diffraction pattern, equal to the incoherent sum of diffraction from rigid single molecular complexes aligned along several discrete crystallographic orientations and hence with an increased information content3. Although such continuous diffraction patterns have long been observed—and are of interest as a source of information about the dynamics of proteins4 —they have not been used for structure determination. Here we show for crystals of the integral membrane protein complex photosystem II that lattice disorder increases the information content and the resolution of the diffraction pattern well beyond the 4.5 Å limit of measurable Bragg peaks, which allows us to directly phase5 the pattern. With the molecular envelope conventionally determined at 4.5 Å as a constraint, we then obtain a static image of the photosystem II dimer at 3.5 Å resolution. This result shows that continuous diffraction can be used to overcome long-supposed resolution limits of macromolecular crystallography, with a method that puts great value in commonly encountered imperfect crystals and opens up the possibility for model-free phasing6,7. PMID:26863980

From crystalline to amorphous calcium pyrophosphates: A solid state Nuclear Magnetic Resonance perspective.

PubMed

Gras, Pierre; Baker, Annabelle; Combes, Christèle; Rey, Christian; Sarda, Stéphanie; Wright, Adrian J; Smith, Mark E; Hanna, John V; Gervais, Christel; Laurencin, Danielle; Bonhomme, Christian

2016-02-01

Hydrated calcium pyrophosphates (CPP, Ca2P2O7·nH2O) are a fundamental family of materials among osteoarticular pathologic calcifications. In this contribution, a comprehensive multinuclear NMR (Nuclear Magnetic Resonance) study of four crystalline and two amorphous phases of this family is presented. (1)H, (31)P and (43)Ca MAS (Magic Angle Spinning) NMR spectra were recorded, leading to informative fingerprints characterizing each compound. In particular, different (1)H and (43)Ca solid state NMR signatures were observed for the amorphous phases, depending on the synthetic procedure used. The NMR parameters of the crystalline phases were determined using the GIPAW (Gauge Including Projected Augmented Wave) DFT approach, based on first-principles calculations. In some cases, relaxed structures were found to improve the agreement between experimental and calculated values, demonstrating the importance of proton positions and pyrophosphate local geometry in this particular NMR crystallography approach. Such calculations serve as a basis for the future ab initio modeling of the amorphous CPP phases. The general concept of NMR crystallography is applied to the detailed study of calcium pyrophosphates (CPP), whether hydrated or not, and whether crystalline or amorphous. CPP are a fundamental family of materials among osteoarticular pathologic calcifications. Their prevalence increases with age, impacting on 17.5% of the population after the age of 80. They are frequently involved or associated with acute articular arthritis such as pseudogout. Current treatments are mainly directed at relieving the symptoms of joint inflammation but not at inhibiting CPP formation nor at dissolving these crystals. The combination of advanced NMR techniques, modeling and DFT based calculation of NMR parameters allows new original insights in the detailed structural description of this important class of biomaterials. Copyright © 2016. Published by Elsevier Ltd.

Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature.

PubMed

Ishigami, Izumi; Zatsepin, Nadia A; Hikita, Masahide; Conrad, Chelsie E; Nelson, Garrett; Coe, Jesse D; Basu, Shibom; Grant, Thomas D; Seaberg, Matthew H; Sierra, Raymond G; Hunter, Mark S; Fromme, Petra; Fromme, Raimund; Yeh, Syun-Ru; Rousseau, Denis L

2017-07-25

Cytochrome c oxidase (C c O), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine C c O (bC c O) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-ray crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a 3 iron atom, with a bent Fe-C-O angle of ∼142°. In contrast, in the synchrotron structure, the Fe-CO bond is cleaved; CO relocates to a new site near Cu B , which, in turn, moves closer to the heme a 3 iron by ∼0.38 Å. Structural comparison reveals that ligand binding to the heme a 3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a 3 , thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.

Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ishigami, Izumi; Zatsepin, Nadia A.; Hikita, Masahide

Here, cytochrome c oxidase (C cO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine C cO (bC cO) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-raymore » crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a3 iron atom, with a bent Fe–C–O angle of ~142°. In contrast, in the synchrotron structure, the Fe–CO bond is cleaved; CO relocates to a new site near Cu B, which, in turn, moves closer to the heme a 3 iron by ~0.38 Å. Structural comparison reveals that ligand binding to the heme a 3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a 3, thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.« less

Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature

DOE PAGES

Ishigami, Izumi; Zatsepin, Nadia A.; Hikita, Masahide; ...

2017-07-11

Here, cytochrome c oxidase (C cO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine C cO (bC cO) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-raymore » crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a3 iron atom, with a bent Fe–C–O angle of ~142°. In contrast, in the synchrotron structure, the Fe–CO bond is cleaved; CO relocates to a new site near Cu B, which, in turn, moves closer to the heme a 3 iron by ~0.38 Å. Structural comparison reveals that ligand binding to the heme a 3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a 3, thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.« less

Effects of heel base size, walking speed, and slope angle on center of pressure trajectory and plantar pressure when wearing high-heeled shoes.

PubMed

Luximon, Yan; Cong, Yan; Luximon, Ameersing; Zhang, Ming

2015-06-01

High-heeled shoes are associated with instability and a high risk of fall, fracture, and ankle sprain. This study investigated the effects of heel base size (HBS) on walking stability under different walking speeds and slope angles. The trajectory of the center of pressure (COP), maximal peak pressure, pressure time integral, contact area, and perceived stability were analyzed. The results revealed that a small HBS increased the COP deviations, shifting the COP more medially at the beginning of the gait cycle. The slope angle mainly affected the COP in the anteroposterior direction. An increased slope angle shifted the COP posterior and caused greater pressure and a larger contact area in the midfoot and rearfoot regions, which can provide more support. Subjective measures on perceived stability were consistent with objective measures. The results suggested that high-heeled shoes with a small HBS did not provide stable plantar support, particularly on a small slope angle. The changes in the COP and pressure pattern caused by a small HBS might increase joint torque and muscle activity and induce lower limb problems. Copyright © 2015 Elsevier B.V. All rights reserved.

Sample manipulation and data assembly for robust microcrystal synchrotron crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guo, Gongrui; Fuchs, Martin R.; Shi, Wuxian

With the recent developments in microcrystal handling, synchrotron microdiffraction beamline instrumentation and data analysis, microcrystal crystallography with crystal sizes of less than 10 µm is appealing at synchrotrons. However, challenges remain in sample manipulation and data assembly for robust microcrystal synchrotron crystallography. Here, the development of micro-sized polyimide well-mounts for the manipulation of microcrystals of a few micrometres in size and the implementation of a robust data-analysis method for the assembly of rotational microdiffraction data sets from many microcrystals are described. Here, the method demonstrates that microcrystals may be routinely utilized for the acquisition and assembly of complete data setsmore » from synchrotron microdiffraction beamlines.« less

Iterative projection algorithms for ab initio phasing in virus crystallography.

PubMed

Lo, Victor L; Kingston, Richard L; Millane, Rick P

2016-12-01

Iterative projection algorithms are proposed as a tool for ab initio phasing in virus crystallography. The good global convergence properties of these algorithms, coupled with the spherical shape and high structural redundancy of icosahedral viruses, allows high resolution phases to be determined with no initial phase information. This approach is demonstrated by determining the electron density of a virus crystal with 5-fold non-crystallographic symmetry, starting with only a spherical shell envelope. The electron density obtained is sufficiently accurate for model building. The results indicate that iterative projection algorithms should be routinely applicable in virus crystallography, without the need for ancillary phase information. Copyright © 2016 Elsevier Inc. All rights reserved.

Beyond crystallography: Diffractive imaging using coherent x-ray light sources

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miao, J.; Ishikawa, T.; Robinson, I. K.

X-ray crystallography has been central to the development of many fields of science over the past century. It has now matured to a point that as long as good-quality crystals are available, their atomic structure can be routinely determined in three dimensions. However, many samples in physics, chemistry, materials science, nanoscience, geology, and biology are noncrystalline, and thus their three-dimensional structures are not accessible by traditional x-ray crystallography. Overcoming this hurdle has required the development of new coherent imaging methods to harness new coherent x-ray light sources. Here we review the revolutionary advances that are transforming x-ray sources and imagingmore » in the 21st century.« less

Racemic protein crystallography.

PubMed

Yeates, Todd O; Kent, Stephen B H

2012-01-01

Although natural proteins are chiral and are all of one "handedness," their mirror image forms can be prepared by chemical synthesis. This opens up new opportunities for protein crystallography. A racemic mixture of the enantiomeric forms of a protein molecule can crystallize in ways that natural proteins cannot. Recent experimental data support a theoretical prediction that this should make racemic protein mixtures highly amenable to crystallization. Crystals obtained from racemic mixtures also offer advantages in structure determination strategies. The relevance of these potential advantages is heightened by advances in synthetic methods, which are extending the size limit for proteins that can be prepared by chemical synthesis. Recent ideas and results in the area of racemic protein crystallography are reviewed.

Sample manipulation and data assembly for robust microcrystal synchrotron crystallography

DOE PAGES

Guo, Gongrui; Fuchs, Martin R.; Shi, Wuxian; ...

2018-04-19

With the recent developments in microcrystal handling, synchrotron microdiffraction beamline instrumentation and data analysis, microcrystal crystallography with crystal sizes of less than 10 µm is appealing at synchrotrons. However, challenges remain in sample manipulation and data assembly for robust microcrystal synchrotron crystallography. Here, the development of micro-sized polyimide well-mounts for the manipulation of microcrystals of a few micrometres in size and the implementation of a robust data-analysis method for the assembly of rotational microdiffraction data sets from many microcrystals are described. Here, the method demonstrates that microcrystals may be routinely utilized for the acquisition and assembly of complete data setsmore » from synchrotron microdiffraction beamlines.« less

Indexing amyloid peptide diffraction from serial femtosecond crystallography: new algorithms for sparse patterns

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brewster, Aaron S.; Sawaya, Michael R.; University of California, Los Angeles, CA 90095-1570

2015-02-01

Special methods are required to interpret sparse diffraction patterns collected from peptide crystals at X-ray free-electron lasers. Bragg spots can be indexed from composite-image powder rings, with crystal orientations then deduced from a very limited number of spot positions. Still diffraction patterns from peptide nanocrystals with small unit cells are challenging to index using conventional methods owing to the limited number of spots and the lack of crystal orientation information for individual images. New indexing algorithms have been developed as part of the Computational Crystallography Toolbox (cctbx) to overcome these challenges. Accurate unit-cell information derived from an aggregate data setmore » from thousands of diffraction patterns can be used to determine a crystal orientation matrix for individual images with as few as five reflections. These algorithms are potentially applicable not only to amyloid peptides but also to any set of diffraction patterns with sparse properties, such as low-resolution virus structures or high-throughput screening of still images captured by raster-scanning at synchrotron sources. As a proof of concept for this technique, successful integration of X-ray free-electron laser (XFEL) data to 2.5 Å resolution for the amyloid segment GNNQQNY from the Sup35 yeast prion is presented.« less

Femtosecond X-ray protein nanocrystallography.

PubMed

Chapman, Henry N; Fromme, Petra; Barty, Anton; White, Thomas A; Kirian, Richard A; Aquila, Andrew; Hunter, Mark S; Schulz, Joachim; DePonte, Daniel P; Weierstall, Uwe; Doak, R Bruce; Maia, Filipe R N C; Martin, Andrew V; Schlichting, Ilme; Lomb, Lukas; Coppola, Nicola; Shoeman, Robert L; Epp, Sascha W; Hartmann, Robert; Rolles, Daniel; Rudenko, Artem; Foucar, Lutz; Kimmel, Nils; Weidenspointner, Georg; Holl, Peter; Liang, Mengning; Barthelmess, Miriam; Caleman, Carl; Boutet, Sébastien; Bogan, Michael J; Krzywinski, Jacek; Bostedt, Christoph; Bajt, Saša; Gumprecht, Lars; Rudek, Benedikt; Erk, Benjamin; Schmidt, Carlo; Hömke, André; Reich, Christian; Pietschner, Daniel; Strüder, Lothar; Hauser, Günter; Gorke, Hubert; Ullrich, Joachim; Herrmann, Sven; Schaller, Gerhard; Schopper, Florian; Soltau, Heike; Kühnel, Kai-Uwe; Messerschmidt, Marc; Bozek, John D; Hau-Riege, Stefan P; Frank, Matthias; Hampton, Christina Y; Sierra, Raymond G; Starodub, Dmitri; Williams, Garth J; Hajdu, Janos; Timneanu, Nicusor; Seibert, M Marvin; Andreasson, Jakob; Rocker, Andrea; Jönsson, Olof; Svenda, Martin; Stern, Stephan; Nass, Karol; Andritschke, Robert; Schröter, Claus-Dieter; Krasniqi, Faton; Bott, Mario; Schmidt, Kevin E; Wang, Xiaoyu; Grotjohann, Ingo; Holton, James M; Barends, Thomas R M; Neutze, Richard; Marchesini, Stefano; Fromme, Raimund; Schorb, Sebastian; Rupp, Daniela; Adolph, Marcus; Gorkhover, Tais; Andersson, Inger; Hirsemann, Helmut; Potdevin, Guillaume; Graafsma, Heinz; Nilsson, Björn; Spence, John C H

2011-02-03

X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction 'snapshots' are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (∼200 nm to 2 μm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.

Measuring Contours of Coal-Seam Cuts

NASA Technical Reports Server (NTRS)

1983-01-01

Angle transducers measure angle between track sections as longwall shearer proceeds along coal face. Distance transducer functions in conjunction with angle transducers to obtain relative angles at known positions. When cut is complete, accumulated data are stored on cassette tape, and track profile is computed and displayed. Micro-processor-based instrument integrates small changes in angle and distance.

Tibial anatomy in normal small breed dogs including anisometry of various extracapsular stabilizing suture attachment sites.

PubMed

Witte, P G

2015-01-01

To investigate proximal tibial anatomy and its influence on anisometry of extracapsular stabilizing sutures in small dog breeds. Mediolateral radiographs of the femora, stifles, and tibiae of 12 small breed dogs were acquired with the stifles positioned at various angles. Measurements taken included tibial plateau angle (TPA), diaphyseal: proximal tibial angle (DPA), patellar tendon angle (PTA), Z-angle, relative tibial tuberosity width (rTTW), and the distance between six combinations of two femoral and three tibial extra-capsular stabilizing suture (ECS) attachment sites. Theoretical strain through stifle range-of-motion was recorded. The TPA (32° ± 5.8°), DPA (10.2° ± 7.3°), PTA (103.7° ± 6.2°), and Z-angle (70.4° ± 9.0°) were positively correlated with one another (R >0.7), but none were correlated with rTTW (0.93 ± 0.10). The F2-T1 combination of ECS attachment sites had lowest strain for nine stifles. The shortest attachment site separation was at a stifle flexion of 50° for nine stifles. Proximal tibial anatomy measurements could not predict optimal attachment site combination, optimal stifle angle for suture placement, or ECS strain. There is individual variation in the optimal attachment site combination and stifle angle for suture placement, which may influence consistency of outcomes with ECS.

3-Dimensional Protein Structure of Influenza

NASA Technical Reports Server (NTRS)

2004-01-01

The loss of productivity due to flu is staggering. Costs range as much as $20 billio a year. High mutation rates of the flu virus have hindered development of new drugs or vaccines. The secret lies in a small molecule which is attached to the host cell's surface. Each flu virus, no matter what strain, must remove this small molecule to escape the host cell to spread infection. Using data from space and earth grown crystals, researchers from the Center of Macromolecular Crystallography (CMC) are desining drugs to bind with this protein's active site. This lock and key fit reduces the spread of flu in the body by blocking its escape route. In collaboration with its corporate partner, the CMC has refined drug structure in preparation for clinical trials. Tested and approved relief is expected to reach drugstores by year 2004.

Two-Dimensional Crystallography Introduced by the Sprinkler Watering Problem

ERIC Educational Resources Information Center

De Toro, Jose A.; Calvo, Gabriel F.; Muniz, Pablo

2012-01-01

The problem of optimizing the number of circular sprinklers watering large fields is used to introduce, from a purely elementary geometrical perspective, some basic concepts in crystallography and comment on a few size effects in condensed matter physics. We examine square and hexagonal lattices to build a function describing the, so-called, dry…

Frontiers of Crystallography: A Project-Based Research-Led Learning Exercise

ERIC Educational Resources Information Center

Wilson, Chick C.; Parkin, Andrew; Thomas, Lynne H.

2012-01-01

A highly interactive research-led learning session for chemistry undergraduates is described, which aims to lead students to an awareness of the applications of crystallography technique through a mentored hands-on crystal structure solution and refinement session. The research-based environment is inherent throughout the 4.5 h program and is…

Forty years of collaborative computational crystallography.

PubMed

Agirre, Jon; Dodson, Eleanor

2018-01-01

A brief overview is provided of the history of collaborative computational crystallography, with an emphasis on the Collaborative Computational Project No. 4. The key steps in its development are outlined, with consideration also given to the underlying reasons which contributed, and ultimately led to, the unprecedented success of this venture. © 2017 The Protein Society.

Fixed Target combined with Spectral Mapping: Approaching 100% Hit Rates for Serial Crystallography

PubMed Central

Pare-Labrosse, Olivier; Kuo, Anling; Marx, Alexander; Epp, Sascha W.; Sherrell, Darren A.; Eger, Bryan T.; Zhong, Yinpeng; Loch, Rolf; Mariani, Valerio; Alonso-Mori, Roberto; Nelson, Silke; Lemke, Henrik T.; Owen, Robin L.; Pearson, Arwen R.; Stuart, David I.; Ernst, Oliver P.; Mueller-Werkmeister, Henrike M.; Miller, R. J. Dwayne

2018-01-01

The advent of ultrafast highly brilliant coherent X-ray Free Electron Laser sources has driven the development of novel structure determination approaches for proteins, and promises visualisation of protein dynamics on the fastest timescales with full atomic resolution. Significant efforts are being applied to the development of sample delivery systems that allow these unique sources to be most efficiently exploited for high throughput serial femtosecond crystallography. We present here the next generation of a fixed target crystallography chip designed for rapid and reliable delivery of up to 11,259 protein crystals with high spatial precision. An experimental scheme for predetermining the positions of crystals in the chip by means of in-situ spectroscopy using a fiducial system for rapid, precise alignment and registration of the crystal positions is presented. This delivers unprecedented performance in serial crystallography experiments at room temperature under atmospheric pressure with a raw hit rate approaching 100% with an effective indexing rate of approximately 50%, increasing the efficiency of beam usage, and allowing the method to be applied to systems where the number of crystals is limited. PMID:27487825

Fixed target combined with spectral mapping: approaching 100% hit rates for serial crystallography.

PubMed

Oghbaey, Saeed; Sarracini, Antoine; Ginn, Helen M; Pare-Labrosse, Olivier; Kuo, Anling; Marx, Alexander; Epp, Sascha W; Sherrell, Darren A; Eger, Bryan T; Zhong, Yinpeng; Loch, Rolf; Mariani, Valerio; Alonso-Mori, Roberto; Nelson, Silke; Lemke, Henrik T; Owen, Robin L; Pearson, Arwen R; Stuart, David I; Ernst, Oliver P; Mueller-Werkmeister, Henrike M; Miller, R J Dwayne

2016-08-01

The advent of ultrafast highly brilliant coherent X-ray free-electron laser sources has driven the development of novel structure-determination approaches for proteins, and promises visualization of protein dynamics on sub-picosecond timescales with full atomic resolution. Significant efforts are being applied to the development of sample-delivery systems that allow these unique sources to be most efficiently exploited for high-throughput serial femtosecond crystallography. Here, the next iteration of a fixed-target crystallography chip designed for rapid and reliable delivery of up to 11 259 protein crystals with high spatial precision is presented. An experimental scheme for predetermining the positions of crystals in the chip by means of in situ spectroscopy using a fiducial system for rapid, precise alignment and registration of the crystal positions is presented. This delivers unprecedented performance in serial crystallography experiments at room temperature under atmospheric pressure, giving a raw hit rate approaching 100% with an effective indexing rate of approximately 50%, increasing the efficiency of beam usage and allowing the method to be applied to systems where the number of crystals is limited.

100 Years Later: Celebrating the Contributions of X-ray Crystallography to Allergy and Clinical Immunology

PubMed Central

Pomés, Anna; Chruszcz, Maksymilian; Gustchina, Alla; Minor, Wladek; Mueller, Geoffrey A.; Pedersen, Lars C.; Wlodawer, Alexander; Chapman, Martin D.

2015-01-01

Current knowledge of molecules involved in immunology and allergic disease results from significant contributions of X-ray crystallography, a discipline that just celebrated its 100th anniversary. The histories of allergens and X-ray crystallography are intimately intertwined. The first enzyme structure to be determined was lysozyme, also known as the chicken food allergen Gal d 4. Crystallography determines the exact three-dimensional positions of atoms in molecules. Structures of molecular complexes in the disciplines of immunology and allergy have revealed the atoms involved in molecular interactions and in mechanisms of disease. These complexes include peptides presented by MHC class II molecules, cytokines bound to their receptors, allergen-antibody complexes, and innate immune receptors with their ligands. The information derived from crystallographic studies provides insights into the function of molecules. Allergen function is one of the determinants of environmental exposure, which is essential for IgE sensitization. Proteolytic activity of allergens or their capacity to bind lipopolysaccharides may also contribute to allergenicity. The atomic positions define the molecular surface that is accessible to antibodies. This surface in turn determines antibody specificity and cross-reactivity that are important factors for the selection of allergen panels used for molecular diagnosis and for the interpretation of clinical symptoms. This review celebrates the contributions of X-ray crystallography to clinical immunology and allergy, focusing on new molecular perspectives that influence the diagnosis and treatment of allergic diseases. PMID:26145985

100 Years later: Celebrating the contributions of x-ray crystallography to allergy and clinical immunology.

PubMed

Pomés, Anna; Chruszcz, Maksymilian; Gustchina, Alla; Minor, Wladek; Mueller, Geoffrey A; Pedersen, Lars C; Wlodawer, Alexander; Chapman, Martin D

2015-07-01

Current knowledge of molecules involved in immunology and allergic disease results from the significant contributions of x-ray crystallography, a discipline that just celebrated its 100th anniversary. The histories of allergens and x-ray crystallography are intimately intertwined. The first enzyme structure to be determined was lysozyme, also known as the chicken food allergen Gal d 4. Crystallography determines the exact 3-dimensional positions of atoms in molecules. Structures of molecular complexes in the disciplines of immunology and allergy have revealed the atoms involved in molecular interactions and mechanisms of disease. These complexes include peptides presented by MHC class II molecules, cytokines bound to their receptors, allergen-antibody complexes, and innate immune receptors with their ligands. The information derived from crystallographic studies provides insights into the function of molecules. Allergen function is one of the determinants of environmental exposure, which is essential for IgE sensitization. Proteolytic activity of allergens or their capacity to bind LPSs can also contribute to allergenicity. The atomic positions define the molecular surface that is accessible to antibodies. In turn, this surface determines antibody specificity and cross-reactivity, which are important factors for the selection of allergen panels used for molecular diagnosis and the interpretation of clinical symptoms. This review celebrates the contributions of x-ray crystallography to clinical immunology and allergy, focusing on new molecular perspectives that influence the diagnosis and treatment of allergic diseases. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. All rights reserved.

The future of crystallography in drug discovery

PubMed Central

Zheng, Heping; Hou, Jing; Zimmerman, Matthew D; Wlodawer, Alexander; Minor, Wladek

2014-01-01

Introduction X-ray crystallography plays an important role in structure-based drug design (SBDD), and accurate analysis of crystal structures of target macromolecules and macromolecule–ligand complexes is critical at all stages. However, whereas there has been significant progress in improving methods of structural biology, particularly in X-ray crystallography, corresponding progress in the development of computational methods (such as in silico high-throughput screening) is still on the horizon. Crystal structures can be overinterpreted and thus bias hypotheses and follow-up experiments. As in any experimental science, the models of macromolecular structures derived from X-ray diffraction data have their limitations, which need to be critically evaluated and well understood for structure-based drug discovery. Areas covered This review describes how the validity, accuracy and precision of a protein or nucleic acid structure determined by X-ray crystallography can be evaluated from three different perspectives: i) the nature of the diffraction experiment; ii) the interpretation of an electron density map; and iii) the interpretation of the structural model in terms of function and mechanism. The strategies to optimally exploit a macromolecular structure are also discussed in the context of ‘Big Data’ analysis, biochemical experimental design and structure-based drug discovery. Expert opinion Although X-ray crystallography is one of the most detailed ‘microscopes’ available today for examining macromolecular structures, the authors would like to re-emphasize that such structures are only simplified models of the target macromolecules. The authors also wish to reinforce the idea that a structure should not be thought of as a set of precise coordinates but rather as a framework for generating hypotheses to be explored. Numerous biochemical and biophysical experiments, including new diffraction experiments, can and should be performed to verify or falsify these hypotheses. X-ray crystallography will find its future application in drug discovery by the development of specific tools that would allow realistic interpretation of the outcome coordinates and/or support testing of these hypotheses. PMID:24372145

Identification of the functional binding pocket for compounds targeting small-conductance Ca2+-activated potassium channels

PubMed Central

Zhang, Miao; Pascal, John M.; Schumann, Marcel; Armen, Roger S.; Zhang, Ji-fang

2012-01-01

Small- and intermediate-conductance Ca2+-activated potassium channels, activated by Ca2+-bound calmodulin, play an important role in regulating membrane excitability. These channels are also linked to clinical abnormalities. A tremendous amount of effort has been devoted to developing small molecule compounds targeting these channels. However, these compounds often suffer from low potency and lack of selectivity, hindering their potentials for clinical use. A key contributing factor is the lack of knowledge of the binding site(s) for these compounds. Here we demonstrate by X-ray crystallography that the binding pocket for the compounds of the 1-EBIO class is located at the calmodulin-channel interface. We show that, based on structure data and molecular docking, mutations of the channel can effectively change the potency of these compounds. Our results provide insight into the molecular nature of the binding pocket and its contribution to the potency and selectivity of the compounds of the 1-EBIO class. PMID:22929778

Identification of the functional binding pocket for compounds targeting small-conductance Ca²⁺-activated potassium channels.

PubMed

Zhang, Miao; Pascal, John M; Schumann, Marcel; Armen, Roger S; Zhang, Ji-Fang

2012-01-01

Small- and intermediate-conductance Ca(2+)-activated potassium channels, activated by Ca(2+)-bound calmodulin, have an important role in regulating membrane excitability. These channels are also linked to clinical abnormalities. A tremendous amount of effort has been devoted to developing small molecule compounds targeting these channels. However, these compounds often suffer from low potency and lack of selectivity, hindering their potential for clinical use. A key contributing factor is the lack of knowledge of the binding site(s) for these compounds. Here we demonstrate by X-ray crystallography that the binding pocket for the compounds of the 1-ethyl-2-benzimidazolinone (1-EBIO) class is located at the calmodulin-channel interface. We show that, based on structure data and molecular docking, mutations of the channel can effectively change the potency of these compounds. Our results provide insight into the molecular nature of the binding pocket and its contribution to the potency and selectivity of the compounds of the 1-EBIO class.

Developments in the Implementation of Acoustic Droplet Ejection for Protein Crystallography.

PubMed

Wu, Ping; Noland, Cameron; Ultsch, Mark; Edwards, Bonnie; Harris, David; Mayer, Robert; Harris, Seth F

2016-02-01

Acoustic droplet ejection (ADE) enables crystallization experiments at the low-nanoliter scale, resulting in rapid vapor diffusion equilibration dynamics and efficient reagent usage in the empirical discovery of structure-enabling protein crystallization conditions. We extend our validation of this technology applied to the diverse physicochemical property space of aqueous crystallization reagents where dynamic fluid analysis coupled to ADE aids in accurate and precise dispensations. Addition of crystallization seed stocks, chemical additives, or small-molecule ligands effectively modulates crystallization, and we here provide examples in optimization of crystal morphology and diffraction quality by the acoustic delivery of ultra-small volumes of these cofactors. Additional applications are discussed, including set up of in situ proteolysis and alternate geometries of crystallization that leverage the small scale afforded by acoustic delivery. Finally, we describe parameters of a system of automation in which the acoustic liquid handler is integrated with a robotic arm, plate centrifuge, peeler, sealer, and stacks, which allows unattended high-throughput crystallization experimentation. © 2015 Society for Laboratory Automation and Screening.

Tables for Supersonic Flow Around Right Circular Cones at Small Angle of Attack

NASA Technical Reports Server (NTRS)

Sims, Joseph L.

1964-01-01

The solution of supersonic flow fields by the method of characteristics requires that starting conditions be known. Ferri, in reference 1, developed a method-of-characteristics solution for axially symmetric bodies of revolution at small angles of attack. With computing machinery that is now available, this has become a feasible method for computing the aerodynamic characteristics of bodies near zero angle of attack. For sharp-nosed bodies of revolution, the required starting line may be obtained by computing the flow field about a cone at a small angle of attack. This calculation is readily performed using Stone's theory in reference 2. Some solutions of this theory are available in reference 3. However, the manner in which these results are presented, namely in a wind-fixed coordinate system, makes their use somewhat cumbersome. Additionally, as pointed out in reference 4, the flow component perpendicular to the meridian planes was computed incorrectly. The results contained herein have been computed in the same basic manner as those of reference 3 with the correct velocity normal to the meridian planes. Also, all results have been transferred into the body-fixed coordinate system. Therefore, the values tabulated herein may be used, in conjunction with the respective zero-angle-of-attack results of reference 5, as starting conditions for the method-of-characteristics solution of the flow field about axially symmetric bodies of revolution at small angles of attack. As in the zero-angle-of-attack case (ref. 5) the present results have been computed using the ideal gas value of 1.4 for the ratio of the specific heats of air. Solutions are given for cone angles from 2.5 deg to 30 deg in increments of 2.5 deg. For each cone angle, results were computed for a constant series of free-stream Mach numbers from 1.5 to 20. In addition, a solution was computed which yielded the minimum free-stream Mach number for a completely supersonic conical flow field. For cone angles of 27.5 deg and 30 deg, this minimum free-stream Mach number was above 1.5. Consequently, solutions at this Mach number were not computed for these two cone angles.

High-Affinity Small-Molecule Inhibitors of the Menin-Mixed Lineage Leukemia (MLL) Interaction Closely Mimic a Natural Protein-Protein Interaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

He, Shihan; Senter, Timothy J.; Pollock, Jonathan

2014-10-02

The protein–protein interaction (PPI) between menin and mixed lineage leukemia (MLL) plays a critical role in acute leukemias, and inhibition of this interaction represents a new potential therapeutic strategy for MLL leukemias. We report development of a novel class of small-molecule inhibitors of the menin–MLL interaction, the hydroxy- and aminomethylpiperidine compounds, which originated from HTS of ~288000 small molecules. We determined menin–inhibitor co-crystal structures and found that these compounds closely mimic all key interactions of MLL with menin. Extensive crystallography studies combined with structure-based design were applied for optimization of these compounds, resulting in MIV-6R, which inhibits the menin–MLL interactionmore » with IC 50 = 56 nM. Treatment with MIV-6 demonstrated strong and selective effects in MLL leukemia cells, validating specific mechanism of action. Our studies provide novel and attractive scaffold as a new potential therapeutic approach for MLL leukemias and demonstrate an example of PPI amenable to inhibition by small molecules.« less

Nanospheres with a smectic hydrophobic core and an amorphous PEG hydrophilic shell: structural changes and implications for drug delivery

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murthy, N. Sanjeeva; Zhang, Zheng; Borsadia, Siddharth

The structural changes in nanospheres with a crystalline core and an amorphous diffuse shell were investigated by small-angle neutron scattering (SANS), small-, medium-, and wide-angle X-ray scattering (SAXS, MAXS and WAXS), and differential scanning calorimetry (DSC).

Note: Magnification of a polarization angle with a Littrow layout brazed grating

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sasao, H., E-mail: sasao.hajime@jaea.go.jp; Kubo, H.; Kawano, Y.

A new method to magnify a small polarization angle with brazed gratings has been developed. In the method, difference in diffraction efficiency for S and P polarization components is used. The magnification dependence on the incident angle can be small by arranging the grating in Littrow layout. A magnification with a factor ∼2.7 has been demonstrated for a 10.6 μm CO{sub 2} laser beam as expected from a calculation. The method is applicable in many polarimetry fields.

Multiple-Fiber-Optic Probe For Light-Scattering Measurements

NASA Technical Reports Server (NTRS)

Dhadwal, Harbans Singh; Ansari, Rafat R.

1996-01-01

Multiple-fiber-optical probe developed for use in measuring light scattered at various angles from specimens of materials. Designed for both static and dynamic light-scattering measurements of colloidal dispersions. Probe compact, rugged unit containing no moving parts and remains stationary during operation. Not restricted to operation in controlled, research-laboratory environment. Positioned inside or outside light-scattering chamber. Provides simultaneous measurements at small angular intervals over range of angles, made to include small scattering angles by orienting probe in appropriate direction.

Coanda-Assisted Spray Manipulation Collar for a Commercial Plasma Spray Gun

NASA Astrophysics Data System (ADS)

Mabey, K.; Smith, B. L.; Whichard, G.; McKechnie, T.

2011-06-01

A Coanda-assisted Spray Manipulation (CSM) collar was retrofitted to a Praxair SG-100 plasma spray gun. The CSM device makes it possible to change the direction of (vector) the plasma jet and powder without moving the gun. The two-piece retrofit device replaces the standard faceplate. Two separate collars were tested: one designed for small vector angles and one for larger vector angles. It was demonstrated that the small-angle device could modify the trajectory of zirconia powder up to several degrees. Doing so could realign the plasma with the powder resulting in increased powder temperature and velocity. The large-angle device was capable of vectoring the plasma jet up to 45°. However, the powder did not vector as much. Under large-angle vectoring, the powder velocity and temperature decreased steadily with vector angle. Both devices were tested using a supersonic configuration to demonstrate that CSM is capable of vectoring supersonic plasmas.

X-ray Structure of Native Scorpion Toxin BmBKTx1 by Racemic Protein Crystallography Using Direct Methods

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mandal, Kalyaneswar; Pentelute, Brad L.; Tereshko, Valentina

2009-04-08

Racemic protein crystallography, enabled by total chemical synthesis, has allowed us to determine the X-ray structure of native scorpion toxin BmBKTx1; direct methods were used for phase determination. This is the first example of a protein racemate that crystallized in space group I41/a.

Beyond crystallography: diffractive imaging using coherent x-ray light sources.

PubMed

Miao, Jianwei; Ishikawa, Tetsuya; Robinson, Ian K; Murnane, Margaret M

2015-05-01

X-ray crystallography has been central to the development of many fields of science over the past century. It has now matured to a point that as long as good-quality crystals are available, their atomic structure can be routinely determined in three dimensions. However, many samples in physics, chemistry, materials science, nanoscience, geology, and biology are noncrystalline, and thus their three-dimensional structures are not accessible by traditional x-ray crystallography. Overcoming this hurdle has required the development of new coherent imaging methods to harness new coherent x-ray light sources. Here we review the revolutionary advances that are transforming x-ray sources and imaging in the 21st century. Copyright © 2015, American Association for the Advancement of Science.

The use of neutron scattering to determine the functional structure of glycoside hydrolase.

PubMed

Nakamura, Akihiko; Ishida, Takuya; Samejima, Masahiro; Igarashi, Kiyohiko

2016-10-01

Neutron diffraction provides different information from X-ray diffraction, because neutrons are scattered by atomic nuclei, whereas X-rays are scattered by electrons. One of the key advantages of neutron crystallography is the ability to visualize hydrogen and deuterium atoms, making it possible to observe the protonation state of amino acid residues, hydrogen bonds, networks of water molecules and proton relay pathways in enzymes. But, because of technical difficulties, less than 100 enzyme structures have been evaluated by neutron crystallography to date. In this review, we discuss the advantages and disadvantages of neutron crystallography as a tool to investigate the functional structure of glycoside hydrolases, with some examples. Copyright © 2016 Elsevier Ltd. All rights reserved.

Fat fraction bias correction using T1 estimates and flip angle mapping.

PubMed

Yang, Issac Y; Cui, Yifan; Wiens, Curtis N; Wade, Trevor P; Friesen-Waldner, Lanette J; McKenzie, Charles A

2014-01-01

To develop a new method of reducing T1 bias in proton density fat fraction (PDFF) measured with iterative decomposition of water and fat with echo asymmetry and least-squares estimation (IDEAL). PDFF maps reconstructed from high flip angle IDEAL measurements were simulated and acquired from phantoms and volunteer L4 vertebrae. T1 bias was corrected using a priori T1 values for water and fat, both with and without flip angle correction. Signal-to-noise ratio (SNR) maps were used to measure precision of the reconstructed PDFF maps. PDFF measurements acquired using small flip angles were then compared to both sets of corrected large flip angle measurements for accuracy and precision. Simulations show similar results in PDFF error between small flip angle measurements and corrected large flip angle measurements as long as T1 estimates were within one standard deviation from the true value. Compared to low flip angle measurements, phantom and in vivo measurements demonstrate better precision and accuracy in PDFF measurements if images were acquired at a high flip angle, with T1 bias corrected using T1 estimates and flip angle mapping. T1 bias correction of large flip angle acquisitions using estimated T1 values with flip angle mapping yields fat fraction measurements of similar accuracy and superior precision compared to low flip angle acquisitions. Copyright © 2013 Wiley Periodicals, Inc.

Use of botulinum toxin in small-angle heterotropia and decompensating heterophoria: a review of the literature.

PubMed

Ripley, L; Rowe, F J

2007-01-01

Botulinum toxin has been used extensively in strabismus management. However, less is published regarding its use in small-angled manifest deviations or decompensating heterophorias, where an alternative to surgery is required. The aim of this review is to look at the use and effectiveness of botulinum toxin in managing small-angled manifest deviations, both constant and intermittent, and decompensating heterophorias. These types of strabismus can prove difficult to manage, as the angle present is often too small for surgery to be advised, but it may still cause a cosmetic or symptom-producing problem. A search of the English speaking literature was undertaken using Medline facilities as well as a limited manual search of non-Medline journals and transactions. A brief overview is provided for mechanisms of action, complications and dose effects, and diagnostic and therapeutic uses of botulinum toxin. The main reported complications are those of ptosis, induced vertical deviation and subconjunctival haemorrhage. The higher the dose, the greater the risk of complications. In small-angle strabismus, botulinum toxin is reported as particularly useful in cases of acquired and acute-onset esotropia in aiding maintenance of binocular vision. It is useful for additional management of surgically under- or over-corrected esotropia, particularly for those with potential for binocular vision. Less effect is reported in primary exotropia versus primary esotropia. It is the management of choice for consecutive exotropia, particularly when patients have had previous multiple surgery and where there is a risk for postoperative diplopia. Botulinum toxin has a specific role in decompensated heterophoria, allowing the visual axes a chance to 'lock on' and subsequently maintain binocular vision. Successful outcomes are reported after 1-2 injections only but the results are best in cases of heterophoria with little near-distance angle disparity.

Numerical study on influence of single control surface on aero elastic behavior of forward-swept wing

NASA Astrophysics Data System (ADS)

Wang, Ning; Su, Xinbing; Ma, Binlin; Zhang, Xiaofei

2017-10-01

In order to study the influence of elastic forward-swept wing (FSW) with single control surface, the computational fluid dynamics/computational structural dynamics (CFD/CSD) loose coupling static aero elastic numerical calculation method was adopted for numerical simulation. The effects of the elastic FSW with leading- or trailing-edge control surface on aero elastic characteristics were calculated and analysed under the condition of high subsonic speed. The result shows that, the deflection of every single control surface could change the aero elastic characteristics of elastic FSW greatly. Compared with the baseline model, when leading-edge control surface deflected up, under the condition of small angles of attack, the aerodynamic characteristics was poor, but the bending and torsional deformation decreased. Under the condition of moderate angles of attack, the aerodynamic characteristics was improved, but bending and torsional deformation increased; When leading-edge control surface deflected down, the aerodynamic characteristics was improved, the bending and torsional deformation decreased/increased under the condition of small/moderate angles of attack. Compared with the baseline model, when trailing-edge control surface deflected down, the aerodynamic characteristics was improved. The bending and torsional deformation increased under the condition of small angles of attack. The bending deformation increased under the condition of small angles of attack, but torsional deformation decreases under the condition of moderate angles of attack. So, for the elastic FSW, the deflection of trailing-edge control surface play a more important role on the improvement of aerodynamic and elastic deformation characteristics.

Informing the improvement of forest products durability using small angle neutron scattering

Treesearch

Nayomi Plaza Rodriguez; Sai Venkatesh Pingali; Shuo Qian; William T. Heller; Joseph E. Jakes

2016-01-01

A better understanding of how wood nanostructure swells with moisture is needed to accelerate the development of forest products with enhanced moisture durability. Despite its suitability to study nanostructures, small angle neutron scattering (SANS) remains an underutilized tool in forest products research. Nanoscale moisture-induced structural changes in intact and...

Correct interpretation of diffraction properties of quartz crystals for X-ray optics applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Xian-Rong; Gog, Thomas; Kim, Jungho

Quartz has hundreds of strong Bragg reflections that may offer a great number of choices for making fixed-angle X-ray analyzers and polarizers at virtually any hard X-ray energies with selectable resolution. However, quartz crystals, unlike silicon and germanium, are chiral and may thus appear in two different forms of handedness that are mirror images. Furthermore, because of the threefold rotational symmetry along thecaxis, the {h 1h 2h 3L} and {h 2h 1h 3L} Bragg reflections may have quite different Darwin bandwidth, reflectivity and angular acceptance, although they have the same Bragg angle. The design of X-ray optics from quartz crystalsmore » therefore requires unambiguous determination of the orientation, handedness and polarity of the crystals. The Laue method and single-axis diffraction technique can provide such information, but the variety of conventions used in the literature to describe quartz structures has caused widespread confusion. The current studies give detailed guidelines for design and fabrication of quartz X-ray optics, with special emphasis on the correct interpretation of Laue patterns in terms of the crystallography and diffraction properties of quartz. Meanwhile, the quartz crystals examined were confirmed by X-ray topography to have acceptably low densities of dislocations and other defects, which is the foundation for developing high-resolution quartz-based X-ray optics.« less

A method of measuring micro-impulse with torsion pendulum based on multi-beam laser heterodyne

NASA Astrophysics Data System (ADS)

Li, Yan-Chao; Wang, Chun-Hui

2012-02-01

In this paper, we propose a novel method of multi-beam laser heterodyne measurement for micro-impulse. The measurement of the micro-impulse, which is converted into the measurement of the small tuning angle of the torsion pendulum, is realized by considering the interaction between pulse laser and working medium. Based on Doppler effect and heterodyne technology, the information regarding the small tuning angle is loaded to the frequency difference of the multi-beam laser heterodyne signal by the frequency modulation of the oscillating mirror, thereby obtaining many values of the small tuning angle after the multi-beam laser heterodyne signal demodulation simultaneously. Processing these values by weighted-average, the small tuning angle can be obtained accurately and the value of the micro-impulse can eventually be calculated. Using Polyvinylchlorid+2%C as a working medium, this novel method is used to simulate the value of the micro-impulse by MATLAB which is generated by considering the interaction between the pulse laser and the working medium, the obtained result shows that the relative error of this method is just 0.5%.

Portable mini-chamber for temperature dependent studies using small angle and wide angle x-ray scattering

NASA Astrophysics Data System (ADS)

Dev, Arun Singh; Kumar, Dileep; Potdar, Satish; Pandit, Pallavi; Roth, Stephan V.; Gupta, Ajay

2018-04-01

The present work describes the design and performance of a vacuum compatible portable mini chamber for temperature dependent GISAXS and GIWAXS studies of thin films and multilayer structures. The water cooled body of the chamber allows sample annealing up to 900 K using ultra high vacuum compatible (UHV) pyrolytic boron nitride heater, thus making it possible to study the temperature dependent evolution of structure and morphology of two-dimensional nanostructured materials. Due to its light weight and small size, the chamber is portable and can be accommodated at synchrotron facilities worldwide. A systematic illustration of the versatility of the chamber has been demonstrated at beamline P03, PETRA-III, DESY, Hamburg, Germany. Temperature dependent grazing incidence small angle x-ray scattering (GISAXS) and grazing incidence wide angle x-ray scattering (GIWAXS) measurements were performed on oblique angle deposited Co/Ag multilayer structure, which jointly revealed that the surface diffusion in Co columns in Co/Ag multilayer enhances by increasing temperature from RT to ˜573 K. This results in a morphology change from columnar tilted structure to densely packed morphological isotropic multilayer.

Thermalization after/during reheating

NASA Astrophysics Data System (ADS)

Harigaya, Keisuke; Mukaida, Kyohei

2014-05-01

If reheating of the Universe takes place via Planck-suppressed decay, it seems that the thermalization of produced particles might be delayed, since they have large energy/small number densities and number violating large angle scatterings which decrease the momentum of particles by large amount are inefficient correspondingly. In this paper, we study the thermalization of such "under occupied" decay products in detail, following recent developments in understanding the thermalization of non-abelian plasma. Contrary to the above naive expectation, it is shown that in most cases thermalization after/during reheating occurs instantaneously by properly taking account of scatterings with small angles and of particles with small momenta. In particular, the condition for instantaneous thermalization before the completion of reheating is found to be , which is much milder than that obtained in previous works with small angle scatterings taken into account.

Ultra-precision fabrication of 500 mm long and laterally graded Ru/C multilayer mirrors for X-ray light sources.

PubMed

Störmer, M; Gabrisch, H; Horstmann, C; Heidorn, U; Hertlein, F; Wiesmann, J; Siewert, F; Rack, A

2016-05-01

X-ray mirrors are needed for beam shaping and monochromatization at advanced research light sources, for instance, free-electron lasers and synchrotron sources. Such mirrors consist of a substrate and a coating. The shape accuracy of the substrate and the layer precision of the coating are the crucial parameters that determine the beam properties required for various applications. In principal, the selection of the layer materials determines the mirror reflectivity. A single layer mirror offers high reflectivity in the range of total external reflection, whereas the reflectivity is reduced considerably above the critical angle. A periodic multilayer can enhance the reflectivity at higher angles due to Bragg reflection. Here, the selection of a suitable combination of layer materials is essential to achieve a high flux at distinct photon energies, which is often required for applications such as microtomography, diffraction, or protein crystallography. This contribution presents the current development of a Ru/C multilayer mirror prepared by magnetron sputtering with a sputtering facility that was designed in-house at the Helmholtz-Zentrum Geesthacht. The deposition conditions were optimized in order to achieve ultra-high precision and high flux in future mirrors. Input for the improved deposition parameters came from investigations by transmission electron microscopy. The X-ray optical properties were investigated by means of X-ray reflectometry using Cu- and Mo-radiation. The change of the multilayer d-spacing over the mirror dimensions and the variation of the Bragg angles were determined. The results demonstrate the ability to precisely control the variation in thickness over the whole mirror length of 500 mm thus achieving picometer-precision in the meter-range.

Ultra-precision fabrication of 500 mm long and laterally graded Ru/C multilayer mirrors for X-ray light sources

DOE Office of Scientific and Technical Information (OSTI.GOV)

Störmer, M., E-mail: michael.stoermer@hzg.de; Gabrisch, H.; Horstmann, C.

2016-05-15

X-ray mirrors are needed for beam shaping and monochromatization at advanced research light sources, for instance, free-electron lasers and synchrotron sources. Such mirrors consist of a substrate and a coating. The shape accuracy of the substrate and the layer precision of the coating are the crucial parameters that determine the beam properties required for various applications. In principal, the selection of the layer materials determines the mirror reflectivity. A single layer mirror offers high reflectivity in the range of total external reflection, whereas the reflectivity is reduced considerably above the critical angle. A periodic multilayer can enhance the reflectivity atmore » higher angles due to Bragg reflection. Here, the selection of a suitable combination of layer materials is essential to achieve a high flux at distinct photon energies, which is often required for applications such as microtomography, diffraction, or protein crystallography. This contribution presents the current development of a Ru/C multilayer mirror prepared by magnetron sputtering with a sputtering facility that was designed in-house at the Helmholtz-Zentrum Geesthacht. The deposition conditions were optimized in order to achieve ultra-high precision and high flux in future mirrors. Input for the improved deposition parameters came from investigations by transmission electron microscopy. The X-ray optical properties were investigated by means of X-ray reflectometry using Cu- and Mo-radiation. The change of the multilayer d-spacing over the mirror dimensions and the variation of the Bragg angles were determined. The results demonstrate the ability to precisely control the variation in thickness over the whole mirror length of 500 mm thus achieving picometer-precision in the meter-range.« less

Large-viewing-angle electroholography by space projection

NASA Astrophysics Data System (ADS)

Sato, Koki; Obana, Kazuki; Okumura, Toshimichi; Kanaoka, Takumi; Nishikawa, Satoko; Takano, Kunihiko

2004-06-01

The specification of hologram image is the full parallax 3D image. In this case we can get more natural 3D image because focusing and convergence are coincident each other. We try to get practical electro-holography system because for conventional electro-holography the image viewing angle is very small. This is due to the limited display pixel size. Now we are developing new method for large viewing angle by space projection method. White color laser is irradiated to single DMD panel ( time shared CGH of RGB three colors ). 3D space screen constructed by very small water particle is used to reconstruct the 3D image with large viewing angle by scattering of water particle.

Celebrating the International Year of Crystallography with a Wisconsin High School Crystal Growing Competition

ERIC Educational Resources Information Center

Guzei, Ilia A.

2014-01-01

In honor of the 2014 International Year of Crystallography, the first Wisconsin Crystal Growing Competition was successfully organized and conducted. High school students from 26 schools across the state competed for prizes by growing large crystals of CuSO[subscript4]·5(H[subscript2]O). This paper describes how the event was planned and carried…

Using the Plan View to Teach Basic Crystallography in General Chemistry

ERIC Educational Resources Information Center

Cushman, Cody V.; Linford, Matthew R.

2015-01-01

The plan view is used in crystallography and materials science to show the positions of atoms in crystal structures. However, it is not widely used in teaching general chemistry. In this contribution, we introduce the plan view, and show these views for the simple cubic, body-centered cubic, face-centered cubic, hexagonal close packed, CsCl, NaCl,…

Crystal and Vibrational Structure of Energetic 3,5-dinitro 1,3,5-oxadiazinane (DOD) by Single Crystal X-ray Diffractometry and Raman Spectroscopy

DTIC Science & Technology

2018-03-19

calculations using a temperature of 298 K. 15. SUBJECT TERMS 3,5-dinitro-1,3,5-oxadiazinane (DOD), X-ray crystallography , Raman, energetic material...X-ray analysis. 2.2 Characterization X-ray Crystallography . DOD crystals were characterized with a SuperNova, Dualflex, EosS2 diffractometer using

Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

PubMed

Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

2016-08-26

Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach.

X-ray structure determination using low-resolution electron microscopy maps for molecular replacement

DOE PAGES

Jackson, Ryan N.; McCoy, Airlie J.; Terwilliger, Thomas C.; ...

2015-07-30

Structures of multi-subunit macromolecular machines are primarily determined by either electron microscopy (EM) or X-ray crystallography. In many cases, a structure for a complex can be obtained at low resolution (at a coarse level of detail) with EM and at higher resolution (with finer detail) by X-ray crystallography. The integration of these two structural techniques is becoming increasingly important for generating atomic models of macromolecular complexes. A low-resolution EM image can be a powerful tool for obtaining the "phase" information that is missing from an X-ray crystallography experiment, however integration of EM and X-ray diffraction data has been technically challenging.more » Here we show a step-by-step protocol that explains how low-resolution EM maps can be placed in the crystallographic unit cell by molecular replacement, and how initial phases computed from the placed EM density are extended to high resolution by averaging maps over non-crystallographic symmetry. As the resolution gap between EM and Xray crystallography continues to narrow, the use of EM maps to help with X-ray crystal structure determination, as described in this protocol, will become increasingly effective.« less

Neutron Crystallography for the Study of Hydrogen Bonds in Macromolecules.

PubMed

Oksanen, Esko; Chen, Julian C-H; Fisher, Suzanne Zoë

2017-04-07

Abstract : The hydrogen bond (H bond) is one of the most important interactions that form the foundation of secondary and tertiary protein structure. Beyond holding protein structures together, H bonds are also intimately involved in solvent coordination, ligand binding, and enzyme catalysis. The H bond by definition involves the light atom, H, and it is very difficult to study directly, especially with X-ray crystallographic techniques, due to the poor scattering power of H atoms. Neutron protein crystallography provides a powerful, complementary tool that can give unambiguous information to structural biologists on solvent organization and coordination, the electrostatics of ligand binding, the protonation states of amino acid side chains and catalytic water species. The method is complementary to X-ray crystallography and the dynamic data obtainable with NMR spectroscopy. Also, as it gives explicit H atom positions, it can be very valuable to computational chemistry where exact knowledge of protonation and solvent orientation can make a large difference in modeling. This article gives general information about neutron crystallography and shows specific examples of how the method has contributed to structural biology, structure-based drug design; and the understanding of fundamental questions of reaction mechanisms.

Neutron crystallography for the study of hydrogen bonds in macromolecules

DOE PAGES

Oksanen, Esko; Chen, Julian C.; Fisher, Zoe

2017-04-07

The hydrogen bond (H bond) is one of the most important interactions that form the foundation of secondary and tertiary protein structure. Beyond holding protein structures together, H bonds are also intimately involved in solvent coordination, ligand binding, and enzyme catalysis. The H bond by definition involves the light atom, H, and it is very difficult to study directly, especially with X-ray crystallographic techniques, due to the poor scattering power of H atoms. Neutron protein crystallography provides a powerful, complementary tool that can give unambiguous information to structural biologists on solvent organization and coordination, the electrostatics of ligand binding, themore » protonation states of amino acid side chains and catalytic water species. The method is complementary to X-ray crystallography and the dynamic data obtainable with NMR spectroscopy. Also, as it gives explicit H atom positions, it can be very valuable to computational chemistry where exact knowledge of protonation and solvent orientation can make a large difference in modeling. Finally, this article gives general information about neutron crystallography and shows specific examples of how the method has contributed to structural biology, structure-based drug design; and the understanding of fundamental questions of reaction mechanisms.« less

Neutron crystallography for the study of hydrogen bonds in macromolecules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oksanen, Esko; Chen, Julian C.; Fisher, Zoe

The hydrogen bond (H bond) is one of the most important interactions that form the foundation of secondary and tertiary protein structure. Beyond holding protein structures together, H bonds are also intimately involved in solvent coordination, ligand binding, and enzyme catalysis. The H bond by definition involves the light atom, H, and it is very difficult to study directly, especially with X-ray crystallographic techniques, due to the poor scattering power of H atoms. Neutron protein crystallography provides a powerful, complementary tool that can give unambiguous information to structural biologists on solvent organization and coordination, the electrostatics of ligand binding, themore » protonation states of amino acid side chains and catalytic water species. The method is complementary to X-ray crystallography and the dynamic data obtainable with NMR spectroscopy. Also, as it gives explicit H atom positions, it can be very valuable to computational chemistry where exact knowledge of protonation and solvent orientation can make a large difference in modeling. Finally, this article gives general information about neutron crystallography and shows specific examples of how the method has contributed to structural biology, structure-based drug design; and the understanding of fundamental questions of reaction mechanisms.« less

Fragment-based discovery of a new family of non-peptidic small-molecule cyclophilin inhibitors with potent antiviral activities

PubMed Central

Ahmed-Belkacem, Abdelhakim; Colliandre, Lionel; Ahnou, Nazim; Nevers, Quentin; Gelin, Muriel; Bessin, Yannick; Brillet, Rozenn; Cala, Olivier; Douguet, Dominique; Bourguet, William; Krimm, Isabelle; Pawlotsky, Jean-Michel; Guichou, Jean- François

2016-01-01

Cyclophilins are peptidyl-prolyl cis/trans isomerases (PPIase) that catalyse the interconversion of the peptide bond at proline residues. Several cyclophilins play a pivotal role in the life cycle of a number of viruses. The existing cyclophilin inhibitors, all derived from cyclosporine A or sanglifehrin A, have disadvantages, including their size, potential for side effects unrelated to cyclophilin inhibition and drug–drug interactions, unclear antiviral spectrum and manufacturing issues. Here we use a fragment-based drug discovery approach using nucleic magnetic resonance, X-ray crystallography and structure-based compound optimization to generate a new family of non-peptidic, small-molecule cyclophilin inhibitors with potent in vitro PPIase inhibitory activity and antiviral activity against hepatitis C virus, human immunodeficiency virus and coronaviruses. This family of compounds has the potential for broad-spectrum, high-barrier-to-resistance treatment of viral infections. PMID:27652979

KRAS G12C Drug Development: Discrimination between Switch II Pocket Configurations Using Hydrogen/Deuterium-Exchange Mass Spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Jia; Harrison, Rane A.; Li, Lianbo

KRAS G12C, the most common RAS mutation found in non-small-cell lung cancer, has been the subject of multiple recent covalent small-molecule inhibitor campaigns including efforts directed at the guanine nucleotide pocket and separate work focused on an inducible pocket adjacent to the switch motifs. Multiple conformations of switch II have been observed, suggesting that switch II pocket (SIIP) binders may be capable of engaging a range of KRAS conformations. Here we report the use of hydrogen/deuterium-exchange mass spectrometry (HDX MS) to discriminate between conformations of switch II induced by two chemical classes of SIIP binders. We investigated the structural basismore » for differences in HDX MS using X-ray crystallography and discovered a new SIIP configuration in response to binding of a quinazoline chemotype. These results have implications for structure-guided drug design targeting the RAS SIIP.« less

On macromolecular refinement at subatomic resolution withinteratomic scatterers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Afonine, Pavel V.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.

2007-11-09

A study of the accurate electron density distribution in molecular crystals at subatomic resolution, better than {approx} 1.0 {angstrom}, requires more detailed models than those based on independent spherical atoms. A tool conventionally used in small-molecule crystallography is the multipolar model. Even at upper resolution limits of 0.8-1.0 {angstrom}, the number of experimental data is insufficient for the full multipolar model refinement. As an alternative, a simpler model composed of conventional independent spherical atoms augmented by additional scatterers to model bonding effects has been proposed. Refinement of these mixed models for several benchmark datasets gave results comparable in quality withmore » results of multipolar refinement and superior of those for conventional models. Applications to several datasets of both small- and macro-molecules are shown. These refinements were performed using the general-purpose macromolecular refinement module phenix.refine of the PHENIX package.« less

On macromolecular refinement at subatomic resolution with interatomic scatterers

PubMed Central

Afonine, Pavel V.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.; Lunin, Vladimir Y.; Urzhumtsev, Alexandre

2007-01-01

A study of the accurate electron-density distribution in molecular crystals at subatomic resolution (better than ∼1.0 Å) requires more detailed models than those based on independent spherical atoms. A tool that is conventionally used in small-molecule crystallography is the multipolar model. Even at upper resolution limits of 0.8–1.0 Å, the number of experimental data is insufficient for full multipolar model refinement. As an alternative, a simpler model composed of conventional independent spherical atoms augmented by additional scatterers to model bonding effects has been proposed. Refinement of these mixed models for several benchmark data sets gave results that were comparable in quality with the results of multipolar refinement and superior to those for conventional models. Applications to several data sets of both small molecules and macromolecules are shown. These refinements were performed using the general-purpose macromolecular refinement module phenix.refine of the PHENIX package. PMID:18007035

On macromolecular refinement at subatomic resolution with interatomic scatterers.

PubMed

Afonine, Pavel V; Grosse-Kunstleve, Ralf W; Adams, Paul D; Lunin, Vladimir Y; Urzhumtsev, Alexandre

2007-11-01

A study of the accurate electron-density distribution in molecular crystals at subatomic resolution (better than approximately 1.0 A) requires more detailed models than those based on independent spherical atoms. A tool that is conventionally used in small-molecule crystallography is the multipolar model. Even at upper resolution limits of 0.8-1.0 A, the number of experimental data is insufficient for full multipolar model refinement. As an alternative, a simpler model composed of conventional independent spherical atoms augmented by additional scatterers to model bonding effects has been proposed. Refinement of these mixed models for several benchmark data sets gave results that were comparable in quality with the results of multipolar refinement and superior to those for conventional models. Applications to several data sets of both small molecules and macromolecules are shown. These refinements were performed using the general-purpose macromolecular refinement module phenix.refine of the PHENIX package.

The minimalist architectures of viroporins and their therapeutic implications

PubMed Central

OuYang, Bo; Chou, James J.

2014-01-01

Many viral genomes encode small, integral membrane proteins that form homo-oligomeric channels in membrane, and they transport protons, cations, and other molecules across the membrane barrier to aid various steps of viral entry and maturation. These viral proteins, collectively named viroporins, are crucial for viral pathogenicity. In the past five years, structures obtained by nuclear magnetic resonance (NMR), X-ray crystallography, and electron microscopy (EM) showed that viroporins often adopt minimalist architectures to achieve their functions. A number of small molecules have been identified to interfere with their channel activity and thereby inhibit viral infection, making viroporins potential drug targets for therapeutic intervention. The known architectures and inhibition mechanisms of viroporins differ significantly from each other, but some common principles are shared between them. This review article summarizes the recent developments in the structural investigation of viroporins and their inhibition by antiviral compounds. PMID:24055819

A rapid alternative to X-ray crystallography for chiral determination: case studies of vibrational circular dichroism (VCD) to advance drug discovery projects.

PubMed

Wesolowski, Steven S; Pivonka, Don E

2013-07-15

The absolute stereochemistry of chiral drugs is usually established via X-ray crystallography. However, vibrational circular dichroism (VCD) spectroscopy coupled with quantum mechanics simulations offers a rapid alternative to crystallography and is readily applied to both crystalline and non-crystalline samples. VCD is an effective complement to X-ray analysis of drug candidates, and it can be used as a high-throughput means of assessing absolute stereochemistry at all phases of the discovery process (hundreds of assignments per year). The practical implementation (or fee-for-service outsourcing) of VCD and selected case studies are illustrated with an emphasis on providing utility and impact to pharmaceutical discovery programs. Copyright © 2013 Elsevier Ltd. All rights reserved.

Liquid sample delivery techniques for serial femtosecond crystallography

PubMed Central

Weierstall, Uwe

2014-01-01

X-ray free-electron lasers overcome the problem of radiation damage in protein crystallography and allow structure determination from micro- and nanocrystals at room temperature. To ensure that consecutive X-ray pulses do not probe previously exposed crystals, the sample needs to be replaced with the X-ray repetition rate, which ranges from 120 Hz at warm linac-based free-electron lasers to 1 MHz at superconducting linacs. Liquid injectors are therefore an essential part of a serial femtosecond crystallography experiment at an X-ray free-electron laser. Here, we compare different techniques of injecting microcrystals in solution into the pulsed X-ray beam in vacuum. Sample waste due to mismatch of the liquid flow rate to the X-ray repetition rate can be addressed through various techniques. PMID:24914163

Long-Wavelength X-Ray Diffraction and Its Applications in Macromolecular Crystallography.

PubMed

Weiss, Manfred S

2017-01-01

For many years, diffraction experiments in macromolecular crystallography at X-ray wavelengths longer than that of Cu-K α (1.54 Å) have been largely underappreciated. Effects caused by increased X-ray absorption result in the fact that these experiments are more difficult than the standard diffraction experiments at short wavelengths. However, due to the also increased anomalous scattering of many biologically relevant atoms, important additional structural information can be obtained. This information, in turn, can be used for phase determination, for substructure identification, in molecular replacement approaches, as well as in structure refinement. This chapter reviews the possibilities and the difficulties associated with such experiments, and it provides a short description of two macromolecular crystallography synchrotron beam lines dedicated to long-wavelength X-ray diffraction experiments.

Opportunities and challenges for time-resolved studies of protein structural dynamics at X-ray free-electron lasers.

PubMed

Neutze, Richard

2014-07-17

X-ray free-electron lasers (XFELs) are revolutionary X-ray sources. Their time structure, providing X-ray pulses of a few tens of femtoseconds in duration; and their extreme peak brilliance, delivering approximately 10(12) X-ray photons per pulse and facilitating sub-micrometre focusing, distinguish XFEL sources from synchrotron radiation. In this opinion piece, I argue that these properties of XFEL radiation will facilitate new discoveries in life science. I reason that time-resolved serial femtosecond crystallography and time-resolved wide angle X-ray scattering are promising areas of scientific investigation that will be advanced by XFEL capabilities, allowing new scientific questions to be addressed that are not accessible using established methods at storage ring facilities. These questions include visualizing ultrafast protein structural dynamics on the femtosecond to picosecond time-scale, as well as time-resolved diffraction studies of non-cyclic reactions. I argue that these emerging opportunities will stimulate a renaissance of interest in time-resolved structural biochemistry.

Reintroducing electrostatics into macromolecular crystallographic refinement: application to neutron crystallography and DNA hydration.

PubMed

Fenn, Timothy D; Schnieders, Michael J; Mustyakimov, Marat; Wu, Chuanjie; Langan, Paul; Pande, Vijay S; Brunger, Axel T

2011-04-13

Most current crystallographic structure refinements augment the diffraction data with a priori information consisting of bond, angle, dihedral, planarity restraints, and atomic repulsion based on the Pauli exclusion principle. Yet, electrostatics and van der Waals attraction are physical forces that provide additional a priori information. Here, we assess the inclusion of electrostatics for the force field used for all-atom (including hydrogen) joint neutron/X-ray refinement. Two DNA and a protein crystal structure were refined against joint neutron/X-ray diffraction data sets using force fields without electrostatics or with electrostatics. Hydrogen-bond orientation/geometry favors the inclusion of electrostatics. Refinement of Z-DNA with electrostatics leads to a hypothesis for the entropic stabilization of Z-DNA that may partly explain the thermodynamics of converting the B form of DNA to its Z form. Thus, inclusion of electrostatics assists joint neutron/X-ray refinements, especially for placing and orienting hydrogen atoms. Copyright © 2011 Elsevier Ltd. All rights reserved.

Reintroducing Electrostatics into Macromolecular Crystallographic Refinement: Application to Neutron Crystallography and DNA Hydration

PubMed Central

Fenn, Timothy D.; Schnieders, Michael J.; Mustyakimov, Marat; Wu, Chuanjie; Langan, Paul; Pande, Vijay S.; Brunger, Axel T.

2011-01-01

Summary Most current crystallographic structure refinements augment the diffraction data with a priori information consisting of bond, angle, dihedral, planarity restraints and atomic repulsion based on the Pauli exclusion principle. Yet, electrostatics and van der Waals attraction are physical forces that provide additional a priori information. Here we assess the inclusion of electrostatics for the force field used for all-atom (including hydrogen) joint neutron/X-ray refinement. Two DNA and a protein crystal structure were refined against joint neutron/X-ray diffraction data sets using force fields without electrostatics or with electrostatics. Hydrogen bond orientation/geometry favors the inclusion of electrostatics. Refinement of Z-DNA with electrostatics leads to a hypothesis for the entropic stabilization of Z-DNA that may partly explain the thermodynamics of converting the B form of DNA to its Z form. Thus, inclusion of electrostatics assists joint neutron/X-ray refinements, especially for placing and orienting hydrogen atoms. PMID:21481775

Portrait of an Enzyme, a Complete Structural Analysis of a Multimodular beta-N-Acetylglucosaminidase from Clostridium perfringens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ficko-Blean, E.; Gregg, K; Adams, J

2009-01-01

Common features of the extracellular carbohydrate-active virulence factors involved in host-pathogen interactions are their large sizes and modular complexities. This has made them recalcitrant to structural analysis, and therefore our understanding of the significance of modularity in these important proteins is lagging. Clostridium perfringens is a prevalent human pathogen that harbors a wide array of large, extracellular carbohydrate-active enzymes and is an excellent and relevant model system to approach this problem. Here we describe the complete structure of C. perfringens GH84C (NagJ), a 1001-amino acid multimodular homolog of the C. perfringens ?-toxin, which was determined using a combination of smallmore » angle x-ray scattering and x-ray crystallography. The resulting structure reveals unprecedented insight into how catalysis, carbohydrate-specific adherence, and the formation of molecular complexes with other enzymes via an ultra-tight protein-protein interaction are spatially coordinated in an enzyme involved in a host-pathogen interaction.« less

Coherent convergent-beam time-resolved X-ray diffraction

PubMed Central

Spence, John C. H.; Zatsepin, Nadia A.; Li, Chufeng

2014-01-01

The use of coherent X-ray lasers for structural biology allows the use of nanometre diameter X-ray beams with large beam divergence. Their application to the structure analysis of protein nanocrystals and single particles raises new challenges and opportunities. We discuss the form of these coherent convergent-beam (CCB) hard X-ray diffraction patterns and their potential use for time-resolved crystallography, normally achieved by Laue (polychromatic) diffraction, for which the monochromatic laser radiation of a free-electron X-ray laser is unsuitable. We discuss the possibility of obtaining single-shot, angle-integrated rocking curves from CCB patterns, and the dependence of the resulting patterns on the focused beam coordinate when the beam diameter is larger or smaller than a nanocrystal, or smaller than one unit cell. We show how structure factor phase information is provided at overlapping interfering orders and how a common phase origin between different shots may be obtained. Their use in refinement of the phase-sensitive intensity between overlapping orders is suggested. PMID:24914153

Identification of phases, symmetries and defects through local crystallography

DOE PAGES

Belianinov, Alex; He, Qian; Kravchenko, Mikhail; ...

2015-07-20

Here we report that advances in electron and probe microscopies allow 10 pm or higher precision in measurements of atomic positions. This level of fidelity is sufficient to correlate the length (and hence energy) of bonds, as well as bond angles to functional properties of materials. Traditionally, this relied on mapping locally measured parameters to macroscopic variables, for example, average unit cell. This description effectively ignores the information contained in the microscopic degrees of freedom available in a high-resolution image. Here we introduce an approach for local analysis of material structure based on statistical analysis of individual atomic neighbourhoods. Clusteringmore » and multivariate algorithms such as principal component analysis explore the connectivity of lattice and bond structure, as well as identify minute structural distortions, thus allowing for chemical description and identification of phases. This analysis lays the framework for building image genomes and structure–property libraries, based on conjoining structural and spectral realms through local atomic behaviour.« less

Single-particle cryo-EM using alignment by classification (ABC): the structure of Lumbricus terrestris haemoglobin

PubMed Central

Seer-Linnemayr, Charlotte; Ravelli, Raimond B. G.; Matadeen, Rishi; De Carlo, Sacha; Alewijnse, Bart; Portugal, Rodrigo V.; Pannu, Navraj S.; Schatz, Michael; van Heel, Marin

2017-01-01

Single-particle cryogenic electron microscopy (cryo-EM) can now yield near-atomic resolution structures of biological complexes. However, the reference-based alignment algorithms commonly used in cryo-EM suffer from reference bias, limiting their applicability (also known as the ‘Einstein from random noise’ problem). Low-dose cryo-EM therefore requires robust and objective approaches to reveal the structural information contained in the extremely noisy data, especially when dealing with small structures. A reference-free pipeline is presented for obtaining near-atomic resolution three-dimensional reconstructions from heterogeneous (‘four-dimensional’) cryo-EM data sets. The methodologies integrated in this pipeline include a posteriori camera correction, movie-based full-data-set contrast transfer function determination, movie-alignment algorithms, (Fourier-space) multivariate statistical data compression and unsupervised classification, ‘random-startup’ three-dimensional reconstructions, four-dimensional structural refinements and Fourier shell correlation criteria for evaluating anisotropic resolution. The procedures exclusively use information emerging from the data set itself, without external ‘starting models’. Euler-angle assignments are performed by angular reconstitution rather than by the inherently slower projection-matching approaches. The comprehensive ‘ABC-4D’ pipeline is based on the two-dimensional reference-free ‘alignment by classification’ (ABC) approach, where similar images in similar orientations are grouped by unsupervised classification. Some fundamental differences between X-ray crystallography versus single-particle cryo-EM data collection and data processing are discussed. The structure of the giant haemoglobin from Lumbricus terrestris at a global resolution of ∼3.8 Å is presented as an example of the use of the ABC-4D procedure. PMID:28989723

A novel β-glucosidase isolated from the microbial metagenome of Lake Poraquê (Amazon, Brazil).

PubMed

Toyama, Danyelle; de Morais, Mariana Abrahão Bueno; Ramos, Felipe Cardoso; Zanphorlin, Letícia Maria; Tonoli, Celisa Caldana Costa; Balula, Augusto Furio; de Miranda, Fernando Pellon; Almeida, Vitor Medeiros; Marana, Sandro Roberto; Ruller, Roberto; Murakami, Mario Tyago; Henrique-Silva, Flavio

2018-04-01

The Amazon region holds most of the biological richness of Brazil. Despite their ecological and biotechnological importance, studies related to microorganisms from this region are limited. Metagenomics leads to exciting discoveries, mainly regarding non-cultivable microorganisms. Herein, we report the discovery of a novel β-glucosidase (glycoside hydrolase family 1) gene from a metagenome from Lake Poraquê in the Amazon region. The gene encodes a protein of 52.9 kDa, named AmBgl-LP, which was recombinantly expressed in Escherichia coli and biochemically and structurally characterized. Although AmBgl-LP hydrolyzed the synthetic substrate p-nitrophenyl-β-d-glucopyranoside (pNPβG) and the natural substrate cellobiose, it showed higher specificity for pNPβG (k cat /K m  = 6 s -1 ·mM -1 ) than cellobiose (k cat /K m  = 0.6 s -1 ·mM -1 ). AmBgl-LP showed maximum activity at 40 °C and pH 6.0 when pNPβG was used as the substrate. Glucose is a competitive inhibitor of AmBgl-LP, presenting a K i of 14 mM. X-ray crystallography and Small Angle X-ray Scattering were used to determine the AmBgl-LP three-dimensional structure and its oligomeric state. Interestingly, despite sharing similar active site architecture with other structurally characterized GH1 family members which are monomeric, AmBgl-LP forms stable dimers in solution. The identification of new GH1 members by metagenomics might extend our understanding of the molecular mechanisms and diversity of these enzymes, besides enabling us to survey their industrial applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Protein Data Bank (PDB): The Single Global Macromolecular Structure Archive

PubMed Central

Burley, Stephen K.; Berman, Helen M.; Kleywegt, Gerard J.; Markley, John L.; Nakamura, Haruki; Velankar, Sameer

2018-01-01

The Protein Data Bank (PDB)—the single global repository of experimentally determined 3D structures of biological macromolecules and their complexes—was established in 1971, becoming the first open-access digital resource in the biological sciences. The PDB archive currently houses ~130,000 entries (May 2017). It is managed by the Worldwide Protein Data Bank organization (wwPDB; wwpdb.org), which includes the RCSB Protein Data Bank (RCSB PDB; rcsb.org), the Protein Data Bank Japan (PDBj; pdbj.org), the Protein Data Bank in Europe (PDBe; pdbe.org), and BioMagResBank (BMRB; www.bmrb.wisc.edu). The four wwPDB partners operate a unified global software system that enforces community-agreed data standards and supports data Deposition, Biocuration, and Validation of ~11,000 new PDB entries annually (deposit.wwpdb.org). The RCSB PDB currently acts as the archive keeper, ensuring disaster recovery of PDB data and coordinating weekly updates. wwPDB partners disseminate the same archival data from multiple FTP sites, while operating complementary websites that provide their own views of PDB data with selected value-added information and links to related data resources. At present, the PDB archives experimental data, associated metadata, and 3D-atomic level structural models derived from three well-established methods: crystallography, nuclear magnetic resonance spectroscopy (NMR), and electron microscopy (3DEM). wwPDB partners are working closely with experts in related experimental areas (small-angle scattering, chemical cross-linking/mass spectrometry, Forster energy resonance transfer or FRET, etc.) to establish a federation of data resources that will support sustainable archiving and validation of 3D structural models and experimental data derived from integrative or hybrid methods. PMID:28573592

Protein Data Bank (PDB): The Single Global Macromolecular Structure Archive.

PubMed

Burley, Stephen K; Berman, Helen M; Kleywegt, Gerard J; Markley, John L; Nakamura, Haruki; Velankar, Sameer

2017-01-01

The Protein Data Bank (PDB)--the single global repository of experimentally determined 3D structures of biological macromolecules and their complexes--was established in 1971, becoming the first open-access digital resource in the biological sciences. The PDB archive currently houses ~130,000 entries (May 2017). It is managed by the Worldwide Protein Data Bank organization (wwPDB; wwpdb.org), which includes the RCSB Protein Data Bank (RCSB PDB; rcsb.org), the Protein Data Bank Japan (PDBj; pdbj.org), the Protein Data Bank in Europe (PDBe; pdbe.org), and BioMagResBank (BMRB; www.bmrb.wisc.edu). The four wwPDB partners operate a unified global software system that enforces community-agreed data standards and supports data Deposition, Biocuration, and Validation of ~11,000 new PDB entries annually (deposit.wwpdb.org). The RCSB PDB currently acts as the archive keeper, ensuring disaster recovery of PDB data and coordinating weekly updates. wwPDB partners disseminate the same archival data from multiple FTP sites, while operating complementary websites that provide their own views of PDB data with selected value-added information and links to related data resources. At present, the PDB archives experimental data, associated metadata, and 3D-atomic level structural models derived from three well-established methods: crystallography, nuclear magnetic resonance spectroscopy (NMR), and electron microscopy (3DEM). wwPDB partners are working closely with experts in related experimental areas (small-angle scattering, chemical cross-linking/mass spectrometry, Forster energy resonance transfer or FRET, etc.) to establish a federation of data resources that will support sustainable archiving and validation of 3D structural models and experimental data derived from integrative or hybrid methods.

The structural basis for the functional comparability of factor VIII and the long-acting variant recombinant factor VIII Fc fusion protein

DOE PAGES

Leksa, N. C.; Chiu, P. -L.; Bou-Assaf, G. M.; ...

2017-05-03

Fusion of the human IgG 1 Fc domain to the C-terminal C2 domain of B-domain-deleted (BDD) factor VIII (FVIII) results in the recombinant FVIII Fc (rFVIIIFc) fusion protein, which has a 1.5-fold longer half-life in humans. To assess the structural properties of rFVIIIFc by comparing its constituent FVIII and Fc elements with their respective isolated components, and evaluating their structural independence within rFVIIIFc. rFVIIIFc and its isolated FVIII and Fc components were compared by the use of hydrogen–deuterium exchange mass spectrometry (HDX-MS). The structure of rFVIIIFc was also evaluated by the use of X-ray crystallography, small-angle X-ray scattering (SAXS), andmore » electron microscopy (EM). The degree of steric interference by the appended Fc domain was assessed by EM and surface plasmon resonance (SPR). HDX-MS analysis of rFVIIIFc revealed that fusion caused no structural perturbations in FVIII or Fc. The rFVIIIFc crystal structure showed that the FVIII component is indistinguishable from published BDD FVIII structures. The Fc domain was not observed, indicating high mobility. SAXS analysis was consistent with an ensemble of rigid-body models in which the Fc domain exists in a largely extended orientation relative to FVIII. Binding of Fab fragments of anti-C2 domain antibodies to BDD FVIII was visualized by EM, and the affinities of the corresponding intact antibodies for BDD FVIII and rFVIIIFc were comparable by SPR analysis. Thus, the FVIII and Fc components of rFVIIIFc are structurally indistinguishable from their isolated constituents, and show a high degree of structural independence, consistent with the functional comparability of rFVIIIFc and unmodified FVIII.« less

The structural basis for the functional comparability of Factor VIII and the long-acting variant recombinant Factor VIII Fc fusion protein

PubMed Central

Leksa, N.C.; Chiu, P.-L.; Bou-Assaf, G.M.; Quan, C.; Liu, Z.; Goodman, A.B.; Chambers, M.G.; Tsutakawa, S.E.; Hammel, M.; Peters, R.T.; Walz, T.; Kulman, J.D.

2017-01-01

SUMMARY Background Fusion of the human IgG1 Fc domain to the C-terminal C2 domain of B domain-deleted (BDD) factor VIII (FVIII) results in the rFVIIIFc fusion protein that has a 1.5-fold longer half-life in humans. Objective To assess the structural properties of rFVIIIFc by comparing its constituent FVIII and Fc elements with their respective isolated components and evaluating their structural independence within rFVIIIFc. Methods rFVIIIFc and its isolated FVIII and Fc components were compared by hydrogen-deuterium exchange mass spectrometry (HDX-MS). The structure of rFVIIIFc was also evaluated by X-ray crystallography, small-angle X-ray scattering (SAXS), and electron microscopy (EM). The degree of steric interference by the appended Fc domain was assessed by EM and surface plasmon resonance (SPR). Results HDX-MS analysis of rFVIIIFc revealed that fusion caused no structural perturbations in FVIII or Fc. The rFVIIIFc crystal structure showed that the FVIII component is indistinguishable from published BDD FVIII structures. The Fc domain was not observed, indicating high mobility. SAXS analysis was consistent with an ensemble of rigid-body models in which the Fc domain exists in a largely extended orientation relative to FVIII. Binding of Fab fragments of anti-C2 domain antibodies to BDD FVIII was visualized by EM, and the affinities of the corresponding intact antibodies for BDD FVIII and rFVIIIFc were comparable by SPR analysis. Conclusions The FVIII and Fc components of rFVIIIFc are structurally indistinguishable from their isolated constituents and exhibit a high degree of structural independence, consistent with the functional comparability of rFVIIIFc and unmodified FVIII. PMID:28397397

Welcoming natural isotopic abundance in solid-state NMR: probing π-stacking and supramolecular structure of organic nanoassemblies using DNP† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc02709a Click here for additional data file.

PubMed Central

Märker, Katharina; Paul, Subhradip; Fernández-de-Alba, Carlos; Lee, Daniel; Mouesca, Jean-Marie; Hediger, Sabine

2017-01-01

The self-assembly of small organic molecules is an intriguing phenomenon, which provides nanoscale structures for applications in numerous fields from medicine to molecular electronics. Detailed knowledge of their structure, in particular on the supramolecular level, is a prerequisite for the rational design of improved self-assembled systems. In this work, we prove the feasibility of a novel concept of NMR-based 3D structure determination of such assemblies in the solid state. The key point of this concept is the deliberate use of samples that contain 13C at its natural isotopic abundance (NA, 1.1%), while exploiting magic-angle spinning dynamic nuclear polarization (MAS-DNP) to compensate for the reduced sensitivity. Since dipolar truncation effects are suppressed to a large extent in NA samples, unique and highly informative spectra can be recorded which are impossible to obtain on an isotopically labeled system. On the self-assembled cyclic diphenylalanine peptide, we demonstrate the detection of long-range internuclear distances up to ∼7 Å, allowing us to observe π-stacking through 13C–13C correlation spectra, providing a powerful tool for the analysis of one of the most important non-covalent interactions. Furthermore, experimental polarization transfer curves are in remarkable agreement with numerical simulations based on the crystallographic structure, and can be fully rationalized as the superposition of intra- and intermolecular contributions. This new approach to NMR crystallography provides access to rich and precise structural information, opening up a new avenue to de novo crystal structure determination by NMR. PMID:28451235

First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer

PubMed Central

Arturo, Emilia C.; Gupta, Kushol; Héroux, Annie; Stith, Linda; Cross, Penelope J.; Parker, Emily J.; Loll, Patrick J.; Jaffe, Eileen K.

2016-01-01

Improved understanding of the relationship among structure, dynamics, and function for the enzyme phenylalanine hydroxylase (PAH) can lead to needed new therapies for phenylketonuria, the most common inborn error of amino acid metabolism. PAH is a multidomain homo-multimeric protein whose conformation and multimerization properties respond to allosteric activation by the substrate phenylalanine (Phe); the allosteric regulation is necessary to maintain Phe below neurotoxic levels. A recently introduced model for allosteric regulation of PAH involves major domain motions and architecturally distinct PAH tetramers [Jaffe EK, Stith L, Lawrence SH, Andrake M, Dunbrack RL, Jr (2013) Arch Biochem Biophys 530(2):73–82]. Herein, we present, to our knowledge, the first X-ray crystal structure for a full-length mammalian (rat) PAH in an autoinhibited conformation. Chromatographic isolation of a monodisperse tetrameric PAH, in the absence of Phe, facilitated determination of the 2.9 Å crystal structure. The structure of full-length PAH supersedes a composite homology model that had been used extensively to rationalize phenylketonuria genotype–phenotype relationships. Small-angle X-ray scattering (SAXS) confirms that this tetramer, which dominates in the absence of Phe, is different from a Phe-stabilized allosterically activated PAH tetramer. The lack of structural detail for activated PAH remains a barrier to complete understanding of phenylketonuria genotype–phenotype relationships. Nevertheless, the use of SAXS and X-ray crystallography together to inspect PAH structure provides, to our knowledge, the first complete view of the enzyme in a tetrameric form that was not possible with prior partial crystal structures, and facilitates interpretation of a wealth of biochemical and structural data that was hitherto impossible to evaluate. PMID:26884182

The structural basis for the functional comparability of factor VIII and the long-acting variant recombinant factor VIII Fc fusion protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leksa, N. C.; Chiu, P. -L.; Bou-Assaf, G. M.

Fusion of the human IgG 1 Fc domain to the C-terminal C2 domain of B-domain-deleted (BDD) factor VIII (FVIII) results in the recombinant FVIII Fc (rFVIIIFc) fusion protein, which has a 1.5-fold longer half-life in humans. To assess the structural properties of rFVIIIFc by comparing its constituent FVIII and Fc elements with their respective isolated components, and evaluating their structural independence within rFVIIIFc. rFVIIIFc and its isolated FVIII and Fc components were compared by the use of hydrogen–deuterium exchange mass spectrometry (HDX-MS). The structure of rFVIIIFc was also evaluated by the use of X-ray crystallography, small-angle X-ray scattering (SAXS), andmore » electron microscopy (EM). The degree of steric interference by the appended Fc domain was assessed by EM and surface plasmon resonance (SPR). HDX-MS analysis of rFVIIIFc revealed that fusion caused no structural perturbations in FVIII or Fc. The rFVIIIFc crystal structure showed that the FVIII component is indistinguishable from published BDD FVIII structures. The Fc domain was not observed, indicating high mobility. SAXS analysis was consistent with an ensemble of rigid-body models in which the Fc domain exists in a largely extended orientation relative to FVIII. Binding of Fab fragments of anti-C2 domain antibodies to BDD FVIII was visualized by EM, and the affinities of the corresponding intact antibodies for BDD FVIII and rFVIIIFc were comparable by SPR analysis. Thus, the FVIII and Fc components of rFVIIIFc are structurally indistinguishable from their isolated constituents, and show a high degree of structural independence, consistent with the functional comparability of rFVIIIFc and unmodified FVIII.« less

Multibeam collimator uses prism stack

NASA Technical Reports Server (NTRS)

Minott, P. O.

1981-01-01

Optical instrument creates many divergent light beams for surveying and machine element alignment applications. Angles and refractive indices of stack of prisms are selected to divert incoming laser beam by small increments, different for each prism. Angles of emerging beams thus differ by small, precisely-controlled amounts. Instrument is nearly immune to vibration, changes in gravitational force, temperature variations, and mechanical distortion.

Small-angle x-ray scattering investigations of extrudates

NASA Astrophysics Data System (ADS)

Pikus, Stanislaw; Jamroz, Jerzy

1997-02-01

The small-angle X-ray scattering investigations of the extrudes are presented. The investigations of the different samples of starch by means of the SAXS indicate the new possibilities for using this method for extrudates examination. Results obtained by SAXS method of close dependance between intensity SAXS scattering and characteristic parameters of the extrudates were shown.

Neutronographic investigations of supramolecular structures on upgraded small-angle spectrometer YuMO

NASA Astrophysics Data System (ADS)

Kuklin, A. I.; Rogachev, A. V.; Soloviov, D. V.; Ivankov, O. I.; Kovalev, Yu S.; Utrobin, P. K.; Kutuzov, S. A.; Soloviev, A. G.; Rulev, M. I.; Gordeliy, V. I.

2017-05-01

Abstract.The work is a review of neutronographic investigations of supramolecular structures on upgraded small-angle spectrometer YuMO. Here, key parameters of small-angle spectrometers are considered. It is shown that two-detector system is the basis of YuMO upgrade. It allows to widen the dynamic q-range twice. In result, the available q-range is widened and dynamic q-range and data collection rate are doubled. The detailed description of YuMO spectrometer is given.The short review of experimental researches made on the spectrometer in the polymers field, biology, material science and physical chemistry is given. The current investigations also have a methodological aspect. It is shown that upgraded spectrometer provides advanced world level of research of supramolecular structures.

Small angle slot divertor concept for long pulse advanced tokamaks

NASA Astrophysics Data System (ADS)

Guo, H. Y.; Sang, C. F.; Stangeby, P. C.; Lao, L. L.; Taylor, T. S.; Thomas, D. M.

2017-04-01

SOLPS-EIRENE edge code analysis shows that a gas-tight slot divertor geometry with a small-angle (glancing-incidence) target, named the small angle slot (SAS) divertor, can achieve cold, dissipative/detached divertor conditions at relatively low values of plasma density at the outside midplane separatrix. SAS exhibits the following key features: (1) strong enhancement of the buildup of neutral density in a localized region near the plasma strike point on the divertor target; (2) spreading of the cooling front across the divertor target with the slot gradually flaring out from the strike point, thus effectively reducing both heat flux and erosion on the entire divertor target surface. Such a divertor may potentially provide a power and particle handling solution for long pulse advanced tokamaks.

Pitch angle scattering of relativistic electrons from stationary magnetic waves: Continuous Markov process and quasilinear theory

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lemons, Don S.

2012-01-15

We develop a Markov process theory of charged particle scattering from stationary, transverse, magnetic waves. We examine approximations that lead to quasilinear theory, in particular the resonant diffusion approximation. We find that, when appropriate, the resonant diffusion approximation simplifies the result of the weak turbulence approximation without significant further restricting the regime of applicability. We also explore a theory generated by expanding drift and diffusion rates in terms of a presumed small correlation time. This small correlation time expansion leads to results valid for relatively small pitch angle and large wave energy density - a regime that may govern pitchmore » angle scattering of high-energy electrons into the geomagnetic loss cone.« less

A SMALL-ANGLE DRILL-HOLE WHIPSTOCK

DOEpatents

Nielsen, D.E.; Olsen, J.L.; Bennett, W.P.

1963-01-29

A small angle whipstock is described for accurately correcting or deviating a drill hole by a very small angle. The whipstock is primarily utilized when drilling extremely accurate, line-of-slight test holes as required for diagnostic studies related to underground nuclear test shots. The invention is constructed of a length of cylindrical pipe or casing, with a whipstock seating spike extending from the lower end. A wedge-shaped segment is secured to the outer circumference of the upper end of the cylinder at a position diametrically opposite the circumferential position of the spike. Pin means are provided for affixing the whipstock to a directional drill bit and stem to alloy orienting and setting the whipstock properly in the drill hole. (AEC)

Binocular function in patients with pseudophakic monovision.

PubMed

Ito, Misae; Shimizu, Kimiya; Niida, Takahiro; Amano, Rie; Ishikawa, Hitoshi

2014-08-01

To evaluate the relationship between ocular deviation and stereopsis and fusion in patients who had pseudophakic monovision surgery. Department of Ophthalmology, Kitasato University Hospital, Kanagawa, Japan. Retrospective comparative case series. Patients had surgical monovision correction with monofocal intraocular lens placement followed by routine postoperative examinations. The alternate prism cover test was used to measure motor alignment. Sensory tests for binocularity included sensory fusion determinations using the Worth 4-dot test, near stereopsis test, and fusion amplitude measured with a prism bar. Patients with monovision were categorized as having small-angle exophoria (≤10.0 prism diopters [Δ]) or moderate-angle exophoria (>10.0 Δ). This study comprised 60 patients with a mean age of 70.2 years ± 7.7 (SD). The difference in the mean stereopsis values between patients with small-angle exophoria and patients with moderate-angle exophoria was statistically significant (P<.001). In the moderate-angle exophoria group, 10 patients (62.5%) developed intermittent exotropia after surgery; however, no serious ocular deviation problems were observed. The fusion amplitudes in patients with pseudophakic monovision were approximately similar to normal values. Patients with moderate-angle exophoria were more likely to fail the Worth 4-dot test than those with small-angle exophoria. In patients with pseudophakic monovision having a near exophoria angle of more than 10.0 Δ, the possibility of changes in ocular deviation and stereopsis after surgery is a concern. Moreover, the application of monovision in patients with a previous moderate-angle exophoria should be carefully considered. No author has a financial or proprietary interest in any material or method mentioned. Copyright © 2014 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

Study of the gel films of Acetobacter Xylinum cellulose and its modified samples by {sup 1}H NMR cryoporometry and small-angle X-ray scattering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Babushkina, T. A.; Klimova, T. P.; Shtykova, E. V.

2010-03-15

Gel films of Acetobacter Xylinum cellulose and its modified samples have been investigated by 1H nuclear magnetic resonance (NMR) cryoporometry and small-angle X-ray scattering. The joint use of these two methods made it possible to characterize the sizes of aqueous pores in gel films and estimate the sizes of structural inhomogeneities before and after the sorption of polyvinylpyrrolidone and Se{sub 0} nanoparticles (stabilized by polyvinylpyrrolidone) into the films. According to small-angle X-ray scattering data, the sizes of inhomogeneities in a gel film change only slightly upon the sorption of polyvinylpyrrolidone and nanoparticles. The impregnated material is sorbed into water-filled cavitiesmore » that are present in the gel film. {sup 1}H NMR cryoporometry allowed us to reveal the details of changes in the sizes of small aqueous pores during modifications.« less

The structure of denisovite, a fibrous nanocrystalline polytypic disordered ‘very complex’ silicate, studied by a synergistic multi-disciplinary approach employing methods of electron crystallography and X-ray powder diffraction

PubMed Central

Schowalter, Marco; Schmidt, Martin U.; Czank, Michael; Depmeier, Wulf; Rosenauer, Andreas

2017-01-01

Denisovite is a rare mineral occurring as aggregates of fibres typically 200–500 nm diameter. It was confirmed as a new mineral in 1984, but important facts about its chemical formula, lattice parameters, symmetry and structure have remained incompletely known since then. Recently obtained results from studies using microprobe analysis, X-ray powder diffraction (XRPD), electron crystallography, modelling and Rietveld refinement will be reported. The electron crystallography methods include transmission electron microscopy (TEM), selected-area electron diffraction (SAED), high-angle annular dark-field imaging (HAADF), high-resolution transmission electron microscopy (HRTEM), precession electron diffraction (PED) and electron diffraction tomography (EDT). A structural model of denisovite was developed from HAADF images and later completed on the basis of quasi-kinematic EDT data by ab initio structure solution using direct methods and least-squares refinement. The model was confirmed by Rietveld refinement. The lattice parameters are a = 31.024 (1), b = 19.554 (1) and c = 7.1441 (5) Å, β = 95.99 (3)°, V = 4310.1 (5) Å3 and space group P12/a1. The structure consists of three topologically distinct dreier silicate chains, viz. two xonotlite-like dreier double chains, [Si6O17]10−, and a tubular loop-branched dreier triple chain, [Si12O30]12−. The silicate chains occur between three walls of edge-sharing (Ca,Na) octahedra. The chains of silicate tetrahedra and the octahedra walls extend parallel to the z axis and form a layer parallel to (100). Water molecules and K+ cations are located at the centre of the tubular silicate chain. The latter also occupy positions close to the centres of eight-membered rings in the silicate chains. The silicate chains are geometrically constrained by neighbouring octahedra walls and present an ambiguity with respect to their z position along these walls, with displacements between neighbouring layers being either Δz = c/4 or −c/4. Such behaviour is typical for polytypic sequences and leads to disorder along [100]. In fact, the diffraction pattern does not show any sharp reflections with l odd, but continuous diffuse streaks parallel to a* instead. Only reflections with l even are sharp. The diffuse scattering is caused by (100) nano­lamellae separated by stacking faults and twin boundaries. The structure can be described according to the order–disorder (OD) theory as a stacking of layers parallel to (100). PMID:28512570

Apparatus and method for nanoflow liquid jet and serial femtosecond x-ray protein crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bogan, Michael J.; Laksmono, Hartawan; Sierra, Raymond G.

Techniques for nanoflow serial femtosecond x-ray protein crystallography include providing a sample fluid by mixing a plurality of a first target of interest with a carrier fluid and injecting the sample fluid into a vacuum chamber at a rate less than about 4 microliters per minute. In some embodiments, the carrier fluid has a viscosity greater than about 3 centipoise.

Deformation and Failure of Protein Materials in Physiologically Extreme Conditions and Disease

DTIC Science & Technology

2009-03-01

resonance (NMR) spectroscopy and X- ray crystallography have advanced our ability to identify 3D protein structures57. Site-specific studies using NMR, a... ray crystallography, providing structural and temporal information about mechanisms of deformation and assembly (for example in intermediate...tens of thousands of 3D atomistic protein structures, identifying the structure of numerous proteins from varying species sources60. X- ray

Combined x-ray crystallography and computational modeling approach to investigate the Hsp90 C-terminal peptide binding to FKBP51.

PubMed

Kumar, Rajnish; Moche, Martin; Winblad, Bengt; Pavlov, Pavel F

2017-10-27

FK506 binding protein of 51 kDa (FKBP51) is a heat shock protein 90 (Hsp90) co-chaperone involved in the regulation of steroid hormone receptors activity. It is known for its role in various regulatory pathways implicated in mood and stress-related disorders, cancer, obesity, Alzheimer's disease and corticosteroid resistant asthma. It consists of two FKBP12 like active peptidyl prolyl isomerase (PPIase) domains (an active FK1 and inactive FK2 domain) and one tetratricopeptide repeat (TPR) domain that mediates interaction with Hsp90 via its C-terminal MEEVD peptide. Here, we report a combined x-ray crystallography and molecular dynamics study to reveal the binding mechanism of Hsp90 MEEVD peptide to the TPR domain of FKBP51. The results demonstrated that the Hsp90 C-terminal peptide binds to the TPR domain of FKBP51 with the help of di-carboxylate clamp involving Lys272, Glu273, Lys352, Asn322, and Lys329 which are conserved throughout several di-carboxylate clamp TPR proteins. Interestingly, the results from molecular dynamics study are also in agreement to the complex structure where all the contacts between these two partners were consistent throughout the simulation period. In a nutshell, our findings provide new opportunity to engage this important protein-protein interaction target by small molecules designed by structure based drug design strategy.

Micro-crystallography developments at GM/CA CAT at the APS.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, S.; Makarov, O.; Benn, R.

2010-01-01

Recently, several important structures have been solved using micro-crystallographic techniques that previously could not have been solved with conventional crystallography. At GM/CA-CAT we continue to develop micro-crystallographic capabilities for difficult problems such as small crystals of large macromolecular complexes or membrane proteins grown in the lipidic cubic phase. This paper will describe three major upgrades to our arsenal of tools, 'mini-beam' collimators, active beamstop, and an improved goniostat. Our 'mini-beam' collimators have evolved to a new triple-collimator fabricated from molybdenum as a uni-body. This has significantly improved the robustness, ease of initial alignment, and reduction of background. More recently, twomore » prototypes of a quad-collimator have been developed and fabricated to provide a selection of mini-beams of 5, 10, 20 {mu}m and a 300 {mu}m scatter-guard on a single body. The smaller beams and samples have increased the demand on the tolerances of our goniostat. To meet these challenges we have designed and implemented a goniostat with a 1-micron peak-to-peak sphere of confusion. This is a significant improvement over the previous 6 micron sphere of confusion of the commercially available air-bearing and XY stages. Finally, an 'active beamstop' has been constructed. This will provide non-invasive, real time feedback at the sample during data collection.« less

Micro-Crystallography Developments at GM/CA-CAT at the APS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu Shenglan; Makarov, Oleg; Benn, Rich

2010-06-23

Recently, several important structures have been solved using micro-crystallographic techniques that previously could not have been solved with conventional crystallography. At GM/CA-CAT we continue to develop micro-crystallographic capabilities for difficult problems such as small crystals of large macromolecular complexes or membrane proteins grown in the lipidic cubic phase. This paper will describe three major upgrades to our arsenal of tools, 'mini-beam' collimators, active beamstop, and an improved goniostat. Our 'mini-beam' collimators have evolved to a new triple-collimator fabricated from molybdenum as a uni-body. This has significantly improved the robustness, ease of initial alignment, and reduction of background. More recently, twomore » prototypes of a quad-collimator have been developed and fabricated to provide a selection of mini-beams of 5, 10, 20 {mu}m and a 300 {mu}m scatter-guard on a single body. The smaller beams and samples have increased the demand on the tolerances of our goniostat. To meet these challenges we have designed and implemented a goniostat with a 1-micron peak-to-peak sphere of confusion. This is a significant improvement over the previous 6 micron sphere of confusion of the commercially available air-bearing and XY stages. Finally, an 'active beamstop' has been constructed. This will provide non-invasive, real time feedback at the sample during data collection.« less

Femtosecond X-ray protein nanocrystallography

PubMed Central

Chapman, Henry N.; Fromme, Petra; Barty, Anton; White, Thomas A.; Kirian, Richard A.; Aquila, Andrew; Hunter, Mark S.; Schulz, Joachim; DePonte, Daniel P.; Weierstall, Uwe; Doak, R. Bruce; Maia, Filipe R. N. C.; Martin, Andrew V.; Schlichting, Ilme; Lomb, Lukas; Coppola, Nicola; Shoeman, Robert L.; Epp, Sascha W.; Hartmann, Robert; Rolles, Daniel; Rudenko, Artem; Foucar, Lutz; Kimmel, Nils; Weidenspointner, Georg; Holl, Peter; Liang, Mengning; Barthelmess, Miriam; Caleman, Carl; Boutet, Sébastien; Bogan, Michael J.; Krzywinski, Jacek; Bostedt, Christoph; Bajt, Saša; Gumprecht, Lars; Rudek, Benedikt; Erk, Benjamin; Schmidt, Carlo; Hömke, André; Reich, Christian; Pietschner, Daniel; Strüder, Lothar; Hauser, Günter; Gorke, Hubert; Ullrich, Joachim; Herrmann, Sven; Schaller, Gerhard; Schopper, Florian; Soltau, Heike; Kühnel, Kai-Uwe; Messerschmidt, Marc; Bozek, John D.; Hau-Riege, Stefan P.; Frank, Matthias; Hampton, Christina Y.; Sierra, Raymond G.; Starodub, Dmitri; Williams, Garth J.; Hajdu, Janos; Timneanu, Nicusor; Seibert, M. Marvin; Andreasson, Jakob; Rocker, Andrea; Jönsson, Olof; Svenda, Martin; Stern, Stephan; Nass, Karol; Andritschke, Robert; Schröter, Claus-Dieter; Krasniqi, Faton; Bott, Mario; Schmidt, Kevin E.; Wang, Xiaoyu; Grotjohann, Ingo; Holton, James M.; Barends, Thomas R. M.; Neutze, Richard; Marchesini, Stefano; Fromme, Raimund; Schorb, Sebastian; Rupp, Daniela; Adolph, Marcus; Gorkhover, Tais; Andersson, Inger; Hirsemann, Helmut; Potdevin, Guillaume; Graafsma, Heinz; Nilsson, Björn; Spence, John C. H.

2012-01-01

X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded1-3. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction ‘snapshots’ are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source4. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes5. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (~200 nm to 2 μm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes6. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage. PMID:21293373

Design of a probe for two-dimensional small angle detection

NASA Astrophysics Data System (ADS)

He, Haixia; Wang, Xuanze; Zhong, Yuning; Yang, Liangen; Cao, Hongduan

2008-10-01

A novel two-dimensional small angle probe is introduced, which is based on principle of auto-collimation and utilizes quadrant Si-photoelectric detector (QPD) as detection device. AC modulation, AC magnification and absolute value demodulation are incorporated to restrain the DC excursion caused by background light and noise etc and to improve the sensitivity and stability of angle detection. To ensure that while the laser is shining, the current signal (converted into voltage signal) of QPD also is linear to the AC modulation voltage, this paper adopted AC modulation signal (5400Hz) with a DC offset. AC magnification circuit with reasonable parameters is designed to inhibit DC drift and the impact of industrial frequency noise and to ensure good amplification to signal frequency at the same time. A piezoelectric-driven micro-angle generator is designed to demarcate the angle. The calibration data are input to single chip, and the measurement of angles can be shown in SMC1602A.

Small-angle neutron scattering study of the structure of mixed micellar solutions based on heptaethylene glycol monotetradecyl ether and cesium dodecyl sulfate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rajewska, A., E-mail: aldonar@jinr.ru; Medrzycka, K.; Hallmann, E.

2016-01-15

The micellization in mixed aqueous systems based on a nonionic surfactant, heptaethylene glycol monotetradecyl ether (C{sub 14}E{sub 7}), and an anionic surfactant, cesium dodecyl sulfate, has been investigated by small-angle neutron scattering. Preliminary data on the behavior of the C{sub 14}E{sub 7} aqueous solutions (with three concentrations, 0.17, 0.5, and 1%) mixed with a small amount of anionic surfactant, cesium dodecyl sulfate, are reported.

PRECISE ANGLE MONITOR BASED ON THE CONCEPT OF PENCIL-BEAM INTERFEROMETRY

DOE Office of Scientific and Technical Information (OSTI.GOV)

QIAN,S.; TAKACS,P.

2000-07-30

The precise angle monitoring is a very important metrology task for research, development and industrial applications. Autocollimator is one of the most powerful and widely applied instruments for small angle monitoring, which is based on the principle of geometric optics. In this paper the authors introduce a new precise angle monitoring system, Pencil-beam Angle Monitor (PAM), base on pencil beam interferometry. Its principle of operation is a combination of physical and geometrical optics. The angle calculation method is similar to the autocollimator. However, the autocollimator creates a cross image but the precise pencil-beam angle monitoring system produces an interference fringemore » on the focal plane. The advantages of the PAM are: high angular sensitivity, long-term stability character making angle monitoring over long time periods possible, high measurement accuracy in the order of sub-microradian, simultaneous measurement ability in two perpendicular directions or on two different objects, dynamic measurement possibility, insensitive to the vibration and air turbulence, automatic display, storage and analysis by use of the computer, small beam diameter making the alignment extremely easy and longer test distance. Some test examples are presented.« less

X-ray diffraction gratings: Precise control of ultra-low blaze angle via anisotropic wet etching

DOE Office of Scientific and Technical Information (OSTI.GOV)

Voronov, Dmitriy L.; Naulleau, Patrick; Gullikson, Eric M.

2016-07-25

Diffraction gratings are used from micron to nanometer wavelengths as dispersing elements in optical instruments. At shorter wavelengths, crystals can be used as diffracting elements, but due to the 3D nature of the interaction with light are wavelength selective rather than wavelength dispersing. There is an urgent need to extend grating technology into the x-ray domain of wavelengths from 1 to 0.1 nm, but this requires the use of gratings that have a faceted surface in which the facet angles are very small, typically less than 1°. Small facet angles are also required in the extreme ultra-violet and soft x-ray energymore » ranges in free electron laser applications, in order to reduce power density below a critical damage threshold. In this work, we demonstrate a technique based on anisotropic etching of silicon designed to produce very small angle facets with a high degree of perfection.« less

Modeling spatial tuning of adaptation of the angular vestibulo-ocular reflex

PubMed Central

Yakushin, Sergei B.

2012-01-01

Gain adaptation of the yaw angular vestibular ocular reflex (aVOR) induced in side-down positions has gravity-independent (global) and -dependent (localized) components. When the head oscillation angles are small during adaptation, localized gain changes are maximal in the approximate position of adaptation. Concurrently, polarization vectors of canal–otolith vestibular neurons adapt their orientations during these small-angle adaptation paradigms. Whether there is orientation adaptation with large amplitude head oscillations, when the head is not localized to a specific position, is unknown. Yaw aVOR gains were decreased by oscillating monkeys about a yaw axis in a side-down position in a subject–stationary visual surround for 2 h. Amplitudes of head oscillation ranged from 15° to 180°. The yaw aVOR gain was tested in darkness at 0.5 Hz, with small angles of oscillation (±15°) while upright and in tilted positions. The peak value of the gain change was highly tuned for small angular oscillations during adaptation and significantly broadened with larger oscillation angles during adaptation. When the orientation of the polarization vectors associated with the gravity-dependent component of the neural network model was adapted toward the direction of gravity, it predicted the localized learning for small angles and the broadening when the orientation adaptation was diminished. The model-based analysis suggests that the otolith orientation adaptation plays an important role in the localized behavior of aVOR as a function of gravity and in regulating the relationship between global and localized adaptation. PMID:22660376

X-ray crystallography and its impact on understanding bacterial cell wall remodeling processes.

PubMed

Büttner, Felix Michael; Renner-Schneck, Michaela; Stehle, Thilo

2015-02-01

The molecular structure of matter defines its properties and function. This is especially true for biological macromolecules such as proteins, which participate in virtually all biochemical processes. A three dimensional structural model of a protein is thus essential for the detailed understanding of its physiological function and the characterization of essential properties such as ligand binding and reaction mechanism. X-ray crystallography is a well-established technique that has been used for many years, but it is still by far the most widely used method for structure determination. A particular strength of this technique is the elucidation of atomic details of molecular interactions, thus providing an invaluable tool for a multitude of scientific projects ranging from the structural classification of macromolecules over the validation of enzymatic mechanisms or the understanding of host-pathogen interactions to structure-guided drug design. In the first part of this review, we describe essential methodological and practical aspects of X-ray crystallography. We provide some pointers that should allow researchers without a background in structural biology to assess the overall quality and reliability of a crystal structure. To highlight its potential, we then survey the impact X-ray crystallography has had on advancing an understanding of a class of enzymes that modify the bacterial cell wall. A substantial number of different bacterial amidase structures have been solved, mostly by X-ray crystallography. Comparison of these structures highlights conserved as well as divergent features. In combination with functional analyses, structural information on these enzymes has therefore proven to be a valuable template not only for understanding their mechanism of catalysis, but also for targeted interference with substrate binding. Copyright © 2015 Elsevier GmbH. All rights reserved.

High-throughput methods for electron crystallography.

PubMed

Stokes, David L; Ubarretxena-Belandia, Iban; Gonen, Tamir; Engel, Andreas

2013-01-01

Membrane proteins play a tremendously important role in cell physiology and serve as a target for an increasing number of drugs. Structural information is key to understanding their function and for developing new strategies for combating disease. However, the complex physical chemistry associated with membrane proteins has made them more difficult to study than their soluble cousins. Electron crystallography has historically been a successful method for solving membrane protein structures and has the advantage of providing a native lipid environment for these proteins. Specifically, when membrane proteins form two-dimensional arrays within a lipid bilayer, electron microscopy can be used to collect images and diffraction and the corresponding data can be combined to produce a three-dimensional reconstruction, which under favorable conditions can extend to atomic resolution. Like X-ray crystallography, the quality of the structures are very much dependent on the order and size of the crystals. However, unlike X-ray crystallography, high-throughput methods for screening crystallization trials for electron crystallography are not in general use. In this chapter, we describe two alternative methods for high-throughput screening of membrane protein crystallization within the lipid bilayer. The first method relies on the conventional use of dialysis for removing detergent and thus reconstituting the bilayer; an array of dialysis wells in the standard 96-well format allows the use of a liquid-handling robot and greatly increases throughput. The second method relies on titration of cyclodextrin as a chelating agent for detergent; a specialized pipetting robot has been designed not only to add cyclodextrin in a systematic way, but to use light scattering to monitor the reconstitution process. In addition, the use of liquid-handling robots for making negatively stained grids and methods for automatically imaging samples in the electron microscope are described.

Conceptual design of novel IP-conveyor-belt Weissenberg-mode data-collection system with multi-readers for macromolecular crystallography. A comparison between Galaxy and Super Galaxy.

PubMed

Sakabe, N; Sakabe, K; Sasaki, K

2004-01-01

Galaxy is a Weissenberg-type high-speed high-resolution and highly accurate fully automatic data-collection system using two cylindrical IP-cassettes each with a radius of 400 mm and a width of 450 mm. It was originally developed for static three-dimensional analysis using X-ray diffraction and was installed on bending-magnet beamline BL6C at the Photon Factory. It was found, however, that Galaxy was also very useful for time-resolved protein crystallography on a time scale of minutes. This has prompted us to design a new IP-conveyor-belt Weissenberg-mode data-collection system called Super Galaxy for time-resolved crystallography with improved time and crystallographic resolution over that achievable with Galaxy. Super Galaxy was designed with a half-cylinder-shaped cassette with a radius of 420 mm and a width of 690 mm. Using 1.0 A incident X-rays, these dimensions correspond to a maximum resolutions of 0.71 A in the vertical direction and 1.58 A in the horizontal. Upper and lower screens can be used to set the frame size of the recorded image. This function is useful not only to reduce the frame exchange time but also to save disk space on the data server. The use of an IP-conveyor-belt and many IP-readers make Super Galaxy well suited for time-resolved, monochromatic X-ray crystallography at a very intense third-generation SR beamline. Here, Galaxy and a conceptual design for Super Galaxy are described, and their suitability for use as data-collection systems for macromolecular time-resolved monochromatic X-ray crystallography are compared.

Film cooling performance of a row of dual-fanned holes at various injection angles

NASA Astrophysics Data System (ADS)

Li, Guangchao; Wang, Haofeng; Zhang, Wei; Kou, Zhihai; Xu, Rangshu

2017-10-01

Film cooling performance about a row of dual-fanned holes with injection angles of 30°, 60 ° and 90° were experimentally investigated at blowing ratios of 1.0 and 2.0. Dual-fanned hole is a novel shaped hole which has both inlet expansion and outlet expansion. A transient thermochromic liquid crystal technique was used to reveal the local values of film cooling effectiveness and heat transfer coefficient. The results show that injection angles have strong influence on the two dimensional distributions of film cooling effectiveness and heat transfer coefficient. For the small injection angle of 30 degree and small blowing ratio of 1.0, there is only a narrow spanwise region covered with film. The increase of injection angle and blowing ratio both leads to the enhanced spanwise film diffusion, but reduced local cooling ability far away from the hole. Injection angles have comprehensive influence on the averaged film cooling effectiveness for various x/d locations. As injection angles are 30 and 60 degree, two bands of high heat transfer coefficients are found in mixing region of the gas and coolant. As injection angle increases to 90 degree, the mixing leads to the enhanced heat transfer region near the film hole. The averaged heat transfer coefficient increases with the increase of injection angle.

Primary Data Treatment Software for Position-Sensitive Detector of Small-Angle Neutron Scattering Spectrometer in the Isotropic Pattern Scattering Case

NASA Astrophysics Data System (ADS)

Soloviev, Alexei; Kutuzov, Sergei; Ivankov, Olexander; Kuklin, Alexander

2018-02-01

A new data converter has been created for the new position-sensitive detector (PSD) of small-angle neutron scattering (SANS) spectrometer YuMO. In the isotropic pattern scattering case, it provides the possibility for processing PSD data with the SAS data processing program that has already been in use.

Control torque generation of a CMG-based small satellite with MTGAC system: a trade-off study

NASA Astrophysics Data System (ADS)

Salleh, M. B.; Suhadis, N. M.; Rajendran, P.; Mazlan, N. M.

2018-05-01

In this paper, the gimbal angle compensation method using magnetic control law has been adopted for a small satellite operating in low earth orbit under disturbance toques influence. Three light weight magnetic torquers have been used to generate the magnetic compensation torque to bring diverge gimbals at preferable angle. The magnetic control torque required to compensate the gimbal angle is based on the gimbal error rate which depends on the gimbal angle converging time. A simulation study has been performed without and with the MTGAC system to investigate the amount of generated control torque as a trade-off between the power consumption, attitude control performance and CMG dynamic performance. Numerical simulations show that the satellite with the MTGAC system generates more control torques which leads to the additional power requirement but in return results in a favorable attitude control performance and gimbal angle management.

Small-angle x-ray scattering in amorphous silicon: A computational study

NASA Astrophysics Data System (ADS)

Paudel, Durga; Atta-Fynn, Raymond; Drabold, David A.; Elliott, Stephen R.; Biswas, Parthapratim

2018-05-01

We present a computational study of small-angle x-ray scattering (SAXS) in amorphous silicon (a -Si) with particular emphasis on the morphology and microstructure of voids. The relationship between the scattering intensity in SAXS and the three-dimensional structure of nanoscale inhomogeneities or voids is addressed by generating large high-quality a -Si networks with 0.1%-0.3% volume concentration of voids, as observed in experiments using SAXS and positron annihilation spectroscopy. A systematic study of the variation of the scattering intensity in the small-angle scattering region with the size, shape, number density, and the spatial distribution of the voids in the networks is presented. Our results suggest that the scattering intensity in the small-angle region is particularly sensitive to the size and the total volume fraction of the voids, but the effect of the geometry or shape of the voids is less pronounced in the intensity profiles. A comparison of the average size of the voids obtained from the simulated values of the intensity, using the Guinier approximation and Kratky plots, with that of the same from the spatial distribution of the atoms in the vicinity of void surfaces is presented.

Open Access Internet Resources for Nano-Materials Physics Education

NASA Astrophysics Data System (ADS)

Moeck, Peter; Seipel, Bjoern; Upreti, Girish; Harvey, Morgan; Garrick, Will

2006-05-01

Because a great deal of nano-material science and engineering relies on crystalline materials, materials physicists have to provide their own specific contributions to the National Nanotechnology Initiative. Here we briefly review two freely accessible internet-based crystallographic databases, the Nano-Crystallography Database (http://nanocrystallography.research.pdx.edu) and the Crystallography Open Database (http://crystallography.net). Information on over 34,000 full structure determinations are stored in these two databases in the Crystallographic Information File format. The availability of such crystallographic data on the internet in a standardized format allows for all kinds of web-based crystallographic calculations and visualizations. Two examples of which that are dealt with in this paper are: interactive crystal structure visualizations in three dimensions and calculations of lattice-fringe fingerprints for the identification of unknown nanocrystals from their atomic-resolution transmission electron microscopy images.

Difficult macromolecular structures determined using X-ray diffraction techniques.

PubMed

Hernández-Santoyo, Alejandra

2012-07-01

Macromolecular crystallography has been, for the last few decades, the main source of structural information of biological macromolecular systems and it is one of the most powerful techniques for the analysis of enzyme mechanisms and macromolecular interactions at the atomic level. In addition, it is also an extremely powerful tool for drug design. Recent technological and methodological developments in macromolecular X-ray crystallography have allowed solving structures that until recently were considered difficult or even impossible, such as structures at atomic or subatomic resolution or large macromolecular complexes and assemblies at low resolution. These developments have also helped to solve the 3D-structure of macromolecules from twin crystals. Recently, this technique complemented with cryo-electron microscopy and neutron crystallography has provided the structure of large macromolecular machines with great precision allowing understanding of the mechanisms of their function.

How cryo‐electron microscopy and X‐ray crystallography complement each other

PubMed Central

Wang, Jia‐Wei

2016-01-01

Abstract With the ability to resolve structures of macromolecules at atomic resolution, X‐ray crystallography has been the most powerful tool in modern structural biology. At the same time, recent technical improvements have triggered a resolution revolution in the single particle cryo‐EM method. While the two methods are different in many respects, from sample preparation to structure determination, they both have the power to solve macromolecular structures at atomic resolution. It is important to understand the unique advantages and caveats of the two methods in solving structures and to appreciate the complementary nature of the two methods in structural biology. In this review we provide some examples, and discuss how X‐ray crystallography and cryo‐EM can be combined in deciphering structures of macromolecules for our full understanding of their biological mechanisms. PMID:27543495

Mix-and-diffuse serial synchrotron crystallography

DOE PAGES

Beyerlein, Kenneth R.; Dierksmeyer, Dennis; Mariani, Valerio; ...

2017-10-09

Unravelling the interaction of biological macromolecules with ligands and substrates at high spatial and temporal resolution remains a major challenge in structural biology. The development of serial crystallography methods at X-ray free-electron lasers and subsequently at synchrotron light sources allows new approaches to tackle this challenge. Here, a new polyimide tape drive designed for mix-and-diffuse serial crystallography experiments is reported. The structure of lysozyme bound by the competitive inhibitor chitotriose was determined using this device in combination with microfluidic mixers. The electron densities obtained from mixing times of 2 and 50 s show clear binding of chitotriose to the enzymemore » at a high level of detail. Here, the success of this approach shows the potential for high-throughput drug screening and even structural enzymology on short timescales at bright synchrotron light sources.« less

Mix-and-diffuse serial synchrotron crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beyerlein, Kenneth R.; Dierksmeyer, Dennis; Mariani, Valerio

Unravelling the interaction of biological macromolecules with ligands and substrates at high spatial and temporal resolution remains a major challenge in structural biology. The development of serial crystallography methods at X-ray free-electron lasers and subsequently at synchrotron light sources allows new approaches to tackle this challenge. Here, a new polyimide tape drive designed for mix-and-diffuse serial crystallography experiments is reported. The structure of lysozyme bound by the competitive inhibitor chitotriose was determined using this device in combination with microfluidic mixers. The electron densities obtained from mixing times of 2 and 50 s show clear binding of chitotriose to the enzymemore » at a high level of detail. Here, the success of this approach shows the potential for high-throughput drug screening and even structural enzymology on short timescales at bright synchrotron light sources.« less

A Bright Future for Serial Femtosecond Crystallography with XFELs.

PubMed

Johansson, Linda C; Stauch, Benjamin; Ishchenko, Andrii; Cherezov, Vadim

2017-09-01

X-ray free electron lasers (XFELs) have the potential to revolutionize macromolecular structural biology due to the unique combination of spatial coherence, extreme peak brilliance, and short duration of X-ray pulses. A recently emerged serial femtosecond (fs) crystallography (SFX) approach using XFEL radiation overcomes some of the biggest hurdles of traditional crystallography related to radiation damage through the diffraction-before-destruction principle. Intense fs XFEL pulses enable high-resolution room-temperature structure determination of difficult-to-crystallize biological macromolecules, while simultaneously opening a new era of time-resolved structural studies. Here, we review the latest developments in instrumentation, sample delivery, data analysis, crystallization methods, and applications of SFX to important biological questions, and conclude with brief insights into the bright future of structural biology using XFELs. Copyright © 2017 Elsevier Ltd. All rights reserved.

How cryo-electron microscopy and X-ray crystallography complement each other.

PubMed

Wang, Hong-Wei; Wang, Jia-Wei

2017-01-01

With the ability to resolve structures of macromolecules at atomic resolution, X-ray crystallography has been the most powerful tool in modern structural biology. At the same time, recent technical improvements have triggered a resolution revolution in the single particle cryo-EM method. While the two methods are different in many respects, from sample preparation to structure determination, they both have the power to solve macromolecular structures at atomic resolution. It is important to understand the unique advantages and caveats of the two methods in solving structures and to appreciate the complementary nature of the two methods in structural biology. In this review we provide some examples, and discuss how X-ray crystallography and cryo-EM can be combined in deciphering structures of macromolecules for our full understanding of their biological mechanisms. © 2016 The Protein Society.

A readout system for X-ray powder crystallography

NASA Astrophysics Data System (ADS)

Loukas, D.; Haralabidis, N.; Pavlidis, A.; Karvelas, E.; Psycharis ^a, K. Misiakos, V.; Mousa, J.; Dre, Ch.

2000-06-01

A system for capturing and processing data, from radiation detectors, in the field of X-ray crystallography has been developed. The system includes a custom-made mixed analog-digital 16-channel VLSI circuit in 50 μm pitch. Each channel comprises a charge amplifier, a shaper, a comparator and a 21-bit counter. The circuit can be scaled in a daisy chain configuration. Data acquisition is performed with a custom made PCI card while the control software is developed with Visual C++ under the MS Windows NT environment. Performance of a fully operational system, in terms of electronic noise, statistical variations and data capture speed is presented. The noise level permits counting of X-rays down to 8 keV while the counting capability is in excess of 200 kHz. The system is intended for X-ray crystallography with silicon detectors.

Determination of X-ray flux using silicon pin diodes

PubMed Central

Owen, Robin L.; Holton, James M.; Schulze-Briese, Clemens; Garman, Elspeth F.

2009-01-01

Accurate measurement of photon flux from an X-ray source, a parameter required to calculate the dose absorbed by the sample, is not yet routinely available at macromolecular crystallography beamlines. The development of a model for determining the photon flux incident on pin diodes is described here, and has been tested on the macromolecular crystallography beamlines at both the Swiss Light Source, Villigen, Switzerland, and the Advanced Light Source, Berkeley, USA, at energies between 4 and 18 keV. These experiments have shown that a simple model based on energy deposition in silicon is sufficient for determining the flux incident on high-quality silicon pin diodes. The derivation and validation of this model is presented, and a web-based tool for the use of the macromolecular crystallography and wider synchrotron community is introduced. PMID:19240326

Mapping the continuous reciprocal space intensity distribution of X-ray serial crystallography.

PubMed

Yefanov, Oleksandr; Gati, Cornelius; Bourenkov, Gleb; Kirian, Richard A; White, Thomas A; Spence, John C H; Chapman, Henry N; Barty, Anton

2014-07-17

Serial crystallography using X-ray free-electron lasers enables the collection of tens of thousands of measurements from an equal number of individual crystals, each of which can be smaller than 1 µm in size. This manuscript describes an alternative way of handling diffraction data recorded by serial femtosecond crystallography, by mapping the diffracted intensities into three-dimensional reciprocal space rather than integrating each image in two dimensions as in the classical approach. We call this procedure 'three-dimensional merging'. This procedure retains information about asymmetry in Bragg peaks and diffracted intensities between Bragg spots. This intensity distribution can be used to extract reflection intensities for structure determination and opens up novel avenues for post-refinement, while observed intensity between Bragg peaks and peak asymmetry are of potential use in novel direct phasing strategies.

Field-induced self-assembly of iron oxide nanoparticles investigated using small-angle neutron scattering.

PubMed

Fu, Zhendong; Xiao, Yinguo; Feoktystov, Artem; Pipich, Vitaliy; Appavou, Marie-Sousai; Su, Yixi; Feng, Erxi; Jin, Wentao; Brückel, Thomas

2016-11-03

The magnetic-field-induced assembly of magnetic nanoparticles (NPs) provides a unique and flexible strategy in the design and fabrication of functional nanostructures and devices. We have investigated the field-induced self-assembly of core-shell iron oxide NPs dispersed in toluene by means of small-angle neutron scattering (SANS). The form factor of the core-shell NPs was characterized and analyzed using SANS with polarized neutrons. Large-scale aggregates of iron oxide NPs formed above 0.02 T as indicated by very-small-angle neutron scattering measurements. A three-dimensional long-range ordered superlattice of iron oxide NPs was revealed under the application of a moderate magnetic field. The crystal structure of the superlattice has been identified to be face-centred cubic.

The effects of burial diagenesis on multiscale porosity in the St. Peter Sandstone: An imaging, small-angle, and ultra-small-angle neutron scattering analysis

DOE PAGES

Anovitz, Lawrence M.; Freiburg, Jared T.; Wasbrough, Matthew; ...

2017-11-06

To examine the effects of burial diagenesis on heirarchical pore structures in sandstone and compare those with the effects of overgrowth formation, we obtained samples of St. Peter Sandstone from drill cores obtained in the Illinois and Michigan Basins. The multiscale pore structure of rocks in sedimentary reservoirs and the mineralogy associated with those pores are critical factors for estimating reservoir properties, including fluid mass in place, permeability, and capillary pressures, as well as geochemical interactions between the rock and the fluid. The combination of small- and ultra-small-angle neutron scattering with backscattered electron or X ray-computed tomographic imaging, or both,more » provided a means by which pore structures were quantified at scales ranging from aproximately 1 nm to 1 cm—seven orders of magnitude. Larger scale (>10 µm) porosity showed the expected logarithmic decrease in porosity with depth, although there was significant variation in each sample group. However, small- and ultra-small-angle neutron scattering data showed that the proportion of small-scale porosity increased with depth. Porosity distributions were not continuous, but consisted of a series of log normal-like distributions at several distinct scales within these rocks. Fractal dimensions at larger scales decreased (surfaces smoothed) with increasing depth, and those at smaller scales increased (surfaces roughened) and pores become more isolated (higher lacunarity). Furthermore, data suggest that changes in pore-size distributions are controlled by both physical (compaction) and chemical effects (precipitation, cementation, dissolution).« less

The effects of burial diagenesis on multiscale porosity in the St. Peter Sandstone: An imaging, small-angle, and ultra-small-angle neutron scattering analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anovitz, Lawrence M.; Freiburg, Jared T.; Wasbrough, Matthew

To examine the effects of burial diagenesis on heirarchical pore structures in sandstone and compare those with the effects of overgrowth formation, we obtained samples of St. Peter Sandstone from drill cores obtained in the Illinois and Michigan Basins. The multiscale pore structure of rocks in sedimentary reservoirs and the mineralogy associated with those pores are critical factors for estimating reservoir properties, including fluid mass in place, permeability, and capillary pressures, as well as geochemical interactions between the rock and the fluid. The combination of small- and ultra-small-angle neutron scattering with backscattered electron or X ray-computed tomographic imaging, or both,more » provided a means by which pore structures were quantified at scales ranging from aproximately 1 nm to 1 cm—seven orders of magnitude. Larger scale (>10 µm) porosity showed the expected logarithmic decrease in porosity with depth, although there was significant variation in each sample group. However, small- and ultra-small-angle neutron scattering data showed that the proportion of small-scale porosity increased with depth. Porosity distributions were not continuous, but consisted of a series of log normal-like distributions at several distinct scales within these rocks. Fractal dimensions at larger scales decreased (surfaces smoothed) with increasing depth, and those at smaller scales increased (surfaces roughened) and pores become more isolated (higher lacunarity). Furthermore, data suggest that changes in pore-size distributions are controlled by both physical (compaction) and chemical effects (precipitation, cementation, dissolution).« less

XAFS and Protein Crystallography Beamline BL38B1 at SPring-8

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tanida, Hajime; Miura, Keiko; Takeshita, Kunikazu

2004-05-12

The SPring-8 bending magnet beamline BL38B1 is designed for R and D of optics, detectors, experiments for XAFS and protein X-ray crystallography (PX). This beamline has a multi-purpose hutch for two experimental stations of XAFS and PX, and removable optical benches used for R and D of detectors and instruments. The design and the performance of the beamline are presented.

System and method for forming synthetic protein crystals to determine the conformational structure by crystallography

DOEpatents

Craig, George D.; Glass, Robert; Rupp, Bernhard

1997-01-01

A method for forming synthetic crystals of proteins in a carrier fluid by use of the dipole moments of protein macromolecules that self-align in the Helmholtz layer adjacent to an electrode. The voltage gradients of such layers easily exceed 10.sup.6 V/m. The synthetic protein crystals are subjected to x-ray crystallography to determine the conformational structure of the protein involved.

Synergic Investigation Of The Self-Assembly Structure And Mechanism Of Retroviral Capsid Proteins By Solid State NMR, Transmission Electron Microscopy And Multiscale simulation

DTIC Science & Technology

2017-03-29

310 helix. Green: this work. Cyans: solution NMR RSV CA structure in PDB entry 1D1D.[18] Magentas: X-ray crystallography structure of flat hexameric...to combine cryo-electron microscopy and X-ray crystallography , Methods, 49 (2009) 174-180. [8] K.Y. Chan, J. Gumbart, R. McGreevy, J.M. Watermeyer

Oil-free hyaluronic acid matrix for serial femtosecond crystallography

NASA Astrophysics Data System (ADS)

Sugahara, Michihiro; Song, Changyong; Suzuki, Mamoru; Masuda, Tetsuya; Inoue, Shigeyuki; Nakane, Takanori; Yumoto, Fumiaki; Nango, Eriko; Tanaka, Rie; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Hatsui, Takaki; Yabashi, Makina; Nureki, Osamu; Numata, Keiji; Iwata, So

2016-04-01

The grease matrix was originally introduced as a microcrystal-carrier for serial femtosecond crystallography and has been expanded to applications for various types of proteins, including membrane proteins. However, the grease-based matrix has limited application for oil-sensitive proteins. Here we introduce a grease-free, water-based hyaluronic acid matrix. Applications for proteinase K and lysozyme proteins were able to produce electron density maps at 2.3-Å resolution.

A technique for determining the deuterium/hydrogen contrast map in neutron macromolecular crystallography.

PubMed

Chatake, Toshiyuki; Fujiwara, Satoru

2016-01-01

A difference in the neutron scattering length between hydrogen and deuterium leads to a high density contrast in neutron Fourier maps. In this study, a technique for determining the deuterium/hydrogen (D/H) contrast map in neutron macromolecular crystallography is developed and evaluated using ribonuclease A. The contrast map between the D2O-solvent and H2O-solvent crystals is calculated in real space, rather than in reciprocal space as performed in previous neutron D/H contrast crystallography. The present technique can thus utilize all of the amplitudes of the neutron structure factors for both D2O-solvent and H2O-solvent crystals. The neutron D/H contrast maps clearly demonstrate the powerful detectability of H/D exchange in proteins. In fact, alternative protonation states and alternative conformations of hydroxyl groups are observed at medium resolution (1.8 Å). Moreover, water molecules can be categorized into three types according to their tendency towards rotational disorder. These results directly indicate improvement in the neutron crystal structure analysis. This technique is suitable for incorporation into the standard structure-determination process used in neutron protein crystallography; consequently, more precise and efficient determination of the D-atom positions is possible using a combination of this D/H contrast technique and standard neutron structure-determination protocols.

Raster-scanning serial protein crystallography using micro- and nano-focused synchrotron beams

DOE Office of Scientific and Technical Information (OSTI.GOV)

Coquelle, Nicolas; CNRS, IBS, 38044 Grenoble; CEA, IBS, 38044 Grenoble

A raster scanning serial protein crystallography approach is presented, that consumes as low ∼200–700 nl of sedimented crystals. New serial data pre-analysis software, NanoPeakCell, is introduced. High-resolution structural information was obtained from lysozyme microcrystals (20 µm in the largest dimension) using raster-scanning serial protein crystallography on micro- and nano-focused beamlines at the ESRF. Data were collected at room temperature (RT) from crystals sandwiched between two silicon nitride wafers, thereby preventing their drying, while limiting background scattering and sample consumption. In order to identify crystal hits, new multi-processing and GUI-driven Python-based pre-analysis software was developed, named NanoPeakCell, that was able tomore » read data from a variety of crystallographic image formats. Further data processing was carried out using CrystFEL, and the resultant structures were refined to 1.7 Å resolution. The data demonstrate the feasibility of RT raster-scanning serial micro- and nano-protein crystallography at synchrotrons and validate it as an alternative approach for the collection of high-resolution structural data from micro-sized crystals. Advantages of the proposed approach are its thriftiness, its handling-free nature, the reduced amount of sample required, the adjustable hit rate, the high indexing rate and the minimization of background scattering.« less

Phosphodiester models for cleavage of nucleic acids

PubMed Central

2018-01-01

Nucleic acids that store and transfer biological information are polymeric diesters of phosphoric acid. Cleavage of the phosphodiester linkages by protein enzymes, nucleases, is one of the underlying biological processes. The remarkable catalytic efficiency of nucleases, together with the ability of ribonucleic acids to serve sometimes as nucleases, has made the cleavage of phosphodiesters a subject of intensive mechanistic studies. In addition to studies of nucleases by pH-rate dependency, X-ray crystallography, amino acid/nucleotide substitution and computational approaches, experimental and theoretical studies with small molecular model compounds still play a role. With small molecules, the importance of various elementary processes, such as proton transfer and metal ion binding, for stabilization of transition states may be elucidated and systematic variation of the basicity of the entering or departing nucleophile enables determination of the position of the transition state on the reaction coordinate. Such data is important on analyzing enzyme mechanisms based on synergistic participation of several catalytic entities. Many nucleases are metalloenzymes and small molecular models offer an excellent tool to construct models for their catalytic centers. The present review tends to be an up to date summary of what has been achieved by mechanistic studies with small molecular phosphodiesters. PMID:29719577

Detailed statistical contact angle analyses; "slow moving" drops on inclining silicon-oxide surfaces.

PubMed

Schmitt, M; Groß, K; Grub, J; Heib, F

2015-06-01

Contact angle determination by sessile drop technique is essential to characterise surface properties in science and in industry. Different specific angles can be observed on every solid which are correlated with the advancing or the receding of the triple line. Different procedures and definitions for the determination of specific angles exist which are often not comprehensible or reproducible. Therefore one of the most important things in this area is to build standard, reproducible and valid methods for determining advancing/receding contact angles. This contribution introduces novel techniques to analyse dynamic contact angle measurements (sessile drop) in detail which are applicable for axisymmetric and non-axisymmetric drops. Not only the recently presented fit solution by sigmoid function and the independent analysis of the different parameters (inclination, contact angle, velocity of the triple point) but also the dependent analysis will be firstly explained in detail. These approaches lead to contact angle data and different access on specific contact angles which are independent from "user-skills" and subjectivity of the operator. As example the motion behaviour of droplets on flat silicon-oxide surfaces after different surface treatments is dynamically measured by sessile drop technique when inclining the sample plate. The triple points, the inclination angles, the downhill (advancing motion) and the uphill angles (receding motion) obtained by high-precision drop shape analysis are independently and dependently statistically analysed. Due to the small covered distance for the dependent analysis (<0.4mm) and the dominance of counted events with small velocity the measurements are less influenced by motion dynamics and the procedure can be called "slow moving" analysis. The presented procedures as performed are especially sensitive to the range which reaches from the static to the "slow moving" dynamic contact angle determination. They are characterised by small deviations of the computed values. Additional to the detailed introduction of this novel analytical approaches plus fit solution special motion relations for the drop on inclined surfaces and detailed relations about the reactivity of the freshly cleaned silicon wafer surface resulting in acceleration behaviour (reactive de-wetting) are presented. Copyright © 2014 Elsevier Inc. All rights reserved.

The gonial angle stripper: an instrument for the treatment of prominent gonial angle.

PubMed

Kyutoku, S; Yanagida, A; Kusumoto, K; Ogawa, Y

1994-12-01

In the Orient, a prominent gonial angle, so-called benign masseteric hypertrophy, is rather common and considered unattractive. Therefore, its surgical correction is one of the most popular forms of facial skeletal contouring. For accurate and safe osteotomy of the mandibular angle region, a gonial angle stripper was specially invented. It has a small projection that will ease identification of the osteotomy line in a narrow operative field. The tool has been clinically used in eight patients to prove its usefulness, especially for a posteriorly developed mandibular angle.

The Pluto System At Small Phase Angles

NASA Astrophysics Data System (ADS)

Verbiscer, Anne J.; Buie, Marc W.; Binzel, Richard; Ennico, Kimberly; Grundy, William M.; Olkin, Catherine B.; Showalter, Mark Robert; Spencer, John R.; Stern, S. Alan; Weaver, Harold A.; Young, Leslie; New Horizons Science Team

2016-10-01

Hubble Space Telescope observations of the Pluto system acquired during the New Horizons encounter epoch (HST Program 13667, M. Buie, PI) span the phase angle range from 0.06 to 1.7 degrees, enabling the measurement and characterization of the opposition effect for Pluto and its satellites at 0.58 microns using HST WFC3/UVIS with the F350LP filter, which has a broadband response and a pivot wavelength of 0.58 microns. At these small phase angles, differences in the opposition effect width and amplitude appear. The small satellites Nix and Hydra both exhibit a very narrow opposition surge, while the considerably larger moon Charon has a broader opposition surge. Microtextural surface properties derived from the shape and magnitude of the opposition surge of each surface contain a record of the collisional history of the system. We combine these small phase angle observations with those made at larger phase angles by the New Horizons Long Range Reconnaissance Imager (LORRI), which also has a broadband response with a pivot wavelength of 0.61 microns, to produce the most complete disk-integrated solar phase curves that we will have for decades to come. Modeling these disk-integrated phase curves generates sets of photometric parameters that will inform spectral modeling of the satellite surfaces as well as terrains on Pluto from spatially resolved New Horizons Ralph Linear Etalon Imaging Spectral Array (LEISA) data from 1.2 to 2.5 microns. Rotationally resolved phase curves of Pluto reveal opposition effects that only appear at phase angles less than 0.1 degree and have widths and amplitudes that are highly dependent on longitude and therefore on Pluto's diverse terrains. The high albedo region informally known as Sputnik Planum dominates the disk-integrated reflectance of Pluto on the New Horizons encounter hemisphere. These results lay the groundwork for observations at true opposition in 2018, when the Pluto system will be observable at phase angles so small that an Earth transit across the solar disk will be visible from Pluto and its satellites.

Flatness metrology based on small-angle deflectometric procedures with electronic tiltmeters

NASA Astrophysics Data System (ADS)

Ehret, G.; Laubach, S.; Schulz, M.

2017-06-01

The measurement of optical flats, e. g. synchrotron or XFEL mirrors, with single nanometer topography uncertainty is still challenging. At PTB, we apply for this task small-angle deflectometry in which the angle between the direction of the beam sent to the surface and the beam detected is small. Conventional deflectometric systems measure the surface angle with autocollimators whose light beam also represents the straightness reference. An advanced flatness metrology system was recently implemented at PTB that separates the straightness reference task from the angle detection task. We call it `Exact Autocollimation Deflectometric Scanning' because the specimen is slightly tilted in such a way that at every scanning position the specimen is `exactly' perpendicular to the reference light beam directed by a pentaprism to the surface under test. The tilt angle of the surface is then measured with an additional autocollimator. The advantage of the EADS method is that the two tasks (straightness reference and measurement of surface slope) are separated and each of these can be optimized independently. The idea presented in this paper is to replace this additional autocollimator by one or more electro-mechanical tiltmeters, which are typically faster and have a higher resolution than highly accurate commercially available autocollimators. We investigate the point stability and the linearity of a highly accurate electronic tiltmeter. The pros and cons of using tiltmeters in flatness metrology are discussed.

Development of Inhibitors of the PAS-B Domain of the HIF-2α Transcription Factor

PubMed Central

Rogers, Jamie L.; Bayeh, Liela; Scheuermann, Thomas H.; Longgood, Jamie; Key, Jason; Naidoo, Jacinth; Melito, Lisa; Shokri, Cameron; Frantz, Doug E.; Bruick, Richard K.; Gardner, Kevin H.; MacMillan, John B.; Tambar, Uttam K.

2013-01-01

Hypoxia Inducible Factors (HIFs) are heterodimeric transcription factors induced in a variety of pathophysiological settings, including cancer. We describe the first detailed structure-activity-relationship study of small molecules designed to inhibit HIF-2α–ARNT heterodimerization by binding an internal cavity of the HIF-2α PAS-B domain. Through a series of biophysical characterizations of inhibitor/protein interactions (NMR and X-ray crystallography), we have established the structural requirements for artificial inhibitors of the HIF-2α–ARNT PAS-B interaction. These results may serve as a foundation for discovering therapeutic agents that function by a novel mode of action. PMID:23363003

Application of advanced shearing techniques to the calibration of autocollimators with small angle generators and investigation of error sources.

PubMed

Yandayan, T; Geckeler, R D; Aksulu, M; Akgoz, S A; Ozgur, B

2016-05-01

The application of advanced error-separating shearing techniques to the precise calibration of autocollimators with Small Angle Generators (SAGs) was carried out for the first time. The experimental realization was achieved using the High Precision Small Angle Generator (HPSAG) of TUBITAK UME under classical dimensional metrology laboratory environmental conditions. The standard uncertainty value of 5 mas (24.2 nrad) reached by classical calibration method was improved to the level of 1.38 mas (6.7 nrad). Shearing techniques, which offer a unique opportunity to separate the errors of devices without recourse to any external standard, were first adapted by Physikalisch-Technische Bundesanstalt (PTB) to the calibration of autocollimators with angle encoders. It has been demonstrated experimentally in a clean room environment using the primary angle standard of PTB (WMT 220). The application of the technique to a different type of angle measurement system extends the range of the shearing technique further and reveals other advantages. For example, the angular scales of the SAGs are based on linear measurement systems (e.g., capacitive nanosensors for the HPSAG). Therefore, SAGs show different systematic errors when compared to angle encoders. In addition to the error-separation of HPSAG and the autocollimator, detailed investigations on error sources were carried out. Apart from determination of the systematic errors of the capacitive sensor used in the HPSAG, it was also demonstrated that the shearing method enables the unique opportunity to characterize other error sources such as errors due to temperature drift in long term measurements. This proves that the shearing technique is a very powerful method for investigating angle measuring systems, for their improvement, and for specifying precautions to be taken during the measurements.

Small-angle neutron scattering investigations of Co-doped iron oxide nanoparticles. Preliminary results

NASA Astrophysics Data System (ADS)

Creanga, Dorina; Balasoiu, Maria; Soloviov, Dmitro; Balasoiu-Gaina, Alexandra-Maria; Puscasu, Emil; Lupu, Nicoleta; Stan, Cristina

2018-03-01

Preliminary small-angle neutron scattering investigations on aqueous suspensions of several cobalt doped ferrites (CoxFe3-xO4, x=0; 0.5; 1) nanoparticles prepared by chemical co-precipitation method, are reported. The measurements were accomplished at the YuMO instrument in function at the IBR-2 reactor. Results of intermediary data treatment are presented and discussed.

Classification and assessment of retrieved electron density maps in coherent X-ray diffraction imaging using multivariate analysis.

PubMed

Sekiguchi, Yuki; Oroguchi, Tomotaka; Nakasako, Masayoshi

2016-01-01

Coherent X-ray diffraction imaging (CXDI) is one of the techniques used to visualize structures of non-crystalline particles of micrometer to submicrometer size from materials and biological science. In the structural analysis of CXDI, the electron density map of a sample particle can theoretically be reconstructed from a diffraction pattern by using phase-retrieval (PR) algorithms. However, in practice, the reconstruction is difficult because diffraction patterns are affected by Poisson noise and miss data in small-angle regions due to the beam stop and the saturation of detector pixels. In contrast to X-ray protein crystallography, in which the phases of diffracted waves are experimentally estimated, phase retrieval in CXDI relies entirely on the computational procedure driven by the PR algorithms. Thus, objective criteria and methods to assess the accuracy of retrieved electron density maps are necessary in addition to conventional parameters monitoring the convergence of PR calculations. Here, a data analysis scheme, named ASURA, is proposed which selects the most probable electron density maps from a set of maps retrieved from 1000 different random seeds for a diffraction pattern. Each electron density map composed of J pixels is expressed as a point in a J-dimensional space. Principal component analysis is applied to describe characteristics in the distribution of the maps in the J-dimensional space. When the distribution is characterized by a small number of principal components, the distribution is classified using the k-means clustering method. The classified maps are evaluated by several parameters to assess the quality of the maps. Using the proposed scheme, structure analysis of a diffraction pattern from a non-crystalline particle is conducted in two stages: estimation of the overall shape and determination of the fine structure inside the support shape. In each stage, the most accurate and probable density maps are objectively selected. The validity of the proposed scheme is examined by application to diffraction data that were obtained from an aggregate of metal particles and a biological specimen at the XFEL facility SACLA using custom-made diffraction apparatus.

TILT ANGLE AND FOOTPOINT SEPARATION OF SMALL AND LARGE BIPOLAR SUNSPOT REGIONS OBSERVED WITH HMI

DOE Office of Scientific and Technical Information (OSTI.GOV)

McClintock, B. H.; Norton, A. A., E-mail: u1049686@umail.usq.edu.au, E-mail: aanorton@stanford.edu

2016-02-10

We investigate bipolar sunspot regions and how tilt angle and footpoint separation vary during emergence and decay. The Helioseismic and Magnetic Imager on board the Solar Dynamic Observatory collects data at a higher cadence than historical records and allows for a detailed analysis of regions over their lifetimes. We sample the umbral tilt angle, footpoint separation, and umbral area of 235 bipolar sunspot regions in Helioseismic and Magnetic Imager—Debrecen Data with an hourly cadence. We use the time when the umbral area peaks as time zero to distinguish between the emergence and decay periods of each region and we limitmore » our analysis of tilt and separation behavior over time to within ±96 hr of time zero. Tilt angle evolution is distinctly different for regions with small (≈30 MSH), midsize (≈50 MSH), and large (≈110 MSH) maximum umbral areas, with 45 and 90 MSH being useful divisions for separating the groups. At the peak umbral area, we determine median tilt angles for small (7.°6), midsize (5.°9), and large (9.°3) regions. Within ±48 hr of the time of peak umbral area, large regions steadily increase in tilt angle, midsize regions are nearly constant, and small regions show evidence of negative tilt during emergence. A period of growth in footpoint separation occurs over a 72-hr period for all of the regions from roughly 40 to 70 Mm. The smallest bipoles (<9 MSH) are outliers in that they do not obey Joy's law and have a much smaller footpoint separation. We confirm the Muñoz-Jaramillo et al. results that the sunspots appear to be two distinct populations.« less

Room-temperature serial crystallography at synchrotron X-ray sources using slowly flowing free-standing high-viscosity microstreams.

PubMed

Botha, Sabine; Nass, Karol; Barends, Thomas R M; Kabsch, Wolfgang; Latz, Beatrice; Dworkowski, Florian; Foucar, Lutz; Panepucci, Ezequiel; Wang, Meitian; Shoeman, Robert L; Schlichting, Ilme; Doak, R Bruce

2015-02-01

Recent advances in synchrotron sources, beamline optics and detectors are driving a renaissance in room-temperature data collection. The underlying impetus is the recognition that conformational differences are observed in functionally important regions of structures determined using crystals kept at ambient as opposed to cryogenic temperature during data collection. In addition, room-temperature measurements enable time-resolved studies and eliminate the need to find suitable cryoprotectants. Since radiation damage limits the high-resolution data that can be obtained from a single crystal, especially at room temperature, data are typically collected in a serial fashion using a number of crystals to spread the total dose over the entire ensemble. Several approaches have been developed over the years to efficiently exchange crystals for room-temperature data collection. These include in situ collection in trays, chips and capillary mounts. Here, the use of a slowly flowing microscopic stream for crystal delivery is demonstrated, resulting in extremely high-throughput delivery of crystals into the X-ray beam. This free-stream technology, which was originally developed for serial femtosecond crystallography at X-ray free-electron lasers, is here adapted to serial crystallography at synchrotrons. By embedding the crystals in a high-viscosity carrier stream, high-resolution room-temperature studies can be conducted at atmospheric pressure using the unattenuated X-ray beam, thus permitting the analysis of small or weakly scattering crystals. The high-viscosity extrusion injector is described, as is its use to collect high-resolution serial data from native and heavy-atom-derivatized lysozyme crystals at the Swiss Light Source using less than half a milligram of protein crystals. The room-temperature serial data allow de novo structure determination. The crystal size used in this proof-of-principle experiment was dictated by the available flux density. However, upcoming developments in beamline optics, detectors and synchrotron sources will enable the use of true microcrystals. This high-throughput, high-dose-rate methodology provides a new route to investigating the structure and dynamics of macromolecules at ambient temperature.

Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography.

PubMed

Weierstall, Uwe; James, Daniel; Wang, Chong; White, Thomas A; Wang, Dingjie; Liu, Wei; Spence, John C H; Bruce Doak, R; Nelson, Garrett; Fromme, Petra; Fromme, Raimund; Grotjohann, Ingo; Kupitz, Christopher; Zatsepin, Nadia A; Liu, Haiguang; Basu, Shibom; Wacker, Daniel; Han, Gye Won; Katritch, Vsevolod; Boutet, Sébastien; Messerschmidt, Marc; Williams, Garth J; Koglin, Jason E; Marvin Seibert, M; Klinker, Markus; Gati, Cornelius; Shoeman, Robert L; Barty, Anton; Chapman, Henry N; Kirian, Richard A; Beyerlein, Kenneth R; Stevens, Raymond C; Li, Dianfan; Shah, Syed T A; Howe, Nicole; Caffrey, Martin; Cherezov, Vadim

2014-01-01

Lipidic cubic phase (LCP) crystallization has proven successful for high-resolution structure determination of challenging membrane proteins. Here we present a technique for extruding gel-like LCP with embedded membrane protein microcrystals, providing a continuously renewed source of material for serial femtosecond crystallography. Data collected from sub-10-μm-sized crystals produced with less than 0.5 mg of purified protein yield structural insights regarding cyclopamine binding to the Smoothened receptor.

Structural dynamics of lipid bilayers using ultrafast electron crystallography

NASA Astrophysics Data System (ADS)

Chen, Songye; Seidel, Marco; Zewail, Ahmed

2007-03-01

The structures and dynamics of bilayers of crystalline fatty acids and phospholipids were studied using ultrafast electron crystallography (UEC). The systems investigated are arachidic (eicosanoic) acid and dimyristoyl phosphatidic acid (DMPA), deposited on a substrate by the Langmuir-Blodgett technique. The atomic structures under different preparation conditions were determined. The structural dynamics following a temperature jump induced by femtosecond laser on the substrates were obtained and compared to the equilibrium temperature dependence.

System and method for forming synthetic protein crystals to determine the conformational structure by crystallography

DOEpatents

Craig, G.D.; Glass, R.; Rupp, B.

1997-01-28

A method is disclosed for forming synthetic crystals of proteins in a carrier fluid by use of the dipole moments of protein macromolecules that self-align in the Helmholtz layer adjacent to an electrode. The voltage gradients of such layers easily exceed 10{sup 6}V/m. The synthetic protein crystals are subjected to x-ray crystallography to determine the conformational structure of the protein involved. 2 figs.

Resolution of structural heterogeneity in dynamic crystallography

PubMed Central

Ren, Zhong; Chan, Peter W. Y.; Moffat, Keith; Pai, Emil F.; Royer, William E.; Šrajer, Vukica; Yang, Xiaojing

2013-01-01

Dynamic behavior of proteins is critical to their function. X-­ray crystallography, a powerful yet mostly static technique, faces inherent challenges in acquiring dynamic information despite decades of effort. Dynamic ‘structural changes’ are often indirectly inferred from ‘structural differences’ by comparing related static structures. In contrast, the direct observation of dynamic structural changes requires the initiation of a biochemical reaction or process in a crystal. Both the direct and the indirect approaches share a common challenge in analysis: how to interpret the structural heterogeneity intrinsic to all dynamic processes. This paper presents a real-space approach to this challenge, in which a suite of analytical methods and tools to identify and refine the mixed structural species present in multiple crystallographic data sets have been developed. These methods have been applied to representative scenarios in dynamic crystallography, and reveal structural information that is otherwise difficult to interpret or inaccessible using conventional methods. PMID:23695239

Fine refinement of solid state structure of racemic form of phospho-tyrosine employing NMR Crystallography approach.

PubMed

Paluch, Piotr; Pawlak, Tomasz; Oszajca, Marcin; Lasocha, Wieslaw; Potrzebowski, Marek J

2015-02-01

We present step by step facets important in NMR Crystallography strategy employing O-phospho-dl-tyrosine as model sample. The significance of three major techniques being components of this approach: solid state NMR (SS NMR), X-ray diffraction of powdered sample (PXRD) and theoretical calculations (Gauge Invariant Projector Augmented Wave; GIPAW) is discussed. Each experimental technique provides different set of structural constraints. From the PXRD measurement the size of the unit cell, space group and roughly refined molecular structure are established. SS NMR provides information about content of crystallographic asymmetric unit, local geometry, molecular motion in the crystal lattice and hydrogen bonding pattern. GIPAW calculations are employed for validation of quality of elucidation and fine refinement of structure. Crystal and molecular structure of O-phospho-dl-tyrosine solved by NMR Crystallography is deposited at Cambridge Crystallographic Data Center under number CCDC 1005924. Copyright © 2014 Elsevier Inc. All rights reserved.

Pink-beam serial crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meents, A.; Wiedorn, M. O.; Srajer, V.

Serial X-ray crystallography allows macromolecular structure determination at both X-ray free electron lasers (XFELs) and, more recently, synchrotron sources. The time resolution for serial synchrotron crystallography experiments has been limited to millisecond timescales with monochromatic beams. The polychromatic, “pink”, beam provides a more than two orders of magnitude increased photon flux and hence allows accessing much shorter timescales in diffraction experiments at synchrotron sources. Here we report the structure determination of two different protein samples by merging pink-beam diffraction patterns from many crystals, each collected with a single 100 ps X-ray pulse exposure per crystal using a setup optimized formore » very low scattering background. In contrast to experiments with monochromatic radiation, data from only 50 crystals were required to obtain complete datasets. The high quality of the diffraction data highlights the potential of this method for studying irreversible reactions at sub-microsecond timescales using high-brightness X-ray facilities.« less

Combining X-ray and neutron crystallography with spectroscopy.

PubMed

Kwon, Hanna; Smith, Oliver; Raven, Emma Lloyd; Moody, Peter C E

2017-02-01

X-ray protein crystallography has, through the determination of the three-dimensional structures of enzymes and their complexes, been essential to the understanding of biological chemistry. However, as X-rays are scattered by electrons, the technique has difficulty locating the presence and position of H atoms (and cannot locate H + ions), knowledge of which is often crucially important for the understanding of enzyme mechanism. Furthermore, X-ray irradiation, through photoelectronic effects, will perturb the redox state in the crystal. By using single-crystal spectrophotometry, reactions taking place in the crystal can be monitored, either to trap intermediates or follow photoreduction during X-ray data collection. By using neutron crystallography, the positions of H atoms can be located, as it is the nuclei rather than the electrons that scatter neutrons, and the scattering length is not determined by the atomic number. Combining the two techniques allows much greater insight into both reaction mechanism and X-ray-induced photoreduction.

Serial Millisecond Crystallography of Membrane Proteins.

PubMed

Jaeger, Kathrin; Dworkowski, Florian; Nogly, Przemyslaw; Milne, Christopher; Wang, Meitian; Standfuss, Joerg

2016-01-01

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) is a powerful method to determine high-resolution structures of pharmaceutically relevant membrane proteins. Recently, the technology has been adapted to carry out serial millisecond crystallography (SMX) at synchrotron sources, where beamtime is more abundant. In an injector-based approach, crystals grown in lipidic cubic phase (LCP) or embedded in viscous medium are delivered directly into the unattenuated beam of a microfocus beamline. Pilot experiments show the application of microjet-based SMX for solving the structure of a membrane protein and compatibility of the method with de novo phasing. Planned synchrotron upgrades, faster detectors and software developments will go hand-in-hand with developments at free-electron lasers to provide a powerful methodology for solving structures from microcrystals at room temperature, ligand screening or crystal optimization for time-resolved studies with minimal or no radiation damage.

Applied and implied semantics in crystallographic publishing

PubMed Central

2012-01-01

Background Crystallography is a data-rich, software-intensive scientific discipline with a community that has undertaken direct responsibility for publishing its own scientific journals. That community has worked actively to develop information exchange standards allowing readers of structure reports to access directly, and interact with, the scientific content of the articles. Results Structure reports submitted to some journals of the International Union of Crystallography (IUCr) can be automatically validated and published through an efficient and cost-effective workflow. Readers can view and interact with the structures in three-dimensional visualization applications, and can access the experimental data should they wish to perform their own independent structure solution and refinement. The journals also layer on top of this facility a number of automated annotations and interpretations to add further scientific value. Conclusions The benefits of semantically rich information exchange standards have revolutionised the scholarly publishing process for crystallography, and establish a model relevant to many other physical science disciplines. PMID:22932420

Resolution of structural heterogeneity in dynamic crystallography.

PubMed

Ren, Zhong; Chan, Peter W Y; Moffat, Keith; Pai, Emil F; Royer, William E; Šrajer, Vukica; Yang, Xiaojing

2013-06-01

Dynamic behavior of proteins is critical to their function. X-ray crystallography, a powerful yet mostly static technique, faces inherent challenges in acquiring dynamic information despite decades of effort. Dynamic `structural changes' are often indirectly inferred from `structural differences' by comparing related static structures. In contrast, the direct observation of dynamic structural changes requires the initiation of a biochemical reaction or process in a crystal. Both the direct and the indirect approaches share a common challenge in analysis: how to interpret the structural heterogeneity intrinsic to all dynamic processes. This paper presents a real-space approach to this challenge, in which a suite of analytical methods and tools to identify and refine the mixed structural species present in multiple crystallographic data sets have been developed. These methods have been applied to representative scenarios in dynamic crystallography, and reveal structural information that is otherwise difficult to interpret or inaccessible using conventional methods.

Pink-beam serial crystallography

DOE PAGES

Meents, A.; Wiedorn, M. O.; Srajer, V.; ...

2017-11-03

Serial X-ray crystallography allows macromolecular structure determination at both X-ray free electron lasers (XFELs) and, more recently, synchrotron sources. The time resolution for serial synchrotron crystallography experiments has been limited to millisecond timescales with monochromatic beams. The polychromatic, “pink”, beam provides a more than two orders of magnitude increased photon flux and hence allows accessing much shorter timescales in diffraction experiments at synchrotron sources. Here we report the structure determination of two different protein samples by merging pink-beam diffraction patterns from many crystals, each collected with a single 100 ps X-ray pulse exposure per crystal using a setup optimized formore » very low scattering background. In contrast to experiments with monochromatic radiation, data from only 50 crystals were required to obtain complete datasets. The high quality of the diffraction data highlights the potential of this method for studying irreversible reactions at sub-microsecond timescales using high-brightness X-ray facilities.« less

Protein crystallization: Eluding the bottleneck of X-ray crystallography

PubMed Central

Holcomb, Joshua; Spellmon, Nicholas; Zhang, Yingxue; Doughan, Maysaa; Li, Chunying; Yang, Zhe

2017-01-01

To date, X-ray crystallography remains the gold standard for the determination of macromolecular structure and protein substrate interactions. However, the unpredictability of obtaining a protein crystal remains the limiting factor and continues to be the bottleneck in determining protein structures. A vast amount of research has been conducted in order to circumvent this issue with limited success. No single method has proven to guarantee the crystallization of all proteins. However, techniques using antibody fragments, lipids, carrier proteins, and even mutagenesis of crystal contacts have been implemented to increase the odds of obtaining a crystal with adequate diffraction. In addition, we review a new technique using the scaffolding ability of PDZ domains to facilitate nucleation and crystal lattice formation. Although in its infancy, such technology may be a valuable asset and another method in the crystallography toolbox to further the chances of crystallizing problematic proteins. PMID:29051919

Quantum crystallography: A perspective.

PubMed

Massa, Lou; Matta, Chérif F

2018-06-30

Extraction of the complete quantum mechanics from X-ray scattering data is the ultimate goal of quantum crystallography. This article delivers a perspective for that possibility. It is desirable to have a method for the conversion of X-ray diffraction data into an electron density that reflects the antisymmetry of an N-electron wave function. A formalism for this was developed early on for the determination of a constrained idempotent one-body density matrix. The formalism ensures pure-state N-representability in the single determinant sense. Applications to crystals show that quantum mechanical density matrices of large molecules can be extracted from X-ray scattering data by implementing a fragmentation method termed the kernel energy method (KEM). It is shown how KEM can be used within the context of quantum crystallography to derive quantum mechanical properties of biological molecules (with low data-to-parameters ratio). © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

X-ray crystallography, an essential tool for the determination of thermodynamic relationships between crystalline polymorphs.

PubMed

Céolin, R; Rietveld, I-B

2016-01-01

After a short review of the controversies surrounding the discovery of crystalline polymorphism in relation to our present day understanding, the methods of how to solve the stability hierarchy of different polymorphs will be briefly discussed. They involve either theoretical calculations, or, more commonly, experimental methods based on classical thermodynamics. The experimental approach is mainly carried out using heat-exchange data associated to the transition of one form into another. It will be demonstrated that work-related data associated to the phase transition should be taken into account and the role of X-ray crystallography therein will be discussed. X-ray crystallography has become increasingly precise and can nowadays provide specific volumes and their differences as a function of temperature, and also as a function of pressure, humidity, and time. Copyright © 2015 Académie Nationale de Pharmacie. Published by Elsevier Masson SAS. All rights reserved.

Correlation between protein sequence similarity and x-ray diffraction quality in the protein data bank.

PubMed

Lu, Hui-Meng; Yin, Da-Chuan; Ye, Ya-Jing; Luo, Hui-Min; Geng, Li-Qiang; Li, Hai-Sheng; Guo, Wei-Hong; Shang, Peng

2009-01-01

As the most widely utilized technique to determine the 3-dimensional structure of protein molecules, X-ray crystallography can provide structure of the highest resolution among the developed techniques. The resolution obtained via X-ray crystallography is known to be influenced by many factors, such as the crystal quality, diffraction techniques, and X-ray sources, etc. In this paper, the authors found that the protein sequence could also be one of the factors. We extracted information of the resolution and the sequence of proteins from the Protein Data Bank (PDB), classified the proteins into different clusters according to the sequence similarity, and statistically analyzed the relationship between the sequence similarity and the best resolution obtained. The results showed that there was a pronounced correlation between the sequence similarity and the obtained resolution. These results indicate that protein structure itself is one variable that may affect resolution when X-ray crystallography is used.

High-speed fixed-target serial virus crystallography

PubMed Central

Roedig, Philip; Ginn, Helen M.; Pakendorf, Tim; Sutton, Geoff; Harlos, Karl; Walter, Thomas S.; Meyer, Jan; Fischer, Pontus; Duman, Ramona; Vartiainen, Ismo; Reime, Bernd; Warmer, Martin; Brewster, Aaron S.; Young, Iris D.; Michels-Clark, Tara; Sauter, Nicholas K.; Kotecha, Abhay; Kelly, James; Rowlands, David J.; Sikorsky, Marcin; Nelson, Silke; Damiani, Daniel S.; Alonso-Mori, Roberto; Ren, Jingshan; Fry, Elizabeth E.; David, Christian; Stuart, David I.; Wagner, Armin; Meents, Alke

2017-01-01

We report a method for serial X-ray crystallography at X-ray free electron lasers (XFELs), which allows for full use of the current 120 Hz repetition rate of the Linear Coherent Light Source (LCLS). Using a micro-patterned silicon chip in combination with the high-speed Roadrunner goniometer for sample delivery we were able to determine the crystal structures of a picornavirus, bovine enterovirus 2 (BEV2), and the cytoplasmic polyhedrosis virus type 18 polyhedrin. Total data collection times were less than 14 and 10 minutes, respectively. Our method requires only micrograms of sample and will therefore broaden the applicability of serial femtosecond crystallography to challenging projects for which only limited sample amounts are available. By synchronizing the sample exchange to the XFEL repetition rate, our method allows for the most efficient use of the limited beamtime available at XFELs and should enable a substantial increase in sample throughput at these facilities. PMID:28628129

Visual field structure in the Empress Leilia, Asterocampa leilia (Lepidoptera, Nymphalidae): dimensions and regional variation in acuity.

PubMed

Rutowski, Ronald L; Warrant, Eric J

2002-02-01

Male Empress Leilia butterflies ( Asterocampa leilia) use a sit-and-wait tactic to locate mates. To see how vision might influence male behavior, we studied the morphology, optics, and receptor physiology of their eyes and found the following. (1) Each eye's visual field is approximately hemispherical with at most a 10 degrees overlap in the fields of the eyes. There are no large sexual differences in visual field dimensions. (2) In both sexes, rhabdoms in the frontal and dorsal ommatidia are longer than those in other eye regions. (3) Interommatidial angles are smallest frontally and around the equator of the eye. Minimum interommatidial angles are 0.9-1 degrees in males and 1.3-1.4 degrees in females. (4) Acceptance angles of ommatidia closely match interommatidial angles in the frontal region of the eye. We conclude that vision in these butterflies is mostly monocular and that males have more acute vision than females, especially in the frontal region (large facets, small interommatidial angles, small acceptance angles, long rhabdoms, and a close match between interommatidial angles and acceptance angles). This study also suggests that perched males direct their most acute vision where females are likely to appear but show no eye modifications that appear clearly related to a mate-locating tactic.

Theoretical study of the transonic lift of a double-wedge profile with detached bow wave

NASA Technical Reports Server (NTRS)

Vincenti, Walter G; Wagoner, Cleo B

1954-01-01

A theoretical study is described of the aerodynamic characteristics at small angle of attack of a thin, double-wedge profile in the range of supersonic flight speed in which the bow wave is detached. The analysis is carried out within the framework of the transonic (nonlinear) small-disturbance theory, and the effects of angle of attack are regarded as a small perturbation on the flow previously calculated at zero angle. The mixed flow about the front half of the profile is calculated by relaxation solution of a suitably defined boundary-value problem for transonic small-disturbance equation in the hodograph plane (i.e., the Tricomi equation). The purely supersonic flow about the rear half is found by an extension of the usual numerical method of characteristics. Analytical results are also obtained, within the framework of the same theory, for the range of speed in which the bow wave is attached and the flow is completely supersonic.

Impacts of gantry angle dependent scanning beam properties on proton PBS treatment

NASA Astrophysics Data System (ADS)

Lin, Yuting; Clasie, Benjamin; Lu, Hsiao-Ming; Flanz, Jacob; Shen, Tim; Jee, Kyung-Wook

2017-01-01

While proton beam models in treatment planning systems are generally assumed invariant with respect to the beam deliveries at different gantry angles. Physical properties of scanning pencil beams can change. The gantry angle dependent properties include the delivered charge to the monitor unit chamber, the spot position and the spot shape. The aim of this study is to investigate the extent of the changes and their dosimetric impacts using historical pencil beam scanning (PBS) treatment data. Online beam delivery records at the time of the patient-specific qualify assurance were retrospectively collected for a total of 34 PBS fields from 28 patients treated at our institution. For each field, proton beam properties at two different gantry angles (the planned and zero gantry angles) were extracted by a newly-developed machine log analysis method and used to reconstruct the delivered dose distributions in the cubic water phantom geometry. The reconstructed doses at the two different angles and a planar dose measurement by a 2D ion-chamber array were compared and the dosimetric impacts of the gantry angle dependency were accessed by a 3D γ-index analysis. In addition, the pencil beam spot size was independently characterized as a function of the gantry angle and the beam energy. The dosimetric effects of the perturbed beam shape were also investigated. Comparisons of spot-by-spot beam positions between both gantry angles show a mean deviation of 0.4 and 0.7 mm and a standard deviation of 0.3 and 0.6 mm for x and y directions, respectively. The delivered giga-protons per spot show a percent mean difference and a standard deviation of 0.01% and 0.3%, respectively, from each planned spot weight. These small deviations lead to an excellent agreement in dose comparisons with an average γ passing rate of 99.1%. When each calculation for both planned and zero gantry angles was compared to the measurement, a high correlation in γ values was also observed, also indicating the dosimetric differences are small when a field is delivered at different gantry angles. Utilizing the online beam delivery records, the gantry angle dependencies of the PBS beam delivery were assessed and quantified. The study confirms the variations of the physical properties to be sufficiently small within the clinical tolerances without taking into account the gantry angle variation.

Theory and tests of a thermal ion detector sensitive only at Near-normal incidence

NASA Technical Reports Server (NTRS)

Robinson, J. W.

1981-01-01

Measurements of thermal ions are influenced by factors such as spacecraft potential, velocity, angle of attack, and sheath size. A theory is presented for the response of an instrument which accepts ions only within a small angle of incidence from normal. Although a more general theory is available and forms the basis of this one, the small angle restriction allows a simpler formulation which does not depend on sheath size. Furthermore, practical instruments are easily designed around this restriction. Laboratory tests verify that such instruments respond as expected and they illustrate how design details influence perturbations from the ideal response characteristics.

Angle amplifier based on multiplexed volume holographic gratings

NASA Astrophysics Data System (ADS)

Cao, Liangcai; Zhao, Yifei; He, Qingsheng; Jin, Guofan

2008-03-01

Angle amplifier of laser beam scanner is a widely used device in optical systems. Volume holographic optical elements can be applied in the angle amplifier. Compared with the traditional angle amplifier, it has the advantages of high angle resolution, high diffraction efficiency, small size, and high angle magnification and flexible design. Bragg anglewavelength- compensating recording method is introduced. Because of the Bragg compensatory relation between angle and wavelength, this device could be recorded at another wavelength. The design of the angle amplifier recording at the wavelength of 514.2nm for the working wavelength of 632.8nm is described. An optical setup for recording the angle amplifier device is designed and discussed. Experimental results in the photorefractive crystal Fe:LiNbO 3 demonstrate the feasibility of the angle amplifier scheme.

Observations of ULF oscillations in the ion fluxes at small pitch angles with ATS 6. [low energy particle detection

NASA Technical Reports Server (NTRS)

Su, S.-Y.; Mcpherron, R. L.; Konradi, A.; Fritz, T. A.

1980-01-01

The ultra-low-frequency modulation of ion flux densities at small pitch angles observed by ATS 6 is examined, with particular attention given to a detailed analysis of a representative event. ULF modulation events with maximum modulation at small pitch angles were identified 14 times during the first eight months of operation of the NOAA low-energy particle detector on ATS 6. For the event of October 23, 1974, maximum flux modulation, with a maximum/minimum intensity ratio of 3.7, was observed in the 100 to 150 keV detector at an angle of 32 deg to the ambient field. Spectral analysis of magnetic field data reveals a right elliptically polarized magnetic perturbation with a 96-sec period and a 5-gamma rms amplitude, propagating in the dipole meridian at an angle of about 15 deg to the ambient field and the dipole axis. Proton flux modulation is found to lag the field by up to 180 deg for the lowest-energy channel. Observations are compared with the drift wave, MHD slow wave, and bounce resonant interaction associated with transverse wave models, and it is found that none of the wave models can adequately account for all of the correlated particle and field oscillations.

Small-Angle Neutron Scattering on Crosslink Distribution of Epoxy Networks.

DTIC Science & Technology

1985-10-01

distinct second phase or heterogeneity has been detected. Small- angle X-ray scattering (SAXS), 1 nuclear magnetic resonance (NMR) ,2 electron ... paramagnetic resonance (EPR),3 and glass transition 4temperature (Tg) measurements reveal a second phase which is attri- . buted to a heterogeneous...FUNDING/SPONSORING lab. OFFICE SYMBOL 9. PROCUREMENT INSTRUMENT IDENTIFICATION NUMBER * ORGANIZATION I (If applticable)j F3361 5-84-C-5020 * Bc ADDRESS

CMB-lensing beyond the Born approximation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marozzi, Giovanni; Fanizza, Giuseppe; Durrer, Ruth

2016-09-01

We investigate the weak lensing corrections to the cosmic microwave background temperature anisotropies considering effects beyond the Born approximation. To this aim, we use the small deflection angle approximation, to connect the lensed and unlensed power spectra, via expressions for the deflection angles up to third order in the gravitational potential. While the small deflection angle approximation has the drawback to be reliable only for multipoles ℓ ∼< 2500, it allows us to consistently take into account the non-Gaussian nature of cosmological perturbation theory beyond the linear level. The contribution to the lensed temperature power spectrum coming from the non-Gaussianmore » nature of the deflection angle at higher order is a new effect which has not been taken into account in the literature so far. It turns out to be the leading contribution among the post-Born lensing corrections. On the other hand, the effect is smaller than corrections coming from non-linearities in the matter power spectrum, and its imprint on CMB lensing is too small to be seen in present experiments.« less

Investigation of steric influences on hydrogen-bonding motifs in cyclic ureas by using X-ray, neutron, and computational methods.

PubMed

McCormick, Laura J; McDonnell-Worth, Ciaran; Platts, James A; Edwards, Alison J; Turner, David R

2013-11-01

A series of urea-derived heterocycles, 5N-substituted hexahydro-1,3,5-triazin-2-ones, has been prepared and their structures have been determined for the first time. This family of compounds only differ in their substituent at the 5-position (which is derived from the corresponding primary amine), that is, methyl (1), ethyl (2), isopropyl (3), tert-butyl (4), benzyl (5), N,N-(diethyl)ethylamine (6), and 2-hydroxyethyl (7). The common heterocyclic core of these molecules is a cyclic urea, which has the potential to form a hydrogen-bonding tape motif that consists of self-associative R₂²(8) dimers. The results from X-ray crystallography and, where possible, Laue neutron crystallography show that the hydrogen-bonding motifs that are observed and the planarity of the hydrogen bonds appear to depend on the steric hindrance at the α-carbon atom of the N substituent. With the less-hindered substituents, methyl and ethyl, the anticipated tape motif is observed. When additional methyl groups are added onto the α-carbon atom, as in the isopropyl and tert-butyl derivatives, a different 2D hydrogen-bonding motif is observed. Despite the bulkiness of the substituents, the benzyl and N,N-(diethyl)ethylamine derivatives have methylene units at the α-carbon atom and, therefore, display the tape motif. The introduction of a competing hydrogen-bond donor/acceptor in the 2-hydroxyethyl derivative disrupts the tape motif, with a hydroxy group interrupting the N-H···O=C interactions. The geometry around the hydrogen-bearing nitrogen atoms, whether planar or non-planar, has been confirmed for compounds 2 and 5 by using Laue neutron diffraction and rationalized by using computational methods, thus demonstrating that distortion of O-C-N-H torsion angles occurs to maintain almost-linear hydrogen-bonding interactions. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

NMR Crystallography of Enzyme Active Sites: Probing Chemically-Detailed, Three-Dimensional Structure in Tryptophan Synthase

PubMed Central

Dunn, Michael F.

2013-01-01

Conspectus NMR crystallography – the synergistic combination of X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry – offers unprecedented insight into three-dimensional, chemically-detailed structure. From its initial role in refining diffraction data of organic and inorganic solids, NMR crystallography is now being developed for application to active sites in biomolecules, where it reveals chemically-rich detail concerning the interactions between enzyme site residues and the reacting substrate that is not achievable when X-ray, NMR, or computational methodologies are applied in isolation. For example, typical X-ray crystal structures (1.5 to 2.5 Å resolution) of enzyme-bound intermediates identify possible hydrogen-bonding interactions between site residues and substrate, but do not directly identify the protonation state of either. Solid-state NMR can provide chemical shifts for selected atoms of enzyme-substrate complexes, but without a larger structural framework in which to interpret them, only empirical correlations with local chemical structure are possible. Ab initio calculations and molecular mechanics can build models for enzymatic processes, but rely on chemical details that must be specified. Together, however, X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry can provide consistent and testable models for structure and function of enzyme active sites: X-ray crystallography provides a coarse framework upon which models of the active site can be developed using computational chemistry; these models can be distinguished by comparison of their calculated NMR chemical shifts with the results of solid-state NMR spectroscopy experiments. Conceptually, each technique is a puzzle piece offering a generous view of the big picture. Only when correctly pieced together, however, can they reveal the big picture at highest resolution. In this Account, we detail our first steps in the development of NMR crystallography for application to enzyme catalysis. We begin with a brief introduction to NMR crystallography and then define the process that we have employed to probe the active site in the β-subunit of tryptophan synthase with unprecedented atomic-level resolution. This approach has resulted in a novel structural hypothesis for the protonation state of the quinonoid intermediate in tryptophan synthase and its surprising role in directing the next step in the catalysis of L-Trp formation. PMID:23537227

On a fast calculation of structure factors at a subatomic resolution.

PubMed

Afonine, P V; Urzhumtsev, A

2004-01-01

In the last decade, the progress of protein crystallography allowed several protein structures to be solved at a resolution higher than 0.9 A. Such studies provide researchers with important new information reflecting very fine structural details. The signal from these details is very weak with respect to that corresponding to the whole structure. Its analysis requires high-quality data, which previously were available only for crystals of small molecules, and a high accuracy of calculations. The calculation of structure factors using direct formulae, traditional for 'small-molecule' crystallography, allows a relatively simple accuracy control. For macromolecular crystals, diffraction data sets at a subatomic resolution contain hundreds of thousands of reflections, and the number of parameters used to describe the corresponding models may reach the same order. Therefore, the direct way of calculating structure factors becomes very time expensive when applied to large molecules. These problems of high accuracy and computational efficiency require a re-examination of computer tools and algorithms. The calculation of model structure factors through an intermediate generation of an electron density [Sayre (1951). Acta Cryst. 4, 362-367; Ten Eyck (1977). Acta Cryst. A33, 486-492] may be much more computationally efficient, but contains some parameters (grid step, 'effective' atom radii etc.) whose influence on the accuracy of the calculation is not straightforward. At the same time, the choice of parameters within safety margins that largely ensure a sufficient accuracy may result in a significant loss of the CPU time, making it close to the time for the direct-formulae calculations. The impact of the different parameters on the computer efficiency of structure-factor calculation is studied. It is shown that an appropriate choice of these parameters allows the structure factors to be obtained with a high accuracy and in a significantly shorter time than that required when using the direct formulae. Practical algorithms for the optimal choice of the parameters are suggested.

Applications of Real Space Crystallography in Characterization of Dislocations in Geological Materials in a Scanning Electron Microscope (SEM)

NASA Astrophysics Data System (ADS)

Kaboli, S.; Burnley, P. C.

2017-12-01

Imaging and characterization of defects in crystalline materials is of significant importance in various disciplines including geoscience, materials science, and applied physics. Linear defects such as dislocations and planar defects such as twins and stacking faults, strongly influence many of the properties of crystalline materials and also reflect the conditions and degree of deformation. Dislocations have been conventionally imaged in thin foils in a transmission electron microscope (TEM). Since the development of field emission scanning electron microscopes (FE-SEM) with high gun brightness and small spot size, extensive efforts have been dedicated to the imaging and characterization of dislocations in semi-conductors using electron channeling contrast imaging (ECCI) in the SEM. The obvious advantages of using SEM over TEM include easier and non-destructive sample preparation and a large field of view enabling statistical examination of the density and distribution of dislocations and other defects. In this contribution, we extend this technique to geological materials and introduce the Real Space Crystallography methodology for imaging and complete characterization of dislocations based on bend contour contrast obtained by ECCI in FE-SEM. Bend contours map out the distortion in the crystal lattice across a deformed grain. The contrast of dislocations is maximum in the vicinity of bend contours where crystal planes diffract at small and positive deviations from the Bragg positions (as defined by Bragg's law of electron diffraction). Imaging is performed in a commercial FE-SEM equipped with a standard silicon photodiode backscattered (BSE) detector and an electron backscatter diffraction (EBSD) system for crystal orientation measurements. We demonstrate the practice of this technique in characterization of a number of geological materials in particular quartz, forsterite olivine and corundum, experimentally deformed at high pressure-temperature conditions. This new approach in microstructure characterization of deformed geologic materials in FE-SEM, without the use of etching or decoration techniques, has valuable applications to both experimentally deformed and naturally deformed specimens.

Tricritical points in a Vicsek model of self-propelled particles with bounded confidence

NASA Astrophysics Data System (ADS)

Romensky, Maksym; Lobaskin, Vladimir; Ihle, Thomas

2014-12-01

We study the orientational ordering in systems of self-propelled particles with selective interactions. To introduce the selectivity we augment the standard Vicsek model with a bounded-confidence collision rule: a given particle only aligns to neighbors who have directions quite similar to its own. Neighbors whose directions deviate more than a fixed restriction angle α are ignored. The collective dynamics of this system is studied by agent-based simulations and kinetic mean-field theory. We demonstrate that the reduction of the restriction angle leads to a critical noise amplitude decreasing monotonically with that angle, turning into a power law with exponent 3/2 for small angles. Moreover, for small system sizes we show that upon decreasing the restriction angle, the kind of the transition to polar collective motion changes from continuous to discontinuous. Thus, an apparent tricritical point with different scaling laws is identified and calculated analytically. We investigate the shifting and vanishing of this point due to the formation of density bands as the system size is increased. Agent-based simulations in small systems with large particle velocities show excellent agreement with the kinetic theory predictions. We also find that at very small interaction angles, the polar ordered phase becomes unstable with respect to the apolar phase. We derive analytical expressions for the dependence of the threshold noise on the restriction angle. We show that the mean-field kinetic theory also permits stationary nematic states below a restriction angle of 0.681 π . We calculate the critical noise, at which the disordered state bifurcates to a nematic state, and find that it is always smaller than the threshold noise for the transition from disorder to polar order. The disordered-nematic transition features two tricritical points: At low and high restriction angle, the transition is discontinuous but continuous at intermediate α . We generalize our results to systems that show fragmentation into more than two groups and obtain scaling laws for the transition lines and the corresponding tricritical points. A numerical method to evaluate the nonlinear Fredholm integral equation for the stationary distribution function is also presented. This method is shown to give excellent agreement with agent-based simulations, even in strongly ordered systems at noise values close to zero.

Angles between orthogonal spd bond orbitals with maximum strength.

PubMed

Pauling, L

1976-05-01

An equation is derived for values of bond angles for two equivalent best spd hybrid bond orbitals with given amounts of s, p, and d character, and is applied in the discussion of structures of transargononic compounds, including the xenon and halogen fluorides. Bond orbitals with a rather small amount of d character tend to lie at angles 90 degrees and 180 degrees , and those with a larger amount, at somewhat smaller angles.

NMR crystallography: structure and properties of materials from solid-state nuclear magnetic resonance observables

PubMed Central

Bryce, David L.

2017-01-01

This topical review provides a brief overview of recent developments in NMR crystallography and related NMR approaches to studying the properties of molecular and ionic solids. Areas of complementarity with diffraction-based methods are underscored. These include the study of disordered systems, of dynamic systems, and other selected examples where NMR can provide unique insights. Highlights from the literature as well as recent work from my own group are discussed. PMID:28875022

Native sulfur/chlorine SAD phasing for serial femtosecond crystallography.

PubMed

Nakane, Takanori; Song, Changyong; Suzuki, Mamoru; Nango, Eriko; Kobayashi, Jun; Masuda, Tetsuya; Inoue, Shigeyuki; Mizohata, Eiichi; Nakatsu, Toru; Tanaka, Tomoyuki; Tanaka, Rie; Shimamura, Tatsuro; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Hatsui, Takaki; Yabashi, Makina; Nureki, Osamu; Iwata, So; Sugahara, Michihiro

2015-12-01

Serial femtosecond crystallography (SFX) allows structures to be determined with minimal radiation damage. However, phasing native crystals in SFX is not very common. Here, the structure determination of native lysozyme from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of sulfur and chlorine at a wavelength of 1.77 Å is successfully demonstrated. This sulfur SAD method can be applied to a wide range of proteins, which will improve the determination of native crystal structures.

NMR Crystallography of a Carbanionic Intermediate in Tryptophan Synthase: Chemical Structure, Tautomerization, and Reaction Specificity.

PubMed

Caulkins, Bethany G; Young, Robert P; Kudla, Ryan A; Yang, Chen; Bittbauer, Thomas J; Bastin, Baback; Hilario, Eduardo; Fan, Li; Marsella, Michael J; Dunn, Michael F; Mueller, Leonard J

2016-11-23

Carbanionic intermediates play a central role in the catalytic transformations of amino acids performed by pyridoxal-5'-phosphate (PLP)-dependent enzymes. Here, we make use of NMR crystallography-the synergistic combination of solid-state nuclear magnetic resonance, X-ray crystallography, and computational chemistry-to interrogate a carbanionic/quinonoid intermediate analogue in the β-subunit active site of the PLP-requiring enzyme tryptophan synthase. The solid-state NMR chemical shifts of the PLP pyridine ring nitrogen and additional sites, coupled with first-principles computational models, allow a detailed model of protonation states for ionizable groups on the cofactor, substrates, and nearby catalytic residues to be established. Most significantly, we find that a deprotonated pyridine nitrogen on PLP precludes formation of a true quinonoid species and that there is an equilibrium between the phenolic and protonated Schiff base tautomeric forms of this intermediate. Natural bond orbital analysis indicates that the latter builds up negative charge at the substrate C α and positive charge at C4' of the cofactor, consistent with its role as the catalytic tautomer. These findings support the hypothesis that the specificity for β-elimination/replacement versus transamination is dictated in part by the protonation states of ionizable groups on PLP and the reacting substrates and underscore the essential role that NMR crystallography can play in characterizing both chemical structure and dynamics within functioning enzyme active sites.

Protein crystallography for aspiring crystallographers or how to avoid pitfalls and traps in macromolecular structure determination.

PubMed

Wlodawer, Alexander; Minor, Wladek; Dauter, Zbigniew; Jaskolski, Mariusz

2013-11-01

The number of macromolecular structures deposited in the Protein Data Bank now approaches 100,000, with the vast majority of them determined by crystallographic methods. Thousands of papers describing such structures have been published in the scientific literature, and 20 Nobel Prizes in chemistry or medicine have been awarded for discoveries based on macromolecular crystallography. New hardware and software tools have made crystallography appear to be an almost routine (but still far from being analytical) technique and many structures are now being determined by scientists with very limited experience in the practical aspects of the field. However, this apparent ease is sometimes illusory and proper procedures need to be followed to maintain high standards of structure quality. In addition, many noncrystallographers may have problems with the critical evaluation and interpretation of structural results published in the scientific literature. The present review provides an outline of the technical aspects of crystallography for less experienced practitioners, as well as information that might be useful for users of macromolecular structures, aiming to show them how to interpret (but not overinterpret) the information present in the coordinate files and in their description. A discussion of the extent of information that can be gleaned from the atomic coordinates of structures solved at different resolution is provided, as well as problems and pitfalls encountered in structure determination and interpretation. © 2013 FEBS.

Protein crystallography for aspiring crystallographers or how to avoid pitfalls and traps in macromolecular structure determination

PubMed Central

Wlodawer, Alexander; Minor, Wladek; Dauter, Zbigniew; Jaskolski, Mariusz

2014-01-01

The number of macromolecular structures deposited in the Protein Data Bank now approaches 100 000, with the vast majority of them determined by crystallographic methods. Thousands of papers describing such structures have been published in the scientific literature, and 20 Nobel Prizes in chemistry or medicine have been awarded for discoveries based on macromolecular crystallography. New hardware and software tools have made crystallography appear to be an almost routine (but still far from being analytical) technique and many structures are now being determined by scientists with very limited experience in the practical aspects of the field. However, this apparent ease is sometimes illusory and proper procedures need to be followed to maintain high standards of structure quality. In addition, many noncrystallographers may have problems with the critical evaluation and interpretation of structural results published in the scientific literature. The present review provides an outline of the technical aspects of crystallography for less experienced practitioners, as well as information that might be useful for users of macromolecular structures, aiming to show them how to interpret (but not overinterpret) the information present in the coordinate files and in their description. A discussion of the extent of information that can be gleaned from the atomic coordinates of structures solved at different resolution is provided, as well as problems and pitfalls encountered in structure determination and interpretation. PMID:24034303

Designing a diverse high-quality library for crystallography-based FBDD screening.

PubMed

Tounge, Brett A; Parker, Michael H

2011-01-01

A well-chosen set of fragments is able to cover a large chemical space using a small number of compounds. The actual size and makeup of the fragment set is dependent on the screening method since each technique has its own practical limits in terms of the number of compounds that can be screened and requirements for compound solubility. In this chapter, an overview of the general requirements for a fragment library is presented for different screening platforms. In the case of the FBDD work at Johnson & Johnson Pharmaceutical Research and Development, L.L.C., our main screening technology is X-ray crystallography. Since every soaked protein crystal needs to be diffracted and a protein structure determined to delineate if a fragment binds, the size of our initial screening library cannot be a rate-limiting factor. For this reason, we have chosen 900 as the appropriate primary fragment library size. To choose the best set, we have developed our own mix of simple property ("Rule of 3") and "bad" substructure filtering. While this gets one a long way in terms of limiting the fragment pool, there are still tens of thousands of compounds to choose from after this initial step. Many of the choices left at this stage are not drug-like, so we have developed an FBDD Score to help select a 900-compound set. The details of this score and the filtering are presented. Copyright © 2011 Elsevier Inc. All rights reserved.

Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography.

PubMed

Mandal, Kalyaneswar; Uppalapati, Maruti; Ault-Riché, Dana; Kenney, John; Lowitz, Joshua; Sidhu, Sachdev S; Kent, Stephen B H

2012-09-11

Total chemical synthesis was used to prepare the mirror image (D-protein) form of the angiogenic protein vascular endothelial growth factor (VEGF-A). Phage display against D-VEGF-A was used to screen designed libraries based on a unique small protein scaffold in order to identify a high affinity ligand. Chemically synthesized D- and L- forms of the protein ligand showed reciprocal chiral specificity in surface plasmon resonance binding experiments: The L-protein ligand bound only to D-VEGF-A, whereas the D-protein ligand bound only to L-VEGF-A. The D-protein ligand, but not the L-protein ligand, inhibited the binding of natural VEGF(165) to the VEGFR1 receptor. Racemic protein crystallography was used to determine the high resolution X-ray structure of the heterochiral complex consisting of {D-protein antagonist + L-protein form of VEGF-A}. Crystallization of a racemic mixture of these synthetic proteins in appropriate stoichiometry gave a racemic protein complex of more than 73 kDa containing six synthetic protein molecules. The structure of the complex was determined to a resolution of 1.6 Å. Detailed analysis of the interaction between the D-protein antagonist and the VEGF-A protein molecule showed that the binding interface comprised a contact surface area of approximately 800 Å(2) in accord with our design objectives, and that the D-protein antagonist binds to the same region of VEGF-A that interacts with VEGFR1-domain 2.

RVX-297- a novel BD2 selective inhibitor of BET bromodomains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kharenko, Olesya A., E-mail: olesya@zenithepigenetics.com; Gesner, Emily M.; Patel, Reena G.

Bromodomains are epigenetic readers that specifically bind to the acetyl lysine residues of histones and transcription factors. Small molecule BET bromodomain inhibitors can disrupt this interaction which leads to potential modulation of several disease states. Here we describe the binding properties of a novel BET inhibitor RVX-297 that is structurally related to the clinical compound RVX-208, currently undergoing phase III clinical trials for the treatment of cardiovascular diseases, but is distinctly different in its biological and pharmacokinetic profiles. We report that RVX-297 preferentially binds to the BD2 domains of the BET bromodomain and Extra Terminal (BET) family of protein. Wemore » demonstrate the differential binding modes of RVX-297 in BD1 and BD2 domains of BRD4 and BRD2 using X-ray crystallography, and describe the structural differences driving the BD2 selective binding of RVX-297. The isothermal titration calorimetry (ITC) data illustrate the related differential thermodynamics of binding of RVX-297 to single as well as dual BET bromodomains. - Highlights: • A novel inhibitor of BET bromodomains, RVX-297 is described. • The differential binding modes of RVX-297 in BD1 and BD2 domains of BRD4 and BRD2 using X-ray crystallography are described. • RVX-297 preferentially binds to the BD2 domains of the BET bromodomains. • The structural and thermodynamic properties of the BD2 selective binding of RVX-297 are characterized.« less

A Compact X-Ray System for Macromolecular Crystallography. 5

NASA Technical Reports Server (NTRS)

Gubarev, Mikhail; Ciszak, Ewa; Ponomarev, Igor; Joy, Marshall

2000-01-01

We describe the design and performance of a high flux x-ray system for macromolecular crystallography that combines a microfocus x-ray generator (40 gm FWHM spot size at a power level of 46.5Watts) and a 5.5 mm focal distance polycapillary optic. The Cu K(sub alpha) X-ray flux produced by this optimized system is 7.0 times above the X-ray flux previously reported. The X-ray flux from the microfocus system is also 3.2 times higher than that produced by the rotating anode generator equipped with a long focal distance graded multilayer monochromator (Green optic; CMF24-48-Cu6) and 30% less than that produced by the rotating anode generator with the newest design of graded multilayer monochromator (Blue optic; CMF12-38-Cu6). Both rotating anode generators operate at a power level of 5000 Watts, dissipating more than 100 times the power of our microfocus x-ray system. Diffraction data collected from small test crystals are of high quality. For example, 42,540 reflections collected at ambient temperature from a lysozyme crystal yielded R(sub sym) 5.0% for the data extending to 1.7A, and 4.8% for the complete set of data to 1.85A. The amplitudes of the reflections were used to calculate difference electron density maps that revealed positions of structurally important ions and water molecules in the crystal of lysozyme using the phases calculated from the protein model.

Plasma Theory and Simulation

DTIC Science & Technology

1988-06-30

equation using finite difference methods. The distribution function is represented by a large number of particles. The particle’s velocities change as a...Small angle Coulomb collisions The FP equation for describing small angle Coulomb collisions can be solved numerically using finite difference techniques...A finite Fourrier transform (FT) is made in z, then we can solve for each k using the following finite difference scheme [5]: 2{r 1 +l1 2 (,,+ 1 - fj

Kinoform optics applied to X-ray photon correlation spectroscopy.

PubMed

Sandy, A R; Narayanan, S; Sprung, M; Su, J-D; Evans-Lutterodt, K; Isakovic, A F; Stein, A

2010-05-01

Moderate-demagnification higher-order silicon kinoform focusing lenses have been fabricated to facilitate small-angle X-ray photon correlation spectroscopy (XPCS) experiments. The geometric properties of such lenses, their focusing performance and their applicability for XPCS measurements are described. It is concluded that one-dimensional vertical X-ray focusing via silicon kinoform lenses significantly increases the usable coherent flux from third-generation storage-ring light sources for small-angle XPCS experiments.

Study of High Temperature Failure Mechanisms in Ceramics

DTIC Science & Technology

1988-06-01

The major experimental 4 techniques employed in the program are the use of small- angle neutron scattering to characterize cavity nucleation and growth...creep crackgrowth. Of particular interest are the development of a stochastic model of grainboundary sliding and a micromechanical model that relates...Accession For NTIS GF.A&I DTIC T,’ IDi st ribut Ion’ ;i Avillii~diii l l= (~~ I. RESEARCH OBJECTIVES I. Utilize small- angle neutron scattering to

Local texture and strongly linked conduction in spray-pyrolyzed TlBa2Ca2Cu3O(8+x) deposits

NASA Astrophysics Data System (ADS)

Kroeger, D. M.; Goyal, A.; Specht, E. D.; Wang, Z. L.; Tkaczyk, J. E.; Sutliff, J. A.; Deluca, J. A.

Local texture in polycrystalline TlBa2Ca2 Cu3O(8+x) deposits has been determined from transmission electron microscopy, electron backscatter diffraction patterns and x-ray diffraction. The small-grained deposits had excellent c-axis alignment and contained colonies of grains with similar but not identical a-axis orientations. Most grain boundaries within a colony have small misorientation angles and should not be weak links. It is proposed that long range conduction utilizes a percolative network of small angle grain boundaries at colony intersections.

Study of insertion force and deformation for suturing with precurved NiTi guidewire.

PubMed

Wang, Yancheng; Chen, Roland K; Tai, Bruce L; Xu, Kai; Shih, Albert J

2015-04-01

This research presents an experimental study evaluating stomach suturing using a precurved nickel-titanium (NiTi) guidewire for an endoscopic minimally invasive obesity treatment. Precise path planning is critical for accurate and effective suturing. A position measurement system utilizing a hand-held magnetic sensor was used to measure the shape of a precurved guidewire and to determine the radius of curvature before and after suturing. Ex vivo stomach suturing experiments using four different guidewire tip designs varying the radius of curvature and bevel angles were conducted. The changes in radius of curvature and suturing force during suturing were measured. A model was developed to predict the guidewire radius of curvature based on the measured suturing force. Results show that a small bevel angle and a large radius of curvature reduce the suturing force and the combination of small bevel angle and small radius of curvature can maintain the shape of guidewire for accurate suturing.

Effective phase function of light scattered at small angles by polydisperse particulate media

NASA Astrophysics Data System (ADS)

Turcu, I.

2008-06-01

Particles with typical dimensions higher than the light wavelength and relative refraction indexes close to one, scatter light mainly in the forward direction where the scattered light intensity has a narrow peak. For particulate media accomplishing these requirements the light scattered at small angles in a far-field detecting set-up can be described analytically by an effective phase function (EPF) even in the multiple scattering regime. The EPF model which was built for monodispersed systems has been extended to polydispersed media. The main ingredients consist in the replacement of the single particle phase function and of the optical thickness with their corresponding averaged values. Using a Gamma particle size distribution (PSD) as a testing model, the effect of polydispersity was systematically investigated. The increase of the average radius or/and of the PSD standard deviation leads to the decrease of the angular spreading of the small angle scattered light.

Emission-angle and polarization-rotation effects in the lensed CMB

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lewis, Antony; Hall, Alex; Challinor, Anthony, E-mail: antony@cosmologist.info, E-mail: ahall@roe.ac.uk, E-mail: a.d.challinor@ast.cam.ac.uk

Lensing of the CMB is an important effect, and is usually modelled by remapping the unlensed CMB fields by a lensing deflection. However the lensing deflections also change the photon path so that the emission angle is no longer orthogonal to the background last-scattering surface. We give the first calculation of the emission-angle corrections to the standard lensing approximation from dipole (Doppler) sources for temperature and quadrupole sources for temperature and polarization. We show that while the corrections are negligible for the temperature and E-mode polarization, additional large-scale B-modes are produced with a white spectrum that dominates those from post-Bornmore » field rotation (curl lensing). On large scales about one percent of the total lensing-induced B-mode amplitude is expected to be due to this effect. However, the photon emission angle does remain orthogonal to the perturbed last-scattering surface due to time delay, and half of the large-scale emission-angle B modes cancel with B modes from time delay to give a total contribution of about half a percent. While not important for planned observations, the signal could ultimately limit the ability of delensing to reveal low amplitudes of primordial gravitational waves. We also derive the rotation of polarization due to multiple deflections between emission and observation. The rotation angle is of quadratic order in the deflection angle, and hence negligibly small: polarization typically rotates by less than an arcsecond, orders of magnitude less than a small-scale image rotates due to post-Born field rotation (which is quadratic in the shear). The field-rotation B modes dominate the other effects on small scales.« less

Angles between orthogonal spd bond orbitals with maximum strength*

PubMed Central

Pauling, Linus

1976-01-01

An equation is derived for values of bond angles for two equivalent best spd hybrid bond orbitals with given amounts of s, p, and d character, and is applied in the discussion of structures of transargononic compounds, including the xenon and halogen fluorides. Bond orbitals with a rather small amount of d character tend to lie at angles 90° and 180°, and those with a larger amount, at somewhat smaller angles. PMID:16592315

Applications of X-Ray Micro-Beam for Data Collection.

PubMed

Sanishvili, Ruslan; Fischetti, Robert F

2017-01-01

Micro-diffraction tools for macromolecular crystallography, first developed at the end of 1990s and now an integral part of many synchrotron beamlines, enable some of the experiments which were not feasible just a decade or so ago. These include data collection from very small samples, just a few micrometers in size; from larger, but severely inhomogeneous samples; and from samples which are optically invisible. Improved micro-diffraction tools led to improved signal-to-noise ratio, to mitigation of radiation damage in some cases, and to better-designed diffraction experiments. Small, micron-scale beams can be attained in different ways and knowing the details of the implementation is important in order to design the diffraction experiment properly. Similarly, precision, reproducibility and stability of the goniometry, and caveats of detection systems need to be taken into account. Lastly, to make micro-diffraction widely applicable, the sophistication, robustness, and user-friendliness of these tools are just as important as the technical capabilities.

Structure-based design, synthesis and crystallization of 2-arylquinazolines as lipid pocket ligands of p38α MAPK

PubMed Central

Bührmann, Mike; Wiedemann, Bianca M.; Müller, Matthias P.; Hardick, Julia; Ecke, Maria

2017-01-01

In protein kinase research, identifying and addressing small molecule binding sites other than the highly conserved ATP-pocket are of intense interest because this line of investigation extends our understanding of kinase function beyond the catalytic phosphotransfer. Such alternative binding sites may be involved in altering the activation state through subtle conformational changes, control cellular enzyme localization, or in mediating and disrupting protein-protein interactions. Small organic molecules that target these less conserved regions might serve as tools for chemical biology research and to probe alternative strategies in targeting protein kinases in disease settings. Here, we present the structure-based design and synthesis of a focused library of 2-arylquinazoline derivatives to target the lipophilic C-terminal binding pocket in p38α MAPK, for which a clear biological function has yet to be identified. The interactions of the ligands with p38α MAPK was analyzed by SPR measurements and validated by protein X-ray crystallography. PMID:28892510

Structural basis of ligand interaction with atypical chemokine receptor 3

NASA Astrophysics Data System (ADS)

Gustavsson, Martin; Wang, Liwen; van Gils, Noortje; Stephens, Bryan S.; Zhang, Penglie; Schall, Thomas J.; Yang, Sichun; Abagyan, Ruben; Chance, Mark R.; Kufareva, Irina; Handel, Tracy M.

2017-01-01

Chemokines drive cell migration through their interactions with seven-transmembrane (7TM) chemokine receptors on cell surfaces. The atypical chemokine receptor 3 (ACKR3) binds chemokines CXCL11 and CXCL12 and signals exclusively through β-arrestin-mediated pathways, without activating canonical G-protein signalling. This receptor is upregulated in numerous cancers making it a potential drug target. Here we collected over 100 distinct structural probes from radiolytic footprinting, disulfide trapping, and mutagenesis to map the structures of ACKR3:CXCL12 and ACKR3:small-molecule complexes, including dynamic regions that proved unresolvable by X-ray crystallography in homologous receptors. The data are integrated with molecular modelling to produce complete and cohesive experimentally driven models that confirm and expand on the existing knowledge of the architecture of receptor:chemokine and receptor:small-molecule complexes. Additionally, we detected and characterized ligand-induced conformational changes in the transmembrane and intracellular regions of ACKR3 that elucidate fundamental structural elements of agonism in this atypical receptor.

One-Micron Beams for Macromolecular Crystallography at GM/CA-CAT

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoder, D. W.; Sanishvili, R.; Xu, S.

2010-06-23

GM/CA-CAT has developed a 1-{mu}m beam for challenging micro-diffraction experiments with macromolecular crystals (e.g. small crystals) and for radiation damage studies. Reflective (Kirkpatrick-Baez mirrors) and diffractive (Fresnel zone plates) optics have been used to focus the beam. Both cases are constrained by the need to maintain a small beam convergence. Using two different zone plates, 1.0x1.0 and 0.8x0.9 {mu}m{sup 2} (VxH,FWHM) beams were created at 15.2 keV and 18.5 keV, respectively. Additionally, by introducing a vertical focusing mirror upstream of the zone plate, a line focus at 15.2 keV was created (28x1.4 {mu}m{sup 2} VxH,FWHM) with the line oriented perpendicularmore » to the X-ray polarization and the crystal rotation axis. Crystal-mounting stages with nanometer resolution have been assembled to profile these beams and to perform diffraction experiments.« less