The development of structural x-ray crystallography

NASA Astrophysics Data System (ADS)

Woolfson, M. M.

2018-03-01

From its birth in 1912, when only the simplest structures could be solved, x-ray structural crystallography is now able to solve macromolecular structures containing many thousands of independent non-hydrogen atoms. This progress has depended on, and been driven by, great technical advances in the development of powerful synchrotron x-ray sources, advanced automated equipment for the collection and storage of large data sets and powerful computers to deal with everything from data processing to running programmes employing complex algorithms for the automatic solution of structures. The sheer number of developments in the subject over the past century makes it impossible for this review to be exhaustive, but it will describe some major developments that will enable the reader to understand how the subject has grown from its humble beginnings to what it is today.

Ultrasonic acoustic levitation for fast frame rate X-ray protein crystallography at room temperature.

PubMed

Tsujino, Soichiro; Tomizaki, Takashi

2016-05-06

Increasing the data acquisition rate of X-ray diffraction images for macromolecular crystals at room temperature at synchrotrons has the potential to significantly accelerate both structural analysis of biomolecules and structure-based drug developments. Using lysozyme model crystals, we demonstrated the rapid acquisition of X-ray diffraction datasets by combining a high frame rate pixel array detector with ultrasonic acoustic levitation of protein crystals in liquid droplets. The rapid spinning of the crystal within a levitating droplet ensured an efficient sampling of the reciprocal space. The datasets were processed with a program suite developed for serial femtosecond crystallography (SFX). The structure, which was solved by molecular replacement, was found to be identical to the structure obtained by the conventional oscillation method for up to a 1.8-Å resolution limit. In particular, the absence of protein crystal damage resulting from the acoustic levitation was carefully established. These results represent a key step towards a fully automated sample handling and measurement pipeline, which has promising prospects for a high acquisition rate and high sample efficiency for room temperature X-ray crystallography.

Ultrasonic acoustic levitation for fast frame rate X-ray protein crystallography at room temperature

NASA Astrophysics Data System (ADS)

Tsujino, Soichiro; Tomizaki, Takashi

2016-05-01

Increasing the data acquisition rate of X-ray diffraction images for macromolecular crystals at room temperature at synchrotrons has the potential to significantly accelerate both structural analysis of biomolecules and structure-based drug developments. Using lysozyme model crystals, we demonstrated the rapid acquisition of X-ray diffraction datasets by combining a high frame rate pixel array detector with ultrasonic acoustic levitation of protein crystals in liquid droplets. The rapid spinning of the crystal within a levitating droplet ensured an efficient sampling of the reciprocal space. The datasets were processed with a program suite developed for serial femtosecond crystallography (SFX). The structure, which was solved by molecular replacement, was found to be identical to the structure obtained by the conventional oscillation method for up to a 1.8-Å resolution limit. In particular, the absence of protein crystal damage resulting from the acoustic levitation was carefully established. These results represent a key step towards a fully automated sample handling and measurement pipeline, which has promising prospects for a high acquisition rate and high sample efficiency for room temperature X-ray crystallography.

Ultrasonic acoustic levitation for fast frame rate X-ray protein crystallography at room temperature

PubMed Central

Tsujino, Soichiro; Tomizaki, Takashi

2016-01-01

Increasing the data acquisition rate of X-ray diffraction images for macromolecular crystals at room temperature at synchrotrons has the potential to significantly accelerate both structural analysis of biomolecules and structure-based drug developments. Using lysozyme model crystals, we demonstrated the rapid acquisition of X-ray diffraction datasets by combining a high frame rate pixel array detector with ultrasonic acoustic levitation of protein crystals in liquid droplets. The rapid spinning of the crystal within a levitating droplet ensured an efficient sampling of the reciprocal space. The datasets were processed with a program suite developed for serial femtosecond crystallography (SFX). The structure, which was solved by molecular replacement, was found to be identical to the structure obtained by the conventional oscillation method for up to a 1.8-Å resolution limit. In particular, the absence of protein crystal damage resulting from the acoustic levitation was carefully established. These results represent a key step towards a fully automated sample handling and measurement pipeline, which has promising prospects for a high acquisition rate and high sample efficiency for room temperature X-ray crystallography. PMID:27150272

X-ray crystallography

NASA Technical Reports Server (NTRS)

2001-01-01

X-rays diffracted from a well-ordered protein crystal create sharp patterns of scattered light on film. A computer can use these patterns to generate a model of a protein molecule. To analyze the selected crystal, an X-ray crystallographer shines X-rays through the crystal. Unlike a single dental X-ray, which produces a shadow image of a tooth, these X-rays have to be taken many times from different angles to produce a pattern from the scattered light, a map of the intensity of the X-rays after they diffract through the crystal. The X-rays bounce off the electron clouds that form the outer structure of each atom. A flawed crystal will yield a blurry pattern; a well-ordered protein crystal yields a series of sharp diffraction patterns. From these patterns, researchers build an electron density map. With powerful computers and a lot of calculations, scientists can use the electron density patterns to determine the structure of the protein and make a computer-generated model of the structure. The models let researchers improve their understanding of how the protein functions. They also allow scientists to look for receptor sites and active areas that control a protein's function and role in the progress of diseases. From there, pharmaceutical researchers can design molecules that fit the active site, much like a key and lock, so that the protein is locked without affecting the rest of the body. This is called structure-based drug design.

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.

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.

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

High Pressure X-Ray Crystallography With the Diamond Cell at NIST/NBS

PubMed Central

Piermarini, Gasper J.

2001-01-01

Scientists in the Crystallography Section at NIST/NBS made several outstanding contributions which greatly promoted the development and advancement of high pressure x-ray crystallography during the second-half of the 20th century. These milestone achievements or “firsts” included: (1) the invention of the lever-arm type diamond anvil cell (DAC) in 1958; (2) the development of DAC technology for powder x-ray diffraction at high pressure in 1960; (3) the introduction of DAC methodology for single crystal x-ray diffraction at high pressure in 1964; (4) the invention of the optical fluorescence ruby method of pressure measurement in 1971; and (5) the discovery of hydrostatic pressure-transmitting media useful to unprecedented pressures for that time. These achievements provided the spark that ignited the explosion of activity in high pressure research that occurred in laboratories throughout the world during the latter part of the 20th century. It is still going on, unabated, today. An estimated 5000 DACs were built during the last 40 years. PMID:27500054

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.

Probing the Complex Architecture of Multimodular Carbohydrate-Active Enzymes Using a Combination of Small Angle X-Ray Scattering and X-Ray Crystallography.

PubMed

Czjzek, Mirjam; Ficko-Blean, Elizabeth

2017-01-01

The various modules in multimodular carbohydrate-active enzymes (CAZymes) may function in catalysis, carbohydrate binding, protein-protein interactions or as linkers. Here, we describe how combining the biophysical techniques of Small Angle X-ray Scattering (SAXS) and macromolecular X-ray crystallography (XRC) provides a powerful tool for examination into questions related to overall structural organization of ultra multimodular CAZymes.

A Compact X-Ray System for Macromolecular Crystallography

NASA Technical Reports Server (NTRS)

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

2000-01-01

We describe the design and performance of a high flux x-ray system for a macromolecular crystallography that combines a microfocus x-ray generator (40 micrometer full width at half maximum spot size at a power level of 46.5 W) and a collimating polycapillary optic. The Cu Ka lpha x-ray flux produced by this optimized system through a 500,um diam orifice is 7.0 times greater than the x-ray flux previously reported by Gubarev et al. [M. Gubarev et al., J. Appl. Crystallogr. 33, 882 (2000)]. The x-ray flux from the microfocus system is also 2.6 times higher than that produced by a rotating anode generator equipped with a graded multilayer monochromator (green optic, Osmic Inc. CMF24-48-Cu6) and 40% less than that produced by a rotating anode generator with the newest design of graded multilayer monochromator (blue optic, Osmic, Inc. CMF12-38-Cu6). Both rotating anode generators operate at a power level of 5000 W, 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 data extending to 1.70 A, and 4.8% for the complete set of data to 1.85 A. The amplitudes of the observed 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.

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.

Cell-free protein synthesis for structure determination by X-ray crystallography.

PubMed

Watanabe, Miki; Miyazono, Ken-ichi; Tanokura, Masaru; Sawasaki, Tatsuya; Endo, Yaeta; Kobayashi, Ichizo

2010-01-01

Structure determination has been difficult for those proteins that are toxic to the cells and cannot be prepared in a large amount in vivo. These proteins, even when biologically very interesting, tend to be left uncharacterized in the structural genomics projects. Their cell-free synthesis can bypass the toxicity problem. Among the various cell-free systems, the wheat-germ-based system is of special interest due to the following points: (1) Because the gene is placed under a plant translational signal, its toxic expression in a bacterial host is reduced. (2) It has only little codon preference and, especially, little discrimination between methionine and selenomethionine (SeMet), which allows easy preparation of selenomethionylated proteins for crystal structure determination by SAD and MAD methods. (3) Translation is uncoupled from transcription, so that the toxicity of the translation product on DNA and its transcription, if any, can be bypassed. We have shown that the wheat-germ-based cell-free protein synthesis is useful for X-ray crystallography of one of the 4-bp cutter restriction enzymes, which are expected to be very toxic to all forms of cells retaining the genome. Our report on its structure represents the first report of structure determination by X-ray crystallography using protein overexpressed with the wheat-germ-based cell-free protein expression system. This will be a method of choice for cytotoxic proteins when its cost is not a problem. Its use will become popular when the crystal structure determination technology has evolved to require only a tiny amount of protein.

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.

Time-Resolved Macromolecular Crystallography at Modern X-Ray Sources.

PubMed

Schmidt, Marius

2017-01-01

Time-resolved macromolecular crystallography unifies protein structure determination with chemical kinetics. With the advent of fourth generation X-ray sources the time-resolution can be on the order of 10-40 fs, which opens the ultrafast time scale to structure determination. Fundamental motions and transitions associated with chemical reactions in proteins can now be observed. Moreover, new experimental approaches at synchrotrons allow for the straightforward investigation of all kind of reactions in biological macromolecules. Here, recent developments in the field are reviewed.

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.

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

Accessing protein conformational ensembles using room-temperature X-ray crystallography

PubMed Central

Fraser, James S.; van den Bedem, Henry; Samelson, Avi J.; Lang, P. Therese; Holton, James M.; Echols, Nathaniel; Alber, Tom

2011-01-01

Modern protein crystal structures are based nearly exclusively on X-ray data collected at cryogenic temperatures (generally 100 K). The cooling process is thought to introduce little bias in the functional interpretation of structural results, because cryogenic temperatures minimally perturb the overall protein backbone fold. In contrast, here we show that flash cooling biases previously hidden structural ensembles in protein crystals. By analyzing available data for 30 different proteins using new computational tools for electron-density sampling, model refinement, and molecular packing analysis, we found that crystal cryocooling remodels the conformational distributions of more than 35% of side chains and eliminates packing defects necessary for functional motions. In the signaling switch protein, H-Ras, an allosteric network consistent with fluctuations detected in solution by NMR was uncovered in the room-temperature, but not the cryogenic, electron-density maps. These results expose a bias in structural databases toward smaller, overpacked, and unrealistically unique models. Monitoring room-temperature conformational ensembles by X-ray crystallography can reveal motions crucial for catalysis, ligand binding, and allosteric regulation. PMID:21918110

Coded diffraction system in X-ray crystallography using a boolean phase coded aperture approximation

NASA Astrophysics Data System (ADS)

Pinilla, Samuel; Poveda, Juan; Arguello, Henry

2018-03-01

Phase retrieval is a problem present in many applications such as optics, astronomical imaging, computational biology and X-ray crystallography. Recent work has shown that the phase can be better recovered when the acquisition architecture includes a coded aperture, which modulates the signal before diffraction, such that the underlying signal is recovered from coded diffraction patterns. Moreover, this type of modulation effect, before the diffraction operation, can be obtained using a phase coded aperture, just after the sample under study. However, a practical implementation of a phase coded aperture in an X-ray application is not feasible, because it is computationally modeled as a matrix with complex entries which requires changing the phase of the diffracted beams. In fact, changing the phase implies finding a material that allows to deviate the direction of an X-ray beam, which can considerably increase the implementation costs. Hence, this paper describes a low cost coded X-ray diffraction system based on block-unblock coded apertures that enables phase reconstruction. The proposed system approximates the phase coded aperture with a block-unblock coded aperture by using the detour-phase method. Moreover, the SAXS/WAXS X-ray crystallography software was used to simulate the diffraction patterns of a real crystal structure called Rhombic Dodecahedron. Additionally, several simulations were carried out to analyze the performance of block-unblock approximations in recovering the phase, using the simulated diffraction patterns. Furthermore, the quality of the reconstructions was measured in terms of the Peak Signal to Noise Ratio (PSNR). Results show that the performance of the block-unblock phase coded apertures approximation decreases at most 12.5% compared with the phase coded apertures. Moreover, the quality of the reconstructions using the boolean approximations is up to 2.5 dB of PSNR less with respect to the phase coded aperture reconstructions.

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.

Hit detection in serial femtosecond crystallography using X-ray spectroscopy of plasma emission.

PubMed

Jönsson, H Olof; Caleman, Carl; Andreasson, Jakob; Tîmneanu, Nicuşor

2017-11-01

Serial femtosecond crystallography is an emerging and promising method for determining protein structures, making use of the ultrafast and bright X-ray pulses from X-ray free-electron lasers. The upcoming X-ray laser sources will produce well above 1000 pulses per second and will pose a new challenge: how to quickly determine successful crystal hits and avoid a high-rate data deluge. Proposed here is a hit-finding scheme based on detecting photons from plasma emission after the sample has been intercepted by the X-ray laser. Plasma emission spectra are simulated for systems exposed to high-intensity femtosecond pulses, for both protein crystals and the liquid carrier systems that are used for sample delivery. The thermal radiation from the glowing plasma gives a strong background in the XUV region that depends on the intensity of the pulse, around the emission lines from light elements (carbon, nitrogen, oxygen). Sample hits can be reliably distinguished from the carrier liquid based on the characteristic emission lines from heavier elements present only in the sample, such as sulfur. For buffer systems with sulfur present, selenomethionine substitution is suggested, where the selenium emission lines could be used both as an indication of a hit and as an aid in phasing and structural reconstruction of the protein.

FreeDam - A webtool for free-electron laser-induced damage in femtosecond X-ray crystallography

NASA Astrophysics Data System (ADS)

Jönsson, H. Olof; Östlin, Christofer; Scott, Howard A.; Chapman, Henry N.; Aplin, Steve J.; Tîmneanu, Nicuşor; Caleman, Carl

2018-03-01

Over the last decade X-ray free-electron laser (XFEL) sources have been made available to the scientific community. One of the most successful uses of these new machines has been protein crystallography. When samples are exposed to the intense short X-ray pulses provided by the XFELs, the sample quickly becomes highly ionized and the atomic structure is affected. Here we present a webtool dubbed FreeDam based on non-thermal plasma simulations, for estimation of radiation damage in free-electron laser experiments in terms of ionization, temperatures and atomic displacements. The aim is to make this tool easily accessible to scientists who are planning and performing experiments at XFELs.

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

UV-Visible Absorption Spectroscopy Enhanced X-ray Crystallography at Synchrotron and X-ray Free Electron Laser Sources.

PubMed

Cohen, Aina E; Doukov, Tzanko; Soltis, Michael S

2016-01-01

This review describes the use of single crystal UV-Visible Absorption micro-Spectrophotometry (UV-Vis AS) to enhance the design and execution of X-ray crystallography experiments for structural investigations of reaction intermediates of redox active and photosensitive proteins. Considerations for UV-Vis AS measurements at the synchrotron and associated instrumentation are described. UV-Vis AS is useful to verify the intermediate state of an enzyme and to monitor the progression of reactions within crystals. Radiation induced redox changes within protein crystals may be monitored to devise effective diffraction data collection strategies. An overview of the specific effects of radiation damage on macromolecular crystals is presented along with data collection strategies that minimize these effects by combining data from multiple crystals used at the synchrotron and with the X-ray free electron laser.

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

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

Study on four polymorphs of bifendate based on X-ray crystallography.

PubMed

Nie, Jinju; Yang, Dezhi; Hu, Kun; Lu, Yang

2016-05-01

Bifendate, a synthetic anti-hepatitis drug, exhibits polycrystalline mode phenomena with 2 polymorphs reported (forms A and B). Single crystals of the known crystalline form B and 3 new crystallosolvates involving bifendate solvated with tetrahydrofuran (C), dioxane (D), and pyridine (E) in a stoichiometric ratio of 1:1 were obtained and characterized by X-ray crystallography, thermal analysis, and Fourier transform infrared (FT-IR) spectroscopy. The differences in molecular conformation, intermolecular interaction and crystal packing arrangement for the four polymorphs were determined and the basis for the polymorphisms was investigated. The rotation of single bonds resulted in different orientations for the biphenyl, methyl ester and methoxyl groups. All guest solvent molecules interacted with the host molecule via an interesting intercalative mode along the [1 0 0] direction in the channel formed by the host molecules through weak aromatic stacking interactions or non-classical hydrogen bonds, of which the volume and planarity played an important role in the intercalation of the host with the guest. The incorporation of solvent-augmented rotation of the C-C bond of the biphenyl group had a striking effect on the host molecular conformation and contributed to the formation of bifendate polymorphs. Moreover, the simulated powder X-ray diffraction (PXRD) patterns for each form were calculated on the basis of the single-crystal data and proved to be unique. The single-crystal structures of the four crystalline forms are reported in this paper.

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.

Integrated description of protein dynamics from room-temperature X-ray crystallography and NMR

PubMed Central

Fenwick, R. Bryn; van den Bedem, Henry; Fraser, James S.; Wright, Peter E.

2014-01-01

Detailed descriptions of atomic coordinates and motions are required for an understanding of protein dynamics and their relation to molecular recognition, catalytic function, and allostery. Historically, NMR relaxation measurements have played a dominant role in the determination of the amplitudes and timescales (picosecond–nanosecond) of bond vector fluctuations, whereas high-resolution X-ray diffraction experiments can reveal the presence of and provide atomic coordinates for multiple, weakly populated substates in the protein conformational ensemble. Here we report a hybrid NMR and X-ray crystallography analysis that provides a more complete dynamic picture and a more quantitative description of the timescale and amplitude of fluctuations in atomic coordinates than is obtainable from the individual methods alone. Order parameters (S2) were calculated from single-conformer and multiconformer models fitted to room temperature and cryogenic X-ray diffraction data for dihydrofolate reductase. Backbone and side-chain order parameters derived from NMR relaxation experiments are in excellent agreement with those calculated from the room-temperature single-conformer and multiconformer models, showing that the picosecond timescale motions observed in solution occur also in the crystalline state. These motions are quenched in the crystal at cryogenic temperatures. The combination of NMR and X-ray crystallography in iterative refinement promises to provide an atomic resolution description of the alternate conformational substates that are sampled through picosecond to nanosecond timescale fluctuations of the protein structure. The method also provides insights into the structural heterogeneity of nonmethyl side chains, aromatic residues, and ligands, which are less commonly analyzed by NMR relaxation measurements. PMID:24474795

Curved position-sensitive detector for X-ray crystallography

NASA Astrophysics Data System (ADS)

Izumi, T.

1980-11-01

A new curved position-sensitive proportional detector has been constructed for X-ray crystallography. A very hard steel wire 0.2 mm in diameter was used as a single anode wire. It was bent to a radius of 6.5 cm and was suspended elastically in a wide 160° 2θ angular aperture. An amplifier and ADC-per-cathode strip system was made in order to encode the position. The spatial resolution is better than 0.37 mm (fwhm) along the curved anode wire, and this value corresponds to an angular resolution of 0.28° in 2θ. It is shown that a thick hard anode wire is quite suitable for use as a curved position-sensitive detector.

Automated identification of functional dynamic networks from X-ray crystallography

PubMed Central

van den Bedem, Henry; Bhabha, Gira; Yang, Kun; Wright, Peter E.; Fraser, James S.

2013-01-01

Protein function often depends on the exchange between conformational substates. Allosteric ligand binding or distal mutations can stabilize specific active site conformations and consequently alter protein function. In addition to comparing independently determined X-ray crystal structures, alternative conformations observed at low levels of electron density have the potential to provide mechanistic insights into conformational dynamics. Here, we report a new multi-conformer contact network algorithm (CONTACT) that identifies networks of conformationally heterogeneous residues directly from high-resolution X-ray crystallography data. Contact networks in Escherichia coli dihydrofolate reductase (ecDHFR) predict the long-range pattern of NMR chemical shift perturbations of an allosteric mutation. A comparison of contact networks in wild type and mutant ecDHFR suggests how mutations that alter optimized networks of coordinated motions can impair catalytic function. Thus, CONTACT-guided mutagenesis will allow the structure-dynamics-function relationship to be exploited in protein engineering and design. PMID:23913260

Metalloprotein structures at ambient conditions and in real-time: biological crystallography and spectroscopy using X-ray free electron lasers

DOE PAGES

Kern, Jan; Yachandra, Vittal K.; Yano, Junko

2015-09-02

We have studied the structure of enzymes and the chemistry at the catalytic sites, intensively and have acquired an understanding of the atomic-scale chemistry which requires a new approach beyond steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the geometric and electronic structure of metallo-enzymes at ambient conditions, while overcoming the severe X-ray-induced changes to the redox active catalytic center, is key for deriving reaction mechanisms. Such studies become possible by the intense and ultra-short femtosecond (fs) X-ray pulses from an X-ray free electron laser (XFEL) by acquiring a signal before the samplemore » is destroyed. Our review describes the recent and pioneering uses of XFELs to study the protein structure and dynamics of metallo-enzymes using crystallography and scattering, as well as the chemical structure and dynamics of the catalytic complexes (charge, spin, and covalency) using spectroscopy during the reaction to understand the electron-transfer processes and elucidate the mechanism.« less

Metalloprotein structures at ambient conditions and in real-time: biological crystallography and spectroscopy using X-ray free electron lasers

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

Kern, Jan; Yachandra, Vittal K.; Yano, Junko

We have studied the structure of enzymes and the chemistry at the catalytic sites, intensively and have acquired an understanding of the atomic-scale chemistry which requires a new approach beyond steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the geometric and electronic structure of metallo-enzymes at ambient conditions, while overcoming the severe X-ray-induced changes to the redox active catalytic center, is key for deriving reaction mechanisms. Such studies become possible by the intense and ultra-short femtosecond (fs) X-ray pulses from an X-ray free electron laser (XFEL) by acquiring a signal before the samplemore » is destroyed. Our review describes the recent and pioneering uses of XFELs to study the protein structure and dynamics of metallo-enzymes using crystallography and scattering, as well as the chemical structure and dynamics of the catalytic complexes (charge, spin, and covalency) using spectroscopy during the reaction to understand the electron-transfer processes and elucidate the mechanism.« less

Femtosecond crystallography with ultrabright electrons and x-rays: capturing chemistry in action.

PubMed

Miller, R J Dwayne

2014-03-07

With the recent advances in ultrabright electron and x-ray sources, it is now possible to extend crystallography to the femtosecond time domain to literally light up atomic motions involved in the primary processes governing structural transitions. This review chronicles the development of brighter and brighter electron and x-ray sources that have enabled atomic resolution to structural dynamics for increasingly complex systems. The primary focus is on achieving sufficient brightness using pump-probe protocols to resolve the far-from-equilibrium motions directing chemical processes that in general lead to irreversible changes in samples. Given the central importance of structural transitions to conceptualizing chemistry, this emerging field has the potential to significantly improve our understanding of chemistry and its connection to driving biological processes.

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.

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.

In meso in situ serial X-ray crystallography of soluble and membrane proteins

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

Huang, Chia-Ying; Olieric, Vincent; Ma, Pikyee

A method for performing high-throughput in situ serial X-ray crystallography with soluble and membrane proteins in the lipid cubic phase is described. It works with microgram quantities of protein and lipid (and ligand when present) and is compatible with the most demanding sulfur SAD phasing. The lipid cubic phase (LCP) continues to grow in popularity as a medium in which to generate crystals of membrane (and soluble) proteins for high-resolution X-ray crystallographic structure determination. To date, the PDB includes 227 records attributed to the LCP or in meso method. Among the listings are some of the highest profile membrane proteins,more » including the β{sub 2}-adrenoreceptor–G{sub s} protein complex that figured in the award of the 2012 Nobel Prize in Chemistry to Lefkowitz and Kobilka. The most successful in meso protocol to date uses glass sandwich crystallization plates. Despite their many advantages, glass plates are challenging to harvest crystals from. However, performing in situ X-ray diffraction measurements with these plates is not practical. Here, an alternative approach is described that provides many of the advantages of glass plates and is compatible with high-throughput in situ measurements. The novel in meso in situ serial crystallography (IMISX) method introduced here has been demonstrated with AlgE and PepT (alginate and peptide transporters, respectively) as model integral membrane proteins and with lysozyme as a test soluble protein. Structures were solved by molecular replacement and by experimental phasing using bromine SAD and native sulfur SAD methods to resolutions ranging from 1.8 to 2.8 Å using single-digit microgram quantities of protein. That sulfur SAD phasing worked is testament to the exceptional quality of the IMISX diffraction data. The IMISX method is compatible with readily available, inexpensive materials and equipment, is simple to implement and is compatible with high-throughput in situ serial data collection at

X-Ray Lasers

ERIC Educational Resources Information Center

Chapline, George; Wood, Lowell

1975-01-01

Outlines the prospects of generating coherent x rays using high-power lasers and indentifies problem areas in their development. Indicates possible applications for coherent x rays in the fields of chemistry, biology, and crystallography. (GS)

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.

Combining NMR and X-ray crystallography in fragment-based drug discovery: discovery of highly potent and selective BACE-1 inhibitors.

PubMed

Wyss, Daniel F; Wang, Yu-Sen; Eaton, Hugh L; Strickland, Corey; Voigt, Johannes H; Zhu, Zhaoning; Stamford, Andrew W

2012-01-01

Fragment-based drug discovery (FBDD) has become increasingly popular over the last decade. We review here how we have used highly structure-driven fragment-based approaches to complement more traditional lead discovery to tackle high priority targets and those struggling for leads. Combining biomolecular nuclear magnetic resonance (NMR), X-ray crystallography, and molecular modeling with structure-assisted chemistry and innovative biology as an integrated approach for FBDD can solve very difficult problems, as illustrated in this chapter. Here, a successful FBDD campaign is described that has allowed the development of a clinical candidate for BACE-1, a challenging CNS drug target. Crucial to this achievement were the initial identification of a ligand-efficient isothiourea fragment through target-based NMR screening and the determination of its X-ray crystal structure in complex with BACE-1, which revealed an extensive H-bond network with the two active site aspartate residues. This detailed 3D structural information then enabled the design and validation of novel, chemically stable and accessible heterocyclic acylguanidines as aspartic acid protease inhibitor cores. Structure-assisted fragment hit-to-lead optimization yielded iminoheterocyclic BACE-1 inhibitors that possess desirable molecular properties as potential therapeutic agents to test the amyloid hypothesis of Alzheimer's disease in a clinical setting.

Substrate specificity of pyrimidine nucleoside phosphorylases of NP-II family probed by X-ray crystallography and molecular modeling

NASA Astrophysics Data System (ADS)

Balaev, V. V.; Lashkov, A. A.; Prokofev, I. I.; Gabdulkhakov, A. G.; Seregina, T. A.; Mironov, A. S.; Betzel, C.; Mikhailov, A. M.

2016-09-01

Pyrimidine nucleoside phosphorylases, which are widely used in the biotechnological production of nucleosides, have different substrate specificity for pyrimidine nucleosides. An interesting feature of these enzymes is that the three-dimensional structure of thymidine-specific nucleoside phosphorylase is similar to the structure of nonspecific pyrimidine nucleoside phosphorylase. The three-dimensional structures of thymidine phosphorylase from Salmonella typhimurium and nonspecific pyrimidine nucleoside phosphorylase from Bacillus subtilis in complexes with a sulfate anion were determined for the first time by X-ray crystallography. An analysis of the structural differences between these enzymes demonstrated that Lys108, which is involved in the phosphate binding in pyrimidine nucleoside phosphorylase, corresponds to Met111 in thymidine phosphorylases. This difference results in a decrease in the charge on one of the hydroxyl oxygens of the phosphate anion in thymidine phosphorylase and facilitates the catalysis through SN2 nucleophilic substitution. Based on the results of X-ray crystallography, the virtual screening was performed for identifying a potent inhibitor (anticancer agent) of nonspecific pyrimidine nucleoside phosphorylase, which does not bind to thymidine phosphorylase. The molecular dynamics simulation revealed the stable binding of the discovered compound—2-pyrimidin-2-yl-1H-imidazole-4-carboxylic acid—to the active site of pyrimidine nucleoside phosphorylase.

Graphene-based microfluidics for serial crystallography.

PubMed

Sui, Shuo; Wang, Yuxi; Kolewe, Kristopher W; Srajer, Vukica; Henning, Robert; Schiffman, Jessica D; Dimitrakopoulos, Christos; Perry, Sarah L

2016-08-02

Microfluidic strategies to enable the growth and subsequent serial crystallographic analysis of micro-crystals have the potential to facilitate both structural characterization and dynamic structural studies of protein targets that have been resistant to single-crystal strategies. However, adapting microfluidic crystallization platforms for micro-crystallography requires a dramatic decrease in the overall device thickness. We report a robust strategy for the straightforward incorporation of single-layer graphene into ultra-thin microfluidic devices. This architecture allows for a total material thickness of only ∼1 μm, facilitating on-chip X-ray diffraction analysis while creating a sample environment that is stable against significant water loss over several weeks. We demonstrate excellent signal-to-noise in our X-ray diffraction measurements using a 1.5 μs polychromatic X-ray exposure, and validate our approach via on-chip structure determination using hen egg white lysozyme (HEWL) as a model system. Although this work is focused on the use of graphene for protein crystallography, we anticipate that this technology should find utility in a wide range of both X-ray and other lab on a chip applications.

X-Ray Crystallography Reagent

NASA Technical Reports Server (NTRS)

Morrison, Dennis R. (Inventor); Mosier, Benjamin (Inventor)

2003-01-01

Microcapsules prepared by encapsulating an aqueous solution of a protein, drug or other bioactive substance inside a semi-permeable membrane by are disclosed. The microcapsules are formed by interfacial coacervation under conditions where the shear forces are limited to 0-100 dynes per square centimeter at the interface. By placing the microcapsules in a high osmotic dewatering solution. the protein solution is gradually made saturated and then supersaturated. and the controlled nucleation and crystallization of the protein is achieved. The crystal-filled microcapsules prepared by this method can be conveniently harvested and stored while keeping the encapsulated crystals in essentially pristine condition due to the rugged. protective membrane. Because the membrane components themselves are x-ray transparent, large crystal-containing microcapsules can be individually selected, mounted in x-ray capillary tubes and subjected to high energy x-ray diffraction studies to determine the 3-D smucture of the protein molecules. Certain embodiments of the microcapsules of the invention have composite polymeric outer membranes which are somewhat elastic, water insoluble, permeable only to water, salts, and low molecular weight molecules and are structurally stable in fluid shear forces typically encountered in the human vascular system.

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.

High-Resolution Detector For X-Ray Diffraction

NASA Technical Reports Server (NTRS)

Carter, Daniel C.; Withrow, William K.; Pusey, Marc L.; Yost, Vaughn H.

1988-01-01

Proposed x-ray-sensitive imaging detector offers superior spatial resolution, counting-rate capacity, and dynamic range. Instrument based on laser-stimulated luminescence and reusable x-ray-sensitive film. Detector scans x-ray film line by line. Extracts latent image in film and simultaneously erases film for reuse. Used primarily for protein crystallography. Principle adapted to imaging detectors for electron microscopy and fluorescence spectroscopy and general use in astronomy, engineering, and medicine.

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

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.

Extending X-Ray Crystallography to Allow the Imaging of Noncrystalline Materials, Cells, and Single Protein Complexes

NASA Astrophysics Data System (ADS)

Miao, Jianwei; Ishikawa, Tetsuya; Shen, Qun; Earnest, Thomas

2008-05-01

In 1999, researchers extended X-ray crystallography to allow the imaging of noncrystalline specimens by measuring the X-ray diffraction pattern of a noncrystalline specimen and then directly phasing it using the oversampling method with iterative algorithms. Since then, the field has evolved moving in three important directions. The first is the 3D structural determination of noncrystalline materials, which includes the localization of the defects and strain field inside nanocrystals, and quantitative 3D imaging of disordered materials such as nanoparticles and biomaterials. The second is the 3D imaging of frozen-hydrated whole cells at a resolution of 10 nm or better. A main thrust is to localize specific multiprotein complexes inside cells. The third is the potential of imaging single large protein complexes using extremely intense and ultrashort X-ray pulses. In this article, we review the principles of this methodology, summarize recent developments in each of the three directions, and illustrate a few examples.

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

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

High-throughput plasmid construction using homologous recombination in yeast: its mechanisms and application to protein production for X-ray crystallography.

PubMed

Mizutani, Kimihiko

2015-01-01

Homologous recombination is a system for repairing the broken genomes of living organisms by connecting two DNA strands at their homologous sequences. Today, homologous recombination in yeast is used for plasmid construction as a substitute for traditional methods using restriction enzymes and ligases. This method has various advantages over the traditional method, including flexibility in the position of DNA insertion and ease of manipulation. Recently, the author of this review reported the construction of plasmids by homologous recombination in the methanol-utilizing yeast Pichia pastoris, which is known to be an excellent expression host for secretory proteins and membrane proteins. The method enabled high-throughput construction of expression systems of proteins using P. pastoris; the constructed expression systems were used to investigate the expression conditions of membrane proteins and to perform X-ray crystallography of secretory proteins. This review discusses the mechanisms and applications of homologous recombination, including the production of proteins for X-ray crystallography.

X-ray free electron lasers motivate bioanalytical characterization of protein nanocrystals: serial femtosecond crystallography.

PubMed

Bogan, Michael J

2013-04-02

Atomic resolution structures of large biomacromolecular complexes can now be recorded at room temperature from crystals with submicrometer dimensions using intense femtosecond pulses delivered by the world's largest and most powerful X-ray machine, a laser called the Linac Coherent Light Source. Abundant opportunities exist for the bioanalytical sciences to help extend this revolutionary advance in structural biology to the ultimate goal of recording molecular-movies of noncrystalline biomacromolecules. This Feature will introduce the concept of serial femtosecond crystallography to the nonexpert, briefly review progress to date, and highlight some potential contributions from the analytical sciences.

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

Combined analysis of 1,3-benzodioxoles by crystalline sponge X-ray crystallography and laser desorption ionization mass spectrometry.

PubMed

Hayashi, Yukako; Ohara, Kazuaki; Taki, Rika; Saeki, Tomomi; Yamaguchi, Kentaro

2018-03-12

The crystalline sponge (CS) method, which employs single-crystal X-ray diffraction to determine the structure of an analyte present as a liquid or an oil and having a low melting point, was used in combination with laser desorption ionization mass spectrometry (LDI-MS). 1,3-Benzodioxole derivatives were encapsulated in CS and their structures were determined by combining X-ray crystallography and MS. After the X-ray analysis, the CS was subjected to imaging mass spectrometry (IMS) with an LDI spiral-time-of-flight mass spectrometer (TOF-MS). The ion detection area matched the microscopic image of the encapsulated CS. In addition, the accumulated 1D mass spectra showed that fragmentation of the guest molecule (hereafter, guest) can be easily visualized without any interference from the fragment ions of CS except for two strong ion peaks derived from the tridentate ligand TPT (2,4,6-tris(4-pyridyl)-1,3,5-triazine) of the CS and its fragment. X-ray analysis clearly showed the presence of the guest as well as the π-π, CH-halogen, and CH-O interactions between the guest and the CS framework. However, some guests remained randomly diffused in the nanopores of CS. In addition, the detection limit was less than sub-pmol order based on the weight and density of CS determined by X-ray analysis. Spectroscopic data, such as UV-vis and NMR, also supported the encapsulation of the guest through the interaction between the guest and CS components. The results denote that the CS-LDI-MS method, which combines CS, X-ray analysis and LDI-MS, is effective for structure determination.

Watching proteins function with 150-ps time-resolved X-ray crystallography

NASA Astrophysics Data System (ADS)

Anfinrud, Philip

2007-03-01

We have used time-resolved Laue crystallography to characterize ligand migration pathways and dynamics in wild-type and several mutant forms of myoglobin (Mb), a ligand-binding heme protein found in muscle tissue. In these pump-probe experiments, which were conducted on the ID09B time-resolved beamline at the European Synchrotron and Radiation Facility, a laser pulse photodissociates CO from an MbCO crystal and a suitably delayed X-ray pulse probes its structure via Laue diffraction. Single-site mutations in the vicinity of the heme pocket docking site were found to have a dramatic effect on ligand migration. To visualize this process, time-resolved electron density maps were stitched together into movies that unveil with <2-å spatial resolution and 150-ps time-resolution the correlated protein motions that accompany and/or mediate ligand migration. These studies help to illustrate at an atomic level relationships between protein structure, dynamics, and function.

Accounting for partiality in serial crystallography using ray-tracing principles

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

Kroon-Batenburg, Loes M. J., E-mail: l.m.j.kroon-batenburg@uu.nl; Schreurs, Antoine M. M.; Ravelli, Raimond B. G.

Serial crystallography generates partial reflections from still diffraction images. Partialities are estimated with EVAL ray-tracing simulations, thereby improving merged reflection data to a similar quality as conventional rotation data. Serial crystallography generates ‘still’ diffraction data sets that are composed of single diffraction images obtained from a large number of crystals arbitrarily oriented in the X-ray beam. Estimation of the reflection partialities, which accounts for the expected observed fractions of diffraction intensities, has so far been problematic. In this paper, a method is derived for modelling the partialities by making use of the ray-tracing diffraction-integration method EVAL. The method estimates partialitiesmore » based on crystal mosaicity, beam divergence, wavelength dispersion, crystal size and the interference function, accounting for crystallite size. It is shown that modelling of each reflection by a distribution of interference-function weighted rays yields a ‘still’ Lorentz factor. Still data are compared with a conventional rotation data set collected from a single lysozyme crystal. Overall, the presented still integration method improves the data quality markedly. The R factor of the still data compared with the rotation data decreases from 26% using a Monte Carlo approach to 12% after applying the Lorentz correction, to 5.3% when estimating partialities by EVAL and finally to 4.7% after post-refinement. The merging R{sub int} factor of the still data improves from 105 to 56% but remains high. This suggests that the accuracy of the model parameters could be further improved. However, with a multiplicity of around 40 and an R{sub int} of ∼50% the merged still data approximate the quality of the rotation data. The presented integration method suitably accounts for the partiality of the observed intensities in still diffraction data, which is a critical step to improve data quality in serial crystallography.« less

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

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.…

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.

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

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

Structural investigation of oxovanadium(IV) Schiff base complexes: X-ray crystallography, electrochemistry and kinetic of thermal decomposition.

PubMed

Asadi, Mozaffar; Asadi, Zahra; Savaripoor, Nooshin; Dusek, Michal; Eigner, Vaclav; Shorkaei, Mohammad Ranjkesh; Sedaghat, Moslem

2015-02-05

A series of new VO(IV) complexes of tetradentate N2O2 Schiff base ligands (L(1)-L(4)), were synthesized and characterized by FT-IR, UV-vis and elemental analysis. The structure of the complex VOL(1)⋅DMF was also investigated by X-ray crystallography which revealed a vanadyl center with distorted octahedral coordination where the 2-aza and 2-oxo coordinating sites of the ligand were perpendicular to the "-yl" oxygen. The electrochemical properties of the vanadyl complexes were investigated by cyclic voltammetry. A good correlation was observed between the oxidation potentials and the electron withdrawing character of the substituents on the Schiff base ligands, showing the following trend: MeO5-H>5-Br>5-Cl. Furthermore, the kinetic parameters of thermal decomposition were calculated by using the Coats-Redfern equation. According to the Coats-Redfern plots the kinetics of thermal decomposition of studied complexes is of the first-order in all stages, the free energy of activation for each following stage is larger than the previous one and the complexes have good thermal stability. The preparation of VOL(1)⋅DMF yielded also another compound, one kind of vanadium oxide [VO]X, with different habitus of crystals, (platelet instead of prisma) and without L(1) ligand, consisting of a V10O28 cage, diaminium moiety and dimethylamonium as a counter ions. Because its crystal structure was also new, we reported it along with the targeted complex. Copyright © 2014 Elsevier B.V. All rights reserved.

Phosphor Scanner For Imaging X-Ray Diffraction

NASA Technical Reports Server (NTRS)

Carter, Daniel C.; Hecht, Diana L.; Witherow, William K.

1992-01-01

Improved optoelectronic scanning apparatus generates digitized image of x-ray image recorded in phosphor. Scanning fiber-optic probe supplies laser light stimulating luminescence in areas of phosphor exposed to x rays. Luminescence passes through probe and fiber to integrating sphere and photomultiplier. Sensitivity and resolution exceed previously available scanners. Intended for use in x-ray crystallography, medical radiography, and molecular biology.

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

The O2-Evolving Complex of Photosystem II: Recent Insights from Quantum Mechanics/Molecular Mechanics (QM/MM), Extended X-ray Absorption Fine Structure (EXAFS), and Femtosecond X-ray Crystallography Data.

PubMed

Askerka, Mikhail; Brudvig, Gary W; Batista, Victor S

2017-01-17

Efficient photoelectrochemical water oxidation may open a way to produce energy from renewable solar power. In biology, generation of fuel due to water oxidation happens efficiently on an immense scale during the light reactions of photosynthesis. To oxidize water, photosynthetic organisms have evolved a highly conserved protein complex, Photosystem II. Within that complex, water oxidation happens at the CaMn 4 O 5 inorganic catalytic cluster, the so-called oxygen-evolving complex (OEC), which cycles through storage "S" states as it accumulates oxidizing equivalents and produces molecular oxygen. In recent years, there has been significant progress in understanding the OEC as it evolves through the catalytic cycle. Studies have combined conventional and femtosecond X-ray crystallography with extended X-ray absorption fine structure (EXAFS) and quantum mechanics/molecular mechanics (QM/MM) methods and have addressed changes in protonation states of μ-oxo bridges and the coordination of substrate water through the analysis of ammonia binding as a chemical analog of water. These advances are thought to be critical to understanding the catalytic cycle since protonation states regulate the relative stability of different redox states and the geometry of the OEC. Therefore, establishing the mechanism for substrate water binding and the nature of protonation/redox state transitions in the OEC is essential for understanding the catalytic cycle of O 2 evolution. The structure of the dark-stable S 1 state has been a target for X-ray crystallography for the past 15 years. However, traditional X-ray crystallography has been hampered by radiation-induced reduction of the OEC. Very recently, a revolutionary X-ray free electron laser (XFEL) technique was applied to PSII to reveal atomic positions at 1.95 Å without radiation damage, which brought us closer than ever to establishing the ultimate structure of the OEC in the S 1 state. However, the atom positions in this crystal

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

Insight into small molecule binding to the neonatal Fc receptor by X-ray crystallography and 100 kHz magic-angle-spinning NMR

PubMed Central

Macpherson, Alex; Smith-Penzel, Susanne; Basse, Nicolas; Lecomte, Fabien; Deboves, Hervé; Taylor, Richard D.; Norman, Tim; Porter, John; Waters, Lorna C.; Westwood, Marta; Cossins, Ben; Cain, Katharine; White, James; Griffin, Robert; Prosser, Christine; Kelm, Sebastian; Sullivan, Amy H.; Fox, David; Carr, Mark D.; Henry, Alistair; Taylor, Richard; Meier, Beat H.; Oschkinat, Hartmut; Lawson, Alastair D.

2018-01-01

Aiming at the design of an allosteric modulator of the neonatal Fc receptor (FcRn)–Immunoglobulin G (IgG) interaction, we developed a new methodology including NMR fragment screening, X-ray crystallography, and magic-angle-spinning (MAS) NMR at 100 kHz after sedimentation, exploiting very fast spinning of the nondeuterated soluble 42 kDa receptor construct to obtain resolved proton-detected 2D and 3D NMR spectra. FcRn plays a crucial role in regulation of IgG and serum albumin catabolism. It is a clinically validated drug target for the treatment of autoimmune diseases caused by pathogenic antibodies via the inhibition of its interaction with IgG. We herein present the discovery of a small molecule that binds into a conserved cavity of the heterodimeric, extracellular domain composed of an α-chain and β2-microglobulin (β2m) (FcRnECD, 373 residues). X-ray crystallography was used alongside NMR at 100 kHz MAS with sedimented soluble protein to explore possibilities for refining the compound as an allosteric modulator. Proton-detected MAS NMR experiments on fully protonated [13C,15N]-labeled FcRnECD yielded ligand-induced chemical-shift perturbations (CSPs) for residues in the binding pocket and allosteric changes close to the interface of the two receptor heterodimers present in the asymmetric unit as well as potentially in the albumin interaction site. X-ray structures with and without ligand suggest the need for an optimized ligand to displace the α-chain with respect to β2m, both of which participate in the FcRnECD–IgG interaction site. Our investigation establishes a method to characterize structurally small molecule binding to nondeuterated large proteins by NMR, even in their glycosylated form, which may prove highly valuable for structure-based drug discovery campaigns. PMID:29782488

Insight into small molecule binding to the neonatal Fc receptor by X-ray crystallography and 100 kHz magic-angle-spinning NMR.

PubMed

Stöppler, Daniel; Macpherson, Alex; Smith-Penzel, Susanne; Basse, Nicolas; Lecomte, Fabien; Deboves, Hervé; Taylor, Richard D; Norman, Tim; Porter, John; Waters, Lorna C; Westwood, Marta; Cossins, Ben; Cain, Katharine; White, James; Griffin, Robert; Prosser, Christine; Kelm, Sebastian; Sullivan, Amy H; Fox, David; Carr, Mark D; Henry, Alistair; Taylor, Richard; Meier, Beat H; Oschkinat, Hartmut; Lawson, Alastair D

2018-05-01

Aiming at the design of an allosteric modulator of the neonatal Fc receptor (FcRn)-Immunoglobulin G (IgG) interaction, we developed a new methodology including NMR fragment screening, X-ray crystallography, and magic-angle-spinning (MAS) NMR at 100 kHz after sedimentation, exploiting very fast spinning of the nondeuterated soluble 42 kDa receptor construct to obtain resolved proton-detected 2D and 3D NMR spectra. FcRn plays a crucial role in regulation of IgG and serum albumin catabolism. It is a clinically validated drug target for the treatment of autoimmune diseases caused by pathogenic antibodies via the inhibition of its interaction with IgG. We herein present the discovery of a small molecule that binds into a conserved cavity of the heterodimeric, extracellular domain composed of an α-chain and β2-microglobulin (β2m) (FcRnECD, 373 residues). X-ray crystallography was used alongside NMR at 100 kHz MAS with sedimented soluble protein to explore possibilities for refining the compound as an allosteric modulator. Proton-detected MAS NMR experiments on fully protonated [13C,15N]-labeled FcRnECD yielded ligand-induced chemical-shift perturbations (CSPs) for residues in the binding pocket and allosteric changes close to the interface of the two receptor heterodimers present in the asymmetric unit as well as potentially in the albumin interaction site. X-ray structures with and without ligand suggest the need for an optimized ligand to displace the α-chain with respect to β2m, both of which participate in the FcRnECD-IgG interaction site. Our investigation establishes a method to characterize structurally small molecule binding to nondeuterated large proteins by NMR, even in their glycosylated form, which may prove highly valuable for structure-based drug discovery campaigns.

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.

New carbocyclic nucleoside analogues with a bicyclo[2.2.1]heptane fragment as sugar moiety; synthesis, X-ray crystallography and anticancer activity.

PubMed

Tănase, Constantin I; Drăghici, Constantin; Căproiu, Miron Teodor; Shova, Sergiu; Mathe, Christophe; Cocu, Florea G; Enache, Cristian; Maganu, Maria

2014-01-01

An amine group was synthesized starting from an optically active bicyclo[2.2.1]heptane compound, which was then used to build the 5 atoms ring of a key 6-chloropurine intermediate. This was then reacted with ammonia and selected amines obtaining new adenine- and 6-substituted adenine conformationally constrained carbocyclic nucleoside analogues with a bicyclo[2.2.1]heptane skeleton in the sugar moiety. X-ray crystallography confirmed an exo-coupling of base to the ring and a L configuration of the nucleoside analogues. The compounds were tested for anticancer activity. Copyright © 2013 Elsevier Ltd. 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

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

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.

1,3-Oxazole-based selective picomolar inhibitors of cytosolic human carbonic anhydrase II alleviate ocular hypertension in rabbits: Potency is supported by X-ray crystallography of two leads.

PubMed

Ferraroni, Marta; Lucarini, Laura; Masini, Emanuela; Korsakov, Mikhail; Scozzafava, Andrea; Supuran, Claudiu T; Krasavin, Mikhail

2017-09-01

Two lead 1,3-oxazole-based carbonic anhydrase inhibitors (CAIs) earlier identified as selective, picomolar inhibitors of hCA II (a cytosolic target for treatment of glaucoma) have been investigated further. Firstly, they were found to be conveniently synthesized on multigram scale, which enables further development. These compounds were found to be comparable in efficacy to dorzolamide eye drops when applied in the eye drop form as well. Finally, the reasons for unusually high potency of these compounds became understood from their high-resolution X-ray crystallography structures. These data significantly expand our understanding of heterocycle-based primary sulfonamides, many of which have recently emerged from our labs - particularly, from the corneal permeability standpoint. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Small-Angle X-ray Scattering (SAXS) Instrument Performance and Validation Using Silver Nanoparticles

DTIC Science & Technology

2016-12-01

Intercalibration of small-angle X- Ray and neutron-scattering data. Journal of Applied Crystallography . 1988;21:629–638. 7. Zhang F, Ilavsky J, Long GG...Materials Transactions A. 2009;41:1151–1158. 8. Kusz J, Bohm H. Performance of a confocal multilayer X-ray optic. Journal of Applied Crystallography ...Journal of Applied Crystallography . 2004;37:369–380. 10. Orthaber D, Bergmann A, Glatter O. SAXS experiments on absolute scale with Kratky systems using

Small Angle X ray Scattering (SAXS) Instrument Performance and Validation Using Silver Nanoparticles

DTIC Science & Technology

2016-12-01

Intercalibration of small-angle X- Ray and neutron-scattering data. Journal of Applied Crystallography . 1988;21:629–638. 7. Zhang F, Ilavsky J, Long GG...Materials Transactions A. 2009;41:1151–1158. 8. Kusz J, Bohm H. Performance of a confocal multilayer X-ray optic. Journal of Applied Crystallography ...Journal of Applied Crystallography . 2004;37:369–380. 10. Orthaber D, Bergmann A, Glatter O. SAXS experiments on absolute scale with Kratky systems using

Insights into the mechanism of X-ray-induced disulfide-bond cleavage in lysozyme crystals based on EPR, optical absorption and X-ray diffraction studies

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

Sutton, Kristin A.; Black, Paul J.; Mercer, Kermit R.

2013-12-01

Electron paramagnetic resonance (EPR) and online UV–visible absorption microspectrophotometry with X-ray crystallography have been used in a complementary manner to follow X-ray-induced disulfide-bond cleavage, to confirm a multi-track radiation-damage process and to develop a model of that process. Electron paramagnetic resonance (EPR) and online UV–visible absorption microspectrophotometry with X-ray crystallography have been used in a complementary manner to follow X-ray-induced disulfide-bond cleavage. Online UV–visible spectroscopy showed that upon X-irradiation, disulfide radicalization appeared to saturate at an absorbed dose of approximately 0.5–0.8 MGy, in contrast to the saturating dose of ∼0.2 MGy observed using EPR at much lower dose rates. Themore » observations suggest that a multi-track model involving product formation owing to the interaction of two separate tracks is a valid model for radiation damage in protein crystals. The saturation levels are remarkably consistent given the widely different experimental parameters and the range of total absorbed doses studied. The results indicate that even at the lowest doses used for structural investigations disulfide bonds are already radicalized. Multi-track considerations offer the first step in a comprehensive model of radiation damage that could potentially lead to a combined computational and experimental approach to identifying when damage is likely to be present, to quantitate it and to provide the ability to recover the native unperturbed structure.« less

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.

Room temperature neutron crystallography of drug resistant HIV-1 protease uncovers limitations of X-ray structural analysis at 100K

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

Gerlits, Oksana O.; Keen, David A.; Blakeley, Matthew P.

HIV-1 protease inhibitors are crucial for treatment of HIV-1/AIDS, but their effectiveness is thwarted by rapid emergence of drug resistance. To better understand binding of clinical inhibitors to resistant HIV-1 protease, we used room-temperature joint X-ray/neutron (XN) crystallography to obtain an atomic-resolution structure of the protease triple mutant (V32I/I47V/V82I) in complex with amprenavir. The XN structure reveals a D+ ion located midway between the inner Oδ1 oxygen atoms of the catalytic aspartic acid residues. Comparison of the current XN structure with our previous XN structure of the wild-type HIV-1 protease-amprenavir complex suggests that the three mutations do not significantly altermore » the drug–enzyme interactions. This is in contrast to the observations in previous 100 K X-ray structures of these complexes that indicated loss of interactions by the drug with the triple mutant protease. These findings, thus, uncover limitations of structural analysis of drug binding using X-ray structures obtained at 100 K.« less

Room temperature neutron crystallography of drug resistant HIV-1 protease uncovers limitations of X-ray structural analysis at 100K

DOE PAGES

Gerlits, Oksana O.; Keen, David A.; Blakeley, Matthew P.; ...

2017-02-14

HIV-1 protease inhibitors are crucial for treatment of HIV-1/AIDS, but their effectiveness is thwarted by rapid emergence of drug resistance. To better understand binding of clinical inhibitors to resistant HIV-1 protease, we used room-temperature joint X-ray/neutron (XN) crystallography to obtain an atomic-resolution structure of the protease triple mutant (V32I/I47V/V82I) in complex with amprenavir. The XN structure reveals a D+ ion located midway between the inner Oδ1 oxygen atoms of the catalytic aspartic acid residues. Comparison of the current XN structure with our previous XN structure of the wild-type HIV-1 protease-amprenavir complex suggests that the three mutations do not significantly altermore » the drug–enzyme interactions. This is in contrast to the observations in previous 100 K X-ray structures of these complexes that indicated loss of interactions by the drug with the triple mutant protease. These findings, thus, uncover limitations of structural analysis of drug binding using X-ray structures obtained at 100 K.« less

Life in the fast lane for protein crystallization and X-ray crystallography

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Liu, Zhi-Jie; Tempel, Wolfram; Praissman, Jeremy; Lin, Dawei; Wang, Bi-Cheng; Gavira, Jose A.; Ng, Joseph D.

2005-01-01

The common goal for structural genomic centers and consortiums is to decipher as quickly as possible the three-dimensional structures for a multitude of recombinant proteins derived from known genomic sequences. Since X-ray crystallography is the foremost method to acquire atomic resolution for macromolecules, the limiting step is obtaining protein crystals that can be useful of structure determination. High-throughput methods have been developed in recent years to clone, express, purify, crystallize and determine the three-dimensional structure of a protein gene product rapidly using automated devices, commercialized kits and consolidated protocols. However, the average number of protein structures obtained for most structural genomic groups has been very low compared to the total number of proteins purified. As more entire genomic sequences are obtained for different organisms from the three kingdoms of life, only the proteins that can be crystallized and whose structures can be obtained easily are studied. Consequently, an astonishing number of genomic proteins remain unexamined. In the era of high-throughput processes, traditional methods in molecular biology, protein chemistry and crystallization are eclipsed by automation and pipeline practices. The necessity for high-rate production of protein crystals and structures has prevented the usage of more intellectual strategies and creative approaches in experimental executions. Fundamental principles and personal experiences in protein chemistry and crystallization are minimally exploited only to obtain "low-hanging fruit" protein structures. We review the practical aspects of today's high-throughput manipulations and discuss the challenges in fast pace protein crystallization and tools for crystallography. Structural genomic pipelines can be improved with information gained from low-throughput tactics that may help us reach the higher-bearing fruits. Examples of recent developments in this area are reported from

Life in the Fast Lane for Protein Crystallization and X-Ray Crystallography

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Liu, Zhi-Jie; Tempel, Wolfram; Praissman, Jeremy; Lin, Dawei; Wang, Bi-Cheng; Gavira, Jose A.; Ng, Joseph D.

2004-01-01

The common goal for structural genomic centers and consortiums is to decipher as quickly as possible the three-dimensional structures for a multitude of recombinant proteins derived from known genomic sequences. Since X-ray crystallography is the foremost method to acquire atomic resolution for macromolecules, the limiting step is obtaining protein crystals that can be useful of structure determination. High-throughput methods have been developed in recent years to clone, express, purify, crystallize and determine the three-dimensional structure of a protein gene product rapidly using automated devices, commercialized kits and consolidated protocols. However, the average number of protein structures obtained for most structural genomic groups has been very low compared to the total number of proteins purified. As more entire genomic sequences are obtained for different organisms from the three kingdoms of life, only the proteins that can be crystallized and whose structures can be obtained easily are studied. Consequently, an astonishing number of genomic proteins remain unexamined. In the era of high-throughput processes, traditional methods in molecular biology, protein chemistry and crystallization are eclipsed by automation and pipeline practices. The necessity for high rate production of protein crystals and structures has prevented the usage of more intellectual strategies and creative approaches in experimental executions. Fundamental principles and personal experiences in protein chemistry and crystallization are minimally exploited only to obtain "low-hanging fruit" protein structures. We review the practical aspects of today s high-throughput manipulations and discuss the challenges in fast pace protein crystallization and tools for crystallography. Structural genomic pipelines can be improved with information gained from low-throughput tactics that may help us reach the higher-bearing fruits. Examples of recent developments in this area are reported from

Deformable complex network for refining low-resolution X-ray structures

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

Zhang, Chong; Wang, Qinghua; Ma, Jianpeng, E-mail: jpma@bcm.edu

2015-10-27

A new refinement algorithm called the deformable complex network that combines a novel angular network-based restraint with a deformable elastic network model in the target function has been developed to aid in structural refinement in macromolecular X-ray crystallography. In macromolecular X-ray crystallography, building more accurate atomic models based on lower resolution experimental diffraction data remains a great challenge. Previous studies have used a deformable elastic network (DEN) model to aid in low-resolution structural refinement. In this study, the development of a new refinement algorithm called the deformable complex network (DCN) is reported that combines a novel angular network-based restraint withmore » the DEN model in the target function. Testing of DCN on a wide range of low-resolution structures demonstrated that it constantly leads to significantly improved structural models as judged by multiple refinement criteria, thus representing a new effective refinement tool for low-resolution structural determination.« less

The O 2 -Evolving Complex of Photosystem II: Recent Insights from Quantum Mechanics/Molecular Mechanics (QM/MM), Extended X-ray Absorption Fine Structure (EXAFS), and Femtosecond X-ray Crystallography Data

DOE PAGES

Askerka, Mikhail; Brudvig, Gary W.; Batista, Victor S.

2016-12-21

Efficient photoelectrochemical water oxidation may open a way to produce energy from renewable solar power. In biology, generation of fuel due to water oxidation happens efficiently on an immense scale during the light reactions of photosynthesis. To oxidize water, photosynthetic organisms have evolved a highly conserved protein complex, Photosystem II. Within that complex, water oxidation happens at the CaMn 4O 5 inorganic catalytic cluster, the so-called oxygen-evolving complex (OEC), which cycles through storage “S” states as it accumulates oxidizing equivalents and produces molecular oxygen. In recent years, there has been significant progress in understanding the OEC as it evolves throughmore » the catalytic cycle. Studies have combined conventional and femtosecond X-ray crystallography with extended X-ray absorption fine structure (EXAFS) and quantum mechanics/molecular mechanics (QM/ MM) methods and have addressed changes in protonation states of μ-oxo bridges and the coordination of substrate water through the analysis of ammonia binding as a chemical analog of water. These advances are thought to be critical to understanding the catalytic cycle since protonation states regulate the relative stability of different redox states and the geometry of the OEC. Therefore, establishing the mechanism for substrate water binding and the nature of protonation/redox state transitions in the OEC is essential for understanding the catalytic cycle of O 2 evolution. The structure of the dark-stable S1 state has been a target for X-ray crystallography for the past 15 years. However, traditional Xray crystallography has been hampered by radiation-induced reduction of the OEC. Very recently, a revolutionary X-ray free electron laser (XFEL) technique was applied to PSII to reveal atomic positions at 1.95 Å without radiation damage, which brought us closer than ever to establishing the ultimate structure of the OEC in the S 1 state. However, the atom positions in this

The O 2 -Evolving Complex of Photosystem II: Recent Insights from Quantum Mechanics/Molecular Mechanics (QM/MM), Extended X-ray Absorption Fine Structure (EXAFS), and Femtosecond X-ray Crystallography Data

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

Askerka, Mikhail; Brudvig, Gary W.; Batista, Victor S.

Efficient photoelectrochemical water oxidation may open a way to produce energy from renewable solar power. In biology, generation of fuel due to water oxidation happens efficiently on an immense scale during the light reactions of photosynthesis. To oxidize water, photosynthetic organisms have evolved a highly conserved protein complex, Photosystem II. Within that complex, water oxidation happens at the CaMn 4O 5 inorganic catalytic cluster, the so-called oxygen-evolving complex (OEC), which cycles through storage “S” states as it accumulates oxidizing equivalents and produces molecular oxygen. In recent years, there has been significant progress in understanding the OEC as it evolves throughmore » the catalytic cycle. Studies have combined conventional and femtosecond X-ray crystallography with extended X-ray absorption fine structure (EXAFS) and quantum mechanics/molecular mechanics (QM/ MM) methods and have addressed changes in protonation states of μ-oxo bridges and the coordination of substrate water through the analysis of ammonia binding as a chemical analog of water. These advances are thought to be critical to understanding the catalytic cycle since protonation states regulate the relative stability of different redox states and the geometry of the OEC. Therefore, establishing the mechanism for substrate water binding and the nature of protonation/redox state transitions in the OEC is essential for understanding the catalytic cycle of O 2 evolution. The structure of the dark-stable S1 state has been a target for X-ray crystallography for the past 15 years. However, traditional Xray crystallography has been hampered by radiation-induced reduction of the OEC. Very recently, a revolutionary X-ray free electron laser (XFEL) technique was applied to PSII to reveal atomic positions at 1.95 Å without radiation damage, which brought us closer than ever to establishing the ultimate structure of the OEC in the S 1 state. However, the atom positions in this

Synthesis, characterization, X-ray crystallography, acetyl cholinesterase inhibition and antioxidant activities of some novel ketone derivatives of gallic hydrazide-derived Schiff bases.

PubMed

Gwaram, Nura Suleiman; Ali, Hapipah Mohd; Abdulla, Mahmood Ameen; Buckle, Michael J C; Sukumaran, Sri Devi; Chung, Lip Yong; Othman, Rozana; Alhadi, Abeer A; Yehye, Wageeh A; Hadi, A Hamid A; Hassandarvish, Pouya; Khaledi, Hamid; Abdelwahab, Siddig Ibrahim

2012-02-28

Alzheimer's disease (AD) is the most common form of dementia among older people and the pathogenesis of this disease is associated with oxidative stress. Acetylcholinesterase inhibitors with antioxidant activities are considered potential treatments for AD. Some novel ketone derivatives of gallic hydrazide-derived Schiff bases were synthesized and examined for their antioxidant activities and in vitro and in silico acetyl cholinesterase inhibition. The compounds were characterized using spectroscopy and X-ray crystallography. The ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays revealed that all the compounds have strong antioxidant activities. N-(1-(5-bromo-2-hydroxyphenyl)-ethylidene)-3,4,5-trihydroxybenzohydrazide (2) was the most potent inhibitor of human acetyl cholinesterase, giving an inhibition rate of 77% at 100 μM. Molecular docking simulation of the ligand-enzyme complex suggested that the ligand may be positioned in the enzyme's active-site gorge, interacting with residues in the peripheral anionic subsite (PAS) and acyl binding pocket (ABP). The current work warrants further preclinical studies to assess the potential for these novel compounds for the treatment of AD.

a-Si:H TFT-silicon hybrid low-energy x-ray detector

DOE PAGES

Shin, Kyung -Wook; Karim, Karim S.

2017-03-15

Direct conversion crystalline silicon X-ray imagers are used for low-energy X-ray photon (4-20 keV) detection in scientific research applications such as protein crystallography. In this paper, we demonstrate a novel pixel architecture that integrates a crystalline silicon X-ray detector with a thin-film transistor amorphous silicon pixel readout circuit. We describe a simplified two-mask process to fabricate a complete imaging array and present preliminary results that show the fabricated pixel to be sensitive to 5.89-keV photons from a low activity Fe-55 gamma source. Furthermore, this paper presented can expedite the development of high spatial resolution, low cost, direct conversion imagers formore » X-ray diffraction and crystallography applications.« less

Enabling X-ray free electron laser crystallography for challenging biological systems from a limited number of crystals

PubMed Central

Uervirojnangkoorn, Monarin; Zeldin, Oliver B; Lyubimov, Artem Y; Hattne, Johan; Brewster, Aaron S; Sauter, Nicholas K; Brunger, Axel T; Weis, William I

2015-01-01

There is considerable potential for X-ray free electron lasers (XFELs) to enable determination of macromolecular crystal structures that are difficult to solve using current synchrotron sources. Prior XFEL studies often involved the collection of thousands to millions of diffraction images, in part due to limitations of data processing methods. We implemented a data processing system based on classical post-refinement techniques, adapted to specific properties of XFEL diffraction data. When applied to XFEL data from three different proteins collected using various sample delivery systems and XFEL beam parameters, our method improved the quality of the diffraction data as well as the resulting refined atomic models and electron density maps. Moreover, the number of observations for a reflection necessary to assemble an accurate data set could be reduced to a few observations. These developments will help expand the applicability of XFEL crystallography to challenging biological systems, including cases where sample is limited. DOI: http://dx.doi.org/10.7554/eLife.05421.001 PMID:25781634

Enabling X-ray free electron laser crystallography for challenging biological systems from a limited number of crystals

DOE PAGES

Uervirojnangkoorn, Monarin; Zeldin, Oliver B.; Lyubimov, Artem Y.; ...

2015-03-17

There is considerable potential for X-ray free electron lasers (XFELs) to enable determination of macromolecular crystal structures that are difficult to solve using current synchrotron sources. Prior XFEL studies often involved the collection of thousands to millions of diffraction images, in part due to limitations of data processing methods. We implemented a data processing system based on classical post-refinement techniques, adapted to specific properties of XFEL diffraction data. When applied to XFEL data from three different proteins collected using various sample delivery systems and XFEL beam parameters, our method improved the quality of the diffraction data as well as themore » resulting refined atomic models and electron density maps. Moreover, the number of observations for a reflection necessary to assemble an accurate data set could be reduced to a few observations. In conclusion, these developments will help expand the applicability of XFEL crystallography to challenging biological systems, including cases where sample is limited.« less

Enabling X-ray free electron laser crystallography for challenging biological systems from a limited number of crystals

DOE PAGES

Uervirojnangkoorn, Monarin; Zeldin, Oliver B.; Lyubimov, Artem Y.; ...

2015-03-17

There is considerable potential for X-ray free electron lasers (XFELs) to enable determination of macromolecular crystal structures that are difficult to solve using current synchrotron sources. Prior XFEL studies often involved the collection of thousands to millions of diffraction images, in part due to limitations of data processing methods. We implemented a data processing system based on classical post-refinement techniques, adapted to specific properties of XFEL diffraction data. When applied to XFEL data from three different proteins collected using various sample delivery systems and XFEL beam parameters, our method improved the quality of the diffraction data as well as themore » resulting refined atomic models and electron density maps. Moreover, the number of observations for a reflection necessary to assemble an accurate data set could be reduced to a few observations. These developments will help expand the applicability of XFEL crystallography to challenging biological systems, including cases where sample is limited.« less

Characterization of X-Ray Diffraction System with a Microfocus X-Ray Source and a Polycapillary Optic

NASA Technical Reports Server (NTRS)

Gubarev, Mikhail; Marshall, Joy K.; Ciszak, Ewa; Ponomarev, Igor

2000-01-01

We present here an optimized microfocus x-ray source and polycapillary optic system designed for diffraction of small protein crystals. The x-ray beam is formed by a 5.5mm focal length capillary collimator coupled with a 40 micron x-ray source operating at 46Watts. Measurements of the x-ray flux, the divergence and the spectral characteristics of the beam are presented, This optimized system provides a seven fold greater flux than our recently reported configuration [M. Gubarev, et al., J. of Applied Crystallography (2000) 33, in press]. We now make a comparison with a 5kWatts rotating anode generator (Rigaku) coupled with confocal multilayer focusing mirrors (Osmic, CMF12- 38Cu6). The microfocus x-ray source and polycapillary collimator system delivers 60% of the x-ray flux from the rotating anode system. Additional ways to improve our microfocus x-ray system, and thus increase the x-ray flux will be discussed.

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

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.

Native State Mass Spectrometry, Surface Plasmon Resonance, and X-ray Crystallography Correlate Strongly as a Fragment Screening Combination.

PubMed

Woods, Lucy A; Dolezal, Olan; Ren, Bin; Ryan, John H; Peat, Thomas S; Poulsen, Sally-Ann

2016-03-10

Fragment-based drug discovery (FBDD) is contingent on the development of analytical methods to identify weak protein-fragment noncovalent interactions. Herein we have combined an underutilized fragment screening method, native state mass spectrometry, together with two proven and popular fragment screening methods, surface plasmon resonance and X-ray crystallography, in a fragment screening campaign against human carbonic anhydrase II (CA II). In an initial fragment screen against a 720-member fragment library (the "CSIRO Fragment Library") seven CA II binding fragments, including a selection of nonclassical CA II binding chemotypes, were identified. A further 70 compounds that comprised the initial hit chemotypes were subsequently sourced from the full CSIRO compound collection and screened. The fragment results were extremely well correlated across the three methods. Our findings demonstrate that there is a tremendous opportunity to apply native state mass spectrometry as a complementary fragment screening method to accelerate drug discovery.

X-ray free electron laser: opportunities for drug discovery.

PubMed

Cheng, Robert K Y; Abela, Rafael; Hennig, Michael

2017-11-08

Past decades have shown the impact of structural information derived from complexes of drug candidates with their protein targets to facilitate the discovery of safe and effective medicines. Despite recent developments in single particle cryo-electron microscopy, X-ray crystallography has been the main method to derive structural information. The unique properties of X-ray free electron laser (XFEL) with unmet peak brilliance and beam focus allow X-ray diffraction data recording and successful structure determination from smaller and weaker diffracting crystals shortening timelines in crystal optimization. To further capitalize on the XFEL advantage, innovations in crystal sample delivery for the X-ray experiment, data collection and processing methods are required. This development was a key contributor to serial crystallography allowing structure determination at room temperature yielding physiologically more relevant structures. Adding the time resolution provided by the femtosecond X-ray pulse will enable monitoring and capturing of dynamic processes of ligand binding and associated conformational changes with great impact to the design of candidate drug compounds. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Bioactive Formylated Flavonoids from Eugenia rigida: Isolation, Synthesis, and X-ray Crystallography.

PubMed

Zaki, Mohamed A; Nanayakkara, N P Dhammika; Hetta, Mona H; Jacob, Melissa R; Khan, Shabana I; Mohammed, Rabab; Ibrahim, Mohamed A; Samoylenko, Volodymyr; Coleman, Christina; Fronczek, Frank R; Ferreira, Daneel; Muhammad, Ilias

2016-09-23

Two new flavonoids, rac-6-formyl-5,7-dihydroxyflavanone (1) and 2',6'-dihydroxy-4'-methoxy-3'-methylchalcone (2), together with five known derivatives, rac-8-formyl-5,7-dihydroxyflavanone (3), 4',6'-dihydroxy-2'-methoxy-3'-methyldihydrochalcone (4), rac-7-hydroxy-5-methoxy-6-methylflavanone (5), 3'-formyl-2',4',6'-trihydroxy-5'-methyldihydrochalcone (6), and 3'-formyl-2',4',6'-trihydroxydihydrochalcone (7), were isolated from the leaves of Eugenia rigida. The individual (S)- and (R)-enantiomers of 1 and 3, together with the corresponding formylated flavones 8 (6-formyl-5,7-dihydroxyflavone) and 9 (8-formyl-5,7-dihydroxyflavone), as well as 2',4',6'-trihydroxychalcone (10), 3'-formyl-2',4',6'-trihydroxychalcone (11), and the corresponding 3'-formyl-2',4',6'-trihydroxydihydrochalcone (7) and 2',4',6'-trihydroxydihydrochalcone (12), were synthesized. The structures of the isolated and synthetic compounds were established via NMR, HRESIMS, and electronic circular dichroism data. In addition, the structures of 3, 5, and 8 were confirmed by single-crystal X-ray diffraction crystallography. The isolated and synthetic flavonoids were evaluated for their antimicrobial and cytotoxic activities against a panel of microorganisms and solid tumor cell lines.

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

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.

Accounting for partiality in serial crystallography using ray-tracing principles

PubMed Central

Kroon-Batenburg, Loes M. J.; Schreurs, Antoine M. M.; Ravelli, Raimond B. G.; Gros, Piet

2015-01-01

Serial crystallography generates ‘still’ diffraction data sets that are composed of single diffraction images obtained from a large number of crystals arbitrarily oriented in the X-ray beam. Estimation of the reflection partialities, which accounts for the expected observed fractions of diffraction intensities, has so far been problematic. In this paper, a method is derived for modelling the partialities by making use of the ray-tracing diffraction-integration method EVAL. The method estimates partialities based on crystal mosaicity, beam divergence, wavelength dispersion, crystal size and the interference function, accounting for crystallite size. It is shown that modelling of each reflection by a distribution of interference-function weighted rays yields a ‘still’ Lorentz factor. Still data are compared with a conventional rotation data set collected from a single lysozyme crystal. Overall, the presented still integration method improves the data quality markedly. The R factor of the still data compared with the rotation data decreases from 26% using a Monte Carlo approach to 12% after applying the Lorentz correction, to 5.3% when estimating partialities by EVAL and finally to 4.7% after post-refinement. The merging R int factor of the still data improves from 105 to 56% but remains high. This suggests that the accuracy of the model parameters could be further improved. However, with a multiplicity of around 40 and an R int of ∼50% the merged still data approximate the quality of the rotation data. The presented integration method suitably accounts for the partiality of the observed intensities in still diffraction data, which is a critical step to improve data quality in serial crystallography. PMID:26327370

Accounting for partiality in serial crystallography using ray-tracing principles.

PubMed

Kroon-Batenburg, Loes M J; Schreurs, Antoine M M; Ravelli, Raimond B G; Gros, Piet

2015-09-01

Serial crystallography generates `still' diffraction data sets that are composed of single diffraction images obtained from a large number of crystals arbitrarily oriented in the X-ray beam. Estimation of the reflection partialities, which accounts for the expected observed fractions of diffraction intensities, has so far been problematic. In this paper, a method is derived for modelling the partialities by making use of the ray-tracing diffraction-integration method EVAL. The method estimates partialities based on crystal mosaicity, beam divergence, wavelength dispersion, crystal size and the interference function, accounting for crystallite size. It is shown that modelling of each reflection by a distribution of interference-function weighted rays yields a `still' Lorentz factor. Still data are compared with a conventional rotation data set collected from a single lysozyme crystal. Overall, the presented still integration method improves the data quality markedly. The R factor of the still data compared with the rotation data decreases from 26% using a Monte Carlo approach to 12% after applying the Lorentz correction, to 5.3% when estimating partialities by EVAL and finally to 4.7% after post-refinement. The merging R(int) factor of the still data improves from 105 to 56% but remains high. This suggests that the accuracy of the model parameters could be further improved. However, with a multiplicity of around 40 and an R(int) of ∼50% the merged still data approximate the quality of the rotation data. The presented integration method suitably accounts for the partiality of the observed intensities in still diffraction data, which is a critical step to improve data quality in serial crystallography.

Racemic crystallography of synthetic protein enantiomers used to determine the X-ray structure of plectasin by direct methods

PubMed Central

Mandal, Kalyaneswar; Pentelute, Brad L; Tereshko, Valentina; Thammavongsa, Vilasak; Schneewind, Olaf; Kossiakoff, Anthony A; Kent, Stephen B H

2009-01-01

We describe the use of racemic crystallography to determine the X-ray structure of the natural product plectasin, a potent antimicrobial protein recently isolated from fungus. The protein enantiomers l-plectasin and d-plectasin were prepared by total chemical synthesis; interestingly, l-plectasin showed the expected antimicrobial activity, while d-plectasin was devoid of such activity. The mirror image proteins were then used for racemic crystallization. Synchrotron X-ray diffraction data were collected to atomic resolution from a racemic plectasin crystal; the racemate crystallized in the achiral centrosymmetric space group with one l-plectasin molecule and one d-plectasin molecule forming the unit cell. Dimer-like intermolecular interactions between the protein enantiomers were observed, which may account for the observed extremely low solvent content (13%–15%) and more highly ordered nature of the racemic crystals. The structure of the plectasin molecule was well defined for all 40 amino acids and was generally similar to the previously determined NMR structure, suggesting minimal impact of the crystal packing on the plectasin conformation. PMID:19472324

Structural Basis of Pullulanase Membrane Binding and Secretion Revealed by X-Ray Crystallography, Molecular Dynamics and Biochemical Analysis.

PubMed

East, Alexandra; Mechaly, Ariel E; Huysmans, Gerard H M; Bernarde, Cédric; Tello-Manigne, Diana; Nadeau, Nathalie; Pugsley, Anthony P; Buschiazzo, Alejandro; Alzari, Pedro M; Bond, Peter J; Francetic, Olivera

2016-01-05

The Klebsiella lipoprotein pullulanase (PulA) is exported to the periplasm, triacylated, and anchored via lipids in the inner membrane (IM) prior to its transport to the bacterial surface through a type II secretion system (T2SS). X-Ray crystallography and atomistic molecular dynamics (MD) simulations of PulA in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) model membrane provided an unprecedented molecular view of an N-terminal unstructured tether and the IM lipoprotein retention signal, and revealed novel interactions with the IM via N-terminal immunoglobulin-like domains in PulA. An efficiently secreted nonacylated variant (PulANA) showed similar peripheral membrane association during MD simulations, consistent with the binding of purified PulANA to liposomes. Remarkably, combined X-ray, MD, and functional studies identified a novel subdomain, Ins, inserted in the α-amylase domain, which is required for PulA secretion. Available data support a model in which PulA binding to the IM promotes interactions with the T2SS, possibly via the Ins subdomain. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

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

AXSIS: Exploring the frontiers in attosecond X-ray science, imaging and spectroscopy.

PubMed

Kärtner, F X; Ahr, F; Calendron, A-L; Çankaya, H; Carbajo, S; Chang, G; Cirmi, G; Dörner, K; Dorda, U; Fallahi, A; Hartin, A; Hemmer, M; Hobbs, R; Hua, Y; Huang, W R; Letrun, R; Matlis, N; Mazalova, V; Mücke, O D; Nanni, E; Putnam, W; Ravi, K; Reichert, F; Sarrou, I; Wu, X; Yahaghi, A; Ye, H; Zapata, L; Zhang, D; Zhou, C; Miller, R J D; Berggren, K K; Graafsma, H; Meents, A; Assmann, R W; Chapman, H N; Fromme, P

2016-09-01

X-ray crystallography is one of the main methods to determine atomic-resolution 3D images of the whole spectrum of molecules ranging from small inorganic clusters to large protein complexes consisting of hundred-thousands of atoms that constitute the macromolecular machinery of life. Life is not static, and unravelling the structure and dynamics of the most important reactions in chemistry and biology is essential to uncover their mechanism. Many of these reactions, including photosynthesis which drives our biosphere, are light induced and occur on ultrafast timescales. These have been studied with high time resolution primarily by optical spectroscopy, enabled by ultrafast laser technology, but they reduce the vast complexity of the process to a few reaction coordinates. In the AXSIS project at CFEL in Hamburg, funded by the European Research Council, we develop the new method of attosecond serial X-ray crystallography and spectroscopy, to give a full description of ultrafast processes atomically resolved in real space and on the electronic energy landscape, from co-measurement of X-ray and optical spectra, and X-ray diffraction. This technique will revolutionize our understanding of structure and function at the atomic and molecular level and thereby unravel fundamental processes in chemistry and biology like energy conversion processes. For that purpose, we develop a compact, fully coherent, THz-driven atto-second X-ray source based on coherent inverse Compton scattering off a free-electron crystal, to outrun radiation damage effects due to the necessary high X-ray irradiance required to acquire diffraction signals. This highly synergistic project starts from a completely clean slate rather than conforming to the specifications of a large free-electron laser (FEL) user facility, to optimize the entire instrumentation towards fundamental measurements of the mechanism of light absorption and excitation energy transfer. A multidisciplinary team formed by laser

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.

Complementary uses of small angle X-ray scattering and X-ray crystallography.

PubMed

Pillon, Monica C; Guarné, Alba

2017-11-01

Most proteins function within networks and, therefore, protein interactions are central to protein function. Although stable macromolecular machines have been extensively studied, dynamic protein interactions remain poorly understood. Small-angle X-ray scattering probes the size, shape and dynamics of proteins in solution at low resolution and can be used to study samples in a large range of molecular weights. Therefore, it has emerged as a powerful technique to study the structure and dynamics of biomolecular systems and bridge fragmented information obtained using high-resolution techniques. Here we review how small-angle X-ray scattering can be combined with other structural biology techniques to study protein dynamics. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.

XRayView: a teaching aid for X-ray crystallography.

PubMed

Phillips, G N

1995-10-01

A software package, XRayView, has been developed that uses interactive computer graphics to introduce basic concepts of x-ray diffraction by crystals, including the reciprocal lattice, the Ewald sphere construction, Laue cones, the wavelength dependence of the reciprocal lattice, primitive and centered lattices and systematic extinctions, rotation photography. Laue photography, space group determination and Laue group symmetry, and the alignment of crystals by examination of reciprocal space. XRayView is designed with "user-friendliness" in mind, using pull-down menus to control the program. Many of the experiences of using real x-ray diffraction equipment to examine crystalline diffraction can be simulated. Exercises are available on-line to guide the users through many typical x-ray diffraction experiments.

Quantum Crystallography: Density Matrix-Density Functional Theory and the X-Ray Diffraction Experiment

NASA Astrophysics Data System (ADS)

Soirat, Arnaud J. A.

Density Matrix Theory is a Quantum Mechanical formalism in which the wavefunction is eliminated and its role taken over by reduced density matrices. The interest of this is that, it allows one, in principle, to calculate any electronic property of a physical system, without having to solve the Schrodinger equation, using only two entities much simpler than an N-body wavefunction: first and second -order reduced density matrices. In practice, though, this very promising possibility faces the tremendous theoretical problem of N-representability, which has been solved for the former, but, until now, voids any hope of theoretically determining the latter. However, it has been shown that single determinant reduced density matrices of any order may be recovered from coherent X-ray diffraction data, if one provides a proper Quantum Mechanical description of the Crystallography experiment. A deeper investigation of this method is the purpose of this work, where we, first, further study the calculation of X-ray reduced density matrices N-representable by a single Slater determinant. In this context, we independently derive necessary and sufficient conditions for the uniqueness of the method. We then show how to account for electron correlation in this model. For the first time, indeed, we derive highly accurate, yet practical, density matrices approximately N-representable by correlated-determinant wavefunctions. The interest of such a result lies in the Quantum Mechanical validity of these density matrices, their property of being entirely obtainable from X-ray coherent diffraction data, their very high accuracy conferred by this known property of the N-representing wavefunction, as well as their definition as explicit functionals of the density. All of these properties are finally used in both a theoretical and a numerical application: in the former, we show that these density matrices may be used in the context of Density Functional Theory to highly accurately determine

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

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

Crystallography with online optical and X-ray absorption spectroscopies demonstrates an ordered mechanism in copper nitrite reductase.

PubMed

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

2008-04-25

Nitrite reductases are key enzymes that perform the first committed step in the denitrification process and reduce nitrite to nitric oxide. In copper nitrite reductases, an electron is delivered from the type 1 copper (T1Cu) centre to the type 2 copper (T2Cu) centre where catalysis occurs. Despite significant structural and mechanistic studies, it remains controversial whether the substrates, nitrite, electron and proton are utilised in an ordered or random manner. We have used crystallography, together with online X-ray absorption spectroscopy and optical spectroscopy, to show that X-rays rapidly and selectively photoreduce the T1Cu centre, but that the T2Cu centre does not photoreduce directly over a typical crystallographic data collection time. Furthermore, internal electron transfer between the T1Cu and T2Cu centres does not occur, and the T2Cu centre remains oxidised. These data unambiguously demonstrate an 'ordered' mechanism in which electron transfer is gated by binding of nitrite to the T2Cu. Furthermore, the use of online multiple spectroscopic techniques shows their value in assessing radiation-induced redox changes at different metal sites and demonstrates the importance of ensuring the correct status of redox centres in a crystal structure determination. Here, optical spectroscopy has shown a very high sensitivity for detecting the change in T1Cu redox state, while X-ray absorption spectroscopy has reported on the redox status of the T2Cu site, as this centre has no detectable optical absorption.

Architectural plasticity of AMPK revealed by electron microscopy and X-ray crystallography

PubMed Central

Ouyang, Yan; Zhu, Li; Li, Yifang; Guo, Miaomiao; Liu, Yang; Cheng, Jin; Zhao, Jing; Wu, Yi

2016-01-01

Mammalian AMP-activated protein kinase (AMPK) acts as an important sensor of cellular energy homeostasis related with AMP/ADP to ATP ratio. The overall architecture of AMPK has been determined in either homotrimer or monomer form by electron microscopy (EM) and X-ray crystallography successively. Accordingly proposed models have consistently revealed a key role of the α subunit linker in sensing adenosine nucleoside binding on the γ subunit and mediating allosteric regulation of kinase domain (KD) activity, whereas there are vital differences in orienting N-terminus of α subunit and locating carbohydrate-binding module (CBM) of β subunit. Given that Mg2+, an indispensable cofactor of AMPK was present in the EM sample preparation buffer however absent when forming crystals, here we carried out further reconstructions without Mg2+ to expectably inspect if this ion may contribute to this difference. However, no essential alteration has been found in this study compared to our early work. Further analyses indicate that the intra-molecular movement of the KD and CBM are most likely due to the flexible linkage of the disordered linkers with the rest portion as well as a contribution from the plasticity in the inter-molecular assembly mode, which might ulteriorly reveal an architectural complication of AMPK. PMID:27063142

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.

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.

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

Metalloprotein Crystallography: More than a Structure.

PubMed

Bowman, Sarah E J; Bridwell-Rabb, Jennifer; Drennan, Catherine L

2016-04-19

Metal ions and metallocofactors play important roles in a broad range of biochemical reactions. Accordingly, it has been estimated that as much as 25-50% of the proteome uses transition metal ions to carry out a variety of essential functions. The metal ions incorporated within metalloproteins fulfill functional roles based on chemical properties, the diversity of which arises as transition metals can adopt different redox states and geometries, dictated by the identity of the metal and the protein environment. The coupling of a metal ion with an organic framework in metallocofactors, such as heme and cobalamin, further expands the chemical functionality of metals in biology. The three-dimensional visualization of metal ions and complex metallocofactors within a protein scaffold is often a starting point for enzymology, highlighting the importance of structural characterization of metalloproteins. Metalloprotein crystallography, however, presents a number of implicit challenges including correctly incorporating the relevant metal or metallocofactor, maintaining the proper environment for the protein to be purified and crystallized (including providing anaerobic, cold, or aphotic environments), and being mindful of the possibility of X-ray induced damage to the proteins or incorporated metal ions. Nevertheless, the incorporated metals or metallocofactors also present unique advantages in metalloprotein crystallography. The significant resonance that metals undergo with X-ray photons at wavelengths used for protein crystallography and the rich electronic properties of metals, which provide intense and spectroscopically unique signatures, allow a metalloprotein crystallographer to use anomalous dispersion to determine phases for structure solution and to use simultaneous or parallel spectroscopic techniques on single crystals. These properties, coupled with the improved brightness of beamlines, the ability to tune the wavelength of the X-ray beam, the availability of

Goniometer-based femtosecond X-ray diffraction of mutant 30S ribosomal subunit crystals

DOE PAGES

Dao, E. Han; Sierra, Raymond G.; Laksmono, Hartawan; ...

2015-04-30

In this work, we collected radiation-damage-free data from a set of cryo-cooled crystals for a novel 30S ribosomal subunit mutant using goniometer-based femtosecond crystallography. Crystal quality assessment for these samples was conducted at the X-ray Pump Probe end-station of the Linac Coherent Light Source (LCLS) using recently introduced goniometer-based instrumentation. These 30S subunit crystals were genetically engineered to omit a 26-residue protein, Thx, which is present in the wild-type Thermus thermophilus 30S ribosomal subunit. We are primarily interested in elucidating the contribution of this ribosomal protein to the overall 30S subunit structure. To assess the viability of this study, femtosecondmore » X-ray diffraction patterns from these crystals were recorded at the LCLS during a protein crystal screening beam time. During our data collection, we successfully observed diffraction from these difficult-to-grow 30S ribosomal subunit crystals. Most of our crystals were found to diffract to low resolution, while one crystal diffracted to 3.2 Å resolution. These data suggest the feasibility of pursuing high-resolution data collection as well as the need to improve sample preparation and handling in order to collect a complete radiation-damage-free data set using an X-ray Free Electron Laser.« 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.

Determination of the X-ray structure of the snake venom protein omwaprin by total chemical synthesis and racemic protein crystallography.

PubMed

Banigan, James R; Mandal, Kalyaneswar; Sawaya, Michael R; Thammavongsa, Vilasak; Hendrickx, Antoni P A; Schneewind, Olaf; Yeates, Todd O; Kent, Stephen B H

2010-10-01

The 50-residue snake venom protein L-omwaprin and its enantiomer D-omwaprin were prepared by total chemical synthesis. Radial diffusion assays were performed against Bacillus megaterium and Bacillus anthracis; both L- and D-omwaprin showed antibacterial activity against B. megaterium. The native protein enantiomer, made of L-amino acids, failed to crystallize readily. However, when a racemic mixture containing equal amounts of L- and D-omwaprin was used, diffraction quality crystals were obtained. The racemic protein sample crystallized in the centrosymmetric space group P2(1)/c and its structure was determined at atomic resolution (1.33 A) by a combination of Patterson and direct methods based on the strong scattering from the sulfur atoms in the eight cysteine residues per protein. Racemic crystallography once again proved to be a valuable method for obtaining crystals of recalcitrant proteins and for determining high-resolution X-ray structures by direct methods.

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.

Rapid X-ray Photoreduction of Dimetal-Oxygen Cofactors in Ribonucleotide Reductase

PubMed Central

Sigfridsson, Kajsa G. V.; Chernev, Petko; Leidel, Nils; Popović-Bijelić, Ana; Gräslund, Astrid; Haumann, Michael

2013-01-01

Prototypic dinuclear metal cofactors with varying metallation constitute a class of O2-activating catalysts in numerous enzymes such as ribonucleotide reductase. Reliable structures are required to unravel the reaction mechanisms. However, protein crystallography data may be compromised by x-ray photoreduction (XRP). We studied XPR of Fe(III)Fe(III) and Mn(III)Fe(III) sites in the R2 subunit of Chlamydia trachomatis ribonucleotide reductase using x-ray absorption spectroscopy. Rapid and biphasic x-ray photoreduction kinetics at 20 and 80 K for both cofactor types suggested sequential formation of (III,II) and (II,II) species and similar redox potentials of iron and manganese sites. Comparing with typical x-ray doses in crystallography implies that (II,II) states are reached in <1 s in such studies. First-sphere metal coordination and metal-metal distances differed after chemical reduction at room temperature and after XPR at cryogenic temperatures, as corroborated by model structures from density functional theory calculations. The inter-metal distances in the XPR-induced (II,II) states, however, are similar to R2 crystal structures. Therefore, crystal data of initially oxidized R2-type proteins mostly contain photoreduced (II,II) cofactors, which deviate from the native structures functional in O2 activation, explaining observed variable metal ligation motifs. This situation may be remedied by novel femtosecond free electron-laser protein crystallography techniques. PMID:23400774

Rapid X-ray photoreduction of dimetal-oxygen cofactors in ribonucleotide reductase.

PubMed

Sigfridsson, Kajsa G V; Chernev, Petko; Leidel, Nils; Popovic-Bijelic, Ana; Gräslund, Astrid; Haumann, Michael

2013-04-05

Prototypic dinuclear metal cofactors with varying metallation constitute a class of O2-activating catalysts in numerous enzymes such as ribonucleotide reductase. Reliable structures are required to unravel the reaction mechanisms. However, protein crystallography data may be compromised by x-ray photoreduction (XRP). We studied XPR of Fe(III)Fe(III) and Mn(III)Fe(III) sites in the R2 subunit of Chlamydia trachomatis ribonucleotide reductase using x-ray absorption spectroscopy. Rapid and biphasic x-ray photoreduction kinetics at 20 and 80 K for both cofactor types suggested sequential formation of (III,II) and (II,II) species and similar redox potentials of iron and manganese sites. Comparing with typical x-ray doses in crystallography implies that (II,II) states are reached in <1 s in such studies. First-sphere metal coordination and metal-metal distances differed after chemical reduction at room temperature and after XPR at cryogenic temperatures, as corroborated by model structures from density functional theory calculations. The inter-metal distances in the XPR-induced (II,II) states, however, are similar to R2 crystal structures. Therefore, crystal data of initially oxidized R2-type proteins mostly contain photoreduced (II,II) cofactors, which deviate from the native structures functional in O2 activation, explaining observed variable metal ligation motifs. This situation may be remedied by novel femtosecond free electron-laser protein crystallography techniques.

Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography*

PubMed Central

Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; Lampe, Jed N.; Nishida, Clinton R.; de Montellano, Paul R. Ortiz

2015-01-01

Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. We used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. The results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states. PMID:25670859

Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography

DOE PAGES

Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; ...

2015-02-10

Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. In this paper, we used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop withmore » various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. Finally, the results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.« less

Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography

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

Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie

Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. In this paper, we used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop withmore » various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. Finally, the results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.« less

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.

Method for spatially modulating X-ray pulses using MEMS-based X-ray optics

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

Lopez, Daniel; Shenoy, Gopal; Wang, Jin

A method and apparatus are provided for spatially modulating X-rays or X-ray pulses using microelectromechanical systems (MEMS) based X-ray optics. A torsionally-oscillating MEMS micromirror and a method of leveraging the grazing-angle reflection property are provided to modulate X-ray pulses with a high-degree of controllability.

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

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

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

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

Analysis of cytochrome P450 CYP119 ligand-dependent conformational dynamics by two-dimensional NMR and X-ray crystallography.

PubMed

Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; Lampe, Jed N; Nishida, Clinton R; de Montellano, Paul R Ortiz

2015-04-17

Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. We used two-dimensional (1)H,(15)N HSQC chemical shift perturbation mapping of (15)N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. The results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging

PubMed Central

Warren, Anna J.; Armour, Wes; Axford, Danny; Basham, Mark; Connolley, Thomas; Hall, David R.; Horrell, Sam; McAuley, Katherine E.; Mykhaylyk, Vitaliy; Wagner, Armin; Evans, Gwyndaf

2013-01-01

The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallize on much smaller scales and are frequently mounted in opaque or highly refractive materials. It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this paper, the use of X-ray microradiography and microtomography is reported as a tool for crystal visualization, location and characterization on the macromolecular crystallography beamlines at the Diamond Light Source. The technique is particularly useful for microcrystals and for crystals mounted in opaque materials such as lipid cubic phase. X-ray diffraction raster scanning can be used in combination with radiography to allow informed decision-making at the beamline prior to diffraction data collection. It is demonstrated that the X-ray dose required for a full tomography measurement is similar to that for a diffraction grid-scan, but for sample location and shape estimation alone just a few radiographic projections may be required. PMID:23793151

Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging.

PubMed

Warren, Anna J; Armour, Wes; Axford, Danny; Basham, Mark; Connolley, Thomas; Hall, David R; Horrell, Sam; McAuley, Katherine E; Mykhaylyk, Vitaliy; Wagner, Armin; Evans, Gwyndaf

2013-07-01

The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallize on much smaller scales and are frequently mounted in opaque or highly refractive materials. It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this paper, the use of X-ray microradiography and microtomography is reported as a tool for crystal visualization, location and characterization on the macromolecular crystallography beamlines at the Diamond Light Source. The technique is particularly useful for microcrystals and for crystals mounted in opaque materials such as lipid cubic phase. X-ray diffraction raster scanning can be used in combination with radiography to allow informed decision-making at the beamline prior to diffraction data collection. It is demonstrated that the X-ray dose required for a full tomography measurement is similar to that for a diffraction grid-scan, but for sample location and shape estimation alone just a few radiographic projections may be required.

Focusing X-ray free-electron laser pulses using Kirkpatrick-Baez mirrors at the NCI hutch of the PAL-XFEL.

PubMed

Kim, Jangwoo; Kim, Hyo Yun; Park, Jaehyun; Kim, Sangsoo; Kim, Sunam; Rah, Seungyu; Lim, Jun; Nam, Ki Hyun

2018-01-01

The Pohang Accelerator Laboratory X-ray Free-Electron Laser (PAL-XFEL) is a recently commissioned X-ray free-electron laser (XFEL) facility that provides intense ultrashort X-ray pulses based on the self-amplified spontaneous emission process. The nano-crystallography and coherent imaging (NCI) hutch with forward-scattering geometry is located at the hard X-ray beamline of the PAL-XFEL and provides opportunities to perform serial femtosecond crystallography and coherent X-ray diffraction imaging. To produce intense high-density XFEL pulses at the interaction positions between the X-rays and various samples, a microfocusing Kirkpatrick-Baez (KB) mirror system that includes an ultra-precision manipulator has been developed. In this paper, the design of a KB mirror system that focuses the hard XFEL beam onto a fixed sample point of the NCI hutch, which is positioned along the hard XFEL beamline, is described. The focusing system produces a two-dimensional focusing beam at approximately 2 µm scale across the 2-11 keV photon energy range. XFEL pulses of 9.7 keV energy were successfully focused onto an area of size 1.94 µm × 2.08 µm FWHM.

Batch crystallization of rhodopsin for structural dynamics using an X-ray free-electron laser

DOE PAGES

Wu, Wenting; Nogly, Przemyslaw; Rheinberger, Jan; ...

2015-06-27

Rhodopsin is a membrane protein from the G protein-coupled receptor family. Together with its ligand retinal, it forms the visual pigment responsible for night vision. In order to perform ultrafast dynamics studies, a time-resolved serial femtosecond crystallography method is required owing to the nonreversible activation of rhodopsin. In such an approach, microcrystals in suspension are delivered into the X-ray pulses of an X-ray free-electron laser (XFEL) after a precise photoactivation delay. Here in this study, a millilitre batch production of high-density microcrystals was developed by four methodical conversion steps starting from known vapour-diffusion crystallization protocols: (i) screening the low-salt crystallizationmore » conditions preferred for serial crystallography by vapour diffusion, (ii) optimization of batch crystallization, (iii) testing the crystal size and quality using second-harmonic generation (SHG) imaging and X-ray powder diffraction and (iv) production of millilitres of rhodopsin crystal suspension in batches for serial crystallography tests; these crystals diffracted at an XFEL at the Linac Coherent Light Source using a liquid-jet setup.« less

Cone-beam x-ray luminescence computed tomography based on x-ray absorption dosage

NASA Astrophysics Data System (ADS)

Liu, Tianshuai; Rong, Junyan; Gao, Peng; Zhang, Wenli; Liu, Wenlei; Zhang, Yuanke; Lu, Hongbing

2018-02-01

With the advances of x-ray excitable nanophosphors, x-ray luminescence computed tomography (XLCT) has become a promising hybrid imaging modality. In particular, a cone-beam XLCT (CB-XLCT) system has demonstrated its potential in in vivo imaging with the advantage of fast imaging speed over other XLCT systems. Currently, the imaging models of most XLCT systems assume that nanophosphors emit light based on the intensity distribution of x-ray within the object, not completely reflecting the nature of the x-ray excitation process. To improve the imaging quality of CB-XLCT, an imaging model that adopts an excitation model of nanophosphors based on x-ray absorption dosage is proposed in this study. To solve the ill-posed inverse problem, a reconstruction algorithm that combines the adaptive Tikhonov regularization method with the imaging model is implemented for CB-XLCT reconstruction. Numerical simulations and phantom experiments indicate that compared with the traditional forward model based on x-ray intensity, the proposed dose-based model could improve the image quality of CB-XLCT significantly in terms of target shape, localization accuracy, and image contrast. In addition, the proposed model behaves better in distinguishing closer targets, demonstrating its advantage in improving spatial resolution.

Cone-beam x-ray luminescence computed tomography based on x-ray absorption dosage.

PubMed

Liu, Tianshuai; Rong, Junyan; Gao, Peng; Zhang, Wenli; Liu, Wenlei; Zhang, Yuanke; Lu, Hongbing

2018-02-01

With the advances of x-ray excitable nanophosphors, x-ray luminescence computed tomography (XLCT) has become a promising hybrid imaging modality. In particular, a cone-beam XLCT (CB-XLCT) system has demonstrated its potential in in vivo imaging with the advantage of fast imaging speed over other XLCT systems. Currently, the imaging models of most XLCT systems assume that nanophosphors emit light based on the intensity distribution of x-ray within the object, not completely reflecting the nature of the x-ray excitation process. To improve the imaging quality of CB-XLCT, an imaging model that adopts an excitation model of nanophosphors based on x-ray absorption dosage is proposed in this study. To solve the ill-posed inverse problem, a reconstruction algorithm that combines the adaptive Tikhonov regularization method with the imaging model is implemented for CB-XLCT reconstruction. Numerical simulations and phantom experiments indicate that compared with the traditional forward model based on x-ray intensity, the proposed dose-based model could improve the image quality of CB-XLCT significantly in terms of target shape, localization accuracy, and image contrast. In addition, the proposed model behaves better in distinguishing closer targets, demonstrating its advantage in improving spatial resolution. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

Diffraction leveraged modulation of X-ray pulses using MEMS-based X-ray optics

DOEpatents

Lopez, Daniel; Shenoy, Gopal; Wang, Jin; Walko, Donald A.; Jung, Il-Woong; Mukhopadhyay, Deepkishore

2016-08-09

A method and apparatus are provided for implementing Bragg-diffraction leveraged modulation of X-ray pulses using MicroElectroMechanical systems (MEMS) based diffractive optics. An oscillating crystalline MEMS device generates a controllable time-window for diffraction of the incident X-ray radiation. The Bragg-diffraction leveraged modulation of X-ray pulses includes isolating a particular pulse, spatially separating individual pulses, and spreading a single pulse from an X-ray pulse-train.

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

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

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.

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.

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.

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.

Synthesis, X-ray crystallography, thermal studies, spectroscopic and electrochemistry investigations of uranyl Schiff base complexes.

PubMed

Asadi, Zahra; Shorkaei, Mohammad Ranjkesh

2013-03-15

Some tetradentate salen type Schiff bases and their uranyl complexes were synthesized and characterized by UV-Vis, NMR, IR, TG, C.H.N. and X-ray crystallographic studies. From these investigations it is confirmed that a solvent molecule occupied the fifth position of the equatorial plane of the distorted pentagonal bipyramidal structure. Also, the kinetics of complex decomposition by using thermo gravimetric methods (TG) was studied. The thermal decomposition reactions are first order for the studied complexes. To examine the properties of uranyl complexes according to the substitutional groups, we have carried out the electrochemical studies. The electrochemical reactions of uranyl Schiff base complexes in acetonitrile were reversible. Copyright © 2012 Elsevier B.V. All rights reserved.

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

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.

Direct detection of x-rays for protein crystallography employing a thick, large area CCD

DOEpatents

Atac, Muzaffer; McKay, Timothy

1999-01-01

An apparatus and method for directly determining the crystalline structure of a protein crystal. The crystal is irradiated by a finely collimated x-ray beam. The interaction of the x-ray beam with the crystal produces scattered x-rays. These scattered x-rays are detected by means of a large area, thick CCD which is capable of measuring a significant number of scattered x-rays which impact its surface. The CCD is capable of detecting the position of impact of the scattered x-ray on the surface of the CCD and the quantity of scattered x-rays which impact the same cell or pixel. This data is then processed in real-time and the processed data is outputted to produce a image of the structure of the crystal. If this crystal is a protein the molecular structure of the protein can be determined from the data received.

IP3-mediated gating mechanism of the IP3 receptor revealed by mutagenesis and X-ray crystallography.

PubMed

Hamada, Kozo; Miyatake, Hideyuki; Terauchi, Akiko; Mikoshiba, Katsuhiko

2017-05-02

The inositol 1,4,5-trisphosphate (IP 3 ) receptor (IP 3 R) is an IP 3 -gated ion channel that releases calcium ions (Ca 2+ ) from the endoplasmic reticulum. The IP 3 -binding sites in the large cytosolic domain are distant from the Ca 2+ conducting pore, and the allosteric mechanism of how IP 3 opens the Ca 2+ channel remains elusive. Here, we identify a long-range gating mechanism uncovered by channel mutagenesis and X-ray crystallography of the large cytosolic domain of mouse type 1 IP 3 R in the absence and presence of IP 3 Analyses of two distinct space group crystals uncovered an IP 3 -dependent global translocation of the curvature α-helical domain interfacing with the cytosolic and channel domains. Mutagenesis of the IP 3 R channel revealed an essential role of a leaflet structure in the α-helical domain. These results suggest that the curvature α-helical domain relays IP 3 -controlled global conformational dynamics to the channel through the leaflet, conferring long-range allosteric coupling from IP 3 binding to the Ca 2+ channel.

IP3-mediated gating mechanism of the IP3 receptor revealed by mutagenesis and X-ray crystallography

PubMed Central

Hamada, Kozo; Miyatake, Hideyuki; Terauchi, Akiko; Mikoshiba, Katsuhiko

2017-01-01

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) is an IP3-gated ion channel that releases calcium ions (Ca2+) from the endoplasmic reticulum. The IP3-binding sites in the large cytosolic domain are distant from the Ca2+ conducting pore, and the allosteric mechanism of how IP3 opens the Ca2+ channel remains elusive. Here, we identify a long-range gating mechanism uncovered by channel mutagenesis and X-ray crystallography of the large cytosolic domain of mouse type 1 IP3R in the absence and presence of IP3. Analyses of two distinct space group crystals uncovered an IP3-dependent global translocation of the curvature α-helical domain interfacing with the cytosolic and channel domains. Mutagenesis of the IP3R channel revealed an essential role of a leaflet structure in the α-helical domain. These results suggest that the curvature α-helical domain relays IP3-controlled global conformational dynamics to the channel through the leaflet, conferring long-range allosteric coupling from IP3 binding to the Ca2+ channel. PMID:28416699

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

Structure-Based Design: Synthesis, X-ray Crystallography, and Biological Evaluation of N-Substituted-4-Hydroxy-2-Quinolone-3-Carboxamides as Potential Cytotoxic Agents.

PubMed

Sabbah, Dima A; Hishmah, Bayan; Sweidan, Kamal; Bardaweel, Sanaa; AlDamen, Murad; Zhong, Haizhen A; Abu Khalaf, Reema; Hasan Ibrahim, Ameerah; Al-Qirim, Tariq; Abu Sheikha, Ghassan; Mubarak, Mohammad S

2018-01-01

Oncogenic potential of phosphatidylinositol 3-kinase (PI3Kα) has been highlighted as a therapeutic target for anticancer drug design. Target compounds were designed to address the effect of different substitution patterns at the N atom of the carboxamide moiety on the bioactivity of this series. Synthesis of the targeted compounds, crystallography, biological evaluation tests against human colon carcinoma (HCT-116), and Glide docking studies. A new series of N-substituted- 4-hydroxy-2-quinolone-3-carboxamides was prepared and characterized by means of FT-IR, 1H and 13C NMR, and elemental analysis. In addition, the identity of the core nucleus 5 was successfully characterized with the aid of X-ray crystallography. Biological activity of prepared compounds was investigated in vitro against human colon carcinoma (HCT-116) cell line. Results revealed that these compounds inhibit cell proliferation and induce apoptosis through an increase in caspase-3 activity and a decrease in DNA cellular content. Compounds 7, 14, and 17 which have H-bond acceptor moiety on p-position displayed promising PI3Kα inhibitory activity. On the other hand, derivatives tailored with bulky and hydrophobic motifs (16 and 18) on o- and m-positions exhibited moderate activity. Molecular docking studies against PI3Kα and caspase-3 showed an agreement between the predicted binding affinity (ΔGobsd) and IC50 values of the derivatives for the caspase-3 model. Furthermore, Glide docking studies against PI3Kα demonstrated that the newly synthesized compounds accommodate PI3Kα kinase catalytic domain and form H-bonding with key binding residues. The series exhibited a potential PI3Kα inhibitory activity in HCT-116 cell line. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Correlations in Scattered X-Ray Laser Pulses Reveal Nanoscale Structural Features of Viruses

NASA Astrophysics Data System (ADS)

Kurta, Ruslan P.; Donatelli, Jeffrey J.; Yoon, Chun Hong; Berntsen, Peter; Bielecki, Johan; Daurer, Benedikt J.; DeMirci, Hasan; Fromme, Petra; Hantke, Max Felix; Maia, Filipe R. N. C.; Munke, Anna; Nettelblad, Carl; Pande, Kanupriya; Reddy, Hemanth K. N.; Sellberg, Jonas A.; Sierra, Raymond G.; Svenda, Martin; van der Schot, Gijs; Vartanyants, Ivan A.; Williams, Garth J.; Xavier, P. Lourdu; Aquila, Andrew; Zwart, Peter H.; Mancuso, Adrian P.

2017-10-01

We use extremely bright and ultrashort pulses from an x-ray free-electron laser (XFEL) to measure correlations in x rays scattered from individual bioparticles. This allows us to go beyond the traditional crystallography and single-particle imaging approaches for structure investigations. We employ angular correlations to recover the three-dimensional (3D) structure of nanoscale viruses from x-ray diffraction data measured at the Linac Coherent Light Source. Correlations provide us with a comprehensive structural fingerprint of a 3D virus, which we use both for model-based and ab initio structure recovery. The analyses reveal a clear indication that the structure of the viruses deviates from the expected perfect icosahedral symmetry. Our results anticipate exciting opportunities for XFEL studies of the structure and dynamics of nanoscale objects by means of angular correlations.

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

Novel X-ray Communication Based XNAV Augmentation Method Using X-ray Detectors

PubMed Central

Song, Shibin; Xu, Luping; Zhang, Hua; Bai, Yuanjie

2015-01-01

The further development of X-ray pulsar-based NAVigation (XNAV) is hindered by its lack of accuracy, so accuracy improvement has become a critical issue for XNAV. In this paper, an XNAV augmentation method which utilizes both pulsar observation and X-ray ranging observation for navigation filtering is proposed to deal with this issue. As a newly emerged concept, X-ray communication (XCOM) shows great potential in space exploration. X-ray ranging, derived from XCOM, could achieve high accuracy in range measurement, which could provide accurate information for XNAV. For the proposed method, the measurement models of pulsar observation and range measurement observation are established, and a Kalman filtering algorithm based on the observations and orbit dynamics is proposed to estimate the position and velocity of a spacecraft. A performance comparison of the proposed method with the traditional pulsar observation method is conducted by numerical experiments. Besides, the parameters that influence the performance of the proposed method, such as the pulsar observation time, the SNR of the ranging signal, etc., are analyzed and evaluated by numerical experiments. PMID:26404295

Simulation tools for analyzer-based x-ray phase contrast imaging system with a conventional x-ray source

NASA Astrophysics Data System (ADS)

Caudevilla, Oriol; Zhou, Wei; Stoupin, Stanislav; Verman, Boris; Brankov, J. G.

2016-09-01

Analyzer-based X-ray phase contrast imaging (ABI) belongs to a broader family of phase-contrast (PC) X-ray imaging modalities. Unlike the conventional X-ray radiography, which measures only X-ray absorption, in PC imaging one can also measures the X-rays deflection induced by the object refractive properties. It has been shown that refraction imaging provides better contrast when imaging the soft tissue, which is of great interest in medical imaging applications. In this paper, we introduce a simulation tool specifically designed to simulate the analyzer-based X-ray phase contrast imaging system with a conventional polychromatic X-ray source. By utilizing ray tracing and basic physical principles of diffraction theory our simulation tool can predicting the X-ray beam profile shape, the energy content, the total throughput (photon count) at the detector. In addition we can evaluate imaging system point-spread function for various system configurations.

In cellulo serial crystallography of alcohol oxidase crystals inside yeast cells

PubMed Central

Jakobi, Arjen J.; Passon, Daniel M.; Knoops, Kèvin; Stellato, Francesco; Liang, Mengning; White, Thomas A.; Seine, Thomas; Messerschmidt, Marc; Chapman, Henry N.; Wilmanns, Matthias

2016-01-01

The possibility of using femtosecond pulses from an X-ray free-electron laser to collect diffraction data from protein crystals formed in their native cellular organelle has been explored. X-ray diffraction of submicrometre-sized alcohol oxidase crystals formed in peroxisomes within cells of genetically modified variants of the methylotrophic yeast Hansenula polymorpha is reported and characterized. The observations are supported by synchrotron radiation-based powder diffraction data and electron microscopy. Based on these findings, the concept of in cellulo serial crystallography on protein targets imported into yeast peroxisomes without the need for protein purification as a requirement for subsequent crystallization is outlined. PMID:27006771

In cellulo serial crystallography of alcohol oxidase crystals inside yeast cells

DOE PAGES

Jakobi, Arjen J.; Passon, Daniel M.; Knoops, Kevin; ...

2016-03-01

The possibility of using femtosecond pulses from an X-ray free-electron laser to collect diffraction data from protein crystals formed in their native cellular organelle has been explored. X-ray diffraction of submicrometre-sized alcohol oxidase crystals formed in peroxisomes within cells of genetically modified variants of the methylotrophic yeast Hansenula polymorpha is reported and characterized. Furthermore, the observations are supported by synchrotron radiation-based powder diffraction data and electron microscopy. Based on these findings, the concept of in cellulo serial crystallography on protein targets imported into yeast peroxisomes without the need for protein purification as a requirement for subsequent crystallization is outlined.

In cellulo serial crystallography of alcohol oxidase crystals inside yeast cells

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

Jakobi, Arjen J.; Passon, Daniel M.; Knoops, Kevin

The possibility of using femtosecond pulses from an X-ray free-electron laser to collect diffraction data from protein crystals formed in their native cellular organelle has been explored. X-ray diffraction of submicrometre-sized alcohol oxidase crystals formed in peroxisomes within cells of genetically modified variants of the methylotrophic yeast Hansenula polymorpha is reported and characterized. Furthermore, the observations are supported by synchrotron radiation-based powder diffraction data and electron microscopy. Based on these findings, the concept of in cellulo serial crystallography on protein targets imported into yeast peroxisomes without the need for protein purification as a requirement for subsequent crystallization is outlined.

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

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

Correlations in Scattered X-Ray Laser Pulses Reveal Nanoscale Structural Features of Viruses

DOE PAGES

Kurta, Ruslan P.; Donatelli, Jeffrey J.; Yoon, Chun Hong; ...

2017-10-12

We use extremely bright and ultrashort pulses from an x-ray free-electron laser (XFEL) to measure correlations in x rays scattered from individual bioparticles. This allows us to go beyond the traditional crystallography and single-particle imaging approaches for structure investigations. We employ angular correlations to recover the three-dimensional (3D) structure of nanoscale viruses from x-ray diffraction data measured at the Linac Coherent Light Source. Correlations provide us with a comprehensive structural fingerprint of a 3D virus, which we use both for model-based and ab initio structure recovery. The analyses reveal a clear indication that the structure of the viruses deviates frommore » the expected perfect icosahedral symmetry. Lastly, our results anticipate exciting opportunities for XFEL studies of the structure and dynamics of nanoscale objects by means of angular correlations.« less

X-ray based extensometry

NASA Technical Reports Server (NTRS)

Jordan, E. H.; Pease, D. M.

1988-01-01

A totally new method of extensometry using an X-ray beam was proposed. The intent of the method is to provide a non-contacting technique that is immune to problems associated with density variations in gaseous environments that plague optical methods. X-rays are virtually unrefractable even by solids. The new method utilizes X-ray induced X-ray fluorescence or X-ray induced optical fluorescence of targets that have melting temperatures of over 3000 F. Many different variations of the basic approaches are possible. In the year completed, preliminary experiments were completed which strongly suggest that the method is feasible. The X-ray induced optical fluorescence method appears to be limited to temperatures below roughly 1600 F because of the overwhelming thermal optical radiation. The X-ray induced X-ray fluorescence scheme appears feasible up to very high temperatures. In this system there will be an unknown tradeoff between frequency response, cost, and accuracy. The exact tradeoff can only be estimated. It appears that for thermomechanical tests with cycle times on the order of minutes a very reasonable system may be feasible. The intended applications involve very high temperatures in both materials testing and monitoring component testing. Gas turbine engines, rocket engines, and hypersonic vehicles (NASP) all involve measurement needs that could partially be met by the proposed technology.

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.

Spherical grating based x-ray Talbot interferometry.

PubMed

Cong, Wenxiang; Xi, Yan; Wang, Ge

2015-11-01

Grating interferometry is a state-of-the-art x-ray imaging approach, which can acquire information on x-ray attenuation, phase shift, and small-angle scattering simultaneously. Phase-contrast imaging and dark-field imaging are very sensitive to microstructural variation and offers superior contrast resolution for biological soft tissues. However, a common x-ray tube is a point-like source. As a result, the popular planar grating imaging configuration seriously restricts the flux of photons and decreases the visibility of signals, yielding a limited field of view. The purpose of this study is to extend the planar x-ray grating imaging theory and methods to a spherical grating scheme for a wider range of preclinical and clinical applications. A spherical grating matches the wave front of a point x-ray source very well, allowing the perpendicular incidence of x-rays on the grating to achieve a higher visibility over a larger field of view than the planer grating counterpart. A theoretical analysis of the Talbot effect for spherical grating imaging is proposed to establish a basic foundation for x-ray spherical gratings interferometry. An efficient method of spherical grating imaging is also presented to extract attenuation, differential phase, and dark-field images in the x-ray spherical grating interferometer. Talbot self-imaging with spherical gratings is analyzed based on the Rayleigh-Sommerfeld diffraction formula, featuring a periodic angular distribution in a polar coordinate system. The Talbot distance is derived to reveal the Talbot self-imaging pattern. Numerical simulation results show the self-imaging phenomenon of a spherical grating interferometer, which is in agreement with the theoretical prediction. X-ray Talbot interferometry with spherical gratings has a significant practical promise. Relative to planar grating imaging, spherical grating based x-ray Talbot interferometry has a larger field of view and improves both signal visibility and dose

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.

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

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.

Discovery and development of x-ray diffraction

NASA Astrophysics Data System (ADS)

Jeong, Yeuncheol; Yin, Ming; Datta, Timir

2013-03-01

In 1912 Max Laue at University of Munich reasoned x-rays to be short wavelength electromagnetic waves and figured interference would occur when scattered off crystals. Arnold Sommerfeld, W. Wien, Ewald and others, raised objections to Laue's idea, but soon Walter Friedrich succeeded in recording x-ray interference patterns off copper sulfate crystals. But the Laue-Ewald's 3-dimensional formula predicted excess spots. Fewer spots were observed. William Lawrence Bragg then 22 year old studying at Cambridge University heard the Munich results from father William Henry Brag, physics professor at Univ of Leeds. Lawrence figured the spots are 2-d interference of x-ray wavelets reflecting off successive atomic planes and derived a simple eponymous equation, the Bragg equation d*sin(theta) = n*lamda. 1913 onward the Braggs dominated the crystallography. Max Laue was awarded the physics Nobel in 1914 and the Braggs shared the same in 1915. Starting with Rontgen's first ever prize in 1901, the importance of x-ray techniques is evident from the four out of a total 16 physics Nobels between 1901-1917. We will outline the historical back ground and importance of x-ray diffraction giving rise to techniques that even in 2013, remain work horses in laboratories all over the globe.

C-shaped diastereomers containing cofacial thiophene-substituted quinoxaline rings: synthesis, photophysical properties, and X-ray crystallography.

PubMed

DeBlase, Catherine R; Finke, Ryan T; Porras, Jonathan A; Tanski, Joseph M; Nadeau, Jocelyn M

2014-05-16

Synthesis and characterization of two diastereomeric C-shaped molecules containing cofacial thiophene-substituted quinoxaline rings are described. A previously known bis-α-diketone was condensed with an excess of 4-bromo-1,2-diaminobenzene in the presence of zinc acetate to give a mixture of two C-shaped diastereomers with cofacial bromine-substituted quinoxaline rings. After chromatographic separation, thiophene rings were installed by a microwave-assisted Suzuki coupling reaction, resulting in highly emissive diastereomeric compounds that were studied by UV-vis, fluorescence, and NMR spectroscopy, as well as X-ray crystallography. The unique symmetry of each diastereomer was confirmed by NMR spectroscopy. NMR data indicated that the syn isomer has restricted rotation about the bond connecting the thiophene and quinoxaline rings, which was also observed in the solid state. The spectroscopic properties of the C-shaped diastereomers were compared to a model compound containing only a single thiophene-substituted quinoxaline ring. Ground state intramolecular π-π interactions in solution were detected by NMR and UV-vis spectroscopy. Red-shifted emission bands, band broadening, and large Stokes shifts were observed, which collectively suggest excited state π-π interactions that produce excimer-like emissions, as well as a remarkable positive emission solvatochromism, indicating charge-transfer character in the excited state.

Microbeam X-ray analysis in Poland - past and future

NASA Astrophysics Data System (ADS)

Kusinski, J.

2010-02-01

The article provides an overview of the development of electron beam X-ray microanalysis (EPMA) in Poland. Since the introduction by Prof. Bojarski of EMPA over 45 years ago, tremendous advances in methodologies and in instrumentation have been made in order to improve the precision of quantitative compositional analysis, spatial resolution and analytical sensitivity. This was possible due to the activity of Applied Crystallography Committee at the Polish Academy of Sciences, as well as the groups of researches working in the Institute for Ferrous Metallurgy (Gliwice), the Technical University of Warsaw, the Silesian Technical University (Katowice), the AGH-University of Sciences and Technology (Krakow), and the Institute of Materials Science and Metallurgy Polish Academy of Sciences (Krakow). Based on the research examples realized by these teams, conferences, seminars and congresses organized, as well as books and academic textbooks issued, the evolution of electron beam X-ray microanalysis in Poland is demonstrated.

Hydrogen atoms can be located accurately and precisely by x-ray crystallography.

PubMed

Woińska, Magdalena; Grabowsky, Simon; Dominiak, Paulina M; Woźniak, Krzysztof; Jayatilaka, Dylan

2016-05-01

Precise and accurate structural information on hydrogen atoms is crucial to the study of energies of interactions important for crystal engineering, materials science, medicine, and pharmacy, and to the estimation of physical and chemical properties in solids. However, hydrogen atoms only scatter x-radiation weakly, so x-rays have not been used routinely to locate them accurately. Textbooks and teaching classes still emphasize that hydrogen atoms cannot be located with x-rays close to heavy elements; instead, neutron diffraction is needed. We show that, contrary to widespread expectation, hydrogen atoms can be located very accurately using x-ray diffraction, yielding bond lengths involving hydrogen atoms (A-H) that are in agreement with results from neutron diffraction mostly within a single standard deviation. The precision of the determination is also comparable between x-ray and neutron diffraction results. This has been achieved at resolutions as low as 0.8 Å using Hirshfeld atom refinement (HAR). We have applied HAR to 81 crystal structures of organic molecules and compared the A-H bond lengths with those from neutron measurements for A-H bonds sorted into bonds of the same class. We further show in a selection of inorganic compounds that hydrogen atoms can be located in bridging positions and close to heavy transition metals accurately and precisely. We anticipate that, in the future, conventional x-radiation sources at in-house diffractometers can be used routinely for locating hydrogen atoms in small molecules accurately instead of large-scale facilities such as spallation sources or nuclear reactors.

Hydrogen atoms can be located accurately and precisely by x-ray crystallography

PubMed Central

Woińska, Magdalena; Grabowsky, Simon; Dominiak, Paulina M.; Woźniak, Krzysztof; Jayatilaka, Dylan

2016-01-01

Precise and accurate structural information on hydrogen atoms is crucial to the study of energies of interactions important for crystal engineering, materials science, medicine, and pharmacy, and to the estimation of physical and chemical properties in solids. However, hydrogen atoms only scatter x-radiation weakly, so x-rays have not been used routinely to locate them accurately. Textbooks and teaching classes still emphasize that hydrogen atoms cannot be located with x-rays close to heavy elements; instead, neutron diffraction is needed. We show that, contrary to widespread expectation, hydrogen atoms can be located very accurately using x-ray diffraction, yielding bond lengths involving hydrogen atoms (A–H) that are in agreement with results from neutron diffraction mostly within a single standard deviation. The precision of the determination is also comparable between x-ray and neutron diffraction results. This has been achieved at resolutions as low as 0.8 Å using Hirshfeld atom refinement (HAR). We have applied HAR to 81 crystal structures of organic molecules and compared the A–H bond lengths with those from neutron measurements for A–H bonds sorted into bonds of the same class. We further show in a selection of inorganic compounds that hydrogen atoms can be located in bridging positions and close to heavy transition metals accurately and precisely. We anticipate that, in the future, conventional x-radiation sources at in-house diffractometers can be used routinely for locating hydrogen atoms in small molecules accurately instead of large-scale facilities such as spallation sources or nuclear reactors. PMID:27386545

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

The Race To X-ray Microbeam and Nanobeam Science

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

Ice, Gene E; Budai, John D; Pang, Judy

2011-01-01

X-ray microbeams are an emerging characterization tool with transformational implications for broad areas of science ranging from materials structure and dynamics, geophysics and environmental science to biophysics and protein crystallography. In this review, we discuss the race toward sub-10 nm- x-ray beams with the ability to penetrate tens to hundreds of microns into most materials and with the ability to determine local (crystal) structure. Examples of science enabled by current micro/nanobeam technologies are presented and we provide a perspective on future directions. Applications highlighted are chosen to illustrate the important features of various submicron beam strategies and to highlight themore » directions of current and future research. While it is clear that x-ray microprobes will impact science broadly, the practical limit for hard x-ray beam size, the limit to trace element sensitivity, and the ultimate limitations associated with near-atomic structure determinations are the subject of ongoing research.« less

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.

Serial femtosecond crystallography at the SACLA: breakthrough to dynamic structural biology.

PubMed

Mizohata, Eiichi; Nakane, Takanori; Fukuda, Yohta; Nango, Eriko; Iwata, So

2018-04-01

X-ray crystallography visualizes the world at the atomic level. It has been used as the most powerful technique for observing the three-dimensional structures of biological macromolecules and has pioneered structural biology. To determine a crystal structure with high resolution, it was traditionally required to prepare large crystals (> 200 μm). Later, synchrotron radiation facilities, such as SPring-8, that produce powerful X-rays were built. They enabled users to obtain good quality X-ray diffraction images even with smaller crystals (ca. 200-50 μm). In recent years, one of the most important technological innovations in structural biology has been the development of X-ray free electron lasers (XFELs). The SPring-8 Angstrom Compact free electron LAser (SACLA) in Japan generates the XFEL beam by accelerating electrons to relativistic speeds and directing them through in-vacuum, short-period undulators. Since user operation started in 2012, we have been involved in the development of serial femtosecond crystallography (SFX) measurement systems using XFEL at the SACLA. The SACLA generates X-rays a billion times brighter than SPring-8. The extremely bright XFEL pulses enable data collection with microcrystals (ca. 50-1 μm). Although many molecular analysis techniques exist, SFX is the only technique that can visualize radiation-damage-free structures of biological macromolecules at room temperature in atomic resolution and fast time resolution. Here, we review the achievements of the SACLA-SFX Project in the past 5 years. In particular, we focus on: (1) the measurement system for SFX; (2) experimental phasing by SFX; (3) enzyme chemistry based on damage-free room-temperature structures; and (4) molecular movie taken by time-resolved SFX.

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

Spherical grating based x-ray Talbot interferometry

PubMed Central

Cong, Wenxiang; Xi, Yan; Wang, Ge

2015-01-01

Purpose: Grating interferometry is a state-of-the-art x-ray imaging approach, which can acquire information on x-ray attenuation, phase shift, and small-angle scattering simultaneously. Phase-contrast imaging and dark-field imaging are very sensitive to microstructural variation and offers superior contrast resolution for biological soft tissues. However, a common x-ray tube is a point-like source. As a result, the popular planar grating imaging configuration seriously restricts the flux of photons and decreases the visibility of signals, yielding a limited field of view. The purpose of this study is to extend the planar x-ray grating imaging theory and methods to a spherical grating scheme for a wider range of preclinical and clinical applications. Methods: A spherical grating matches the wave front of a point x-ray source very well, allowing the perpendicular incidence of x-rays on the grating to achieve a higher visibility over a larger field of view than the planer grating counterpart. A theoretical analysis of the Talbot effect for spherical grating imaging is proposed to establish a basic foundation for x-ray spherical gratings interferometry. An efficient method of spherical grating imaging is also presented to extract attenuation, differential phase, and dark-field images in the x-ray spherical grating interferometer. Results: Talbot self-imaging with spherical gratings is analyzed based on the Rayleigh–Sommerfeld diffraction formula, featuring a periodic angular distribution in a polar coordinate system. The Talbot distance is derived to reveal the Talbot self-imaging pattern. Numerical simulation results show the self-imaging phenomenon of a spherical grating interferometer, which is in agreement with the theoretical prediction. Conclusions: X-ray Talbot interferometry with spherical gratings has a significant practical promise. Relative to planar grating imaging, spherical grating based x-ray Talbot interferometry has a larger field of view and

Spherical grating based x-ray Talbot interferometry

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

Cong, Wenxiang, E-mail: congw@rpi.edu, E-mail: xiy2@rpi.edu, E-mail: wangg6@rpi.edu; Xi, Yan, E-mail: congw@rpi.edu, E-mail: xiy2@rpi.edu, E-mail: wangg6@rpi.edu; Wang, Ge, E-mail: congw@rpi.edu, E-mail: xiy2@rpi.edu, E-mail: wangg6@rpi.edu

2015-11-15

Purpose: Grating interferometry is a state-of-the-art x-ray imaging approach, which can acquire information on x-ray attenuation, phase shift, and small-angle scattering simultaneously. Phase-contrast imaging and dark-field imaging are very sensitive to microstructural variation and offers superior contrast resolution for biological soft tissues. However, a common x-ray tube is a point-like source. As a result, the popular planar grating imaging configuration seriously restricts the flux of photons and decreases the visibility of signals, yielding a limited field of view. The purpose of this study is to extend the planar x-ray grating imaging theory and methods to a spherical grating scheme formore » a wider range of preclinical and clinical applications. Methods: A spherical grating matches the wave front of a point x-ray source very well, allowing the perpendicular incidence of x-rays on the grating to achieve a higher visibility over a larger field of view than the planer grating counterpart. A theoretical analysis of the Talbot effect for spherical grating imaging is proposed to establish a basic foundation for x-ray spherical gratings interferometry. An efficient method of spherical grating imaging is also presented to extract attenuation, differential phase, and dark-field images in the x-ray spherical grating interferometer. Results: Talbot self-imaging with spherical gratings is analyzed based on the Rayleigh–Sommerfeld diffraction formula, featuring a periodic angular distribution in a polar coordinate system. The Talbot distance is derived to reveal the Talbot self-imaging pattern. Numerical simulation results show the self-imaging phenomenon of a spherical grating interferometer, which is in agreement with the theoretical prediction. Conclusions: X-ray Talbot interferometry with spherical gratings has a significant practical promise. Relative to planar grating imaging, spherical grating based x-ray Talbot interferometry has a larger field of view and

Insights into the mechanism of X-ray-induced disulfide-bond cleavage in lysozyme crystals based on EPR, optical absorption and X-ray diffraction studies.

PubMed

Sutton, Kristin A; Black, Paul J; Mercer, Kermit R; Garman, Elspeth F; Owen, Robin L; Snell, Edward H; Bernhard, William A

2013-12-01

Electron paramagnetic resonance (EPR) and online UV-visible absorption microspectrophotometry with X-ray crystallography have been used in a complementary manner to follow X-ray-induced disulfide-bond cleavage. Online UV-visible spectroscopy showed that upon X-irradiation, disulfide radicalization appeared to saturate at an absorbed dose of approximately 0.5-0.8 MGy, in contrast to the saturating dose of ∼0.2 MGy observed using EPR at much lower dose rates. The observations suggest that a multi-track model involving product formation owing to the interaction of two separate tracks is a valid model for radiation damage in protein crystals. The saturation levels are remarkably consistent given the widely different experimental parameters and the range of total absorbed doses studied. The results indicate that even at the lowest doses used for structural investigations disulfide bonds are already radicalized. Multi-track considerations offer the first step in a comprehensive model of radiation damage that could potentially lead to a combined computational and experimental approach to identifying when damage is likely to be present, to quantitate it and to provide the ability to recover the native unperturbed structure.

Insights into the mechanism of X-ray-induced disulfide-bond cleavage in lysozyme crystals based on EPR, optical absorption and X-ray diffraction studies

PubMed Central

Sutton, Kristin A.; Black, Paul J.; Mercer, Kermit R.; Garman, Elspeth F.; Owen, Robin L.; Snell, Edward H.; Bernhard, William A.

2013-01-01

Electron paramagnetic resonance (EPR) and online UV–visible absorption microspectrophotometry with X-ray crystallography have been used in a complementary manner to follow X-ray-induced disulfide-bond cleavage. Online UV–visible spectroscopy showed that upon X-irradiation, disulfide radicalization appeared to saturate at an absorbed dose of approximately 0.5–0.8 MGy, in contrast to the saturating dose of ∼0.2 MGy observed using EPR at much lower dose rates. The observations suggest that a multi-track model involving product formation owing to the interaction of two separate tracks is a valid model for radiation damage in protein crystals. The saturation levels are remarkably consistent given the widely different experimental parameters and the range of total absorbed doses studied. The results indicate that even at the lowest doses used for structural investigations disulfide bonds are already radicalized. Multi-track considerations offer the first step in a comprehensive model of radiation damage that could potentially lead to a combined computational and experimental approach to identifying when damage is likely to be present, to quantitate it and to provide the ability to recover the native unperturbed structure. PMID:24311579

Accurate macromolecular structures using minimal measurements from X-ray free-electron lasers

PubMed Central

Hattne, Johan; Echols, Nathaniel; Tran, Rosalie; Kern, Jan; Gildea, Richard J.; Brewster, Aaron S.; Alonso-Mori, Roberto; Glöckner, Carina; Hellmich, Julia; Laksmono, Hartawan; Sierra, Raymond G.; Lassalle-Kaiser, Benedikt; Lampe, Alyssa; Han, Guangye; Gul, Sheraz; DiFiore, Dörte; Milathianaki, Despina; Fry, Alan R.; Miahnahri, Alan; White, William E.; Schafer, Donald W.; Seibert, M. Marvin; Koglin, Jason E.; Sokaras, Dimosthenis; Weng, Tsu-Chien; Sellberg, Jonas; Latimer, Matthew J.; Glatzel, Pieter; Zwart, Petrus H.; Grosse-Kunstleve, Ralf W.; Bogan, Michael J.; Messerschmidt, Marc; Williams, Garth J.; Boutet, Sébastien; Messinger, Johannes; Zouni, Athina; Yano, Junko; Bergmann, Uwe; Yachandra, Vittal K.; Adams, Paul D.; Sauter, Nicholas K.

2014-01-01

X-ray free-electron laser (XFEL) sources enable the use of crystallography to solve three-dimensional macromolecular structures under native conditions and free from radiation damage. Results to date, however, have been limited by the challenge of deriving accurate Bragg intensities from a heterogeneous population of microcrystals, while at the same time modeling the X-ray spectrum and detector geometry. Here we present a computational approach designed to extract statistically significant high-resolution signals from fewer diffraction measurements. PMID:24633409

An Excel Spreadsheet for a One-Dimensional Fourier Map in X-ray Crystallography

ERIC Educational Resources Information Center

Clegg, William

2004-01-01

The teaching of crystal structure determination with single-crystal X-ray diffraction at undergraduate level faces numerous challenges. Single-crystal X-ray diffraction is used in a vast range of chemical research projects and forms the basis for a high proportion of structural results that are presented to high-school, undergraduate, and graduate…

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.

Lab-based x-ray nanoCT imaging

NASA Astrophysics Data System (ADS)

Müller, Mark; Allner, Sebastian; Ferstl, Simone; Dierolf, Martin; Tuohimaa, Tomi; Pfeiffer, Franz

2017-03-01

Due to the recent development of transmission X-ray tubes with very small focal spot sizes, laboratory-based CT imaging with sub-micron resolutions is nowadays possible. We recently developed a novel X-ray nanoCT setup featuring a prototype nanofocus X-ray source and a single-photon counting detector. The system is based on mere geometrical magnification and can reach resolutions of 200 nm. To demonstrate the potential of the nanoCT system for biomedical applications we show high resolution nanoCT data of a small piece of human tooth comprising coronal dentin. The reconstructed CT data clearly visualize the dentin tubules within the tooth piece.

O-Alkylated heavy atom carbohydrate probes for protein X-ray crystallography: Studies towards the synthesis of methyl 2-O-methyl-L-selenofucopyranoside.

PubMed

Sommer, Roman; Hauck, Dirk; Varrot, Annabelle; Imberty, Anne; Künzler, Markus; Titz, Alexander

2016-01-01

Selenoglycosides are used as reactive glycosyl donors in the syntheses of oligosaccharides. In addition, such heavy atom analogs of natural glycosides are useful tools for structure determination of their lectin receptors using X-ray crystallography. Some lectins, e.g., members of the tectonin family, only bind to carbohydrate epitopes with O-alkylated ring hydroxy groups. In this context, we report the first synthesis of an O -methylated selenoglycoside, specifically methyl 2- O -methyl-L-selenofucopyranoside, a ligand of the lectin tectonin-2 from the mushroom Laccaria bicolor . The synthetic route required a strategic revision and further optimization due to the intrinsic lability of alkyl selenoglycosides, in particular for the labile fucose. Here, we describe a successful synthetic access to methyl 2- O -methyl-L-selenofucopyranoside in 9 linear steps and 26% overall yield starting from allyl L-fucopyranoside.

An Exercise in X-Ray Diffraction Using the Polymorphic Transition of Nickel Chromite.

ERIC Educational Resources Information Center

Chipman, David W.

1980-01-01

Describes a laboratory experiment appropriate for a course in either x-ray crystallography or mineralogy. The experiment permits the direct observation of a polymorphic transition in nickel chromite without the use of a special heating stage or heating camera. (Author/GS)

xMDFF: molecular dynamics flexible fitting of low-resolution X-ray structures.

PubMed

McGreevy, Ryan; Singharoy, Abhishek; Li, Qufei; Zhang, Jingfen; Xu, Dong; Perozo, Eduardo; Schulten, Klaus

2014-09-01

X-ray crystallography remains the most dominant method for solving atomic structures. However, for relatively large systems, the availability of only medium-to-low-resolution diffraction data often limits the determination of all-atom details. A new molecular dynamics flexible fitting (MDFF)-based approach, xMDFF, for determining structures from such low-resolution crystallographic data is reported. xMDFF employs a real-space refinement scheme that flexibly fits atomic models into an iteratively updating electron-density map. It addresses significant large-scale deformations of the initial model to fit the low-resolution density, as tested with synthetic low-resolution maps of D-ribose-binding protein. xMDFF has been successfully applied to re-refine six low-resolution protein structures of varying sizes that had already been submitted to the Protein Data Bank. Finally, via systematic refinement of a series of data from 3.6 to 7 Å resolution, xMDFF refinements together with electrophysiology experiments were used to validate the first all-atom structure of the voltage-sensing protein Ci-VSP.

Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers.

PubMed

Fuller, Franklin D; Gul, Sheraz; Chatterjee, Ruchira; Burgie, E Sethe; Young, Iris D; Lebrette, Hugo; Srinivas, Vivek; Brewster, Aaron S; Michels-Clark, Tara; Clinger, Jonathan A; Andi, Babak; Ibrahim, Mohamed; Pastor, Ernest; de Lichtenberg, Casper; Hussein, Rana; Pollock, Christopher J; Zhang, Miao; Stan, Claudiu A; Kroll, Thomas; Fransson, Thomas; Weninger, Clemens; Kubin, Markus; Aller, Pierre; Lassalle, Louise; Bräuer, Philipp; Miller, Mitchell D; Amin, Muhamed; Koroidov, Sergey; Roessler, Christian G; Allaire, Marc; Sierra, Raymond G; Docker, Peter T; Glownia, James M; Nelson, Silke; Koglin, Jason E; Zhu, Diling; Chollet, Matthieu; Song, Sanghoon; Lemke, Henrik; Liang, Mengning; Sokaras, Dimosthenis; Alonso-Mori, Roberto; Zouni, Athina; Messinger, Johannes; Bergmann, Uwe; Boal, Amie K; Bollinger, J Martin; Krebs, Carsten; Högbom, Martin; Phillips, George N; Vierstra, Richard D; Sauter, Nicholas K; Orville, Allen M; Kern, Jan; Yachandra, Vittal K; Yano, Junko

2017-04-01

X-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy, both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing insights into the interplay between the protein structure and dynamics and the chemistry at an active site. The implementation of such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly affects the data quality. We present here a robust way of delivering controlled sample amounts on demand using acoustic droplet ejection coupled with a conveyor belt drive that is optimized for crystallography and spectroscopy measurements of photochemical and chemical reactions over a wide range of time scales. Studies with photosystem II, the phytochrome photoreceptor, and ribonucleotide reductase R2 illustrate the power and versatility of this method.

Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers

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

Fuller, Franklin D.; Gul, Sheraz; Chatterjee, Ruchira

X-ray crystallography at X-ray free-electron laser (XFEL) sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy (XES), both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing new insights into the interplay between the protein structure/dynamics and chemistry at an active site. However, implementing such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly impacts the data quality. We present here a new, robust way of delivering controlled sample amountsmore » on demand using acoustic droplet ejection coupled with a conveyor belt drive that is optimized for crystallography and spectroscopy measurements of photochemical and chemical reactions over a wide range of time scales. Studies with photosystem II, the phytochrome photoreceptor, and ribonucleotide reductase R2 illustrate the power and versatility of this method.« less

Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers

DOE PAGES

Fuller, Franklin D.; Gul, Sheraz; Chatterjee, Ruchira; ...

2017-02-27

X-ray crystallography at X-ray free-electron laser (XFEL) sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy (XES), both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing new insights into the interplay between the protein structure/dynamics and chemistry at an active site. However, implementing such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly impacts the data quality. We present here a new, robust way of delivering controlled sample amountsmore » on demand using acoustic droplet ejection coupled with a conveyor belt drive that is optimized for crystallography and spectroscopy measurements of photochemical and chemical reactions over a wide range of time scales. Studies with photosystem II, the phytochrome photoreceptor, and ribonucleotide reductase R2 illustrate the power and versatility of this method.« less

Mitigation of X-ray damage in macromolecular crystallography by submicrometre line focusing.

PubMed

Finfrock, Y Zou; Stern, Edward A; Alkire, R W; Kas, Joshua J; Evans-Lutterodt, Kenneth; Stein, Aaron; Duke, Norma; Lazarski, Krzysztof; Joachimiak, Andrzej

2013-08-01

Reported here are measurements of the penetration depth and spatial distribution of photoelectron (PE) damage excited by 18.6 keV X-ray photons in a lysozyme crystal with a vertical submicrometre line-focus beam of 0.7 µm full-width half-maximum (FWHM). The experimental results determined that the penetration depth of PEs is 5 ± 0.5 µm with a monotonically decreasing spatial distribution shape, resulting in mitigation of diffraction signal damage. This does not agree with previous theoretical predication that the mitigation of damage requires a peak of damage outside the focus. A new improved calculation provides some qualitative agreement with the experimental results, but significant errors still remain. The mitigation of radiation damage by line focusing was measured experimentally by comparing the damage in the X-ray-irradiated regions of the submicrometre focus with the large-beam case under conditions of equal exposure and equal volumes of the protein crystal, and a mitigation factor of 4.4 ± 0.4 was determined. The mitigation of radiation damage is caused by spatial separation of the dominant PE radiation-damage component from the crystal region of the line-focus beam that contributes the diffraction signal. The diffraction signal is generated by coherent scattering of incident X-rays (which introduces no damage), while the overwhelming proportion of damage is caused by PE emission as X-ray photons are absorbed.

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

Dynamic X-ray diffraction sampling for protein crystal positioning

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

Scarborough, Nicole M.; Godaliyadda, G. M. Dilshan P.; Ye, Dong Hye

A sparse supervised learning approach for dynamic sampling (SLADS) is described for dose reduction in diffraction-based protein crystal positioning. Crystal centering is typically a prerequisite for macromolecular diffraction at synchrotron facilities, with X-ray diffraction mapping growing in popularity as a mechanism for localization. In X-ray raster scanning, diffraction is used to identify the crystal positions based on the detection of Bragg-like peaks in the scattering patterns; however, this additional X-ray exposure may result in detectable damage to the crystal prior to data collection. Dynamic sampling, in which preceding measurements inform the next most information-rich location to probe for image reconstruction,more » significantly reduced the X-ray dose experienced by protein crystals during positioning by diffraction raster scanning. The SLADS algorithm implemented herein is designed for single-pixel measurements and can select a new location to measure. In each step of SLADS, the algorithm selects the pixel, which, when measured, maximizes the expected reduction in distortion given previous measurements. Ground-truth diffraction data were obtained for a 5 µm-diameter beam and SLADS reconstructed the image sampling 31% of the total volume and only 9% of the interior of the crystal greatly reducing the X-ray dosage on the crystal. Furthermore, by usingin situtwo-photon-excited fluorescence microscopy measurements as a surrogate for diffraction imaging with a 1 µm-diameter beam, the SLADS algorithm enabled image reconstruction from a 7% sampling of the total volume and 12% sampling of the interior of the crystal. When implemented into the beamline at Argonne National Laboratory, without ground-truth images, an acceptable reconstruction was obtained with 3% of the image sampled and approximately 5% of the crystal. The incorporation of SLADS into X-ray diffraction acquisitions has the potential to significantly minimize the impact of X-ray exposure

Dynamic X-ray diffraction sampling for protein crystal positioning

PubMed Central

Scarborough, Nicole M.; Godaliyadda, G. M. Dilshan P.; Ye, Dong Hye; Kissick, David J.; Zhang, Shijie; Newman, Justin A.; Sheedlo, Michael J.; Chowdhury, Azhad U.; Fischetti, Robert F.; Das, Chittaranjan; Buzzard, Gregery T.; Bouman, Charles A.; Simpson, Garth J.

2017-01-01

A sparse supervised learning approach for dynamic sampling (SLADS) is described for dose reduction in diffraction-based protein crystal positioning. Crystal centering is typically a prerequisite for macromolecular diffraction at synchrotron facilities, with X-ray diffraction mapping growing in popularity as a mechanism for localization. In X-ray raster scanning, diffraction is used to identify the crystal positions based on the detection of Bragg-like peaks in the scattering patterns; however, this additional X-ray exposure may result in detectable damage to the crystal prior to data collection. Dynamic sampling, in which preceding measurements inform the next most information-rich location to probe for image reconstruction, significantly reduced the X-ray dose experienced by protein crystals during positioning by diffraction raster scanning. The SLADS algorithm implemented herein is designed for single-pixel measurements and can select a new location to measure. In each step of SLADS, the algorithm selects the pixel, which, when measured, maximizes the expected reduction in distortion given previous measurements. Ground-truth diffraction data were obtained for a 5 µm-diameter beam and SLADS reconstructed the image sampling 31% of the total volume and only 9% of the interior of the crystal greatly reducing the X-ray dosage on the crystal. Using in situ two-photon-excited fluorescence microscopy measurements as a surrogate for diffraction imaging with a 1 µm-diameter beam, the SLADS algorithm enabled image reconstruction from a 7% sampling of the total volume and 12% sampling of the interior of the crystal. When implemented into the beamline at Argonne National Laboratory, without ground-truth images, an acceptable reconstruction was obtained with 3% of the image sampled and approximately 5% of the crystal. The incorporation of SLADS into X-ray diffraction acquisitions has the potential to significantly minimize the impact of X-ray exposure on the crystal by

Dynamic X-ray diffraction sampling for protein crystal positioning.

PubMed

Scarborough, Nicole M; Godaliyadda, G M Dilshan P; Ye, Dong Hye; Kissick, David J; Zhang, Shijie; Newman, Justin A; Sheedlo, Michael J; Chowdhury, Azhad U; Fischetti, Robert F; Das, Chittaranjan; Buzzard, Gregery T; Bouman, Charles A; Simpson, Garth J

2017-01-01

A sparse supervised learning approach for dynamic sampling (SLADS) is described for dose reduction in diffraction-based protein crystal positioning. Crystal centering is typically a prerequisite for macromolecular diffraction at synchrotron facilities, with X-ray diffraction mapping growing in popularity as a mechanism for localization. In X-ray raster scanning, diffraction is used to identify the crystal positions based on the detection of Bragg-like peaks in the scattering patterns; however, this additional X-ray exposure may result in detectable damage to the crystal prior to data collection. Dynamic sampling, in which preceding measurements inform the next most information-rich location to probe for image reconstruction, significantly reduced the X-ray dose experienced by protein crystals during positioning by diffraction raster scanning. The SLADS algorithm implemented herein is designed for single-pixel measurements and can select a new location to measure. In each step of SLADS, the algorithm selects the pixel, which, when measured, maximizes the expected reduction in distortion given previous measurements. Ground-truth diffraction data were obtained for a 5 µm-diameter beam and SLADS reconstructed the image sampling 31% of the total volume and only 9% of the interior of the crystal greatly reducing the X-ray dosage on the crystal. Using in situ two-photon-excited fluorescence microscopy measurements as a surrogate for diffraction imaging with a 1 µm-diameter beam, the SLADS algorithm enabled image reconstruction from a 7% sampling of the total volume and 12% sampling of the interior of the crystal. When implemented into the beamline at Argonne National Laboratory, without ground-truth images, an acceptable reconstruction was obtained with 3% of the image sampled and approximately 5% of the crystal. The incorporation of SLADS into X-ray diffraction acquisitions has the potential to significantly minimize the impact of X-ray exposure on the crystal by

Dynamic X-ray diffraction sampling for protein crystal positioning

DOE PAGES

Scarborough, Nicole M.; Godaliyadda, G. M. Dilshan P.; Ye, Dong Hye; ...

2017-01-01

A sparse supervised learning approach for dynamic sampling (SLADS) is described for dose reduction in diffraction-based protein crystal positioning. Crystal centering is typically a prerequisite for macromolecular diffraction at synchrotron facilities, with X-ray diffraction mapping growing in popularity as a mechanism for localization. In X-ray raster scanning, diffraction is used to identify the crystal positions based on the detection of Bragg-like peaks in the scattering patterns; however, this additional X-ray exposure may result in detectable damage to the crystal prior to data collection. Dynamic sampling, in which preceding measurements inform the next most information-rich location to probe for image reconstruction,more » significantly reduced the X-ray dose experienced by protein crystals during positioning by diffraction raster scanning. The SLADS algorithm implemented herein is designed for single-pixel measurements and can select a new location to measure. In each step of SLADS, the algorithm selects the pixel, which, when measured, maximizes the expected reduction in distortion given previous measurements. Ground-truth diffraction data were obtained for a 5 µm-diameter beam and SLADS reconstructed the image sampling 31% of the total volume and only 9% of the interior of the crystal greatly reducing the X-ray dosage on the crystal. Furthermore, by usingin situtwo-photon-excited fluorescence microscopy measurements as a surrogate for diffraction imaging with a 1 µm-diameter beam, the SLADS algorithm enabled image reconstruction from a 7% sampling of the total volume and 12% sampling of the interior of the crystal. When implemented into the beamline at Argonne National Laboratory, without ground-truth images, an acceptable reconstruction was obtained with 3% of the image sampled and approximately 5% of the crystal. The incorporation of SLADS into X-ray diffraction acquisitions has the potential to significantly minimize the impact of X-ray exposure

CCD sensors in synchrotron X-ray detectors

NASA Astrophysics Data System (ADS)

Strauss, M. G.; Naday, I.; Sherman, I. S.; Kraimer, M. R.; Westbrook, E. M.; Zaluzec, N. J.

1988-04-01

The intense photon flux from advanced synchrotron light sources, such as the 7-GeV synchrotron being designed at Argonne, require integrating-type detectors. Charge-coupled devices (CCDs) are well suited as synchrotron X-ray detectors. When irradiated indirectly via a phosphor followed by reducing optics, diffraction patterns of 100 cm 2 can be imaged on a 2 cm 2 CCD. With a conversion efficiency of ˜ 1 CCD electron/X-ray photon, a peak saturation capacity of > 10 6 X-rays can be obtained. A programmable CCD controller operating at a clock frequency of 20 MHz has been developed. The readout rate is 5 × 10 6 pixels/s and the shift rate in the parallel registers is 10 6 lines/s. The test detector was evaluated in two experiments. In protein crystallography diffraction patterns have been obtained from a lysozyme crystal using a conventional rotating anode X-ray generator. Based on these results we expect to obtain at a synchrotron diffraction images at a rate of ˜ 1 frame/s or a complete 3-dimensional data set from a single crystal in ˜ 2 min. In electron energy-loss spectroscopy (EELS), the CCD was used in a parallel detection mode which is similar to the mode array detectors are used in dispersive EXAFS. With a beam current corresponding to 3 × 10 9 electron/s on the detector, a series of 64 spectra were recorded on the CCD in a continuous sequence without interruption due to readout. The frame-to-frame pixel signal fluctuations had σ = 0.4% from which DQE = 0.4 was obtained, where the detector conversion efficiency was 2.6 CCD electrons/X-ray photon. These multiple frame series also showed the time-resolved modulation of the electron microscope optics by stray magnetic fields.

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

Single-crystal Raman spectroscopy and X-ray crystallography at beamline X26-C of the NSLS

PubMed Central

Stoner-Ma, Deborah; Skinner, John M.; Schneider, Dieter K.; Cowan, Matt; Sweet, Robert M.; Orville, Allen M.

2011-01-01

Three-dimensional structures derived from X-ray diffraction of protein crystals provide a wealth of information. Features and interactions important for the function of macromolecules can be deduced and catalytic mechanisms postulated. Still, many questions can remain, for example regarding metal oxidation states and the interpretation of ‘mystery density’, i.e. ambiguous or unknown features within the electron density maps, especially at ∼2 Å resolutions typical of most macromolecular structures. Beamline X26-C at the National Synchrotron Light Source (NSLS), Brookhaven National Laboratory (BNL), provides researchers with the opportunity to not only determine the atomic structure of their samples but also to explore the electronic and vibrational characteristics of the sample before, during and after X-ray diffraction data collection. When samples are maintained under cryo-conditions, an opportunity to promote and follow photochemical reactions in situ as a function of X-ray exposure is also provided. Plans are in place to further expand the capabilities at beamline X26-C and to develop beamlines at NSLS-II, currently under construction at BNL, which will provide users access to a wide array of complementary spectroscopic methods in addition to high-quality X-ray diffraction data. PMID:21169688

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

Structural insights into binding of inhibitors to soluble epoxide hydrolase gained by fragment screening and X-ray crystallography.

PubMed

Amano, Yasushi; Yamaguchi, Tomohiko; Tanabe, Eiki

2014-04-15

Soluble epoxide hydrolase (sEH) is a component of the arachidonic acid cascade and is a candidate target for therapies for hypertension or inflammation. Although many sEH inhibitors are available, their scaffolds are not structurally diverse, and knowledge of their specific interactions with sEH is limited. To obtain detailed structural information about protein-ligand interactions, we conducted fragment screening of sEH, analyzed the fragments using high-throughput X-ray crystallography, and determined 126 fragment-bound structures at high resolution. Aminothiazole and benzimidazole derivatives were identified as novel scaffolds that bind to the catalytic triad of sEH with good ligand efficiency. We further identified fragment hits that bound to subpockets of sEH called the short and long branches. The water molecule conserved in the structure plays an important role in binding to the long branch, whereas Asp496 and the main chain of Phe497 form hydrogen bonds with fragment hits in the short branch. Fragment hits and their crystal structures provide structural insights into ligand binding to sEH that will facilitate the discovery of novel and potent inhibitors of sEH. Copyright © 2014 Elsevier Ltd. 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.

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.…

BioCARS: a synchrotron resource for time-resolved X-ray science

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

Graber, T.; Anderson, S.; Brewer, H.

2011-08-16

BioCARS, a NIH-supported national user facility for macromolecular time-resolved X-ray crystallography at the Advanced Photon Source (APS), has recently completed commissioning of an upgraded undulator-based beamline optimized for single-shot laser-pump X-ray-probe measurements with time resolution as short as 100 ps. The source consists of two in-line undulators with periods of 23 and 27 mm that together provide high-flux pink-beam capability at 12 keV as well as first-harmonic coverage from 6.8 to 19 keV. A high-heat-load chopper reduces the average power load on downstream components, thereby preserving the surface figure of a Kirkpatrick-Baez mirror system capable of focusing the X-ray beammore » to a spot size of 90 {micro}m horizontal by 20 {micro}m vertical. A high-speed chopper isolates single X-ray pulses at 1 kHz in both hybrid and 24-bunch modes of the APS storage ring. In hybrid mode each isolated X-ray pulse delivers up to {approx}4 x 10{sup 10} photons to the sample, thereby achieving a time-averaged flux approaching that of fourth-generation X-FEL sources. A new high-power picosecond laser system delivers pulses tunable over the wavelength range 450-2000 nm. These pulses are synchronized to the storage-ring RF clock with long-term stability better than 10 ps RMS. Monochromatic experimental capability with Biosafety Level 3 certification has been retained.« less

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

SIBYLS - a SAXS and Protein Crystallography Beamline at the ALS

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

Trame, C.; MacDowell, A.A.; Celestre, R.S.

2004-05-12

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 2x1011 hv/sec through a 100{mu}m pinhole at 12.4KeV. For SAXS the flux is up to 3x1013photons/sec at 10KeV with all apertures open when using the multilayer monochromator elements. The performance characteristics of this unique beamline will be described.« less

Emerging opportunities in structural biology with X-ray free-electron lasers

PubMed Central

Schlichting, Ilme; Miao, Jianwei

2012-01-01

X-ray free-electron lasers (X-FELs) produce X-ray pulses with extremely brilliant peak intensity and ultrashort pulse duration. It has been proposed that radiation damage can be “outrun” by using an ultra intense and short X-FEL pulse that passes a biological sample before the onset of significant radiation damage. The concept of “diffraction-before-destruction” has been demonstrated recently at the Linac Coherent Light Source, the first operational hard X-ray FEL, for protein nanocrystals and giant virus particles. The continuous diffraction patterns from single particles allow solving the classical “phase problem” by the oversampling method with iterative algorithms. If enough data are collected from many identical copies of a (biological) particle, its three-dimensional structure can be reconstructed. We review the current status and future prospects of serial femtosecond crystallography (SFX) and single-particle coherent diffraction imaging (CDI) with X-FELs. PMID:22922042

Monitoring X-Ray Emission from X-Ray Bursters

NASA Technical Reports Server (NTRS)

Halpern, Jules P.; Kaaret, Philip

1999-01-01

The scientific goal of this project was to monitor a selected sample of x-ray bursters using data from the All-Sky Monitor (ASM) on the Rossi X-Ray Timing Explorer together with data from the Burst and Transient Source Experiment (BATSE) on the Compton Gamma-Ray Observatory to study the long-term temporal evolution of these sources in the x-ray and hard x-ray bands. The project was closely related to "Long-Term Hard X-Ray Monitoring of X-Ray Bursters", NASA project NAG5-3891, and and "Hard x-ray emission of x-ray bursters", NASA project NAG5-4633, and shares publications in common with both of these. The project involved preparation of software for use in monitoring and then the actual monitoring itself. These efforts have lead to results directly from the ASM data and also from Target of Opportunity Observations (TOO) made with the Rossi X-Ray Timing Explorer based on detection of transient hard x-ray outbursts with the ASM and BATSE.

A mirror for lab-based quasi-monochromatic parallel x-rays

NASA Astrophysics Data System (ADS)

Nguyen, Thanhhai; Lu, Xun; Lee, Chang Jun; Jung, Jin-Ho; Jin, Gye-Hwan; Kim, Sung Youb; Jeon, Insu

2014-09-01

A multilayered parabolic mirror with six W/Al bilayers was designed and fabricated to generate monochromatic parallel x-rays using a lab-based x-ray source. Using this mirror, curved bright bands were obtained in x-ray images as reflected x-rays. The parallelism of the reflected x-rays was investigated using the shape of the bands. The intensity and monochromatic characteristics of the reflected x-rays were evaluated through measurements of the x-ray spectra in the band. High intensity, nearly monochromatic, and parallel x-rays, which can be used for high resolution x-ray microscopes and local radiation therapy systems, were obtained.

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

Towards phasing using high X-ray intensity

DOE PAGES

Galli, Lorenzo; Son, Sang -Kil; Barends, Thomas R. M.; ...

2015-09-30

X-ray free-electron lasers (XFELs) show great promise for macromolecular structure determination from sub-micrometre-sized crystals, using the emerging method of serial femtosecond crystallography. The extreme brightness of the XFEL radiation can multiply ionize most, if not all, atoms in a protein, causing their scattering factors to change during the pulse, with a preferential `bleaching' of heavy atoms. This paper investigates the effects of electronic damage on experimental data collected from a Gd derivative of lysozyme microcrystals at different X-ray intensities, and the degree of ionization of Gd atoms is quantified from phased difference Fourier maps. In conclusion, a pattern sorting schememore » is proposed to maximize the ionization contrast and the way in which the local electronic damage can be used for a new experimental phasing method is discussed.« less

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

Covering complete proteomes with X-ray structures: A current snapshot

DOE PAGES

Mizianty, Marcin J.; Fan, Xiao; Yan, Jing; ...

2014-10-23

Structural genomics programs have developed and applied structure-determination pipelines to a wide range of protein targets, facilitating the visualization of macromolecular interactions and the understanding of their molecular and biochemical functions. The fundamental question of whether three-dimensional structures of all proteins and all functional annotations can be determined using X-ray crystallography is investigated. A first-of-its-kind large-scale analysis of crystallization propensity for all proteins encoded in 1953 fully sequenced genomes was performed. It is shown that current X-ray crystallographic knowhow combined with homology modeling can provide structures for 25% of modeling families (protein clusters for which structural models can be obtainedmore » through homology modeling), with at least one structural model produced for each Gene Ontology functional annotation. The coverage varies between superkingdoms, with 19% for eukaryotes, 35% for bacteria and 49% for archaea, and with those of viruses following the coverage values of their hosts. It is shown that the crystallization propensities of proteomes from the taxonomic superkingdoms are distinct. The use of knowledge-based target selection is shown to substantially increase the ability to produce X-ray structures. It is demonstrated that the human proteome has one of the highest attainable coverage values among eukaryotes, and GPCR membrane proteins suitable for X-ray structure determination were determined.« less

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.

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

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.

Micromirror-based manipulation of synchrotron x-ray beams

NASA Astrophysics Data System (ADS)

Walko, D. A.; Chen, Pice; Jung, I. W.; Lopez, D.; Schwartz, C. P.; Shenoy, G. K.; Wang, Jin

2017-08-01

Synchrotron beamlines typically use macroscopic, quasi-static optics to manipulate x-ray beams. We present the use of dynamic microelectromechanical systems-based optics (MEMS) to temporally modulate synchrotron x-ray beams. We demonstrate this concept using single-crystal torsional MEMS micromirrors oscillating at frequencies of 75 kHz. Such a MEMS micromirror, with lateral dimensions of a few hundred micrometers, can interact with x rays by operating in grazing-incidence reflection geometry; x rays are deflected only when an x-ray pulse is incident on the rotating micromirror under appropriate conditions, i.e., at an angle less than the critical angle for reflectivity. The time window for such deflections depends on the frequency and amplitude of the MEMS rotation. We demonstrate that reflection geometry can produce a time window of a few microseconds. We further demonstrate that MEMS optics can isolate x rays from a selected synchrotron bunch or group of bunches. With ray-trace simulations we explain the currently achievable time windows and suggest a path toward improvements.

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.

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

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

High-Resolution Protein Structure Determination by Serial Femtosecond Crystallography

PubMed Central

Boutet, Sébastien; Lomb, Lukas; Williams, Garth J.; Barends, Thomas R. M.; Aquila, Andrew; Doak, R. Bruce; Weierstall, Uwe; DePonte, Daniel P.; Steinbrener, Jan; Shoeman, Robert L.; Messerschmidt, Marc; Barty, Anton; White, Thomas A.; Kassemeyer, Stephan; Kirian, Richard A.; Seibert, M. Marvin; Montanez, Paul A.; Kenney, Chris; Herbst, Ryan; Hart, Philip; Pines, Jack; Haller, Gunther; Gruner, Sol M.; Philipp, Hugh T.; Tate, Mark W.; Hromalik, Marianne; Koerner, Lucas J.; van Bakel, Niels; Morse, John; Ghonsalves, Wilfred; Arnlund, David; Bogan, Michael J.; Caleman, Carl; Fromme, Raimund; Hampton, Christina Y.; Hunter, Mark S.; Johansson, Linda C.; Katona, Gergely; Kupitz, Christopher; Liang, Mengning; Martin, Andrew V.; Nass, Karol; Redecke, Lars; Stellato, Francesco; Timneanu, Nicusor; Wang, Dingjie; Zatsepin, Nadia A.; Schafer, Donald; Defever, James; Neutze, Richard; Fromme, Petra; Spence, John C. H.; Chapman, Henry N.; Schlichting, Ilme

2013-01-01

Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules. PMID:22653729

Three-dimensional imaging of nanoscale materials by using coherent x-rays

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

Miao, Jianwei

X-ray crystallography is currently the primary methodology used to determine the 3D structure of materials and macromolecules. However, many nanostructures, disordered materials, biomaterials, hybrid materials and biological specimens are noncrystalline and, hence, their structures are not accessible by X-ray crystallography. Probing these structures therefore requires the employment of different approaches. A very promising technique currently under rapid development is X-ray diffraction microscopy (or lensless imaging), in which the coherent X-ray diffraction pattern of a noncrystalline specimen is measured and then directly phased to obtain a high-resolution image. Through the DOE support over the past three years, we have applied X-raymore » diffraction microscopy to quantitative imaging of GaN quantum dot particles, and revealed the internal GaN-Ga2O3 core shell structure in three dimensions. By exploiting the abrupt change in the scattering cross-section near electronic resonances, we carried out the first experimental demonstration of resonant X-ray diffraction microscopy for element specific imaging. We performed nondestructive and quantitative imaging of buried Bi structures inside a Si crystal by directly phasing coherent X-ray diffraction patterns acquired below and above the Bi M5 edge. We have also applied X-ray diffraction microscopy to nondestructive imaging of mineral crystals inside biological composite materials - intramuscular fish bone - at the nanometer scale resolution. We identified mineral crystals in collagen fibrils at different stages of mineralization and proposed a dynamic mechanism to account for the nucleation and growth of mineral crystals in the collagen matrix. In addition, we have also discovered a novel 3D imaging modality, denoted ankylography, which allows for complete 3D structure determination without the necessity of sample titling or scanning. We showed that when the diffraction pattern of a finite object is sampled at a

Selenium single-wavelength anomalous diffraction de novo phasing using an X-ray-free electron laser

DOE PAGES

Hunter, Mark S.; Yoon, Chun Hong; DeMirci, Hasan; ...

2016-11-04

Structural information about biological macromolecules near the atomic scale provides important insight into the functions of these molecules. To date, X-ray crystallography has been the predominant method used for macromolecular structure determination. However, challenges exist when solving structures with X-rays, including the phase problem and radiation damage. X-ray-free electron lasers (X-ray FELs) have enabled collection of diffraction information before the onset of radiation damage, yet the majority of structures solved at X-ray FELs have been phased using external information via molecular replacement. De novo phasing at X-ray FELs has proven challenging due in part to per-pulse variations in intensity andmore » wavelength. Here we report the solution of a selenobiotinyl-streptavidin structure using phases obtained by the anomalous diffraction of selenium measured at a single wavelength (Se-SAD) at the Linac Coherent Light Source. Finally, our results demonstrate Se-SAD, routinely employed at synchrotrons for novel structure determination, is now possible at X-ray FELs.« less

Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography.

PubMed

Roessler, Christian G; Agarwal, Rakhi; Allaire, Marc; Alonso-Mori, Roberto; Andi, Babak; Bachega, José F R; Bommer, Martin; Brewster, Aaron S; Browne, Michael C; Chatterjee, Ruchira; Cho, Eunsun; Cohen, Aina E; Cowan, Matthew; Datwani, Sammy; Davidson, Victor L; Defever, Jim; Eaton, Brent; Ellson, Richard; Feng, Yiping; Ghislain, Lucien P; Glownia, James M; Han, Guangye; Hattne, Johan; Hellmich, Julia; Héroux, Annie; Ibrahim, Mohamed; Kern, Jan; Kuczewski, Anthony; Lemke, Henrik T; Liu, Pinghua; Majlof, Lars; McClintock, William M; Myers, Stuart; Nelsen, Silke; Olechno, Joe; Orville, Allen M; Sauter, Nicholas K; Soares, Alexei S; Soltis, S Michael; Song, Heng; Stearns, Richard G; Tran, Rosalie; Tsai, Yingssu; Uervirojnangkoorn, Monarin; Wilmot, Carrie M; Yachandra, Vittal; Yano, Junko; Yukl, Erik T; Zhu, Diling; Zouni, Athina

2016-04-05

X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples. Copyright © 2016 Elsevier Ltd. All rights reserved.

Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography

DOE PAGES

Roessler, Christian G.; Agarwal, Rakhi; Allaire, Marc; ...

2016-03-17

X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallizationmore » conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. Lastly, we report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). In addition, samples were screened to demonstrate that these methods can be applied to rare samples« less

Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography

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

Roessler, Christian G.; Agarwal, Rakhi; Allaire, Marc

X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallizationmore » conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.« less

Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography

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

Roessler, Christian G.; Agarwal, Rakhi; Allaire, Marc

X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallizationmore » conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. Lastly, we report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). In addition, samples were screened to demonstrate that these methods can be applied to rare samples« less

Multiple pinhole collimator based X-ray luminescence computed tomography

PubMed Central

Zhang, Wei; Zhu, Dianwen; Lun, Michael; Li, Changqing

2016-01-01

X-ray luminescence computed tomography (XLCT) is an emerging hybrid imaging modality, which is able to improve the spatial resolution of optical imaging to hundreds of micrometers for deep targets by using superfine X-ray pencil beams. However, due to the low X-ray photon utilization efficiency in a single pinhole collimator based XLCT, it takes a long time to acquire measurement data. Herein, we propose a multiple pinhole collimator based XLCT, in which multiple X-ray beams are generated to scan a sample at multiple positions simultaneously. Compared with the single pinhole based XLCT, the multiple X-ray beam scanning method requires much less measurement time. Numerical simulations and phantom experiments have been performed to demonstrate the feasibility of the multiple X-ray beam scanning method. In one numerical simulation, we used four X-ray beams to scan a cylindrical object with 6 deeply embedded targets. With measurements from 6 angular projections, all 6 targets have been reconstructed successfully. In the phantom experiment, we generated two X-ray pencil beams with a collimator manufactured in-house. Two capillary targets with 0.6 mm edge-to-edge distance embedded in a cylindrical phantom have been reconstructed successfully. With the two beam scanning, we reduced the data acquisition time by 50%. From the reconstructed XLCT images, we found that the Dice similarity of targets is 85.11% and the distance error between two targets is less than 3%. We have measured the radiation dose during XLCT scan and found that the radiation dose, 1.475 mSv, is in the range of a typical CT scan. We have measured the changes of the collimated X-ray beam size and intensity at different distances from the collimator. We have also studied the effects of beam size and intensity in the reconstruction of XLCT. PMID:27446686

A Newly Designed Microspectrofluorometer for Kinetic Studies on Protein Crystals in Combination with X-Ray Diffraction

PubMed Central

Klink, Björn U.; Goody, Roger S.; Scheidig, Axel J.

2006-01-01

We present a new design for a fluorescence microspectrophotometer for use in kinetic crystallography in combination with x-ray diffraction experiments. The FLUMIX device (Fluorescence spectroscopy to monitor intermediates in x-ray crystallography) is built for 0° fluorescence detection, which has several advantages in comparison to a conventional fluorometer with 90° design. Due to the reduced spatial requirements and the need for only one objective, the system is highly versatile, easy to handle, and can be used for many different applications. In combination with a conventional stereomicroscope, fluorescence measurements or reaction initiation can be performed directly in a hanging drop crystallization setup. The FLUMIX device can be combined with most x-ray sources, normally without the need of a specialized mechanical support. As a biological model system, we have used H-Ras p21 with an artificially introduced photo-labile GTP precursor (caged GTP) and a covalently attached fluorophore (IANBD amide). Using the FLUMIX system, detailed information about the state of photolyzed crystals of the modified H-Ras p21 (p21(mod)) could be obtained. Measurements in combination with a synchrotron beamline showed significant fluorescence changes in p21(mod) crystals even within a few seconds of x-ray exposure at 100 K. PMID:16698776

Insights into photosystem II from isomorphous difference Fourier maps of femtosecond X-ray diffraction data and quantum mechanics/molecular mechanics structural models

DOE PAGES

Wang, Jimin; Askerka, Mikhail; Brudvig, Gary W.; ...

2017-01-12

Understanding structure–function relations in photosystem II (PSII) is important for the development of biomimetic photocatalytic systems. X-ray crystallography, computational modeling, and spectroscopy have played central roles in elucidating the structure and function of PSII. Recent breakthroughs in femtosecond X-ray crystallography offer the possibility of collecting diffraction data from the X-ray free electron laser (XFEL) before radiation damage of the sample, thereby overcoming the main challenge of conventional X-ray diffraction methods. However, the interpretation of XFEL data from PSII intermediates is challenging because of the issues regarding data-processing, uncertainty on the precise positions of light oxygen atoms next to heavy metalmore » centers, and different kinetics of the S-state transition in microcrystals compared to solution. Lastly, we summarize recent advances and outstanding challenges in PSII structure–function determination with emphasis on the implementation of quantum mechanics/molecular mechanics techniques combined with isomorphous difference Fourier maps, direct methods, and high-resolution spectroscopy.« less

Insights into Photosystem II from Isomorphous Difference Fourier Maps of Femtosecond X-ray Diffraction Data and Quantum Mechanics/Molecular Mechanics Structural Models.

PubMed

Wang, Jimin; Askerka, Mikhail; Brudvig, Gary W; Batista, Victor S

2017-02-10

Understanding structure-function relations in photosystem II (PSII) is important for the development of biomimetic photocatalytic systems. X-ray crystallography, computational modeling, and spectroscopy have played central roles in elucidating the structure and function of PSII. Recent breakthroughs in femtosecond X-ray crystallography offer the possibility of collecting diffraction data from the X-ray free electron laser (XFEL) before radiation damage of the sample, thereby overcoming the main challenge of conventional X-ray diffraction methods. However, the interpretation of XFEL data from PSII intermediates is challenging because of the issues regarding data-processing, uncertainty on the precise positions of light oxygen atoms next to heavy metal centers, and different kinetics of the S-state transition in microcrystals compared to solution. Here, we summarize recent advances and outstanding challenges in PSII structure-function determination with emphasis on the implementation of quantum mechanics/molecular mechanics techniques combined with isomorphous difference Fourier maps, direct methods, and high-resolution spectroscopy.

Insights into photosystem II from isomorphous difference Fourier maps of femtosecond X-ray diffraction data and quantum mechanics/molecular mechanics structural models

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

Wang, Jimin; Askerka, Mikhail; Brudvig, Gary W.

Understanding structure–function relations in photosystem II (PSII) is important for the development of biomimetic photocatalytic systems. X-ray crystallography, computational modeling, and spectroscopy have played central roles in elucidating the structure and function of PSII. Recent breakthroughs in femtosecond X-ray crystallography offer the possibility of collecting diffraction data from the X-ray free electron laser (XFEL) before radiation damage of the sample, thereby overcoming the main challenge of conventional X-ray diffraction methods. However, the interpretation of XFEL data from PSII intermediates is challenging because of the issues regarding data-processing, uncertainty on the precise positions of light oxygen atoms next to heavy metalmore » centers, and different kinetics of the S-state transition in microcrystals compared to solution. Lastly, we summarize recent advances and outstanding challenges in PSII structure–function determination with emphasis on the implementation of quantum mechanics/molecular mechanics techniques combined with isomorphous difference Fourier maps, direct methods, and high-resolution spectroscopy.« less

Incoherent Diffractive Imaging via Intensity Correlations of Hard X Rays

NASA Astrophysics Data System (ADS)

Classen, Anton; Ayyer, Kartik; Chapman, Henry N.; Röhlsberger, Ralf; von Zanthier, Joachim

2017-08-01

Established x-ray diffraction methods allow for high-resolution structure determination of crystals, crystallized protein structures, or even single molecules. While these techniques rely on coherent scattering, incoherent processes like fluorescence emission—often the predominant scattering mechanism—are generally considered detrimental for imaging applications. Here, we show that intensity correlations of incoherently scattered x-ray radiation can be used to image the full 3D arrangement of the scattering atoms with significantly higher resolution compared to conventional coherent diffraction imaging and crystallography, including additional three-dimensional information in Fourier space for a single sample orientation. We present a number of properties of incoherent diffractive imaging that are conceptually superior to those of coherent methods.

X ray based displacement measurement for hostile environments

NASA Technical Reports Server (NTRS)

Canistraro, Howard A.; Jordon, Eric H.; Pease, Douglas M.; Fralick, Gustave C.

1992-01-01

A new method on noncontacting, high temperature extensometry based on the focus and scanning of x rays is currently under development and shows great promise of overcoming limitations associated with available techniques. The chief advantage is the ability to make undisturbed measurements through stratified or flowing gases, smoke, and flame. The system is based on the ability to focus and scan low energy, hard x rays such as those emanating from copper or molybdenum sources. The x rays are focused into a narrow and intense line image which can be scanned onto targets that fluoresce secondary x ray radiation. The final goal of the system is the ability to conduct macroscopic strain measurements in hostile environments by utilizing two or more fluorescing targets. Current work is limited to displacement measurement of a single target with a resolution of 1.25 micro-m and a target temperature of 1200 C, directly through an open flame. The main advantage of the technique lies in the penetrating nature of x rays which are not affected by the presence of refracting gas layers, smoke, flame, or intense thermal radiation, all of which could render conventional extensometry methods inoperative or greatly compromise their performance.

Development of X-ray CCD camera based X-ray micro-CT system

NASA Astrophysics Data System (ADS)

Sarkar, Partha S.; Ray, N. K.; Pal, Manoj K.; Baribaddala, Ravi; Agrawal, Ashish; Kashyap, Y.; Sinha, A.; Gadkari, S. C.

2017-02-01

Availability of microfocus X-ray sources and high resolution X-ray area detectors has made it possible for high resolution microtomography studies to be performed outside the purview of synchrotron. In this paper, we present the work towards the use of an external shutter on a high resolution microtomography system using X-ray CCD camera as a detector. During micro computed tomography experiments, the X-ray source is continuously ON and owing to the readout mechanism of the CCD detector electronics, the detector registers photons reaching it during the read-out period too. This introduces a shadow like pattern in the image known as smear whose direction is defined by the vertical shift register. To resolve this issue, the developed system has been incorporated with a synchronized shutter just in front of the X-ray source. This is positioned in the X-ray beam path during the image readout period and out of the beam path during the image acquisition period. This technique has resulted in improved data quality and hence the same is reflected in the reconstructed images.

Fast two-dimensional grid and transmission X-ray microscopy scanning methods for visualizing and characterizing protein crystals

PubMed Central

Wojdyla, Justyna Aleksandra; Panepucci, Ezequiel; Martiel, Isabelle; Ebner, Simon; Huang, Chia-Ying; Caffrey, Martin; Bunk, Oliver; Wang, Meitian

2016-01-01

A fast continuous grid scan protocol has been incorporated into the Swiss Light Source (SLS) data acquisition and analysis software suite on the macromolecular crystallography (MX) beamlines. Its combination with fast readout single-photon counting hybrid pixel array detectors (PILATUS and EIGER) allows for diffraction-based identification of crystal diffraction hotspots and the location and centering of membrane protein microcrystals in the lipid cubic phase (LCP) in in meso in situ serial crystallography plates and silicon nitride supports. Diffraction-based continuous grid scans with both still and oscillation images are supported. Examples that include a grid scan of a large (50 nl) LCP bolus and analysis of the resulting diffraction images are presented. Scanning transmission X-ray microscopy (STXM) complements and benefits from fast grid scanning. STXM has been demonstrated at the SLS beamline X06SA for near-zero-dose detection of protein crystals mounted on different types of sample supports at room and cryogenic temperatures. Flash-cooled crystals in nylon loops were successfully identified in differential and integrated phase images. Crystals of just 10 µm thickness were visible in integrated phase images using data collected with the EIGER detector. STXM offers a truly low-dose method for locating crystals on solid supports prior to diffraction data collection at both synchrotron microfocusing and free-electron laser X-ray facilities. PMID:27275141

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.

X-ray binaries

NASA Technical Reports Server (NTRS)

1976-01-01

Satellite X-ray experiments and ground-based programs aimed at observation of X-ray binaries are discussed. Experiments aboard OAO-3, OSO-8, Ariel 5, Uhuru, and Skylab are included along with rocket and ground-based observations. Major topics covered are: Her X-1, Cyg X-3, Cen X-3, Cyg X-1, the transient source A0620-00, other possible X-ray binaries, and plans and prospects for future observational programs.

BioCARS: a synchrotron resource for time-resolved X-ray science

PubMed Central

Graber, T.; Anderson, S.; Brewer, H.; Chen, Y.-S.; Cho, H. S.; Dashdorj, N.; Henning, R. W.; Kosheleva, I.; Macha, G.; Meron, M.; Pahl, R.; Ren, Z.; Ruan, S.; Schotte, F.; Šrajer, V.; Viccaro, P. J.; Westferro, F.; Anfinrud, P.; Moffat, K.

2011-01-01

BioCARS, a NIH-supported national user facility for macromolecular time-resolved X-ray crystallography at the Advanced Photon Source (APS), has recently completed commissioning of an upgraded undulator-based beamline optimized for single-shot laser-pump X-ray-probe measurements with time resolution as short as 100 ps. The source consists of two in-line undulators with periods of 23 and 27 mm that together provide high-flux pink-beam capability at 12 keV as well as first-harmonic coverage from 6.8 to 19 keV. A high-heat-load chopper reduces the average power load on downstream components, thereby preserving the surface figure of a Kirkpatrick–Baez mirror system capable of focusing the X-ray beam to a spot size of 90 µm horizontal by 20 µm vertical. A high-speed chopper isolates single X-ray pulses at 1 kHz in both hybrid and 24-bunch modes of the APS storage ring. In hybrid mode each isolated X-ray pulse delivers up to ∼4 × 1010 photons to the sample, thereby achieving a time-averaged flux approaching that of fourth-generation X-FEL sources. A new high-power picosecond laser system delivers pulses tunable over the wavelength range 450–2000 nm. These pulses are synchronized to the storage-ring RF clock with long-term stability better than 10 ps RMS. Monochromatic experimental capability with Biosafety Level 3 certification has been retained. PMID:21685684

Ground-based x-ray calibration of the Astro-H/Hitomi soft x-ray telescopes

NASA Astrophysics Data System (ADS)

Iizuka, Ryo; Hayashi, Takayuki; Maeda, Yoshitomo; Ishida, Manabu; Tomikawa, Kazuki; Sato, Toshiki; Kikuchi, Naomichi; Okajima, Takashi; Soong, Yang; Serlemitsos, Peter J.; Mori, Hideyuki; Izumiya, Takanori; Minami, Sari

2018-01-01

We present the summary of the on-ground calibration of two soft x-ray telescopes (SXT-I and SXT-S), developed by NASA's Goddard Space Flight Center (GSFC), onboard Astro-H/Hitomi. After the initial x-ray measurements with a diverging beam at the GSFC 100-m beamline, we performed the full calibration of the x-ray performance, using the 30-m x-ray beamline facility at the Institute of Space and Astronautical Science of Japan Aerospace Exploration Agency in Japan. We adopted a raster scan method with a narrow x-ray pencil beam with a divergence of ˜15″. The on-axis effective area (EA), half-power diameter, and vignetting function were measured at several energies between 1.5 and 17.5 keV. The detailed results appear in tables and figures in this paper. We measured and evaluated the performance of the SXT-S and the SXT-I with regard to the detector-limited field-of-view and the pixel size of the paired flight detector, i.e., SXS and the SXI, respectively. The primary items measured are the EA, image quality, and stray light for on-axis and off-axis sources. The accurate measurement of these parameters is vital to make the precise response function of the ASTRO-H SXTs. This paper presents the definitive results of the ground-based calibration of the ASTRO-H SXTs.

A combined solid-state NMR and X-ray crystallography study of the bromide ion environments in triphenylphosphonium bromides.

PubMed

Burgess, Kevin M N; Korobkov, Ilia; Bryce, David L

2012-04-27

Multinuclear ((31)P and (79/81)Br), multifield (9.4, 11.75, and 21.1 T) solid-state nuclear magnetic resonance experiments are performed for seven phosphonium bromides bearing the triphenylphosphonium cation, a molecular scaffold found in many applications in chemistry. This is undertaken to fully characterise their bromine electric field gradient (EFG) tensors, as well as the chemical shift (CS) tensors of both the halogen and the phosphorus nuclei, providing a rare and novel insight into the local electronic environments surrounding them. New crystal structures, obtained from single-crystal X-ray diffraction, are reported for six compounds to aid in the interpretation of the NMR data. Among them is a new structure of BrPPh(4), because the previously reported one was inconsistent with our magnetic resonance data, thereby demonstrating how NMR data of non-standard nuclei can correct or improve X-ray diffraction data. Our results indicate that, despite sizable quadrupolar interactions, (79/81)Br magnetic resonance spectroscopy is a powerful characterisation tool that allows for the differentiation between chemically similar bromine sites, as shown through the range in the characteristic NMR parameters. (35/37)Cl solid-state NMR data, obtained for an analogous phosphonium chloride sample, provide insight into the relationship between unit cell volume, nuclear quadrupolar coupling constants, and Sternheimer antishielding factors. The experimental findings are complemented by gauge-including projector-augmented wave (GIPAW) DFT calculations, which substantiate our experimentally determined strong dependence of the largest component of the bromine CS tensor, δ(11), on the shortest Br-P distance in the crystal structure, a finding that has possible application in the field of NMR crystallography. This trend is explained in terms of Ramsey's theory on paramagnetic shielding. Overall, this work demonstrates how careful NMR studies of underexploited exotic nuclides, such

Advanced High Brilliance X-Ray Source

NASA Technical Reports Server (NTRS)

Gibson, Walter M.

1998-01-01

The possibility to dramatically increase the efficiency of laboratory based protein structure measurements through the use of polycapillary X-ray optics was investigated. This project initiated April 1, 1993 and concluded December 31, 1996 (including a no cost extension from June 31, 1996). This is a final report of the project. The basis for the project is the ability to collect X-rays from divergent electron bombardment laboratory X-ray sources and redirect them into quasiparallel or convergent (focused) beams. For example, a 0.1 radian (approx. 6 deg) portion of a divergent beam collected by a polycapillary collimator and transformed into a quasiparallel beam of 3 millradian (0.2 deg) could give a gain of 6(exp 2)/0.2(exp 2) x T for the intensity of a diffracted beam from a crystal with a 0.2 deg diffraction width. T is the transmission efficiency of the polycapillary diffraction optic, and for T=0.5, the gain would be 36/0.04 x O.5=45. In practice, the effective collection angle will depend on the source spot size, the input focal length of the optic (usually limited by the source spot-to-window distance on the x-ray tube) and the size of the crystal relative to the output diameter of the optic. The transmission efficiency, T, depends on the characteristics (fractional open area, surface roughness, shape and channel diameter) of the polycapillary optic and is typically in the range 0.2-0.4. These effects could substantially reduce the expected efficiency gain. During the course of this study, the possibility to use a weakly focused beam (0.5 deg convergence) was suggested which could give an additional 10-20 X efficiency gain for small samples . Weakly focused beams from double focusing mirrors are frequently used for macromolecular crystallography studies. Furthermore the crystals are typically oscillated by as much as 2 deg during each X-ray exposure in order to increase the reciprocal space (number of crystal planes) sampled and use of a slightly convergent

FPGA-Based X-Ray Detection and Measurement for an X-Ray Polarimeter

NASA Technical Reports Server (NTRS)

Gregory, Kyle; Hill, Joanne; Black, Kevin; Baumgartner, Wayne

2013-01-01

This technology enables detection and measurement of x-rays in an x-ray polarimeter using a field-programmable gate array (FPGA). The technology was developed for the Gravitational and Extreme Magnetism Small Explorer (GEMS) mission. It performs precision energy and timing measurements, as well as rejection of non-x-ray events. It enables the GEMS polarimeter to detect precisely when an event has taken place so that additional measurements can be made. The technology also enables this function to be performed in an FPGA using limited resources so that mass and power can be minimized while reliability for a space application is maximized and precise real-time operation is achieved. This design requires a low-noise, charge-sensitive preamplifier; a highspeed analog to digital converter (ADC); and an x-ray detector with a cathode terminal. It functions by computing a sum of differences for time-samples whose difference exceeds a programmable threshold. A state machine advances through states as a programmable number of consecutive samples exceeds or fails to exceed this threshold. The pulse height is recorded as the accumulated sum. The track length is also measured based on the time from the start to the end of accumulation. For track lengths longer than a certain length, the algorithm estimates the barycenter of charge deposit by comparing the accumulator value at the midpoint to the final accumulator value. The design also employs a number of techniques for rejecting background events. This innovation enables the function to be performed in space where it can operate autonomously with a rapid response time. This implementation combines advantages of computing system-based approaches with those of pure analog approaches. The result is an implementation that is highly reliable, performs in real-time, rejects background events, and consumes minimal power.

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

Structural dissection of human metapneumovirus phosphoprotein using small angle x-ray scattering.

PubMed

Renner, Max; Paesen, Guido C; Grison, Claire M; Granier, Sébastien; Grimes, Jonathan M; Leyrat, Cédric

2017-11-01

The phosphoprotein (P) is the main and essential cofactor of the RNA polymerase (L) of non-segmented, negative-strand RNA viruses. P positions the viral polymerase onto its nucleoprotein-RNA template and acts as a chaperone of the nucleoprotein (N), thereby preventing nonspecific encapsidation of cellular RNAs. The phosphoprotein of human metapneumovirus (HMPV) forms homotetramers composed of a stable oligomerization domain (P core ) flanked by large intrinsically disordered regions (IDRs). Here we combined x-ray crystallography of P core with small angle x-ray scattering (SAXS)-based ensemble modeling of the full-length P protein and several of its fragments to provide a structural description of P that captures its dynamic character, and highlights the presence of varyingly stable structural elements within the IDRs. We discuss the implications of the structural properties of HMPV P for the assembly and functioning of the viral transcription/replication machinery.

Building lab-scale x-ray tube based irradiators

USDA-ARS?s Scientific Manuscript database

The construction of economical x-ray tube based irradiators in a variety of configurations is described using 1000 Watt x-ray tubes. Single tube, double tube, and four tube designs are described, as well as various cabinet construction techniques. Relatively high dose rates were achieved for small s...

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.

Capillary Optics Based X-Ray Micro-Imaging Elemental Analysis

NASA Astrophysics Data System (ADS)

Hampai, D.; Dabagov, S. B.; Cappuccio, G.; Longoni, A.; Frizzi, T.; Cibin, G.

2010-04-01

A rapidly developed during the last few years micro-X-ray fluorescence spectrometry (μXRF) is a promising multi-elemental technique for non-destructive analysis. Typically it is rather hard to perform laboratory μXRF analysis because of the difficulty of producing an original small-size X-ray beam as well as its focusing. Recently developed for X-ray beam focusing polycapillary optics offers laboratory X-ray micro probes. The combination of polycapillary lens and fine-focused micro X-ray tube can provide high intensity radiation flux on a sample that is necessary in order to perform the elemental analysis. In comparison to a pinhole, an optimized "X-ray source-op tics" system can result in radiation density gain of more than 3 orders by the value. The most advanced way to get that result is to use the confocal configuration based on two X-ray lenses, one for the fluorescence excitation and the other for the detection of secondary emission from a sample studied. In case of X-ray capillary microfocusing a μXRF instrument designed in the confocal scheme allows us to obtain a 3D elemental mapping. In this work we will show preliminary results obtained with our prototype, a portable X-ray microscope for X-ray both imaging and fluorescence analysis; it enables μXRF elemental mapping simultaneously with X-ray imaging. A prototype of compact XRF spectrometer with a spatial resolution less than 100 μm has been designed.

Confocal total reflection X-ray fluorescence technology based on an elliptical monocapillary and a parallel polycapillary X-ray optics.

PubMed

Zhu, Yu; Wang, Yabing; Sun, Tianxi; Sun, Xuepeng; Zhang, Xiaoyun; Liu, Zhiguo; Li, Yufei; Zhang, Fengshou

2018-07-01

A total reflection X-ray fluorescence (TXRF) spectrometer based on an elliptical monocapillary X-ray lens (MXRL) and a parallel polycapillary X-ray lens (PPXRL) was designed. This TXRF instrument has micro focal spot, low divergence and high intensity of incident X-ray beam. The diameter of the focal spot of MXRL was 16.5 µm, and the divergence of the incident X-ray beam was 3.4 mrad. We applied this TXRF instrument to the micro analysis of a single-layer film containing Ni deposited on a Si substrate by metal vapor vacuum arc ion source. Copyright © 2018 Elsevier Ltd. All rights reserved.

Non Destructive 3D X-Ray Imaging of Nano Structures & Composites at Sub-30 NM Resolution, With a Novel Lab Based X-Ray Microscope

DTIC Science & Technology

2006-11-01

NON DESTRUCTIVE 3D X-RAY IMAGING OF NANO STRUCTURES & COMPOSITES AT SUB-30 NM RESOLUTION, WITH A NOVEL LAB BASED X- RAY MICROSCOPE S H Lau...article we describe a 3D x-ray microscope based on a laboratory x-ray source operating at 2.7, 5.4 or 8.0 keV hard x-ray energies. X-ray computed...tomography (XCT) is used to obtain detailed 3D structural information inside optically opaque materials with sub-30 nm resolution. Applications include

Secure UNIX socket-based controlling system for high-throughput protein crystallography experiments.

PubMed

Gaponov, Yurii; Igarashi, Noriyuki; Hiraki, Masahiko; Sasajima, Kumiko; Matsugaki, Naohiro; Suzuki, Mamoru; Kosuge, Takashi; Wakatsuki, Soichi

2004-01-01

A control system for high-throughput protein crystallography experiments has been developed based on a multilevel secure (SSL v2/v3) UNIX socket under the Linux operating system. Main features of protein crystallography experiments (purification, crystallization, loop preparation, data collecting, data processing) are dealt with by the software. All information necessary to perform protein crystallography experiments is stored (except raw X-ray data, that are stored in Network File Server) in a relational database (MySQL). The system consists of several servers and clients. TCP/IP secure UNIX sockets with four predefined behaviors [(a) listening to a request followed by a reply, (b) sending a request and waiting for a reply, (c) listening to a broadcast message, and (d) sending a broadcast message] support communications between all servers and clients allowing one to control experiments, view data, edit experimental conditions and perform data processing remotely. The usage of the interface software is well suited for developing well organized control software with a hierarchical structure of different software units (Gaponov et al., 1998), which will pass and receive different types of information. All communication is divided into two parts: low and top levels. Large and complicated control tasks are split into several smaller ones, which can be processed by control clients independently. For communicating with experimental equipment (beamline optical elements, robots, and specialized experimental equipment etc.), the STARS server, developed at the Photon Factory, is used (Kosuge et al., 2002). The STARS server allows any application with an open socket to be connected with any other clients that control experimental equipment. Majority of the source code is written in C/C++. GUI modules of the system were built mainly using Glade user interface builder for GTK+ and Gnome under Red Hat Linux 7.1 operating system.

Serial Femtosecond Crystallography of G Protein-Coupled Receptors

PubMed Central

Liu, Wei; Wacker, Daniel; Gati, Cornelius; Han, Gye Won; James, Daniel; Wang, Dingjie; Nelson, Garrett; Weierstall, Uwe; Katritch, Vsevolod; Barty, Anton; Zatsepin, Nadia A.; Li, Dianfan; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J.; Koglin, Jason E.; Seibert, M. Marvin; Wang, Chong; Shah, Syed T.A.; Basu, Shibom; Fromme, Raimund; Kupitz, Christopher; Rendek, Kimberley N.; Grotjohann, Ingo; Fromme, Petra; Kirian, Richard A.; Beyerlein, Kenneth R.; White, Thomas A.; Chapman, Henry N.; Caffrey, Martin; Spence, John C.H.; Stevens, Raymond C.; Cherezov, Vadim

2014-01-01

X-ray crystallography of G protein-coupled receptors and other membrane proteins is hampered by difficulties associated with growing sufficiently large crystals that withstand radiation damage and yield high-resolution data at synchrotron sources. Here we used an x-ray free-electron laser (XFEL) with individual 50-fs duration x-ray pulses to minimize radiation damage and obtained a high-resolution room temperature structure of a human serotonin receptor using sub-10 µm microcrystals grown in a membrane mimetic matrix known as lipidic cubic phase. Compared to the structure solved by traditional microcrystallography from cryo-cooled crystals of about two orders of magnitude larger volume, the room temperature XFEL structure displays a distinct distribution of thermal motions and conformations of residues that likely more accurately represent the receptor structure and dynamics in a cellular environment. PMID:24357322

Serial femtosecond crystallography: A revolution in structural biology.

PubMed

Martin-Garcia, Jose M; Conrad, Chelsie E; Coe, Jesse; Roy-Chowdhury, Shatabdi; Fromme, Petra

2016-07-15

Macromolecular crystallography at synchrotron sources has proven to be the most influential method within structural biology, producing thousands of structures since its inception. While its utility has been instrumental in progressing our knowledge of structures of molecules, it suffers from limitations such as the need for large, well-diffracting crystals, and radiation damage that can hamper native structural determination. The recent advent of X-ray free electron lasers (XFELs) and their implementation in the emerging field of serial femtosecond crystallography (SFX) has given rise to a remarkable expansion upon existing crystallographic constraints, allowing structural biologists access to previously restricted scientific territory. SFX relies on exceptionally brilliant, micro-focused X-ray pulses, which are femtoseconds in duration, to probe nano/micrometer sized crystals in a serial fashion. This results in data sets comprised of individual snapshots, each capturing Bragg diffraction of single crystals in random orientations prior to their subsequent destruction. Thus structural elucidation while avoiding radiation damage, even at room temperature, can now be achieved. This emerging field has cultivated new methods for nanocrystallogenesis, sample delivery, and data processing. Opportunities and challenges within SFX are reviewed herein. Published by Elsevier Inc.

Synthesis, X-ray characterisation and reactions of a trigonal planar palladium(0) carbonyl complex, (tbpx)PdCO.

PubMed

Bellabarba, Ronan M; Tooze, Robert P; Slawin, Alexandra M Z

2003-08-07

The novel complex (tbpx)PdCO (1), the first example of a structurally characterised sixteen electron, trigonal planar palladium(0) carbonyl complex, was prepared, characterised by NMR spectroscopy and X-ray crystallography, and some unusual aspects of its reactivity were studied.

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

X-ray luminescence computed tomography imaging based on X-ray distribution model and adaptively split Bregman method

PubMed Central

Chen, Dongmei; Zhu, Shouping; Cao, Xu; Zhao, Fengjun; Liang, Jimin

2015-01-01

X-ray luminescence computed tomography (XLCT) has become a promising imaging technology for biological application based on phosphor nanoparticles. There are mainly three kinds of XLCT imaging systems: pencil beam XLCT, narrow beam XLCT and cone beam XLCT. Narrow beam XLCT can be regarded as a balance between the pencil beam mode and the cone-beam mode in terms of imaging efficiency and image quality. The collimated X-ray beams are assumed to be parallel ones in the traditional narrow beam XLCT. However, we observe that the cone beam X-rays are collimated into X-ray beams with fan-shaped broadening instead of parallel ones in our prototype narrow beam XLCT. Hence we incorporate the distribution of the X-ray beams in the physical model and collected the optical data from only two perpendicular directions to further speed up the scanning time. Meanwhile we propose a depth related adaptive regularized split Bregman (DARSB) method in reconstruction. The simulation experiments show that the proposed physical model and method can achieve better results in the location error, dice coefficient, mean square error and the intensity error than the traditional split Bregman method and validate the feasibility of method. The phantom experiment can obtain the location error less than 1.1 mm and validate that the incorporation of fan-shaped X-ray beams in our model can achieve better results than the parallel X-rays. PMID:26203388

Measuring and modeling diffuse scattering in protein X-ray crystallography

PubMed Central

Van Benschoten, Andrew H.; Liu, Lin; Gonzalez, Ana; Brewster, Aaron S.; Sauter, Nicholas K.; Wall, Michael E.

2016-01-01

X-ray diffraction has the potential to provide rich information about the structural dynamics of macromolecules. To realize this potential, both Bragg scattering, which is currently used to derive macromolecular structures, and diffuse scattering, which reports on correlations in charge density variations, must be measured. Until now, measurement of diffuse scattering from protein crystals has been scarce because of the extra effort of collecting diffuse data. Here, we present 3D measurements of diffuse intensity collected from crystals of the enzymes cyclophilin A and trypsin. The measurements were obtained from the same X-ray diffraction images as the Bragg data, using best practices for standard data collection. To model the underlying dynamics in a practical way that could be used during structure refinement, we tested translation–libration–screw (TLS), liquid-like motions (LLM), and coarse-grained normal-modes (NM) models of protein motions. The LLM model provides a global picture of motions and was refined against the diffuse data, whereas the TLS and NM models provide more detailed and distinct descriptions of atom displacements, and only used information from the Bragg data. Whereas different TLS groupings yielded similar Bragg intensities, they yielded different diffuse intensities, none of which agreed well with the data. In contrast, both the LLM and NM models agreed substantially with the diffuse data. These results demonstrate a realistic path to increase the number of diffuse datasets available to the wider biosciences community and indicate that dynamics-inspired NM structural models can simultaneously agree with both Bragg and diffuse scattering. PMID:27035972

Measuring and modeling diffuse scattering in protein X-ray crystallography

DOE PAGES

Van Benschoten, Andrew H.; Liu, Lin; Gonzalez, Ana; ...

2016-03-28

X-ray diffraction has the potential to provide rich information about the structural dynamics of macromolecules. To realize this potential, both Bragg scattering, which is currently used to derive macromolecular structures, and diffuse scattering, which reports on correlations in charge density variations, must be measured. Until now, measurement of diffuse scattering from protein crystals has been scarce because of the extra effort of collecting diffuse data. Here, we present 3D measurements of diffuse intensity collected from crystals of the enzymes cyclophilin A and trypsin. The measurements were obtained from the same X-ray diffraction images as the Bragg data, using best practicesmore » for standard data collection. To model the underlying dynamics in a practical way that could be used during structure refinement, we tested translation–libration–screw (TLS), liquid-like motions (LLM), and coarse-grained normal-modes (NM) models of protein motions. The LLM model provides a global picture of motions and was refined against the diffuse data, whereas the TLS and NM models provide more detailed and distinct descriptions of atom displacements, and only used information from the Bragg data. Whereas different TLS groupings yielded similar Bragg intensities, they yielded different diffuse intensities, none of which agreed well with the data. In contrast, both the LLM and NM models agreed substantially with the diffuse data. In conclusion, these results demonstrate a realistic path to increase the number of diffuse datasets available to the wider biosciences community and indicate that dynamics-inspired NM structural models can simultaneously agree with both Bragg and diffuse scattering.« less

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.

Full-field transmission x-ray imaging with confocal polycapillary x-ray optics

PubMed Central

Sun, Tianxi; MacDonald, C. A.

2013-01-01

A transmission x-ray imaging setup based on a confocal combination of a polycapillary focusing x-ray optic followed by a polycapillary collimating x-ray optic was designed and demonstrated to have good resolution, better than the unmagnified pixel size and unlimited by the x-ray tube spot size. This imaging setup has potential application in x-ray imaging for small samples, for example, for histology specimens. PMID:23460760

X-ray laser diffraction for structure determination of the rhodopsin-arrestin complex

NASA Astrophysics Data System (ADS)

Zhou, X. Edward; Gao, Xiang; Barty, Anton; Kang, Yanyong; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; White, Thomas A.; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W.; Suino-Powell, Kelly M.; Boutet, Sébastien; Williams, Garth J.; Wang, Meitian; Li, Dianfan; Caffrey, Martin; Chapman, Henry N.; Spence, John C. H.; Fromme, Petra; Weierstall, Uwe; Stevens, Raymond C.; Cherezov, Vadim; Melcher, Karsten; Xu, H. Eric

2016-04-01

Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solved with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes.

X-ray laser diffraction for structure determination of the rhodopsin-arrestin complex.

PubMed

Zhou, X Edward; Gao, Xiang; Barty, Anton; Kang, Yanyong; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; White, Thomas A; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W; Suino-Powell, Kelly M; Boutet, Sébastien; Williams, Garth J; Wang, Meitian; Li, Dianfan; Caffrey, Martin; Chapman, Henry N; Spence, John C H; Fromme, Petra; Weierstall, Uwe; Stevens, Raymond C; Cherezov, Vadim; Melcher, Karsten; Xu, H Eric

2016-04-12

Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solved with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes.

X-ray laser diffraction for structure determination of the rhodopsin-arrestin complex

PubMed Central

Zhou, X. Edward; Gao, Xiang; Barty, Anton; Kang, Yanyong; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; White, Thomas A.; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W.; Suino-Powell, Kelly M.; Boutet, Sébastien; Williams, Garth J.; Wang, Meitian; Li, Dianfan; Caffrey, Martin; Chapman, Henry N.; Spence, John C.H.; Fromme, Petra; Weierstall, Uwe; Stevens, Raymond C.; Cherezov, Vadim; Melcher, Karsten; Xu, H. Eric

2016-01-01

Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solved with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes. PMID:27070998

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.

Development of a micro-X-ray fluorescence system based on polycapillary X-ray optics for non-destructive analysis of archaeological objects

NASA Astrophysics Data System (ADS)

Cheng, Lin; Ding, Xunliang; Liu, Zhiguo; Pan, Qiuli; Chu, Xuelian

2007-08-01

A new micro-X-ray fluorescence (micro-XRF) system based on rotating anode X-ray generator and polycapillary X-ray optics has been set up in XOL Lab, BNU, China, in order to be used for analysis of archaeological objects. The polycapillary X-ray optics used here can focus the primary X-ray beam down to tens of micrometers in diameter that allows for non-destructive and local analysis of sub-mm samples with minor/trace level sensitivity. The analytical characteristics and potential of this micro-XRF system in archaeological research are discussed. Some described uses of this instrument include studying Chinese ancient porcelain.

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

Review: Serial Femtosecond Crystallography: A Revolution in Structural Biology

PubMed Central

Martin-Garcia, Jose M.; Conrad, Chelsie E.; Coe, Jesse; Roy-Chowdhury, Shatabdi; Fromme, Petra

2016-01-01

Macromolecular crystallography at synchrotron sources has proven to be the most influential method within structural biology, producing thousands of structures since its inception. While its utility has been instrumental in progressing our knowledge of structures of molecules, it suffers from limitations such as the need for large, well-diffracting crystals, and radiation damage that can hamper native structural determination. The recent advent of X-ray free electron lasers (XFELs) and their implementation in the emerging field of serial femtosecond crystallography (SFX) has given rise to a remarkable expansion upon existing crystallographic constraints, allowing structural biologists access to previously restricted scientific territory. SFX relies on exceptionally brilliant, micro-focused X-ray pulses, which are femtoseconds in duration, to probe nano/micrometer sized crystals in a serial fashion. This results in data sets comprised of individual snapshots, each capturing Bragg diffraction of single crystals in random orientations prior to their subsequent destruction. Thus structural elucidation while avoiding radiation damage, even at room temperature, can now be achieved. This emerging field has cultivated new methods for nanocrystallogenesis, sample delivery, and data processing. Opportunities and challenges within SFX are reviewed herein. PMID:27143509

Unambiguous determination of H-atom positions: comparing results from neutron and high-resolution X-ray crystallography.

PubMed

Gardberg, Anna S; Del Castillo, Alexis Rae; Weiss, Kevin L; Meilleur, Flora; Blakeley, Matthew P; Myles, Dean A A

2010-05-01

The locations of H atoms in biological structures can be difficult to determine using X-ray diffraction methods. Neutron diffraction offers a relatively greater scattering magnitude from H and D atoms. Here, 1.65 A resolution neutron diffraction studies of fully perdeuterated and selectively CH(3)-protonated perdeuterated crystals of Pyrococcus furiosus rubredoxin (D-rubredoxin and HD-rubredoxin, respectively) at room temperature (RT) are described, as well as 1.1 A resolution X-ray diffraction studies of the same protein at both RT and 100 K. The two techniques are quantitatively compared in terms of their power to directly provide atomic positions for D atoms and analyze the role played by atomic thermal motion by computing the sigma level at the D-atom coordinate in simulated-annealing composite D-OMIT maps. It is shown that 1.65 A resolution RT neutron data for perdeuterated rubredoxin are approximately 8 times more likely overall to provide high-confidence positions for D atoms than 1.1 A resolution X-ray data at 100 K or RT. At or above the 1.0sigma level, the joint X-ray/neutron (XN) structures define 342/378 (90%) and 291/365 (80%) of the D-atom positions for D-rubredoxin and HD-rubredoxin, respectively. The X-ray-only 1.1 A resolution 100 K structures determine only 19/388 (5%) and 8/388 (2%) of the D-atom positions above the 1.0sigma level for D-rubredoxin and HD-rubredoxin, respectively. Furthermore, the improved model obtained from joint XN refinement yielded improved electron-density maps, permitting the location of more D atoms than electron-density maps from models refined against X-ray data only.

Does crystallography need a new name?

DOE PAGES

Argryriou, Dimitri

2017-07-01

The discovery of X-rays and their use in the observation of diffraction from crystals placed crystallography at the forefront of science at the beginning of the last century. The combination of this new tool, together with the emerging understanding of the symmetry of crystals, exposed the locations of atoms in matter and allowed us to start understanding macroscopic properties from an atomic perspective for the first time. These discoveries transformed physics and chemistry bringing to light new scientific fields such as materials science and structural biology.

Does crystallography need a new name?

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

Argryriou, Dimitri

The discovery of X-rays and their use in the observation of diffraction from crystals placed crystallography at the forefront of science at the beginning of the last century. The combination of this new tool, together with the emerging understanding of the symmetry of crystals, exposed the locations of atoms in matter and allowed us to start understanding macroscopic properties from an atomic perspective for the first time. These discoveries transformed physics and chemistry bringing to light new scientific fields such as materials science and structural biology.

Biochemical, spectroscopic and X-ray structural analysis of deuterated multicopper oxidase CueO prepared from a new expression construct for neutron crystallography.

PubMed

Akter, Mahfuza; Inoue, Chika; Komori, Hirofumi; Matsuda, Nana; Sakurai, Takeshi; Kataoka, Kunishige; Higuchi, Yoshiki; Shibata, Naoki

2016-10-01

Multicopper oxidases oxidize various phenolic and nonphenolic compounds by using molecular oxygen as an electron acceptor to produce water. A multicopper oxidase protein, CueO, from Escherichia coli is involved in copper homeostasis in the bacterial cell. Although X-ray crystallographic studies have been conducted, the reduction mechanism of oxygen and the proton-transfer pathway remain unclear owing to the difficulty in identifying H atoms from X-ray diffraction data alone. To elucidate the reaction mechanism using neutron crystallography, a preparation system for obtaining large, high-quality single crystals of deuterated CueO was developed. Tiny crystals were obtained from the deuterated CueO initially prepared from the original construct. The X-ray crystal structure of the deuterated CueO showed that the protein contained an incompletely truncated signal sequence at the N-terminus, which resulted in the heterogeneity of the protein sample for crystallization. Here, a new CueO expression system that had an HRV3C cleavage site just after the signal sequence was constructed. Deuterated CueO from the new construct was expressed in cells cultured in deuterated algae-extract medium and the signal sequence was completely eliminated by HRV3C protease. The deuteration level of the purified protein was estimated by MALDI-TOF mass spectrometry to be at least 83.2% compared with nondeuterated protein. Nondeuterated CueO crystallized in space group P2 1 , with unit-cell parameters a = 49.51, b = 88.79, c = 53.95 Å, β = 94.24°, and deuterated CueO crystallized in space group P2 1 2 1 2 1 , with unit-cell parameters a = 49.91, b = 106.92, c = 262.89 Å. The crystallographic parameters for the crystals of the new construct were different from those previously reported for nondeuterated crystals. The nondeuterated and deuterated CueO from the new construct had similar UV-Vis spectra, enzymatic activities and overall structure and geometry of the ligands of the Cu atoms

Laser plasma x-ray source for ultrafast time-resolved x-ray absorption spectroscopy

DOE PAGES

Miaja-Avila, L.; O'Neil, G. C.; Uhlig, J.; ...

2015-03-02

We describe a laser-driven x-ray plasma source designed for ultrafast x-ray absorption spectroscopy. The source is comprised of a 1 kHz, 20 W, femtosecond pulsed infrared laser and a water target. We present the x-ray spectra as a function of laser energy and pulse duration. Additionally, we investigate the plasma temperature and photon flux as we vary the laser energy. We obtain a 75 μm FWHM x-ray spot size, containing ~10 6 photons/s, by focusing the produced x-rays with a polycapillary optic. Since the acquisition of x-ray absorption spectra requires the averaging of measurements from >10 7 laser pulses, wemore » also present data on the source stability, including single pulse measurements of the x-ray yield and the x-ray spectral shape. In single pulse measurements, the x-ray flux has a measured standard deviation of 8%, where the laser pointing is the main cause of variability. Further, we show that the variability in x-ray spectral shape from single pulses is low, thus justifying the combining of x-rays obtained from different laser pulses into a single spectrum. Finally, we show a static x-ray absorption spectrum of a ferrioxalate solution as detected by a microcalorimeter array. Altogether, our results demonstrate that this water-jet based plasma source is a suitable candidate for laboratory-based time-resolved x-ray absorption spectroscopy experiments.« less

Compact X-ray sources: X-rays from self-reflection

NASA Astrophysics Data System (ADS)

Mangles, Stuart P. D.

2012-05-01

Laser-based particle acceleration offers a way to reduce the size of hard-X-ray sources. Scientists have now developed a simple scheme that produces a bright flash of hard X-rays by using a single laser pulse both to generate and to scatter an electron beam.

X-ray laser diffraction for structure determination of the rhodopsin-arrestin complex

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

Zhou, X. Edward; Gao, Xiang; Barty, Anton

Here, serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solvedmore » with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes.« less

X-ray laser diffraction for structure determination of the rhodopsin-arrestin complex

DOE PAGES

Zhou, X. Edward; Gao, Xiang; Barty, Anton; ...

2016-04-12

Here, serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solvedmore » with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes.« less

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.

Synthesis and x-ray crystallographic analysis of 4,6-di-O-acetyl-2,3-dideoxy-α-D-threo-hexopyranosyl cyanide.

PubMed

Rotella, Madeline; Giovine, Matthew; Dougherty, William; Boyko, Walter; Kassel, Scott; Giuliano, Robert

2016-04-29

The glycopyranosyl cyanide 4,6-di-O-acetyl-2,3-dideoxy-α-D-threo-hexopyranosyl cyanide has been synthesized from tri-O-acetyl-D-galactal by reaction with trimethylsilyl cyanide in the presence of boron trifluoride diethyl etherate followed by catalytic hydrogenation. The synthesis provides the α-anomer stereoselectively, the structure of which was assigned based on 2D NMR techniques and x-ray crystallography. Copyright © 2016 Elsevier Ltd. All rights reserved.

X-ray Imaging and preliminary studies of the X-ray self-emission from an innovative plasma-trap based on the Bernstein waves heating mechanism

NASA Astrophysics Data System (ADS)

Caliri, C.; Romano, F. P.; Mascali, D.; Gammino, S.; Musumarra, A.; Castro, G.; Celona, L.; Neri, L.; Altana, C.

2013-10-01

Electron Cyclotron Resonance Ion Sources (ECRIS) are based on ECR heated plasmas emitting high fluxes of X-rays. Here we illustrate a pilot study of the X-ray emission from a compact plasma-trap in which an off-resonance microwave-plasma interaction has been attempted, highlighting a possible Bernstein-Waves based heating mechanism. EBWs-heating is obtained via the inner plasma EM-to-ES wave conversion and enables to reach densities much larger than the cut-off ones. At LNS-INFN, an innovative diagnostic technique based on the design of a Pinhole Camera (PHC) coupled to a CCD device for X-ray Imaging of the plasma (XRI) has been developed, in order to integrate X-ray traditional diagnostics (XRS). The complementary use of electrostatic probes measurements and X-ray diagnostics enabled us to gain knowledge about the high energy electrons density and temperature and about the spatial structure of the source. The combination of the experimental data with appropriate modeling of the plasma-source allowed to estimate the X-ray emission intensity in different energy domains (ranging from EUV up to Hard X-rays). The use of ECRIS as X-ray source for multidisciplinary applications, is now a concrete perspective due to the intense fluxes produced by the new plasma heating mechanism.

X-ray luminescence computed tomography using a focused x-ray beam.

PubMed

Zhang, Wei; Lun, Michael C; Nguyen, Alex Anh-Tu; Li, Changqing

2017-11-01

Due to the low x-ray photon utilization efficiency and low measurement sensitivity of the electron multiplying charge coupled device camera setup, the collimator-based narrow beam x-ray luminescence computed tomography (XLCT) usually requires a long measurement time. We, for the first time, report a focused x-ray beam-based XLCT imaging system with measurements by a single optical fiber bundle and a photomultiplier tube (PMT). An x-ray tube with a polycapillary lens was used to generate a focused x-ray beam whose x-ray photon density is 1200 times larger than a collimated x-ray beam. An optical fiber bundle was employed to collect and deliver the emitted photons on the phantom surface to the PMT. The total measurement time was reduced to 12.5 min. For numerical simulations of both single and six fiber bundle cases, we were able to reconstruct six targets successfully. For the phantom experiment, two targets with an edge-to-edge distance of 0.4 mm and a center-to-center distance of 0.8 mm were successfully reconstructed by the measurement setup with a single fiber bundle and a PMT. (2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

An efficient intensity-based ready-to-use X-ray image stitcher.

PubMed

Wang, Junchen; Zhang, Xiaohui; Sun, Zhen; Yuan, Fuzhen

2018-06-14

The limited field of view of the X-ray image intensifier makes it difficult to cover a large target area with a single X-ray image. X-ray image stitching techniques have been proposed to produce a panoramic X-ray image. This paper presents an efficient intensity-based X-ray image stitcher, which does not rely on accurate C-arm motion control or auxiliary devices and hence is ready to use in clinic. The stitcher consumes sequentially captured X-ray images with overlap areas and automatically produces a panoramic image. The gradient information for optimization of image alignment is obtained using a back-propagation scheme so that it is convenient to adopt various image warping models. The proposed stitcher has the following advantages over existing methods: (1) no additional hardware modification or auxiliary markers are needed; (2) it is robust against feature-based approaches; (3) arbitrary warping models and shapes of the region of interest are supported; (4) seamless stitching is achieved using multi-band blending. Experiments have been performed to confirm the effectiveness of the proposed method. The proposed X-ray image stitcher is efficient, accurate and ready to use in clinic. Copyright © 2018 John Wiley & Sons, Ltd.

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

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.

Engineering solar cells based on correlative X-ray microscopy

DOE PAGES

Stuckelberger, Michael; West, Bradley; Nietzold, Tara; ...

2017-05-01

In situ and operando measurement techniques combined with nanoscale resolution have proven invaluable in multiple fields of study. We argue that evaluating device performance as well as material behavior by correlative X-ray microscopy with <100 nm resolution can radically change the approach for optimizing absorbers, interfaces and full devices in solar cell research. Here, we thoroughly discuss the measurement technique of X-ray beam induced current and point out fundamental differences between measurements of wafer-based silicon and thin-film solar cells. Based on reports of the last years, we showcase the potential that X-ray microscopy measurements have in combination with in situmore » and operando approaches throughout the solar cell lifecycle: from the growth of individual layers to the performance under operating conditions and degradation mechanisms. Enabled by new developments in synchrotron beamlines, the combination of high spatial resolution with high brilliance and a safe working distance allows for the insertion of measurement equipment that can pave the way for a new class of experiments. When applied to photovoltaics research, we highlight today’s opportunities and challenges in the field of nanoscale X-ray microscopy, and give an outlook on future developments.« less

Tables of X-ray absorption corrections and dispersion corrections: the new versus the old

NASA Astrophysics Data System (ADS)

Creagh, Dudley

1990-11-01

This paper compares the data on X-ray absorption coefficients calculated by Creagh and Hubbell and tabulated in International Tables for Crystallography, vol. C, ed. A.J.C. Wilson (1990) section 4.2.4 [1] with empirical (Saloman, Hubbell and Scofield, At. Data and Nucl. Data Tables 38 (1988) 1, [6]) and semi-empirical (Hubbell, McMaster, Kerr Del Grande and Mallett, in: International Tables for Crystallography, vol. IV, eds. Ibers and Hamilton (Kynoch, Birmingham, 1974) [2]) tabulations as well as the renormalized relativistic Dirac-Hartree-Fock calculations of Scofield [6]. It also makes comparisons of the real part of the dispersion correction ƒ‧(ω, 0) and tabulated in ref. [1], with theoretical data sets (Cromer and Liberman, J. Chem. Phys. 53 (1970) 1891, and Acta Crystallogr. A37 (1981) 267 [4,5]; Wang, Phys. Rev. A34 (1986) 636 [85]; Kissel, in: Workshop Report on New Dimensions in X-ray Scattering, CONF-870459 (Livermore, 1987) p. 9 [86]) and data collected using a variety of experimental techniques. In both cases the data tabulated in ref. [1] is shown to give improved self-consistency and agreement with experiment.

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.

"X-Ray Transients in Star-Forming Regions" and "Hard X-Ray Emission from X-Ray Bursters"

NASA Technical Reports Server (NTRS)

Halpern, Jules P.; Kaaret, Philip

1999-01-01

This grant funded work on the analysis of data obtained with the Burst and Transient Experiment (BATSE) on the Compton Gamma-Ray Observatory. The goal of the work was to search for hard x-ray transients in star forming regions using the all-sky hard x-ray monitoring capability of BATSE. Our initial work lead to the discovery of a hard x-ray transient, GRO J1849-03. Follow-up observations of this source made with the Wide Field Camera on BeppoSAX showed that the source should be identified with the previously known x-ray pulsar GS 1843-02 which itself is identified with the x-ray source X1845-024 originally discovered with the SAS-3 satellite. Our identification of the source and measurement of the outburst recurrence time, lead to the identification of the source as a Be/X-ray binary with a spin period of 94.8 s and an orbital period of 241 days. The funding was used primarily for partial salary and travel support for John Tomsick, then a graduate student at Columbia University. John Tomsick, now Dr. Tomsick, received his Ph.D. from Columbia University in July 1999, based partially on results obtained under this investigation. He is now a postdoctoral research scientist at the University of California, San Diego.

Application of MEMS-based x-ray optics as tuneable nanosecond choppers

NASA Astrophysics Data System (ADS)

Chen, Pice; Walko, Donald A.; Jung, Il Woong; Li, Zhilong; Gao, Ya; Shenoy, Gopal K.; Lopez, Daniel; Wang, Jin

2017-08-01

Time-resolved synchrotron x-ray measurements often rely on using a mechanical chopper to isolate a set of x-ray pulses. We have started the development of micro electromechanical systems (MEMS)-based x-ray optics, as an alternate method to manipulate x-ray beams. In the application of x-ray pulse isolation, we recently achieved a pulse-picking time window of half a nanosecond, which is more than 100 times faster than mechanical choppers can achieve. The MEMS device consists of a comb-drive silicon micromirror, designed for efficiently diffracting an x-ray beam during oscillation. The MEMS devices were operated in Bragg geometry and their oscillation was synchronized to x-ray pulses, with a frequency matching subharmonics of the cycling frequency of x-ray pulses. The microscale structure of the silicon mirror in terms of the curvature and the quality of crystallinity ensures a narrow angular spread of the Bragg reflection. With the discussion of factors determining the diffractive time window, this report showed our approaches to narrow down the time window to half a nanosecond. The short diffractive time window will allow us to select single x-ray pulse out of a train of pulses from synchrotron radiation facilities.

Integrated Controlling System and Unified Database for High Throughput Protein Crystallography Experiments

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

Gaponov, Yu.A.; Igarashi, N.; Hiraki, M.

2004-05-12

An integrated controlling system and a unified database for high throughput protein crystallography experiments have been developed. Main features of protein crystallography experiments (purification, crystallization, crystal harvesting, data collection, data processing) were integrated into the software under development. All information necessary to perform protein crystallography experiments is stored (except raw X-ray data that are stored in a central data server) in a MySQL relational database. The database contains four mutually linked hierarchical trees describing protein crystals, data collection of protein crystal and experimental data processing. A database editor was designed and developed. The editor supports basic database functions to view,more » create, modify and delete user records in the database. Two search engines were realized: direct search of necessary information in the database and object oriented search. The system is based on TCP/IP secure UNIX sockets with four predefined sending and receiving behaviors, which support communications between all connected servers and clients with remote control functions (creating and modifying data for experimental conditions, data acquisition, viewing experimental data, and performing data processing). Two secure login schemes were designed and developed: a direct method (using the developed Linux clients with secure connection) and an indirect method (using the secure SSL connection using secure X11 support from any operating system with X-terminal and SSH support). A part of the system has been implemented on a new MAD beam line, NW12, at the Photon Factory Advanced Ring for general user experiments.« less

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.

Low-dose fixed-target serial synchrotron crystallography.

PubMed

Owen, Robin L; Axford, Danny; Sherrell, Darren A; Kuo, Anling; Ernst, Oliver P; Schulz, Eike C; Miller, R J Dwayne; Mueller-Werkmeister, Henrike M

2017-04-01

The development of serial crystallography has been driven by the sample requirements imposed by X-ray free-electron lasers. Serial techniques are now being exploited at synchrotrons. Using a fixed-target approach to high-throughput serial sampling, it is demonstrated that high-quality data can be collected from myoglobin crystals, allowing room-temperature, low-dose structure determination. The combination of fixed-target arrays and a fast, accurate translation system allows high-throughput serial data collection at high hit rates and with low sample consumption.

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens.

PubMed

MacLachlan, Bruce J; Greenshields-Watson, Alexander; Mason, Georgina H; Schauenburg, Andrea J; Bianchi, Valentina; Rizkallah, Pierre J; Sewell, Andrew K; Fuller, Anna; Cole, David K

2017-02-06

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

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.

Racemic & quasi-racemic protein crystallography enabled by chemical protein synthesis.

PubMed

Kent, Stephen Bh

2018-04-04

A racemic protein mixture can be used to form centrosymmetric crystals for structure determination by X-ray diffraction. Both the unnatural d-protein and the corresponding natural l-protein are made by total chemical synthesis based on native chemical ligation-chemoselective condensation of unprotected synthetic peptide segments. Racemic protein crystallography is important for structure determination of the many natural protein molecules that are refractory to crystallization. Racemic mixtures facilitate the crystallization of recalcitrant proteins, and give diffraction-quality crystals. Quasi-racemic crystallization, using a single d-protein molecule, can facilitate the determination of the structures of a series of l-protein analog molecules. Copyright © 2018 Elsevier Ltd. All rights reserved.

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 microscopy using reflection targets based on SEM with tungsten filament

NASA Astrophysics Data System (ADS)

Liu, Junbiao; Ma, Yutian; Zhao, Weixia; Niu, Geng; Chu, Mingzhang; Yin, Bohua; Han, Li; Liu, Baodong

2016-10-01

X-ray MicroandNano imaging is developed based on the conventional x-ray tomography, it can not only provide nondestructive testing with higher resolution measurement, but also be used to examine the material or the structure with low atomic number and low density. The source with micro-focal spot size is one of the key components of x-ray MicroandNano imaging. The focused electron beam from SEM bombarding the metal target can generate x-ray with ultra-small size. It is convenient to set up x-ray microscopy based on SEM for laboratory use. This paper describes a new x-ray microscopy using reflection targets based on FEI Quanta600 SEM with tungsten filament. The flat panel detector is placed outside of the vacuum chamber with 300μm thickness Be-window to isolate vacuum from the air. A stage with 3 DOFs is added to adjust the positions of the target, the SEM's sample stage is used to move sample. And the shape of target is designed as cone with 60° half cone angle to get the maximum x-ray dosage. The attenuation coefficient of Bewindow for x-ray is about 25%. Finally, the line pair card is used to evaluate the resolution and the result shows that the resolution of the system can receive less than 750nm, when the acceleration voltage is 30keV, the beam current is 160nA, the SEM working distance is 5mm and the acquisition time of the detector is 60s.

X-Ray Emission from the Soft X-Ray Transient Aquila X-1

NASA Technical Reports Server (NTRS)

Tavani, Marco

1998-01-01

Aquila X-1 is the most prolific of soft X-ray transients. It is believed to contain a rapidly spinning neutron star sporadically accreting near the Eddington limit from a low-mass companion star. The interest in studying the repeated X-ray outbursts from Aquila X-1 is twofold: (1) studying the relation between optical, soft and hard X-ray emission during the outburst onset, development and decay; (2) relating the spectral component to thermal and non-thermal processes occurring near the magnetosphere and in the boundary layer of a time-variable accretion disk. Our investigation is based on the BATSE monitoring of Aquila X-1 performed by our group. We observed Aquila X-1 in 1997 and re-analyzed archival information obtained in April 1994 during a period of extraordinary outbursting activity of the source in the hard X-ray range. Our results allow, for the first time for this important source, to obtain simultaneous spectral information from 2 keV to 200 keV. A black body (T = 0.8 keV) plus a broken power-law spectrum describe accurately the 1994 spectrum. Substantial hard X-ray emission is evident in the data, confirming that the accretion phase during sub-Eddington limit episodes is capable of producing energetic hard emission near 5 x 10(exp 35) ergs(exp -1). A preliminary paper summarizes our results, and a more comprehensive account is being written. We performed a theoretical analysis of possible emission mechanisms, and confirmed that a non-thermal emission mechanism triggered in a highly sheared magnetosphere at the accretion disk inner boundary can explain the hard X-ray emission. An anticorrelation between soft and hard X-ray emission is indeed prominently observed as predicted by this model.

X-ray based displacement and strain measurements for hostile environments

NASA Technical Reports Server (NTRS)

Canistraro, Howard A.; Jordan, Eric H.; Pease, Douglas M.

1993-01-01

A completely new method of non-contacting, hostile environment displacement and strain measurement based on the focus and scanning of x-rays, has been developed and demonstrated. The new technique has the ability to overcome many of the limitations associated with available methods. The system is based on the focus and scanning of low energy, hard x-rays such as those emanating from table top copper or molybdenum sources. The x-rays are focused into a narrow and intense line image which can be swept onto targets that fluoresce secondary x-ray radiation. By monitoring the secondary radiation intensity and comparing it with the focused x-ray image's position as it is swept over the target edge, the position of the target edge relative to the focused image can be determined. The present system has a resolution of 0.5 micron, which has been shown to be limited by bearing backlash (or 'yaw' error) in the linear translation table. Its use has been demonstrated in the presence of an open flame with a resultant target temperature in excess of 2000 degrees Fahrenheit (1000 degrees Celsius). Strain measurements have been conducted in a laboratory environment at both room temperature and at a specimen temperature of 1300 degrees Fahrenheit, with an accuracy of within 20 microstrain (primarily a function of the 0.5 micron resolution limit). The main advantage of the technique lies in the penetrating, non-refractive nature of x-rays, which are virtually immune to the presence of refracting gas layers, smoke, flame or intense thermal radiation.

Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography

PubMed Central

Mandal, Kalyaneswar; Uppalapati, Maruti; Ault-Riché, Dana; Kenney, John; Lowitz, Joshua; Sidhu, Sachdev S.; Kent, Stephen B.H.

2012-01-01

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 VEGF165 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 ofVEGF-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 Å2 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. PMID:22927390

New Beamlines For Protein Crystallography At The EMBL-Outstation Hamburg

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

Hermes, C.; Gehrmann, T.; Jahn, D.

2004-05-12

Three new beamlines for Protein Crystallography were built up on a bending magnet fan of the DORIS III storage ring. A 10 mrad wide fan of white Synchrotron Radiation (SR) is evenly distributed among 3 individual stations: X12, a central, wavelength-tunable station intended for anomalous scattering experiments (MAD) and fixed wavelength, high intensity stations symmetrically on either side. The fixed wavelength beamlines X11 and X13 comprise triangular, asymmetrically cut Si (111) monochromators as horizontally focusing optical elements. The tunable station is based on a fixed-exit, horizontally focusing double crystal monochromator system. Vertical focusing is achieved on all three lines bymore » trapezoidal shaped, continuous Rh-coated mirrors which can be dynamically bent. In all three lines the X-ray beam can be examined at various points on its way through the optical system by removable screens and PIN-diode based intensity monitors. Purpose built crystallographic end-stations complete the set-up. The design of individual components and their performance will be described.« less

A vacuum-sealed miniature X-ray tube based on carbon nanotube field emitters

NASA Astrophysics Data System (ADS)

Heo, Sung Hwan; Kim, Hyun Jin; Ha, Jun Mok; Cho, Sung Oh

2012-05-01

A vacuum-sealed miniature X-ray tube based on a carbon nanotube field-emission electron source has been demonstrated. The diameter of the X-ray tube is 10 mm; the total length of the tube is 50 mm, and no external vacuum pump is required for the operation. The maximum tube voltage reaches up to 70 kV, and the X-ray tube generates intense X-rays with the air kerma strength of 108 Gy·cm2 min-1. In addition, X-rays produced from the miniature X-ray tube have a comparatively uniform spatial dose distribution.

SPring-8 BL41XU, a high-flux macromolecular crystallography beamline

PubMed Central

Hasegawa, Kazuya; Shimizu, Nobutaka; Okumura, Hideo; Mizuno, Nobuhiro; Baba, Seiki; Hirata, Kunio; Takeuchi, Tomoyuki; Yamazaki, Hiroshi; Senba, Yasunori; Ohashi, Haruhiko; Yamamoto, Masaki; Kumasaka, Takashi

2013-01-01

SPring-8 BL41XU is a high-flux macromolecular crystallography beamline using an in-vacuum undulator as a light source. The X-rays are monochromated by a liquid-nitrogen-cooling Si double-crystal monochromator, and focused by Kirkpatrick–Baez mirror optics. The focused beam size at the sample is 80 µm (H) × 22 µm (V) with a photon flux of 1.1 × 1013 photons s−1. A pinhole aperture is used to collimate the beam in the range 10–50 µm. This high-flux beam with variable size provides opportunities not only for micro-crystallography but also for data collection effectively making use of crystal volume. The beamline also provides high-energy X-rays covering 20.6–35.4 keV which allows ultra-high-resolution data to be obtained and anomalous diffraction using the K-edge of Xe and I. Upgrade of BL41XU for more rapid and accurate data collection is proceeding. Here, details of BL41XU are given and an outline of the upgrade project is documented. PMID:24121338

The Mapping X-ray Fluorescence Spectrometer (MapX)

NASA Astrophysics Data System (ADS)

Sarrazin, P.; Blake, D. F.; Marchis, F.; Bristow, T.; Thompson, K.

2017-12-01

Many planetary surface processes leave traces of their actions as features in the size range 10s to 100s of microns. The Mapping X-ray Fluorescence Spectrometer (MapX) will provide elemental imaging at 100 micron spatial resolution, yielding elemental chemistry at a scale where many relict physical, chemical, or biological features can be imaged and interpreted in ancient rocks on planetary bodies and planetesimals. MapX is an arm-based instrument positioned on a rock or regolith with touch sensors. During an analysis, an X-ray source (tube or radioisotope) bombards the sample with X-rays or alpha-particles / gamma-rays, resulting in sample X-ray Fluorescence (XRF). X-rays emitted in the direction of an X-ray sensitive CCD imager pass through a 1:1 focusing lens (X-ray micro-pore Optic (MPO)) that projects a spatially resolved image of the X-rays onto the CCD. The CCD is operated in single photon counting mode so that the energies and positions of individual X-ray photons are recorded. In a single analysis, several thousand frames are both stored and processed in real-time. Higher level data products include single-element maps with a lateral spatial resolution of 100 microns and quantitative XRF spectra from ground- or instrument- selected Regions of Interest (ROI). XRF spectra from ROI are compared with known rock and mineral compositions to extrapolate the data to rock types and putative mineralogies. When applied to airless bodies and implemented with an appropriate radioisotope source for alpha-particle excitation, MapX will be able to analyze biogenic elements C, N, O, P, S, in addition to the cations of the rock-forming elements >Na, accessible with either X-ray or gamma-ray excitation. The MapX concept has been demonstrated with a series of lab-based prototypes and is currently under refinement and TRL maturation.

Comparing pharmacophore models derived from crystallography and NMR ensembles

NASA Astrophysics Data System (ADS)

Ghanakota, Phani; Carlson, Heather A.

2017-11-01

NMR and X-ray crystallography are the two most widely used methods for determining protein structures. Our previous study examining NMR versus X-Ray sources of protein conformations showed improved performance with NMR structures when used in our Multiple Protein Structures (MPS) method for receptor-based pharmacophores (Damm, Carlson, J Am Chem Soc 129:8225-8235, 2007). However, that work was based on a single test case, HIV-1 protease, because of the rich data available for that system. New data for more systems are available now, which calls for further examination of the effect of different sources of protein conformations. The MPS technique was applied to Growth factor receptor bound protein 2 (Grb2), Src SH2 homology domain (Src-SH2), FK506-binding protein 1A (FKBP12), and Peroxisome proliferator-activated receptor-γ (PPAR-γ). Pharmacophore models from both crystal and NMR ensembles were able to discriminate between high-affinity, low-affinity, and decoy molecules. As we found in our original study, NMR models showed optimal performance when all elements were used. The crystal models had more pharmacophore elements compared to their NMR counterparts. The crystal-based models exhibited optimum performance only when pharmacophore elements were dropped. This supports our assertion that the higher flexibility in NMR ensembles helps focus the models on the most essential interactions with the protein. Our studies suggest that the "extra" pharmacophore elements seen at the periphery in X-ray models arise as a result of decreased protein flexibility and make very little contribution to model performance.

Total chemical synthesis and X-ray structure of kaliotoxin by racemic protein crystallography.

PubMed

Pentelute, Brad L; Mandal, Kalyaneswar; Gates, Zachary P; Sawaya, Michael R; Yeates, Todd O; Kent, Stephen B H

2010-11-21

Here we report the total synthesis of kaliotoxin by 'one pot' native chemical ligation of three synthetic peptides. A racemic mixture of D- and L-kaliotoxin synthetic protein molecules gave crystals in the centrosymmetric space group P1 that diffracted to atomic-resolution (0.95 Å), enabling the X-ray structure of kaliotoxin to be determined by direct methods.

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

Synchrotron-based X-ray microscopic studies for bioeffects of nanomaterials.

PubMed

Zhu, Ying; Cai, Xiaoqing; Li, Jiang; Zhong, Zengtao; Huang, Qing; Fan, Chunhai

2014-04-01

There have been increasing interests in studying biological effects of nanomaterials, which are nevertheless faced up with many challenges due to the nanoscale dimensions and unique chemical properties of nanomaterials. Synchrotron-based X-ray microscopy, an advanced imaging technology with high spatial resolution and excellent elemental specificity, provides a new platform for studying interactions between nanomaterials and living systems. In this article, we review the recent progress of X-ray microscopic studies on bioeffects of nanomaterials in several living systems including cells, model organisms, animals and plants. We aim to provide an overview of the state of the art, and the advantages of using synchrotron-based X-ray microscopy for characterizing in vitro and in vivo behaviors and biodistribution of nanomaterials. We also expect that the use of a combination of new synchrotron techniques should offer unprecedented opportunities for better understanding complex interactions at the nano-biological interface and accounting for unique bioeffects of nanomaterials. Synchrotron-based X-ray microscopy is a non-destructive imaging technique that enables high resolution spatial mapping of metals with elemental level detection methods. This review summarizes the current use and perspectives of this novel technique in studying the biology and tissue interactions of nanomaterials. Copyright © 2014 Elsevier Inc. All rights reserved.

New carbocyclic N(6)-substituted adenine and pyrimidine nucleoside analogues with a bicyclo[2.2.1]heptane fragment as sugar moiety; synthesis, antiviral, anticancer activity and X-ray crystallography.

PubMed

Tănase, Constantin I; Drăghici, Constantin; Cojocaru, Ana; Galochkina, Anastasia V; Orshanskaya, Jana R; Zarubaev, Vladimir V; Shova, Sergiu; Enache, Cristian; Maganu, Maria

2015-10-01

New nucleoside analogues with an optically active bicyclo[2.2.1]heptane skeleton as sugar moiety and 6-substituted adenine were synthesized by alkylation of 6-chloropurine intermediate. Thymine and uracil analogs were synthesized by building the pyrimidine ring on amine 1. X-ray crystallography confirmed an exo-coupling of the thymine to the ring and an L configuration of the nucleoside analogue. The library of compounds was tested for their inhibitory activity against influenza virus A∖California/07/09 (H1N1)pdm09 and coxsackievirus B4 in cell culture. Compounds 13a and 13d are the most promising for their antiviral activity against influenza, and compound 3c against coxsackievirus B4. Compounds 3b and 3g were tested for anticancer activity. Copyright © 2015 Elsevier Ltd. All rights reserved.

X-ray filter for x-ray powder diffraction

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

Sinsheimer, John Jay; Conley, Raymond P.; Bouet, Nathalie C. D.

Technologies are described for apparatus, methods and systems effective for filtering. The filters may comprise a first plate. The first plate may include an x-ray absorbing material and walls defining first slits. The first slits may include arc shaped openings through the first plate. The walls of the first plate may be configured to absorb at least some of first x-rays when the first x-rays are incident on the x-ray absorbing material, and to output second x-rays. The filters may comprise a second plate spaced from the first plate. The second plate may include the x-ray absorbing material and wallsmore » defining second slits. The second slits may include arc shaped openings through the second plate. The walls of the second plate may be configured to absorb at least some of second x-rays and to output third x-rays.« less

In situ X-ray-based imaging of nano materials

DOE PAGES

Weker, Johanna Nelson; Huang, Xiaojing; Toney, Michael F.

2016-02-13

We study functional nanomaterials that are heterogeneous and understanding their behavior during synthesis and operation requires high resolution diagnostic imaging tools that can be used in situ. Over the past decade, huge progress has been made in the development of X-ray based imaging, including full field and scanning microscopy and their analogs in coherent diffractive imaging. Currently, spatial resolution of about 10 nm and time resolution of sub-seconds are achievable. For catalysis, X-ray imaging allows tracking of particle chemistry under reaction conditions. In energy storage, in situ X-ray imaging of electrode particles is providing important insight into degradation processes. Recently,more » both spatial and temporal resolutions are improving to a few nm and milliseconds and these developments will open up unprecedented opportunities.« less

A von Hamos x-ray spectrometer based on a segmented-type diffraction crystal for single-shot x-ray emission spectroscopy and time-resolved resonant inelastic x-ray scattering studies

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

Szlachetko, J.; Institute of Physics, Jan Kochanowski University, 25-406 Kielce; Nachtegaal, M.

2012-10-15

We report on the design and performance of a wavelength-dispersive type spectrometer based on the von Hamos geometry. The spectrometer is equipped with a segmented-type crystal for x-ray diffraction and provides an energy resolution in the order of 0.25 eV and 1 eV over an energy range of 8000 eV-9600 eV. The use of a segmented crystal results in a simple and straightforward crystal preparation that allows to preserve the spectrometer resolution and spectrometer efficiency. Application of the spectrometer for time-resolved resonant inelastic x-ray scattering and single-shot x-ray emission spectroscopy is demonstrated.

Hydrogen atoms in protein structures: high-resolution X-ray diffraction structure of the DFPase

PubMed Central

2013-01-01

Background Hydrogen atoms represent about half of the total number of atoms in proteins and are often involved in substrate recognition and catalysis. Unfortunately, X-ray protein crystallography at usual resolution fails to access directly their positioning, mainly because light atoms display weak contributions to diffraction. However, sub-Ångstrom diffraction data, careful modeling and a proper refinement strategy can allow the positioning of a significant part of hydrogen atoms. Results A comprehensive study on the X-ray structure of the diisopropyl-fluorophosphatase (DFPase) was performed, and the hydrogen atoms were modeled, including those of solvent molecules. This model was compared to the available neutron structure of DFPase, and differences in the protein and the active site solvation were noticed. Conclusions A further examination of the DFPase X-ray structure provides substantial evidence about the presence of an activated water molecule that may constitute an interesting piece of information as regard to the enzymatic hydrolysis mechanism. PMID:23915572

Development of polycapillary x-ray optics for x-ray spectroscopy

NASA Astrophysics Data System (ADS)

Adams, Bernhard W.; Attenkofer, Klaus; Bond, Justin L.; Craven, Christopher A.; Cremer, Till; O'Mahony, Aileen; Minot, Michael J.; Popecki, Mark A.

2016-09-01

Bundles of hollow glass capillaries can be tapered to produce quasi-focusing x-ray optics. These optics are known as Kumakhov lenses. These optics are interesting for lab-based sources because they can be used to collimate and concentrate x-rays originating from a point, such as a laser focus or an electron-beam focus in a microtube.

Nonlinear X-Ray and Auger Spectroscopy at X-Ray Free-Electron Laser Sources

NASA Astrophysics Data System (ADS)

Rohringer, Nina

2015-05-01

X-ray free-electron lasers (XFELs) open the pathway to transfer non-linear spectroscopic techniques to the x-ray domain. A promising all x-ray pump probe technique is based on coherent stimulated electronic x-ray Raman scattering, which was recently demonstrated in atomic neon. By tuning the XFEL pulse to core-excited resonances, a few seed photons in the spectral tail of the XFEL pulse drive an avalanche of resonant inelastic x-ray scattering events, resulting in exponential amplification of the scattering signal by of 6-7 orders of magnitude. Analysis of the line profile of the emitted radiation permits to demonstrate the cross over from amplified spontaneous emission to coherent stimulated resonance scattering. In combination with statistical covariance mapping, a high-resolution spectrum of the resonant inelastic scattering process can be obtained, opening the path to coherent stimulated x-ray Raman spectroscopy. An extension of these ideas to molecules and a realistic feasibility study of stimulated electronic x-ray Raman scattering in CO will be presented. Challenges to realizing stimulated electronic x-ray Raman scattering at present-day XFEL sources will be discussed, corroborated by results of a recent experiment at the LCLS XFEL. Due to the small gain cross section in molecular targets, other nonlinear spectroscopic techniques such as nonlinear Auger spectroscopy could become a powerful alternative. Theory predictions of a novel pump probe technique based on resonant nonlinear Auger spectroscopic will be discussed and the method will be compared to stimulated x-ray Raman spectroscopy.

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

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.

Exploring the palladium- and platinum-bis(pyridine) complex motif by NMR spectroscopy, X-ray crystallography, (tandem) mass spectrometry, and isothermal titration calorimetry: do substituent effects follow chemical intuition?

PubMed

Weilandt, Torsten; Löw, Nora L; Schnakenburg, Gregor; Daniels, Jörg; Nieger, Martin; Schalley, Christoph A; Lützen, Arne

2012-12-21

A series of ten palladium-bis(pyridine) complexes, as well as their corresponding platinum complexes, have been synthesized. The pyridine ligands in each series carried different σ-donor and/or π-acceptor/donor substituents at the para-position of their pyridine rings. These complexes were analysed by NMR spectroscopy, X-ray crystallography, (tandem) MS, and isothermal titration calorimetry (ITC) to validate whether these methods allowed us to obtain a concise and systematic picture of the relative and absolute thermodynamic stabilities of the complexes, as determined by the electronic effects of the substituents. Interestingly, the NMR spectroscopic data hardly correlated with the expected substituent effects but the heteronuclear platinum-phosphorus coupling constants did. Crystallographic data were found to be blurred by packing effects. Instead, tandem MS and ITC data were in line with each other and followed the expected trends. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Probing the interaction of archaeal DNA polymerases with deaminated bases using X-ray crystallography and non-hydrogen bonding isosteric base analogues.

PubMed

Killelea, Tom; Ghosh, Samantak; Tan, Samuel S; Heslop, Pauline; Firbank, Susan J; Kool, Eric T; Connolly, Bernard A

2010-07-13

Archaeal family-B DNA polymerases stall replication on encountering the pro-mutagenic bases uracil and hypoxanthine. This publication describes an X-ray crystal structure of Thermococcus gorgonarius polymerase in complex with a DNA containing hypoxanthine in the single-stranded region of the template, two bases ahead of the primer-template junction. Full details of the specific recognition of hypoxanthine are revealed, allowing a comparison with published data that describe uracil binding. The two bases are recognized by the same pocket, in the N-terminal domain, and make very similar protein-DNA interactions. Specificity for hypoxanthine (and uracil) arises from a combination of polymerase-base hydrogen bonds and shape fit between the deaminated bases and the pocket. The structure with hypoxanthine at position 2 explains the stimulation of the polymerase 3'-5' proofreading exonuclease, observed with deaminated bases at this location. A beta-hairpin element, involved in partitioning the primer strand between the polymerase and exonuclease active sites, inserts between the two template bases at the extreme end of the double-stranded DNA. This denatures the two complementary primer bases and directs the resulting 3' single-stranded extension toward the exonuclease active site. Finally, the relative importance of hydrogen bonding and shape fit in determining selectivity for deaminated bases has been examined using nonpolar isosteres. Affinity for both 2,4-difluorobenzene and fluorobenzimidazole, non-hydrogen bonding shape mimics of uracil and hypoxanthine, respectively, is strongly diminished, suggesting polar protein-base contacts are important. However, residual interaction with 2,4-difluorobenzene is seen, confirming a role for shape recognition.

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

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.

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

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

A structural study of bone changes in knee osteoarthritis by synchrotron-based X-ray fluorescence and X-ray absorption spectroscopy techniques

NASA Astrophysics Data System (ADS)

Sindhupakorn, Bura; Thienpratharn, Suwittaya; Kidkhunthod, Pinit

2017-10-01

Osteoarthritis (OA) is characterized by degeneration of articular cartilage and thickening of subchondral bone. The present study investigated the changing of biochemical components of cartilage and bone compared between normal and OA people. Using Synchrotron-based X-ray fluorescence (SR-XRF) and X-ray absorption spectroscopy (XAS) techniquesincluding X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) were employed for the bone changes in kneeosteoarthritisstudies. The bone samples were collected from various osteoarthritis patients with both male and female in the ages range between 20 and 74 years old. SR-XRF results excited at 4240 eV for Ca elements show a majority three main groups, based on their XRF intensities, 20-36 years, 40-60 years and over 70 years, respectively. By employing XAS techniques, XANES features can be used to clearly explain in term of electronic transitions occurring in bone samples which are affected from osteoarthritis symptoms. Moreover, a structural change around Ca ions in bone samples is obviously obtained by EXAFS results indicating an increase of Ca-amorphous phase when the ages increase.

Development of an online UV-visible microspectrophotometer for a macromolecular crystallography beamline.

PubMed

Shimizu, Nobutaka; Shimizu, Tetsuya; Baba, Seiki; Hasegawa, Kazuya; Yamamoto, Masaki; Kumasaka, Takashi

2013-11-01

Measurement of the UV-visible absorption spectrum is a convenient technique for detecting chemical changes of proteins, and it is therefore useful to combine spectroscopy and diffraction studies. An online microspectrophotometer for the UV-visible region was developed and installed on the macromolecular crystallography beamline, BL38B1, at SPring-8. This spectrophotometer is equipped with a difference dispersive double monochromator, a mercury-xenon lamp as the light source, and a photomultiplier as the detector. The optical path is mostly constructed using mirrors, in order to obtain high brightness in the UV region, and the confocal optics are assembled using a cross-slit diaphragm like an iris to eliminate stray light. This system can measure optical densities up to a maximum of 4.0. To study the effect of radiation damage, preliminary measurements of glucose isomerase and thaumatin crystals were conducted in the UV region. Spectral changes dependent on X-ray dose were observed at around 280 nm, suggesting that structural changes involving Trp or Tyr residues occurred in the protein crystal. In the case of the thaumatin crystal, a broad peak around 400 nm was also generated after X-ray irradiation, suggesting the cleavage of a disulfide bond. Dose-dependent spectral changes were also observed in cryo-solutions alone, and these changes differed with the composition of the cryo-solution. These responses in the UV region are informative regarding the state of the sample; consequently, this device might be useful for X-ray crystallography.

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

Measurement and Interpretation of Diffuse Scattering in X-Ray Diffraction for Macromolecular Crystallography

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

Wall, Michael E.

X-ray diffraction from macromolecular crystals includes both sharply peaked Bragg reflections and diffuse intensity between the peaks. The information in Bragg scattering reflects the mean electron density in the unit cells of the crystal. The diffuse scattering arises from correlations in the variations of electron density that may occur from one unit cell to another, and therefore contains information about collective motions in proteins.

Implications of the focal beam profile in serial femtosecond crystallography

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

Galli, Lorenzo; Chapman, Henry N.; Metcalf, Peter

The photon density profile of an X-ray free-electron laser (XFEL) beam at the focal position is a critical parameter for serial femtosecond crystallography (SFX), but is difficult to measure because of the destructive power of the beam. A novel high intensity radiation induced phasing method (HIRIP) has been proposed as a general experimental approach for protein structure determination, but has proved to be sensitive to variations of the X-ray intensity, with uniform incident fluence desired for best performance. Here we show that experimental SFX data collected at the nano-focus chamber of the Coherent X-ray Imaging end-station at the Linac Coherentmore » Light Source using crystals with a limited size distribution suggests an average profile of the X-ray beam that has a large variation of intensity. We propose a new method to improve the quality of high fluence data for HI-RIP, by identifying and removing diffraction patterns from crystals exposed to the low intensity region of the beam. The method requires crystals of average size comparable to the width of the focal spot.« less

Bone cartilage imaging with x-ray interferometry using a practical x-ray tube

NASA Astrophysics Data System (ADS)

Kido, Kazuhiro; Makifuchi, Chiho; Kiyohara, Junko; Itou, Tsukasa; Honda, Chika; Momose, Atsushi

2010-04-01

The purpose of this study was to design an X-ray Talbot-Lau interferometer for the imaging of bone cartilage using a practical X-ray tube and to develop that imaging system for clinical use. Wave-optics simulation was performed to design the interferometer with a practical X-ray tube, a source grating, two X-ray gratings, and an X-ray detector. An imaging system was created based on the results of the simulation. The specifications were as follows: the focal spot size was 0.3 mm of an X-ray tube with a tungsten anode (Toshiba, Tokyo, Japan). The tube voltage was set at 40 kVp with an additive aluminum filter, and the mean energy was 31 keV. The pixel size of the X-ray detector, a Condor 486 (Fairchild Imaging, California, USA), was 15 μm. The second grating was a Ronchi-type grating whose pitch was 5.3 μm. Imaging performance of the system was examined with X-ray doses of 0.5, 3 and 9 mGy so that the bone cartilage of a chicken wing was clearly depicted with X-ray doses of 3 and 9 mGy. This was consistent with the simulation's predictions. The results suggest that X-ray Talbot-Lau interferometry would be a promising tool in detecting soft tissues in the human body such as bone cartilage for the X-ray image diagnosis of rheumatoid arthritis. Further optimization of the system will follow to reduce the X-ray dose for clinical use.

High-speed fixed-target serial virus crystallography

DOE PAGES

Roedig, Philip; Ginn, Helen M.; Pakendorf, Tim; ...

2017-06-19

Here, 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 micropatterned silicon chip in combination with the high-speed Roadrunner goniometer for sample delivery, we were able to determine the crystal structures of the picornavirus bovine enterovirus 2 (BEV2) and the cytoplasmic polyhedrosis virus type 18 polyhedrin, with total data collection times of less than 14 and 10 min, respectively. Our method requires only micrograms of sample and should therefore broaden the applicability of serial femtosecond crystallographymore » 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 most efficient use of the limited beam time available at XFELs and should enable a substantial increase in sample throughput at these facilities.« less

High-speed fixed-target serial virus crystallography

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

Roedig, Philip; Ginn, Helen M.; Pakendorf, Tim

Here, 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 micropatterned silicon chip in combination with the high-speed Roadrunner goniometer for sample delivery, we were able to determine the crystal structures of the picornavirus bovine enterovirus 2 (BEV2) and the cytoplasmic polyhedrosis virus type 18 polyhedrin, with total data collection times of less than 14 and 10 min, respectively. Our method requires only micrograms of sample and should therefore broaden the applicability of serial femtosecond crystallographymore » 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 most efficient use of the limited beam time available at XFELs and should enable a substantial increase in sample throughput at these facilities.« less

Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser

PubMed Central

Kupitz, Christopher; Basu, Shibom; Grotjohann, Ingo; Fromme, Raimund; Zatsepin, Nadia A.; Rendek, Kimberly N.; Hunter, Mark S.; Shoeman, Robert L.; White, Thomas A.; Wang, Dingjie; James, Daniel; Yang, Jay-How; Cobb, Danielle E.; Reeder, Brenda; Sierra, Raymond G.; Liu, Haiguang; Barty, Anton; Aquila, Andrew L.; Deponte, Daniel; Kirian, Richard A.; Bari, Sadia; Bergkamp, Jesse J.; Beyerlein, Kenneth R.; Bogan, Michael J.; Caleman, Carl; Chao, Tzu-Chiao; Conrad, Chelsie E.; Davis, Katherine M.; Fleckenstein, Holger; Galli, Lorenzo; Hau-Riege, Stefan P.; Kassemeyer, Stephan; Laksmono, Hartawan; Liang, Mengning; Lomb, Lukas; Marchesini, Stefano; Martin, Andrew V.; Messerschmidt, Marc; Milathianaki, Despina; Nass, Karol; Ros, Alexandra; Roy-Chowdhury, Shatabdi; Schmidt, Kevin; Seibert, Marvin; Steinbrener, Jan; Stellato, Francesco; Yan, Lifen; Yoon, Chunhong; Moore, Thomas A.; Moore, Ana L.; Pushkar, Yulia; Williams, Garth J.; Boutet, Sébastien; Doak, R. Bruce; Weierstall, Uwe; Frank, Matthias; Chapman, Henry N.; Spence, John C. H.; Fromme, Petra

2015-01-01

Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth’s oxygenic atmosphere1. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S0 to S4, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed2 technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S1 state and after double laser excitation (putative S3 state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn4CaO5 core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the ‘dangler’ Mn) and the Mn3CaOx cubane in the S2 to S3 transition, as predicted by spectroscopic and computational studies3,4. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules. PMID:25043005

A marker-based watershed method for X-ray image segmentation.

PubMed

Zhang, Xiaodong; Jia, Fucang; Luo, Suhuai; Liu, Guiying; Hu, Qingmao

2014-03-01

Digital X-ray images are the most frequent modality for both screening and diagnosis in hospitals. To facilitate subsequent analysis such as quantification and computer aided diagnosis (CAD), it is desirable to exclude image background. A marker-based watershed segmentation method was proposed to segment background of X-ray images. The method consisted of six modules: image preprocessing, gradient computation, marker extraction, watershed segmentation from markers, region merging and background extraction. One hundred clinical direct radiograph X-ray images were used to validate the method. Manual thresholding and multiscale gradient based watershed method were implemented for comparison. The proposed method yielded a dice coefficient of 0.964±0.069, which was better than that of the manual thresholding (0.937±0.119) and that of multiscale gradient based watershed method (0.942±0.098). Special means were adopted to decrease the computational cost, including getting rid of few pixels with highest grayscale via percentile, calculation of gradient magnitude through simple operations, decreasing the number of markers by appropriate thresholding, and merging regions based on simple grayscale statistics. As a result, the processing time was at most 6s even for a 3072×3072 image on a Pentium 4 PC with 2.4GHz CPU (4 cores) and 2G RAM, which was more than one time faster than that of the multiscale gradient based watershed method. The proposed method could be a potential tool for diagnosis and quantification of X-ray images. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Indus-2 X-ray lithography beamline for X-ray optics and material science applications

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

Dhamgaye, V. P., E-mail: vishal@rrcat.gov.in; Lodha, G. S., E-mail: vishal@rrcat.gov.in

2014-04-24

X-ray lithography is an ideal technique by which high aspect ratio and high spatial resolution micro/nano structures are fabricated using X-rays from synchrotron radiation source. The technique has been used for fabricating optics (X-ray, visible and infrared), sensors and actuators, fluidics and photonics. A beamline for X-ray lithography is operational on Indus-2. The beamline offers wide lithographic window from 1-40keV photon energy and wide beam for producing microstructures in polymers upto size ∼100mm × 100mm. X-ray exposures are possible in air, vacuum and He gas environment. The air based exposures enables the X-ray irradiation of resist for lithography and alsomore » irradiation of biological and liquid samples.« less

Native chemical ligation at Asx-Cys, Glx-Cys: chemical synthesis and high-resolution X-ray structure of ShK toxin by racemic protein crystallography.

PubMed

Dang, Bobo; Kubota, Tomoya; Mandal, Kalyaneswar; Bezanilla, Francisco; Kent, Stephen B H

2013-08-14

We have re-examined the utility of native chemical ligation at -Gln/Glu-Cys- [Glx-Cys] and -Asn/Asp-Cys- [Asx-Cys] sites. Using the improved thioaryl catalyst 4-mercaptophenylacetic acid (MPAA), native chemical ligation could be performed at -Gln-Cys- and Asn-Cys- sites without side reactions. After optimization, ligation at a -Glu-Cys- site could also be used as a ligation site, with minimal levels of byproduct formation. However, -Asp-Cys- is not appropriate for use as a site for native chemical ligation because of formation of significant amounts of β-linked byproduct. The feasibility of native chemical ligation at -Gln-Cys- enabled a convergent total chemical synthesis of the enantiomeric forms of the ShK toxin protein molecule. The D-ShK protein molecule was ~50,000-fold less active in blocking the Kv1.3 channel than the L-ShK protein molecule. Racemic protein crystallography was used to obtain high-resolution X-ray diffraction data for ShK toxin. The structure was solved by direct methods and showed significant differences from the previously reported NMR structures in some regions of the ShK protein molecule.

Optimization-Based Approach for Joint X-Ray Fluorescence and Transmission Tomographic Inversion

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

Di, Zichao; Leyffer, Sven; Wild, Stefan M.

2016-01-01

Fluorescence tomographic reconstruction, based on the detection of photons coming from fluorescent emission, can be used for revealing the internal elemental composition of a sample. On the other hand, conventional X-ray transmission tomography can be used for reconstructing the spatial distribution of the absorption coefficient inside a sample. In this work, we integrate both X-ray fluorescence and X-ray transmission data modalities and formulate a nonlinear optimization-based approach for reconstruction of the elemental composition of a given object. This model provides a simultaneous reconstruction of both the quantitative spatial distribution of all elements and the absorption effect in the sample. Mathematicallymore » speaking, we show that compared with the single-modality inversion (i.e., the X-ray transmission or fluorescence alone), the joint inversion provides a better-posed problem, which implies a better recovery. Therefore, the challenges in X-ray fluorescence tomography arising mainly from the effects of self-absorption in the sample are partially mitigated. The use of this technique is demonstrated on the reconstruction of several synthetic samples.« less

Rapid simulation of X-ray transmission imaging for baggage inspection via GPU-based ray-tracing

NASA Astrophysics Data System (ADS)

Gong, Qian; Stoian, Razvan-Ionut; Coccarelli, David S.; Greenberg, Joel A.; Vera, Esteban; Gehm, Michael E.

2018-01-01

We present a pipeline that rapidly simulates X-ray transmission imaging for arbitrary system architectures using GPU-based ray-tracing techniques. The purpose of the pipeline is to enable statistical analysis of threat detection in the context of airline baggage inspection. As a faster alternative to Monte Carlo methods, we adopt a deterministic approach for simulating photoelectric absorption-based imaging. The highly-optimized NVIDIA OptiX API is used to implement ray-tracing, greatly speeding code execution. In addition, we implement the first hierarchical representation structure to determine the interaction path length of rays traversing heterogeneous media described by layered polygons. The accuracy of the pipeline has been validated by comparing simulated data with experimental data collected using a heterogenous phantom and a laboratory X-ray imaging system. On a single computer, our approach allows us to generate over 400 2D transmission projections (125 × 125 pixels per frame) per hour for a bag packed with hundreds of everyday objects. By implementing our approach on cloud-based GPU computing platforms, we find that the same 2D projections of approximately 3.9 million bags can be obtained in a single day using 400 GPU instances, at a cost of only 0.001 per bag.

Probing the interaction of archaeal DNA polymerases with deaminated bases using X-ray crystallography and non-hydrogen bonding isosteric base analogues†

PubMed Central

Killelea, Tom; Ghosh, Samantak; Tan, Samuel S.; Heslop, Pauline; Firbank, Susan; Kool, Eric T.; Connolly, Bernard A.

2010-01-01

Archaeal family-B DNA polymerases stall replication on encountering the pro-mutagenic bases uracil and hypoxanthine. This publication describes an X-ray crystal structure of Thermococcus gorgonarius polymerase in complex with a DNA containing hypoxanthine in the single-stranded region of the template, two bases ahead of the primer-template junction. Full details of the specific recognition of hypoxanthine are revealed, allowing a comparison with published data that describes uracil binding. The two bases are recognized by the same pocket, in the N-terminal domain, and make very similar protein-DNA interactions. Specificity for hypoxanthine (and uracil) arises from a combination of polymerase-base hydrogen bonds and shape fit between the deaminated bases and the pocket. The structure with hypoxanthine at the +2 position explains the stimulation of the polymerase 3′-5′ proof reading exonuclease, observed with deaminated bases at this location. A β hairpin element, involved in partitioning the primer strand between the polymerase and exonuclease active sites, inserts between the two template bases at the extreme end of the double stranded DNA. This denatures the two complementary primer bases and directs the resulting 3′ single-stranded extension towards the exonuclease active site. Finally the relative importance of hydrogen bonding and shape fit in determining selectivity for deaminated bases has been examined using non-polar isosteres. Affinity for both 2,4 difluorobenzene and fluorobenzimidazole, non-hydrogen bonding shape mimics of uracil and hypoxanthine respectively, is strongly diminished, suggesting polar protein-base contacts are important. However, residual interaction with 2,4 difluorobenzene is seen, confirming a role for shape recognition. PMID:20527806

Synchrotron-based ambient pressure X-ray photoelectron spectroscopy of hydrogen and helium

NASA Astrophysics Data System (ADS)

Zhong, Jian-Qiang; Wang, Mengen; Hoffmann, William H.; van Spronsen, Matthijs A.; Lu, Deyu; Boscoboinik, J. Anibal

2018-02-01

Contrary to popular belief, it is possible to obtain X-ray photoelectron spectra for elements lighter than lithium, namely hydrogen and helium. The literature is plagued with claims of this impossibility, which holds true for lab-based X-ray sources. However, this limitation is merely technical and is related mostly to the low X-ray photoionization cross-sections of the 1s orbitals of hydrogen and helium. In this letter, we show that, using ambient pressure X-ray photoelectron spectroscopy (XPS), a bright-enough X-ray source allows the study of these elusive elements. This has important implications in the understanding of the limitations of one of the most useful techniques in materials science, and moreover, it potentially opens the possibility of using XPS to directly study the most abundant element in the universe.

Hafnium-Based Contrast Agents for X-ray Computed Tomography.

PubMed

Berger, Markus; Bauser, Marcus; Frenzel, Thomas; Hilger, Christoph Stephan; Jost, Gregor; Lauria, Silvia; Morgenstern, Bernd; Neis, Christian; Pietsch, Hubertus; Sülzle, Detlev; Hegetschweiler, Kaspar

2017-05-15

Heavy-metal-based contrast agents (CAs) offer enhanced X-ray absorption for X-ray computed tomography (CT) compared to the currently used iodinated CAs. We report the discovery of new lanthanide and hafnium azainositol complexes and their optimization with respect to high water solubility and stability. Our efforts culminated in the synthesis of BAY-576, an uncharged hafnium complex with 3:2 stoichiometry and broken complex symmetry. The superior properties of this asymmetrically substituted hafnium CA were demonstrated by a CT angiography study in rabbits that revealed excellent signal contrast enhancement.

Fabrication of absorption gratings with X-ray lithography for X-ray phase contrast imaging

NASA Astrophysics Data System (ADS)

Wang, Bo; Wang, Yu-Ting; Yi, Fu-Ting; Zhang, Tian-Chong; Liu, Jing; Zhou, Yue

2018-05-01

Grating-based X-ray phase contrast imaging is promising especially in the medical area. Two or three gratings are involved in grating-based X-ray phase contrast imaging in which the absorption grating of high-aspect-ratio is the most important device and the fabrication process is a great challenge. The material with large atomic number Z is used to fabricate the absorption grating for excellent absorption of X-ray, and Au is usually used. The fabrication process, which involves X-ray lithography, development and gold electroplating, is described in this paper. The absorption gratings with 4 μm period and about 100 μm height are fabricated and the high-aspect-ratio is 50.

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.

A large format membrane-based x-ray mask for microfluidic chip fabrication

NASA Astrophysics Data System (ADS)

Wang, Lin; Zhang, Min; Desta, Yohannes; Melzak, J.; Wu, C. H.; Peng, Zhengchun

2006-02-01

X-ray lithography is a very good option for the fabrication of micro-devices especially when high aspect ratio patterns are required. Membrane-based x-ray masks are commonly used for high-resolution x-ray lithography. A thin layer of silicon nitride (Si3N4) or silicon carbide (SiC) film (1-2 µm) is normally used as the membrane material for x-ray mask fabrication (Wells G M, Reilly M, Nachman R, Cerrina F, El-Khakani M A and Chaker M 1993 Mater. Res. Soc. Conf. Proc. 306 81-9 Shoki T, Nagasawa H, Kosuga H, Yamaguchi Y, Annaka N, Amemiya I and Nagarekawa O 1993 SPIE Proc. 1924 450-6). The freestanding membrane window of an x-ray mask, which defines the exposing area of the x-ray mask, can be obtained by etching a pre-defined area on a silicon wafer from the backside (Wang L, Desta Y, Fettig R K, Goettert J, Hein H, Jakobs P and Chulz J 2004 J. Micromech. Microeng. 14 722-6). Usually, the window size of an x-ray mask is around 20 × 20 mm because of the low tensile stress of the membrane (10-100 MPa), and the larger window dimension of an x-ray mask may cause the deformation of membranes and lower the mask quality. However, x-ray masks with larger windows are preferred for micro-device fabrication in order to increase the productivity. We analyzed the factors which influence the flatness of large format x-ray masks and fabricated x-ray masks with a window size of 55 × 55 mm and 46 × 65 mm on 1 µm thick membranes by increasing the tensile stress of the membranes (>300 MPa) and optimizing the stress of the absorber layer. The large format x-ray mask was successfully applied for the fabrication of microfluidic chips.

Time-lapse crystallography snapshots of a double-strand break repair polymerase in action.

PubMed

Jamsen, Joonas A; Beard, William A; Pedersen, Lars C; Shock, David D; Moon, Andrea F; Krahn, Juno M; Bebenek, Katarzyna; Kunkel, Thomas A; Wilson, Samuel H

2017-08-15

DNA polymerase (pol) μ is a DNA-dependent polymerase that incorporates nucleotides during gap-filling synthesis in the non-homologous end-joining pathway of double-strand break repair. Here we report time-lapse X-ray crystallography snapshots of catalytic events during gap-filling DNA synthesis by pol μ. Unique catalytic intermediates and active site conformational changes that underlie catalysis are uncovered, and a transient third (product) metal ion is observed in the product state. The product manganese coordinates phosphate oxygens of the inserted nucleotide and PP i . The product metal is not observed during DNA synthesis in the presence of magnesium. Kinetic analyses indicate that manganese increases the rate constant for deoxynucleoside 5'-triphosphate insertion compared to magnesium. The likely product stabilization role of the manganese product metal in pol μ is discussed. These observations provide insight on structural attributes of this X-family double-strand break repair polymerase that impact its biological function in genome maintenance.DNA polymerase (pol) μ functions in DNA double-strand break repair. Here the authors use time-lapse X-ray crystallography to capture the states of pol µ during the conversion from pre-catalytic to product complex and observe a third transiently bound metal ion in the product state.

Absolute x-ray energy calibration and monitoring using a diffraction-based method

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

Hong, Xinguo, E-mail: xhong@bnl.gov; Weidner, Donald J.; Duffy, Thomas S.

2016-07-27

In this paper, we report some recent developments of the diffraction-based absolute X-ray energy calibration method. In this calibration method, high spatial resolution of the measured detector offset is essential. To this end, a remotely controlled long-translation motorized stage was employed instead of the less convenient gauge blocks. It is found that the precision of absolute X-ray energy calibration (ΔE/E) is readily achieved down to the level of 10{sup −4} for high-energy monochromatic X-rays (e.g. 80 keV). Examples of applications to pair distribution function (PDF) measurements and energy monitoring for high-energy X-rays are presented.

Multi-Layer Organic Squaraine-Based Photodiode for Indirect X-Ray Detection

NASA Astrophysics Data System (ADS)

Iacchetti, Antonio; Binda, Maddalena; Natali, Dario; Giussani, Mattia; Beverina, Luca; Fiorini, Carlo; Peloso, Roberta; Sampietro, Marco

2012-10-01

The paper presents an organic-based photodiode coupled to a CsI(Tl) scintillator to realize an X-ray detector. A suitable blend of an indolic squaraine derivative and of fullerene derivative has been used for the photodiode, thus allowing external quantum efficiency in excess of 10% at a wavelength of 570 nm, well matching the scintillator output spectrum. Thanks to the additional deposition of a 15 nm thin layer of a suitable low electron affinity polymer, carriers injection from the metal into the organic semiconductor has been suppressed, and dark current density as low as has been obtained, which is comparable to standard Si-based photodiodes. By using a collimated X-ray beam impinging onto the scintillator mounted over the photodiode we have been able to measure current variations in the order of 150 pA on a dark current floor of less than 50 pA when operating the X-ray tube in switching mode, thus proving the feasibility of indirect X-ray detection by means of organic semiconductors.

Study of Cr/Sc-based multilayer reflecting mirrors using soft x-ray reflectivity and standing wave-enhanced x-ray fluorescence

NASA Astrophysics Data System (ADS)

Wu, Meiyi; Burcklen, Catherine; André, Jean-Michel; Guen, Karine Le; Giglia, Angelo; Koshmak, Konstantin; Nannarone, Stefano; Bridou, Françoise; Meltchakov, Evgueni; Rossi, Sébastien de; Delmotte, Franck; Jonnard, Philippe

2017-11-01

We study Cr/Sc-based multilayer mirrors designed to work in the water window range using hard and soft x-ray reflectivity as well as x-ray fluorescence enhanced by standing waves. Samples differ by the elemental composition of the stack, the thickness of each layer, and the order of deposition. This paper mainly consists of two parts. In the first part, the optical performances of different Cr/Sc-based multilayers are reported, and in the second part, we extend further the characterization of the structural parameters of the multilayers, which can be extracted by comparing the experimental data with simulations. The methodology is detailed in the case of Cr/B4C/Sc sample for which a three-layer model is used. Structural parameters determined by fitting reflectivity curve are then introduced as fixed parameters to plot the x-ray standing wave curve, to compare with the experiment, and confirm the determined structure of the stack.

Synthesis and X-ray Crystallography of [Mg(H2O)6][AnO2(C2H5COO)3]2 (An = U, Np, or Pu).

PubMed

Serezhkin, Viktor N; Grigoriev, Mikhail S; Abdulmyanov, Aleksey R; Fedoseev, Aleksandr M; Savchenkov, Anton V; Serezhkina, Larisa B

2016-08-01

Synthesis and X-ray crystallography of single crystals of [Mg(H2O)6][AnO2(C2H5COO)3]2, where An = U (I), Np (II), or Pu (III), are reported. Compounds I-III are isostructural and crystallize in the trigonal crystal system. The structures of I-III are built of hydrated magnesium cations [Mg(H2O)6](2+) and mononuclear [AnO2(C2H5COO)3](-) complexes, which belong to the AB(01)3 crystallochemical group of uranyl complexes (A = AnO2(2+), B(01) = C2H5COO(-)). Peculiarities of intermolecular interactions in the structures of [Mg(H2O)6][UO2(L)3]2 complexes depending on the carboxylate ion L (acetate, propionate, or n-butyrate) are investigated using the method of molecular Voronoi-Dirichlet polyhedra. Actinide contraction in the series of U(VI)-Np(VI)-Pu(VI) in compounds I-III is reflected in a decrease in the mean An═O bond lengths and in the volume and sphericity degree of Voronoi-Dirichlet polyhedra of An atoms.

Exposing hidden alternative backbone conformations in X-ray crystallography using qFit

DOE PAGES

Keedy, Daniel A.; Fraser, James S.; van den Bedem, Henry; ...

2015-10-27

Proteins must move between different conformations of their native ensemble to perform their functions. Crystal structures obtained from high-resolution X-ray diffraction data reflect this heterogeneity as a spatial and temporal conformational average. Although movement between natively populated alternative conformations can be critical for characterizing molecular mechanisms, it is challenging to identify these conformations within electron density maps. Alternative side chain conformations are generally well separated into distinct rotameric conformations, but alternative backbone conformations can overlap at several atomic positions. Our model building program qFit uses mixed integer quadratic programming (MIQP) to evaluate an extremely large number of combinations of sidechainmore » conformers and backbone fragments to locally explain the electron density. Here, we describe two major modeling enhancements to qFit: peptide flips and alternative glycine conformations. We find that peptide flips fall into four stereotypical clusters and are enriched in glycine residues at the n+1 position. The potential for insights uncovered by new peptide flips and glycine conformations is exemplified by HIV protease, where different inhibitors are associated with peptide flips in the “flap” regions adjacent to the inhibitor binding site. Our results paint a picture of peptide flips as conformational switches, often enabled by glycine flexibility, that result in dramatic local rearrangements. Our results furthermore demonstrate the power of large-scale computational analysis to provide new insights into conformational heterogeneity. Furthermore, improved modeling of backbone heterogeneity with high-resolution X-ray data will connect dynamics to the structure-function relationship and help drive new design strategies for inhibitors of biomedically important systems.« less

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

Virtual Screening and X-ray Crystallography for Human Kallikrein 6 Inhibitors with an Amidinothiophene P1 Group.

PubMed

Liang, Guyan; Chen, Xin; Aldous, Suzanne; Pu, Su-Fen; Mehdi, Shujaath; Powers, Elaine; Giovanni, Andrew; Kongsamut, Sathapana; Xia, Tianhui; Zhang, Ying; Wang, Rachel; Gao, Zhongli; Merriman, Gregory; McLean, Larry R; Morize, Isabelle

2012-02-09

A series of compounds with an amidinothiophene P1 group and a pyrrolidinone-sulphonamide scaffold linker was identified as potent inhibitors of human kallikrein 6 by structure-based virtual screening based on the union accessible binding space of serine proteases. As the first series of potent nonmechanism-based hK6 inhibitors, they may be used as tool compounds for target validation. An X-ray structure of a representative compound complexed with hK6, resolved at a resolution of 1.88 Å, revealed that the amidinothiophene moiety bound in the S1 pocket and the pyrrolidinone-sulphonamide linker projected the aromatic tail into the S' pocket.

Synchrotron-based ambient pressure X-ray photoelectron spectroscopy of hydrogen and helium

DOE PAGES

Zhong, Jian-Qiang; Wang, Mengen; Hoffmann, William H.; ...

2018-03-01

Contrary to popular belief, it is possible to obtain X-ray photoelectron spectra for elements lighter than lithium, namely hydrogen and helium. The literature is plagued with claims of this impossibility, which holds true for lab-based X-ray sources. However, this limitation is merely technical and is related mostly to the low X-ray photoionization cross-sections of the 1s orbitals of hydrogen and helium. Here, we show that, using ambient pressure X-ray photoelectron spectroscopy (XPS), a bright-enough X-ray source allows the study of these elusive elements. This has important implications in the understanding of the limitations of one of the most useful techniquesmore » in materials science, and moreover, it potentially opens the possibility of using XPS to directly study the most abundant element in the universe.« less

Synchrotron-based ambient pressure X-ray photoelectron spectroscopy of hydrogen and helium

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

Zhong, Jian-Qiang; Wang, Mengen; Hoffmann, William H.

Contrary to popular belief, it is possible to obtain X-ray photoelectron spectra for elements lighter than lithium, namely hydrogen and helium. The literature is plagued with claims of this impossibility, which holds true for lab-based X-ray sources. However, this limitation is merely technical and is related mostly to the low X-ray photoionization cross-sections of the 1s orbitals of hydrogen and helium. Here, we show that, using ambient pressure X-ray photoelectron spectroscopy (XPS), a bright-enough X-ray source allows the study of these elusive elements. This has important implications in the understanding of the limitations of one of the most useful techniquesmore » in materials science, and moreover, it potentially opens the possibility of using XPS to directly study the most abundant element in the universe.« less

Method for improve x-ray diffraction determinations of residual stress in nickel-base alloys

DOEpatents

Berman, Robert M.; Cohen, Isadore

1990-01-01

A process for improving the technique of measuring residual stress by x-ray diffraction in pieces of nickel-base alloys which comprises covering part of a predetermined area of the surface of a nickel-base alloy with a dispersion, exposing the covered and uncovered portions of the surface of the alloy to x-rays by way of an x-ray diffractometry apparatus, making x-ray diffraction determinations of the exposed surface, and measuring the residual stress in the alloy based on these determinations. The dispersion is opaque to x-rays and serves a dual purpose since it masks off unsatisfactory signals such that only a small portion of the surface is measured, and it supplies an internal standard by providing diffractogram peaks comparable to the peaks of the nickel alloy so that the alloy peaks can be very accurately located regardless of any sources of error external to the sample.

X-ray Magnetic Scattering From Surfaces^*

NASA Astrophysics Data System (ADS)

Gibbs, Doon

1997-03-01

In the last several years, there have been continuing efforts to probe long-ranged magnetic order at surfaces by x-ray and neutron diffraction, following many earlier studies by low energy electron diffraction. The main motivation has been to discover how bulk magnetic structures are modified near a surface, where the crystal symmetry is broken. In this talk, we describe x-ray scattering studies of the magnetic structure observed near the (001) surface of the antiferromagnet uranium dioxide.(G. M. Watson, Doon Gibbs, G. H. Lander, B. D. Gaulin, L.E. Berman, Hj. Matzke and W. Ellis, Phys. Rev. Lett. 77), 751 (1996). Within about 50 Åof the surface, the intensity of the magnetic scattering decreases continuously as the bulk Neel temperature is approached from below. This contrasts with the bulk magnetic ordering transition which is discontinuous. Recent measurements of the specular magnetic reflectivity suggest that the width of the magnetic interface diverges as a power-law in reduced temperature reminiscent of surface induced disorder. Related experiments concerned with magnetic crystallography of Co_3-Pt(111) surfaces(S. Ferrer, P. Fajardo, F. de Bergevin, J. Alvarez, X. Torrelles, H. A. van der Vegt and V. H. Etgens, Phys. Rev. Lett. 77), 747 (1996). and interfacial magnetic roughness of Co/Cu multilayers(J. F. MacKay, C. Teichert, D.E. Savage and M.G. Lagally, Phys. Rev. Lett. 77), 3925 (1996). will also be discussed. ^* Work at Brookhaven National Laboratory is supported by the U.S. DOE under Contract No. DE-AC02-CH7600016.

Active x-ray optics for Generation-X, the next high resolution x-ray observatory

NASA Astrophysics Data System (ADS)

Elvis, Martin; Brissenden, R. J.; Fabbiano, G.; Schwartz, D. A.; Reid, P.; Podgorski, W.; Eisenhower, M.; Juda, M.; Phillips, J.; Cohen, L.; Wolk, S.

2006-06-01

X-rays provide one of the few bands through which we can study the epoch of reionization, when the first galaxies, black holes and stars were born. To reach the sensitivity required to image these first discrete objects in the universe needs a major advance in X-ray optics. Generation-X (Gen-X) is currently the only X-ray astronomy mission concept that addresses this goal. Gen-X aims to improve substantially on the Chandra angular resolution and to do so with substantially larger effective area. These two goals can only be met if a mirror technology can be developed that yields high angular resolution at much lower mass/unit area than the Chandra optics, matching that of Constellation-X (Con-X). We describe an approach to this goal based on active X-ray optics that correct the mid-frequency departures from an ideal Wolter optic on-orbit. We concentrate on the problems of sensing figure errors, calculating the corrections required, and applying those corrections. The time needed to make this in-flight calibration is reasonable. A laboratory version of these optics has already been developed by others and is successfully operating at synchrotron light sources. With only a moderate investment in these optics the goals of Gen-X resolution can be realized.

CVD-diamond-based position sensitive photoconductive detector for high-flux x-rays and gamma rays.

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

Shu, D.

1999-04-19

A position-sensitive photoconductive detector (PSPCD) using insulating-type CVD diamond as its substrate material has been developed at the Advanced Photon Source (APS). Several different configurations, including a quadrant pattern for a x-ray-transmitting beam position monitor (TBPM) and 1-D and 2-D arrays for PSPCD beam profilers, have been developed. Tests on different PSPCD devices with high-heat-flux undulator white x-ray beam, as well as with gamma-ray beams from {sup 60}Co sources have been done at the APS and National Institute of Standards and Technology (NIST). It was proven that the insulating-type CVD diamond can be used to make a hard x-ray andmore » gamma-ray position-sensitive detector that acts as a solid-state ion chamber. These detectors are based on the photoconductivity principle. A total of eleven of these TBPMs have been installed on the APS front ends for commissioning use. The linear array PSPCD beam profiler has been routinely used for direct measurements of the undulator white beam profile. More tests with hard x-rays and gamma rays are planned for the CVD-diamond 2-D imaging PSPCD. Potential applications include a high-dose-rate beam profiler for fourth-generation synchrotrons radiation facilities, such as free-electron lasers.« less

Pinpointing the base of the AGN jets through general relativistic X-ray reverberation studies

NASA Astrophysics Data System (ADS)

Emmanoulopoulos, D.

2015-03-01

Many theoretical models of Active Galactic Nuclei (AGN) predict that the X-ray corona, lying above the black hole, constitutes the base of the X-ray jet. Thus, by studying the exact geometry of the close black hole environment, we can pinpoint the launching site of the jet. Detection of negative X-ray reverberation time delays (i.e. soft band X-ray variations lagging behind the corresponding hard band X-ray variations) can yield significant information about the geometrical properties of the AGN, such as the location of the X-ray source, as well as the physical properties of the the black hole, such as its mass and spin. In the frame-work of the lamp-post geometry, I present the first systematic X-ray time-lag modelling results of an ensemble of 12 AGN, using a fully general relativistic (GR) ray tracing approach for the estimation of the systems' response functions. By combing these state-of-the art GR response models with statistically innovative fitting routines, I derive the geometrical layout of the close BH environment for each source, unveiling the position of the AGN jet-base.

ISPyB: an information management system for synchrotron macromolecular crystallography.

PubMed

Delagenière, Solange; Brenchereau, Patrice; Launer, Ludovic; Ashton, Alun W; Leal, Ricardo; Veyrier, Stéphanie; Gabadinho, José; Gordon, Elspeth J; Jones, Samuel D; Levik, Karl Erik; McSweeney, Seán M; Monaco, Stéphanie; Nanao, Max; Spruce, Darren; Svensson, Olof; Walsh, Martin A; Leonard, Gordon A

2011-11-15

Individual research groups now analyze thousands of samples per year at synchrotron macromolecular crystallography (MX) resources. The efficient management of experimental data is thus essential if the best possible experiments are to be performed and the best possible data used in downstream processes in structure determination pipelines. Information System for Protein crystallography Beamlines (ISPyB), a Laboratory Information Management System (LIMS) with an underlying data model allowing for the integration of analyses down-stream of the data collection experiment was developed to facilitate such data management. ISPyB is now a multisite, generic LIMS for synchrotron-based MX experiments. Its initial functionality has been enhanced to include improved sample tracking and reporting of experimental protocols, the direct ranking of the diffraction characteristics of individual samples and the archiving of raw data and results from ancillary experiments and post-experiment data processing protocols. This latter feature paves the way for ISPyB to play a central role in future macromolecular structure solution pipelines and validates the application of the approach used in ISPyB to other experimental techniques, such as biological solution Small Angle X-ray Scattering and spectroscopy, which have similar sample tracking and data handling requirements.

Au133(SPh-tBu)52 nanomolecules: X-ray crystallography, optical, electrochemical, and theoretical analysis.

PubMed

Dass, Amala; Theivendran, Shevanuja; Nimmala, Praneeth Reddy; Kumara, Chanaka; Jupally, Vijay Reddy; Fortunelli, Alessandro; Sementa, Luca; Barcaro, Giovanni; Zuo, Xiaobing; Noll, Bruce C

2015-04-15

Crystal structure determination has revolutionized modern science in biology, chemistry, and physics. However, the difficulty in obtaining periodic crystal lattices which are needed for X-ray crystal analysis has hindered the determination of atomic structure in nanomaterials, known as the "nanostructure problem". Here, by using rigid and bulky ligands, we have overcome this limitation and successfully solved the X-ray crystallographic structure of the largest reported thiolated gold nanomolecule, Au133S52. The total composition, Au133(SPh-tBu)52, was verified using high resolution electrospray ionization mass spectrometry (ESI-MS). The experimental and simulated optical spectra show an emergent surface plasmon resonance that is more pronounced than in the slightly larger Au144(SCH2CH2Ph)60. Theoretical analysis indicates that the presence of rigid and bulky ligands is the key to the successful crystal formation.

Single crystal X-ray structure of the artists' pigment zinc yellow

NASA Astrophysics Data System (ADS)

Simonsen, Kim Pilkjær; Christiansen, Marie Bitsch; Vinum, Morten Gotthold; Sanyova, Jana; Bendix, Jesper

2017-08-01

The artists' pigment zinc yellow is in general described as a complex potassium zinc chromate with the empirical formula 4ZnCrO4·K2O·3H2O. Even though the pigment has been in use since the second half of the 19th century also in large-scale industrial applications, the exact structure had hitherto been unknown. In this work, zinc yellow was synthesised by precipitation from an aqueous solution of zinc nitrate and potassium chromate under both neutral and basic conditions, and the products were compared with the pigment used in industrial paints. Analyses by Raman microscopy (MRS), scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDS), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), and powder X-ray diffraction (PXRD), showed that the synthesised products and the industrial pigment were identical. Single-crystal X-ray crystallography determined the structure of zinc yellow as KZn2(CrO4)2(H2O)(OH) or as KZn2(CrO4)2(H3O2) emphasizing the μ-H3O2- moiety. Notably, the zinc yellow is isostructural to the recently structurally characterized cadmium analog and both belong to the natrochalcite structure type.

Method for improving x-ray diffraction determinations of residual stress in nickel-base alloys

DOEpatents

Berman, R.M.; Cohen, I.

1988-04-26

A process for improving the technique of measuring residual stress by x-ray diffraction in pieces of nickel-base alloys is discussed. Part of a predetermined area of the surface of a nickel-base alloy is covered with a dispersion. This exposes the covered and uncovered portions of the surface of the alloy to x-rays by way of an x-ray diffractometry apparatus, making x-ray diffraction determinations of the exposed surface, and measuring the residual stress in the alloy based on these determinations. The dispersion is opaque to x-rays and serves a dual purpose, since it masks off unsatisfactory signals such that only a small portion of the surface is measured, and it supplies an internal standard by providing diffractogram peaks comparable to the peaks of the nickel alloy so that the alloy peaks can be very accurately located regardless of any sources of error external to the sample. 2 figs.

Simulation of a compact analyzer-based imaging system with a regular x-ray source

NASA Astrophysics Data System (ADS)

Caudevilla, Oriol; Zhou, Wei; Stoupin, Stanislav; Verman, Boris; Brankov, J. G.

2017-03-01

Analyzer-based Imaging (ABI) belongs to a broader family of phase-contrast (PC) X-ray techniques. PC measures X-ray deflection phenomena when interacting with a sample, which is known to provide higher contrast images of soft tissue than other X-ray methods. This is of high interest in the medical field, in particular for mammogram applications. This paper presents a simulation tool for table-top ABI systems using a conventional polychromatic X-ray source.

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).

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

Low- Z polymer sample supports for fixed-target serial femtosecond X-ray crystallography

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

Feld, Geoffrey K.; Heymann, Michael; Benner, W. Henry

X-ray free-electron lasers (XFELs) offer a new avenue to the structural probing of complex materials, including biomolecules. Delivery of precious sample to the XFEL beam is a key consideration, as the sample of interest must be serially replaced after each destructive pulse. The fixed-target approach to sample delivery involves depositing samples on a thin-film support and subsequent serial introduction via a translating stage. Some classes of biological materials, including two-dimensional protein crystals, must be introduced on fixed-target supports, as they require a flat surface to prevent sample wrinkling. A series of wafer and transmission electron microscopy (TEM)-style grid supports constructedmore » of low- Z plastic have been custom-designed and produced. Aluminium TEM grid holders were engineered, capable of delivering up to 20 different conventional or plastic TEM grids using fixed-target stages available at the Linac Coherent Light Source (LCLS). As proof-of-principle, X-ray diffraction has been demonstrated from two-dimensional crystals of bacteriorhodopsin and three-dimensional crystals of anthrax toxin protective antigen mounted on these supports at the LCLS. In conclusion, the benefits and limitations of these low- Z fixed-target supports are discussed; it is the authors' belief that they represent a viable and efficient alternative to previously reported fixed-target supports for conducting diffraction studies with XFELs.« less

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

"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.

High-resolution X-ray crystal structure of bovine H-protein using the high-pressure cryocooling method.

PubMed

Higashiura, Akifumi; Ohta, Kazunori; Masaki, Mika; Sato, Masaru; Inaka, Koji; Tanaka, Hiroaki; Nakagawa, Atsushi

2013-11-01

Recently, many technical improvements in macromolecular X-ray crystallography have increased the number of structures deposited in the Protein Data Bank and improved the resolution limit of protein structures. Almost all high-resolution structures have been determined using a synchrotron radiation source in conjunction with cryocooling techniques, which are required in order to minimize radiation damage. However, optimization of cryoprotectant conditions is a time-consuming and difficult step. To overcome this problem, the high-pressure cryocooling method was developed (Kim et al., 2005) and successfully applied to many protein-structure analyses. In this report, using the high-pressure cryocooling method, the X-ray crystal structure of bovine H-protein was determined at 0.86 Å resolution. Structural comparisons between high- and ambient-pressure cryocooled crystals at ultra-high resolution illustrate the versatility of this technique. This is the first ultra-high-resolution X-ray structure obtained using the high-pressure cryocooling method.

A Practical Approach to Protein Crystallography.

PubMed

Ilari, Andrea; Savino, Carmelinda

2017-01-01

Macromolecular crystallography is a powerful tool for structural biology. The resolution of a protein crystal structure is becoming much easier than in the past, thanks to developments in computing, automation of crystallization techniques and high-flux synchrotron sources to collect diffraction datasets. The aim of this chapter is to provide practical procedures to determine a protein crystal structure, illustrating the new techniques, experimental methods, and software that have made protein crystallography a tool accessible to a larger scientific community.It is impossible to give more than a taste of what the X-ray crystallographic technique entails in one brief chapter and there are different ways to solve a protein structure. Since the number of structures available in the Protein Data Bank (PDB) is becoming ever larger (the protein data bank now contains more than 100,000 entries) and therefore the probability to find a good model to solve the structure is ever increasing, we focus our attention on the Molecular Replacement method. Indeed, whenever applicable, this method allows the resolution of macromolecular structures starting from a single data set and a search model downloaded from the PDB, with the aid only of computer work.

Au133(SPh-tBu)52 Nanomolecules: X-ray Crystallography, Optical, Electrochemical, and Theoretical Analysis

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

Dass, Amala; Theivendran, Shevanuja; Nimmala, Praneeth Reddy

2015-04-15

Crystal structure determination has revolutionized modern science in biology, chemistry, and physics. However, the difficulty in obtaining periodic crystal lattices which are needed for X-ray crystal analysis has hindered the determination of atomic structure in nanomaterials, known as the “nanostructure problem”. Here, by using rigid and bulky ligands, we have overcome this limitation and successfully solved the X-ray crystallographic structure of the largest reported thiolated gold nanomolecule, Au133S52. The total composition, Au133(SPh-tBu)52, was verified using high resolution electrospray ionization mass spectrometry (ESI-MS). The experimental and simulated optical spectra show an emergent surface plasmon resonance that is more pronounced than inmore » the slightly larger Au144(SCH2CH2Ph)60. Theoretical analysis indicates that the presence of rigid and bulky ligands is the key to the successful crystal formation.« less

Au 133 (SPh - t Bu) 52 Nanomolecules: X-ray Crystallography, Optical, Electrochemical, and Theoretical Analysis

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

Dass, Amala; Theivendran, Shevanuja; Nimmala, Praneeth Reddy

2015-04-15

Crystal structure determination has revolutionized modern science in biology, chemistry, and physics. However, the difficulty in obtaining periodic crystal lattices which are needed for X-ray crystal analysis has hindered the determination of atomic structure in nanomaterials, known as the "nanostructure problem". Here, by using rigid and bulky ligands, we have overcome this limitation and successfully solved the X-ray crystallographic structure of the largest reported thiolated gold nanomolecule, Au133S52. The total composition, Au-133(SPh-tBu)(52), was verified using high resolution electrospray ionization mass spectrometry (ESI-MS). The experimental and simulated optical spectra show an emergent surface plasmon resonance that is more pronounced than inmore » the slightly larger Au-144(SCH2CH2Ph)(60). Theoretical analysis indicates that the presence of rigid and bulky ligands is the key to the successful crystal formation.« less

THz-pump and X-ray-probe sources based on an electron linac

NASA Astrophysics Data System (ADS)

Setiniyaz, Sadiq; Park, Seong Hee; Kim, Hyun Woo; Vinokurov, Nikolay A.; Jang, Kyu-Ha; Lee, Kitae; Baek, In Hyung; Jeong, Young Uk

2017-11-01

We describe a compact THz-pump and X-ray-probe beamline, based on an electron linac, for ultrafast time-resolved diffraction applications. Two high-energy electron (γ > 50) bunches, 5 ns apart, impinge upon a single-foil or multifoil radiator and generate THz radiation and X-rays simultaneously. The THz pulse from the first bunch is synchronized to the X-ray beam of the second bunch by using an adjustable optical delay of a THz pulse. The peak power of THz radiation from the multifoil radiator is estimated to be 0.14 GW for a 200 pC well-optimized electron bunch. GEANT4 simulations show that a carbon foil with a thickness of 0.5-1.0 mm has the highest yield of 10-20 keV hard X-rays for a 25 MeV beam, which is approximately 103 photons/(keV pC-electrons) within a few degrees of the polar angle. A carbon multifoil radiator with 35 foils (25 μm thick each) can generate close to 103 hard X-rays/(keV pC-electrons) within a 2° acceptance angle. With 200 pC charge and a 100 Hz repetition rate, we can generate 107 X-rays per 1 keV energy bin per second or 105 X-rays per 1 keV energy bin per pulse. The longitudinal time profile of an X-ray pulse ranges from 400 to 600 fs depending on the acceptance angle. The broadening of the time duration of an X-ray pulse is observed owing to its diverging effect. A double-crystal monochromator will be used to select and transport the desired X-rays to the sample. The heating of the radiators by an electron beam is negligible because of the low beam current.

THz-pump and X-ray-probe sources based on an electron linac.

PubMed

Setiniyaz, Sadiq; Park, Seong Hee; Kim, Hyun Woo; Vinokurov, Nikolay A; Jang, Kyu-Ha; Lee, Kitae; Baek, In Hyung; Jeong, Young Uk

2017-11-01

We describe a compact THz-pump and X-ray-probe beamline, based on an electron linac, for ultrafast time-resolved diffraction applications. Two high-energy electron (γ > 50) bunches, 5 ns apart, impinge upon a single-foil or multifoil radiator and generate THz radiation and X-rays simultaneously. The THz pulse from the first bunch is synchronized to the X-ray beam of the second bunch by using an adjustable optical delay of a THz pulse. The peak power of THz radiation from the multifoil radiator is estimated to be 0.14 GW for a 200 pC well-optimized electron bunch. GEANT4 simulations show that a carbon foil with a thickness of 0.5-1.0 mm has the highest yield of 10-20 keV hard X-rays for a 25 MeV beam, which is approximately 10 3 photons/(keV pC-electrons) within a few degrees of the polar angle. A carbon multifoil radiator with 35 foils (25 μm thick each) can generate close to 10 3 hard X-rays/(keV pC-electrons) within a 2° acceptance angle. With 200 pC charge and a 100 Hz repetition rate, we can generate 10 7 X-rays per 1 keV energy bin per second or 10 5 X-rays per 1 keV energy bin per pulse. The longitudinal time profile of an X-ray pulse ranges from 400 to 600 fs depending on the acceptance angle. The broadening of the time duration of an X-ray pulse is observed owing to its diverging effect. A double-crystal monochromator will be used to select and transport the desired X-rays to the sample. The heating of the radiators by an electron beam is negligible because of the low beam current.

Nanomaterial-based x-ray sources

NASA Astrophysics Data System (ADS)

Cole, Matthew T.; Parmee, R. J.; Milne, William I.

2016-02-01

Following the recent global excitement and investment in the emerging, and rapidly growing, classes of one and two-dimensional nanomaterials, we here present a perspective on one of the viable applications of such materials: field electron emission based x-ray sources. These devices, which have a notable history in medicine, security, industry and research, to date have almost exclusively incorporated thermionic electron sources. Since the middle of the last century, field emission based cathodes were demonstrated, but it is only recently that they have become practicable. We outline some of the technological achievements of the past two decades, and describe a number of the seminal contributions. We explore the foremost market hurdles hindering their roll-out and broader industrial adoption and summarise the recent progress in miniaturised, pulsed and multi-source devices.

Synthesis, X-ray crystallography, spectroscopic (FT-IR, 1H &13C NMR and UV), computational (DFT/B3LYP) and enzymes inhibitory studies of 7-hydroximinocholest-5-en-3-ol acetate

NASA Astrophysics Data System (ADS)

Ahmad, Faheem; Parveen, Mehtab; Alam, Mahboob; Azaz, Shaista; Malla, Ali Mohammed; Alam, Mohammad Jane; Lee, Dong-Ung; Ahmad, Shabbir

2016-07-01

The present study reports the synthesis of 7-Hydroximinocholest-5-en-3-ol acetate (syn. 3β-acetoxycholest-5-en-7-one oxime; in general, steroidal oxime). The identity of steroidal molecule was confirmed by NMR, FT-IR, MS, CHN microanalysis and X-ray crystallography. DFT calculations on the titled molecule have been performed. The molecular structure and spectra interpreted by Gaussian hybrid computational analysis theory (B3LYP) are found to be in good correlation with the experimental data obtained from the various spectrophotometric techniques. The vibrational bands appearing in the FTIR are assigned with great accuracy using harmonic frequencies along with intensities and animated modes. Molecular properties like HOMO-LUMO analysis, chemical reactivity descriptors, MEP mapping, dipole moment and natural atomic charges have been presented at the same level of theory. Moreover, the Hirshfeld analysis was carried out to ascertain the secondary interactions and associated 2D fingerprint plots. The percentages of various interactions are pictorialized by fingerprint plots of Hirshfeld surface. Steroidal oxime exhibited promising inhibitory activity against acetylcholinesterase (AChE) as compared to the reference drug, tacrine. Molecular docking was performed to introduce steroidal molecules into the X-ray crystal structures of acetylcholinesterase at the active site to find out the probable binding mode. The results of molecular docking admitted that steroidal oxime may exhibit enzyme inhibitor activity.

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

X-Rays

MedlinePlus

X-rays are a type of radiation called electromagnetic waves. X-ray imaging creates pictures of the inside of ... different amounts of radiation. Calcium in bones absorbs x-rays the most, so bones look white. Fat ...

Cone Beam X-ray Luminescence Computed Tomography Based on Bayesian Method.

PubMed

Zhang, Guanglei; Liu, Fei; Liu, Jie; Luo, Jianwen; Xie, Yaoqin; Bai, Jing; Xing, Lei

2017-01-01

X-ray luminescence computed tomography (XLCT), which aims to achieve molecular and functional imaging by X-rays, has recently been proposed as a new imaging modality. Combining the principles of X-ray excitation of luminescence-based probes and optical signal detection, XLCT naturally fuses functional and anatomical images and provides complementary information for a wide range of applications in biomedical research. In order to improve the data acquisition efficiency of previously developed narrow-beam XLCT, a cone beam XLCT (CB-XLCT) mode is adopted here to take advantage of the useful geometric features of cone beam excitation. Practically, a major hurdle in using cone beam X-ray for XLCT is that the inverse problem here is seriously ill-conditioned, hindering us to achieve good image quality. In this paper, we propose a novel Bayesian method to tackle the bottleneck in CB-XLCT reconstruction. The method utilizes a local regularization strategy based on Gaussian Markov random field to mitigate the ill-conditioness of CB-XLCT. An alternating optimization scheme is then used to automatically calculate all the unknown hyperparameters while an iterative coordinate descent algorithm is adopted to reconstruct the image with a voxel-based closed-form solution. Results of numerical simulations and mouse experiments show that the self-adaptive Bayesian method significantly improves the CB-XLCT image quality as compared with conventional methods.

Cone Beam X-ray Luminescence Computed Tomography Based on Bayesian Method

PubMed Central

Liu, Fei; Luo, Jianwen; Xie, Yaoqin; Bai, Jing

2017-01-01

X-ray luminescence computed tomography (XLCT), which aims to achieve molecular and functional imaging by X-rays, has recently been proposed as a new imaging modality. Combining the principles of X-ray excitation of luminescence-based probes and optical signal detection, XLCT naturally fuses functional and anatomical images and provides complementary information for a wide range of applications in biomedical research. In order to improve the data acquisition efficiency of previously developed narrow-beam XLCT, a cone beam XLCT (CB-XLCT) mode is adopted here to take advantage of the useful geometric features of cone beam excitation. Practically, a major hurdle in using cone beam X-ray for XLCT is that the inverse problem here is seriously ill-conditioned, hindering us to achieve good image quality. In this paper, we propose a novel Bayesian method to tackle the bottleneck in CB-XLCT reconstruction. The method utilizes a local regularization strategy based on Gaussian Markov random field to mitigate the ill-conditioness of CB-XLCT. An alternating optimization scheme is then used to automatically calculate all the unknown hyperparameters while an iterative coordinate descent algorithm is adopted to reconstruct the image with a voxel-based closed-form solution. Results of numerical simulations and mouse experiments show that the self-adaptive Bayesian method significantly improves the CB-XLCT image quality as compared with conventional methods. PMID:27576245

Solid state structural investigations of the bis(chalcone) compound with single crystal X-ray crystallography, DFT, gamma-ray spectroscopy and chemical spectroscopy methods

NASA Astrophysics Data System (ADS)

Yakalı, Gül; Biçer, Abdullah; Eke, Canel; Cin, Günseli Turgut

2018-04-01

A bis(chalcone), (2E,6E)-2,6-bis((E)-3phenylallidene)cyclohexanone, was characterized by 1H NMR, 13C NMR, FTIR, UV-Vis spectroscopy, gamma-ray spectroscopy and single crystal X- ray structural analysis. The optimized molecular structure of the compound is calculated using DFT/B3LYP with 6-31G (d,p) level. The calculated geometrical parameters are in good agreement with the experimental data obtained from our reported X-ray structure. The powder and single crystal compounds were gama-irradiated using clinical electron linear accelerator and 60Co gamma-ray source, respectively. Spectral studies (1H NMR, 13C NMR, FTIR and UV-Vis) of powder chalcone compound were also investigated before and after irradiation. Depending on the irradiation notable changes were observed in spectral features powder sample. Single crystal X-ray diffraction investigation shows that both unirradiated and irradiated single crystal samples crystallizes in a orthorhombic crystal system in the centrosymmetric space group Pbcn and exhibits an C-H..O intramolecular and intermolecular hydrogen bonds. The crystal packing is stabilised by strong intermolecular bifurcate C-H..O hydrogen bonds and π…π stacking interactions. The asymmetric unit of the title compound contains one-half of a molecule. The other half of the molecule is generated with (1-x,y,-3/2-z) symmetry operator. The molecule is almost planar due to having π conjugated system of chalcones. However, irradiated single crystal compound showed significant changes lattice parameters, crystal volume and density. According to results of gamma-ray spectroscopy, radioactive elements of powder compound which are 123Sb(n,g),124Sb,57Fe(g,p),56Mn, 55Mn(g,n), and 54Mn were determined using photoactivation analysis. However, the most intensive gamma-ray energy signals are 124Sb.

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

PubMed Central

Kang, Yanyong; Zhou, X. Edward; Gao, Xiang; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; Barty, Anton; White, Thomas A.; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W.; Ke, Jiyuan; Eileen Tan, M. H.; Zhang, Chenghai; Moeller, Arne; West, Graham M.; Pascal, Bruce; Van Eps, Ned; Caro, Lydia N.; Vishnivetskiy, Sergey A.; Lee, Regina J.; Suino-Powell, Kelly M.; Gu, Xin; Pal, Kuntal; Ma, Jinming; Zhi, Xiaoyong; Boutet, Sébastien; Williams, Garth J.; Messerschmidt, Marc; Gati, Cornelius; Zatsepin, Nadia A.; Wang, Dingjie; James, Daniel; Basu, Shibom; Roy-Chowdhury, Shatabdi; Conrad, Chelsie; Coe, Jesse; Liu, Haiguang; Lisova, Stella; Kupitz, Christopher; Grotjohann, Ingo; Fromme, Raimund; Jiang, Yi; Tan, Minjia; Yang, Huaiyu; Li, Jun; Wang, Meitian; Zheng, Zhong; Li, Dianfan; Howe, Nicole; Zhao, Yingming; Standfuss, Jörg; Diederichs, Kay; Dong, Yuhui; Potter, Clinton S; Carragher, Bridget; Caffrey, Martin; Jiang, Hualiang; Chapman, Henry N.; Spence, John C. H.; Fromme, Petra; Weierstall, Uwe; Ernst, Oliver P.; Katritch, Vsevolod; Gurevich, Vsevolod V.; Griffin, Patrick R.; Hubbell, Wayne L.; Stevens, Raymond C.; Cherezov, Vadim; Melcher, Karsten; Xu, H. Eric

2015-01-01

G protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signaling to numerous G protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly, in which rhodopsin uses distinct structural elements, including TM7 and Helix 8 to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ~20° rotation between the N- and C- domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signaling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology. PMID:26200343

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

PubMed

Kang, Yanyong; Zhou, X Edward; Gao, Xiang; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; Barty, Anton; White, Thomas A; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W; Ke, Jiyuan; Tan, M H Eileen; Zhang, Chenghai; Moeller, Arne; West, Graham M; Pascal, Bruce D; Van Eps, Ned; Caro, Lydia N; Vishnivetskiy, Sergey A; Lee, Regina J; Suino-Powell, Kelly M; Gu, Xin; Pal, Kuntal; Ma, Jinming; Zhi, Xiaoyong; Boutet, Sébastien; Williams, Garth J; Messerschmidt, Marc; Gati, Cornelius; Zatsepin, Nadia A; Wang, Dingjie; James, Daniel; Basu, Shibom; Roy-Chowdhury, Shatabdi; Conrad, Chelsie E; Coe, Jesse; Liu, Haiguang; Lisova, Stella; Kupitz, Christopher; Grotjohann, Ingo; Fromme, Raimund; Jiang, Yi; Tan, Minjia; Yang, Huaiyu; Li, Jun; Wang, Meitian; Zheng, Zhong; Li, Dianfan; Howe, Nicole; Zhao, Yingming; Standfuss, Jörg; Diederichs, Kay; Dong, Yuhui; Potter, Clinton S; Carragher, Bridget; Caffrey, Martin; Jiang, Hualiang; Chapman, Henry N; Spence, John C H; Fromme, Petra; Weierstall, Uwe; Ernst, Oliver P; Katritch, Vsevolod; Gurevich, Vsevolod V; Griffin, Patrick R; Hubbell, Wayne L; Stevens, Raymond C; Cherezov, Vadim; Melcher, Karsten; Xu, H Eric

2015-07-30

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.

Compact "diode-based" multi-energy soft x-ray diagnostic for NSTX.

PubMed

Tritz, K; Clayton, D J; Stutman, D; Finkenthal, M

2012-10-01

A novel and compact, diode-based, multi-energy soft x-ray (ME-SXR) diagnostic has been developed for the National Spherical Tokamak Experiment. The new edge ME-SXR system tested on NSTX consists of a set of vertically stacked diode arrays, each viewing the plasma tangentially through independent pinholes and filters providing an overlapping view of the plasma midplane which allows simultaneous SXR measurements with coarse sub-sampling of the x-ray spectrum. Using computed x-ray spectral emission data, combinations of filters can provide fast (>10 kHz) measurements of changes in the electron temperature and density profiles providing a method to "fill-in" the gaps of the multi-point Thomson scattering system.

Recent advances in synchrotron-based hard x-ray phase contrast imaging

NASA Astrophysics Data System (ADS)

Liu, Y.; Nelson, J.; Holzner, C.; Andrews, J. C.; Pianetta, P.

2013-12-01

Ever since the first demonstration of phase contrast imaging (PCI) in the 1930s by Frits Zernike, people have realized the significant advantage of phase contrast over conventional absorption-based imaging in terms of sensitivity to ‘transparent’ features within specimens. Thus, x-ray phase contrast imaging (XPCI) holds great potential in studies of soft biological tissues, typically containing low Z elements such as C, H, O and N. Particularly when synchrotron hard x-rays are employed, the favourable brightness, energy tunability, monochromatic characteristics and penetration depth have dramatically enhanced the quality and variety of XPCI methods, which permit detection of the phase shift associated with 3D geometry of relatively large samples in a non-destructive manner. In this paper, we review recent advances in several synchrotron-based hard x-ray XPCI methods. Challenges and key factors in methodological development are discussed, and biological and medical applications are presented.

High-Mass X-ray Binaries in hard X- rays

NASA Astrophysics Data System (ADS)

Lutovinov, Alexander

We present a review of the latest results of the all-sky survey, performed with the INTEGRAL observatory. The deep exposure spent by INTEGRAL in the Galactic plane region, as well as for nearby galaxies allowed us to obtain a flux limited sample for High Mass X-ray Binaries in the Local Galactic Group and measure their physical properties, like a luminosity function, spatial density distribution, etc. Particularly, it was determined the most accurate up to date spatial density distribution of HMXBs in the Galaxy and its correlation with the star formation rate distribution. Based on the measured value of the vertical distribution of HMXBs (a scale-height h~85 pc) we also estimated a kinematical age of HMXBs. Properties of the population of HMXBs are explained in the framework of the population synthesis model. Based on this model we argue that a flaring activity of so-called supergiant fast X-ray transients (SFXTs), the recently recognized sub-sample of HMXBs, is likely related with the magnetic arrest of their accretion. The resulted global characteristics of the HMXB population are used for predictions of sources number counts in sky surveys of future X-ray missions.

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.

NMR crystallography of 2-acylamino-6-[1 H]-pyridones: Solid-state NMR, GIPAW computational, and single crystal X-ray diffraction studies

NASA Astrophysics Data System (ADS)

Ośmiałowski, Borys; Kolehmainen, Erkki; Ikonen, Satu; Ahonen, Kari; Löfman, Miika

2011-12-01

2-Acylamino-6-[1 H]-pyridones [acyl = RCO, where R = methyl ( 1), ethyl ( 2), iso-propyl ( 3), tert-butyl ( 4), and 1-adamantyl ( 5)] have been synthesized and characterized by NMR spectroscopy. From three congeners, 2, 3 and 5, also single crystal X-ray structures have been solved. For these derivatives GIPAW calculations acts as a "bridge" between solid-state NMR data and calculated chemical shifts based on X-ray determined geometry. In crystals all three compounds exist as pyridone tautomers possessing similar six-membered ring structure stabilized by intramolecular C dbnd O⋯HN hydrogen bond. Theoretical GIPAW calculated and experimental 13C and 15N CPMAS NMR shifts are in excellent agreement with each other.

Synthesis, spectroscopy, X-ray crystallography, DFT calculations, DNA binding and molecular docking of a propargyl arms containing Schiff base.

PubMed

Balakrishnan, C; Subha, L; Neelakantan, M A; Mariappan, S S

2015-11-05

A propargyl arms containing Schiff base (L) was synthesized by the condensation of 1-[2-hydroxy-4-(prop-2-yn-1-yloxy)phenyl]ethanone with trans-1,2-diaminocyclohexane. The structure of L was characterized by IR, (1)H NMR, (13)C NMR and UV-Vis spectroscopy and by single crystal X-ray diffraction analysis. The UV-Visible spectral behavior of L in different solvents exhibits positive solvatochromism. Density functional calculation of the L in gas phase was performed by using DFT (B3LYP) method with 6-31G basis set. The computed vibrational frequencies and NMR signals of L were compared with the experimental data. Tautomeric stability study inferred that the enolimine is more stable than the ketoamine form. The charge delocalization has been analyzed using natural bond orbital (NBO) analysis. Electronic absorption and emission spectral studies were used to study the binding of L with CT-DNA. The molecular docking was done to identify the interaction of L with A-DNA and B-DNA. Copyright © 2015 Elsevier B.V. All rights reserved.

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).

Speckle-based at-wavelength metrology of X-ray mirrors with super accuracy

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

Kashyap, Yogesh; Wang, Hongchang; Sawhney, Kawal, E-mail: kawal.sawhney@diamond.ac.uk

2016-05-15

X-ray active mirrors, such as bimorph and mechanically bendable mirrors, are increasingly being used on beamlines at modern synchrotron source facilities to generate either focused or “tophat” beams. As well as optical tests in the metrology lab, it is becoming increasingly important to optimise and characterise active optics under actual beamline operating conditions. Recently developed X-ray speckle-based at-wavelength metrology technique has shown great potential. The technique has been established and further developed at the Diamond Light Source and is increasingly being used to optimise active mirrors. Details of the X-ray speckle-based at-wavelength metrology technique and an example of its applicabilitymore » in characterising and optimising a micro-focusing bimorph X-ray mirror are presented. Importantly, an unprecedented angular sensitivity in the range of two nanoradians for measuring the slope error of an optical surface has been demonstrated. Such a super precision metrology technique will be beneficial to the manufacturers of polished mirrors and also in optimization of beam shaping during experiments.« less

Evolution of Cygnus X-3 through its Radio and X-ray States

NASA Astrophysics Data System (ADS)

Szostek, A.; Zdziarski, A. A.; McCollough, M. L.

2009-05-01

Based on X-ray spectra and studies of the long-term correlated behavior between radio and soft X-ray, we present a detailed evolution of Cyg X-3 through its radio and X-ray states. We comment on the nature of the hard X-ray tail and possible Simbol X contribution in constraining the models.

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.

Predicting X-ray diffuse scattering from translation–libration–screw structural ensembles

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

Van Benschoten, Andrew H.; Afonine, Pavel V.; Terwilliger, Thomas C.

2015-07-28

A method of simulating X-ray diffuse scattering from multi-model PDB files is presented. Despite similar agreement with Bragg data, different translation–libration–screw refinement strategies produce unique diffuse intensity patterns. Identifying the intramolecular motions of proteins and nucleic acids is a major challenge in macromolecular X-ray crystallography. Because Bragg diffraction describes the average positional distribution of crystalline atoms with imperfect precision, the resulting electron density can be compatible with multiple models of motion. Diffuse X-ray scattering can reduce this degeneracy by reporting on correlated atomic displacements. Although recent technological advances are increasing the potential to accurately measure diffuse scattering, computational modeling andmore » validation tools are still needed to quantify the agreement between experimental data and different parameterizations of crystalline disorder. A new tool, phenix.diffuse, addresses this need by employing Guinier’s equation to calculate diffuse scattering from Protein Data Bank (PDB)-formatted structural ensembles. As an example case, phenix.diffuse is applied to translation–libration–screw (TLS) refinement, which models rigid-body displacement for segments of the macromolecule. To enable the calculation of diffuse scattering from TLS-refined structures, phenix.tls-as-xyz builds multi-model PDB files that sample the underlying T, L and S tensors. In the glycerophosphodiesterase GpdQ, alternative TLS-group partitioning and different motional correlations between groups yield markedly dissimilar diffuse scattering maps with distinct implications for molecular mechanism and allostery. These methods demonstrate how, in principle, X-ray diffuse scattering could extend macromolecular structural refinement, validation and analysis.« less

X-ray Binaries and the Galaxy Structure in Hard X-rays

NASA Astrophysics Data System (ADS)

Lutovinov, Alexander

The Galaxy structure in the hard X-ray energy band (¿20 keV) was studied using data of the INTEGRAL observatory. A deep and nearly uniform coverage of the galactic plane allowed to increase significantly the sensitivity of the survey and discover several dozens new galac-tic sources. The follow-up observations with XMM-Newton and CHANDRA observatories in X-rays and ground-based telescopes in optical and infrared wavebands gave us a possibility to determine optical counterparts and distances for number of new and already known faint sources. That, in turn, allowed us to build the spatial distribution of different classes of galactic X-ray binaries and obtain preliminary results of the structure of the further part of the Galaxy.

In meso in situ serial X-ray crystallography of soluble and membrane proteins at cryogenic temperatures

PubMed Central

Huang, Chia-Ying; Olieric, Vincent; Ma, Pikyee; Howe, Nicole; Vogeley, Lutz; Liu, Xiangyu; Warshamanage, Rangana; Weinert, Tobias; Panepucci, Ezequiel; Kobilka, Brian; Diederichs, Kay; Wang, Meitian; Caffrey, Martin

2016-01-01

Here, a method for presenting crystals of soluble and membrane proteins growing in the lipid cubic or sponge phase for in situ diffraction data collection at cryogenic temperatures is introduced. The method dispenses with the need for the technically demanding and inefficient crystal-harvesting step that is an integral part of the lipid cubic phase or in meso method of growing crystals. Crystals are dispersed in a bolus of mesophase sandwiched between thin plastic windows. The bolus contains tens to hundreds of crystals, visible with an in-line microscope at macromolecular crystallography synchrotron beamlines and suitably disposed for conventional or serial crystallographic data collection. Wells containing the crystal-laden boluses are removed individually from hermetically sealed glass plates in which crystallization occurs, affixed to pins on goniometer bases and excess precipitant is removed from around the mesophase. The wells are snap-cooled in liquid nitrogen, stored and shipped in Dewars, and manually or robotically mounted on a goniometer in a cryostream for diffraction data collection at 100 K, as is performed routinely with standard, loop-harvested crystals. The method is a variant on the recently introduced in meso in situ serial crystallography (IMISX) method that enables crystallo­graphic measurements at cryogenic temperatures where crystal lifetimes are enormously enhanced whilst reducing protein consumption dramatically. The new approach has been used to generate high-resolution crystal structures of a G-protein-coupled receptor, α-helical and β-barrel transporters and an enzyme as model integral membrane proteins. Insulin and lysozyme were used as test soluble proteins. The quality of the data that can be generated by this method was attested to by performing sulfur and bromine SAD phasing with two of the test proteins. PMID:26894538

Transforming Our Understanding of the X-ray Universe: The Imaging X-ray Polarimeter Explorer (IXPE)

NASA Technical Reports Server (NTRS)

Weisskopf, Martin C.; Bellazzini, Ronaldo; Costa, Enrico; Matt, Giorgio; Marshall, Herman; ODell, Stephen L.; Pavlov, George; Ramsey, Brian; Romani, Roger

2014-01-01

Accurate X-ray polarimetry can provide unique information on high-energy-astrophysical processes and sources. As there have been no meaningful X-ray polarization measurements of cosmic sources since our pioneering work in the 1970's, the time is ripe to explore this new parameter space in X-ray astronomy. To accomplish this requires a well-calibrated and well understood system that-particularly for an Explorer mission-has technical, cost, and schedule credibility. The system that we shall present satisfies these conditions, being based upon completely calibrated imaging- and polarization-sensitive detectors and proven X-ray-telescope technology.

Temporal characteristic analysis of laser-modulated pulsed X-ray source for space X-ray communication

NASA Astrophysics Data System (ADS)

Hang, Shuang; Liu, Yunpeng; Li, Huan; Tang, Xiaobin; Chen, Da

2018-04-01

X-ray communication (XCOM) is a new communication type and is expected to realize high-speed data transmission in some special communication scenarios, such as deep space communication and blackout communication. This study proposes a high-speed modulated X-ray source scheme based on the laser-to-X-ray conversion. The temporal characteristics of the essential components of the proposed laser-modulated pulsed X-ray source (LMPXS) were analyzed to evaluate its pulse emission performance. Results show that the LMPXS can provide a maximum modulation rate up to 100 Mbps which is expected to significantly improve the data rate of XCOM.

X-ray absorption spectroscopy: EXAFS (Extended X-ray Absorption Fine Structure) and XANES (X-ray Absorption Near Edge Structure)