A readout system for X-ray powder crystallography

NASA Astrophysics Data System (ADS)

Loukas, D.; Haralabidis, N.; Pavlidis, A.; Karvelas, E.; Psycharis ^a, K. Misiakos, V.; Mousa, J.; Dre, Ch.

2000-06-01

A system for capturing and processing data, from radiation detectors, in the field of X-ray crystallography has been developed. The system includes a custom-made mixed analog-digital 16-channel VLSI circuit in 50 μm pitch. Each channel comprises a charge amplifier, a shaper, a comparator and a 21-bit counter. The circuit can be scaled in a daisy chain configuration. Data acquisition is performed with a custom made PCI card while the control software is developed with Visual C++ under the MS Windows NT environment. Performance of a fully operational system, in terms of electronic noise, statistical variations and data capture speed is presented. The noise level permits counting of X-rays down to 8 keV while the counting capability is in excess of 200 kHz. The system is intended for X-ray crystallography with silicon detectors.

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

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

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

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

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

Beyond crystallography: diffractive imaging using coherent x-ray light sources.

PubMed

Miao, Jianwei; Ishikawa, Tetsuya; Robinson, Ian K; Murnane, Margaret M

2015-05-01

X-ray crystallography has been central to the development of many fields of science over the past century. It has now matured to a point that as long as good-quality crystals are available, their atomic structure can be routinely determined in three dimensions. However, many samples in physics, chemistry, materials science, nanoscience, geology, and biology are noncrystalline, and thus their three-dimensional structures are not accessible by traditional x-ray crystallography. Overcoming this hurdle has required the development of new coherent imaging methods to harness new coherent x-ray light sources. Here we review the revolutionary advances that are transforming x-ray sources and imaging in the 21st century. Copyright © 2015, American Association for the Advancement of Science.

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.

A Compact X-Ray System for Support of High Throughput Crystallography

NASA Technical Reports Server (NTRS)

Ciszak, Ewa; Gubarev, Mikhail; Gibson, Walter M.; Joy, Marshall K.; Whitaker, Ann F. (Technical Monitor)

2001-01-01

Standard x-ray systems for crystallography rely on massive generators coupled with optics that guide X-ray beams onto the crystal sample. Optics for single-crystal diffractometry include total reflection mirrors, polycapillary optics or graded multilayer monochromators. The benefit of using polycapillary optic is that it can collect x-rays over tile greatest solid angle, and thus most efficiently, utilize the greatest portion of X-rays emitted from the Source, The x-ray generator has to have a small anode spot, and thus its size and power requirements can be substantially reduced We present the design and results from the first high flux x-ray system for crystallography that combine's a microfocus X-ray generator (40microns FWHM Spot size at a power of 45 W) and a collimating, polycapillary optic. Diffraction data collected from small test crystals with cell dimensions up to 160A (lysozyme and thaumatin) are of high quality. For example, diffraction data collected from a lysozyme crystal at RT yielded R=5.0% for data extending to 1.70A. We compare these results with measurements taken from standard crystallographic systems. Our current microfocus X-ray diffraction system is attractive for supporting crystal growth research in the standard crystallography laboratory as well as in remote, automated crystal growth laboratory. Its small volume, light-weight, and low power requirements are sufficient to have it installed in unique environments, i.e.. on-board International Space Station.

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

Watching proteins function with time-resolved x-ray crystallography

NASA Astrophysics Data System (ADS)

Šrajer, Vukica; Schmidt, Marius

2017-09-01

Macromolecular crystallography was immensely successful in the last two decades. To a large degree this success resulted from use of powerful third generation synchrotron x-ray sources. An expansive database of more than 100 000 protein structures, of which many were determined at resolution better than 2 Å, is available today. With this achievement, the spotlight in structural biology is shifting from determination of static structures to elucidating dynamic aspects of protein function. A powerful tool for addressing these aspects is time-resolved crystallography, where a genuine biological function is triggered in the crystal with a goal of capturing molecules in action and determining protein kinetics and structures of intermediates (Schmidt et al 2005a Methods Mol. Biol. 305 115-54, Schmidt 2008 Ultrashort Laser Pulses in Biology and Medicine (Berlin: Springer) pp 201-41, Neutze and Moffat 2012 Curr. Opin. Struct. Biol. 22 651-9, Šrajer 2014 The Future of Dynamic Structural Science (Berlin: Springer) pp 237-51). In this approach, short and intense x-ray pulses are used to probe intermediates in real time and at room temperature, in an ongoing reaction that is initiated synchronously and rapidly in the crystal. Time-resolved macromolecular crystallography with 100 ps time resolution at synchrotron x-ray sources is in its mature phase today, particularly for studies of reversible, light-initiated reactions. The advent of the new free electron lasers for hard x-rays (XFELs; 5-20 keV), which provide exceptionally intense, femtosecond x-ray pulses, marks a new frontier for time-resolved crystallography. The exploration of ultra-fast events becomes possible in high-resolution structural detail, on sub-picosecond time scales (Tenboer et al 2014 Science 346 1242-6, Barends et al 2015 Science 350 445-50, Pande et al 2016 Science 352 725-9). We review here state-of-the-art time-resolved crystallographic experiments both at synchrotrons and XFELs. We also outline

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

NASA Technical Reports Server (NTRS)

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

1999-01-01

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

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.

Watching proteins function with time-resolved x-ray crystallography

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

Šrajer, Vukica; Schmidt, Marius

Macromolecular crystallography was immensely successful in the last two decades. To a large degree this success resulted from use of powerful third generation synchrotron x-ray sources. An expansive database of more than 100 000 protein structures, of which many were determined at resolution better than 2 Å, is available today. With this achievement, the spotlight in structural biology is shifting from determination of static structures to elucidating dynamic aspects of protein function. A powerful tool for addressing these aspects is time-resolved crystallography, where a genuine biological function is triggered in the crystal with a goal of capturing molecules in actionmore » and determining protein kinetics and structures of intermediates (Schmidt et al 2005a Methods Mol. Biol. 305 115–54, Schmidt 2008 Ultrashort Laser Pulses in Biology and Medicine (Berlin: Springer) pp 201–41, Neutze and Moffat 2012 Curr. Opin. Struct. Biol. 22 651–9, Šrajer 2014 The Future of Dynamic Structural Science (Berlin: Springer) pp 237–51). In this approach, short and intense x-ray pulses are used to probe intermediates in real time and at room temperature, in an ongoing reaction that is initiated synchronously and rapidly in the crystal. Time-resolved macromolecular crystallography with 100 ps time resolution at synchrotron x-ray sources is in its mature phase today, particularly for studies of reversible, light-initiated reactions. The advent of the new free electron lasers for hard x-rays (XFELs; 5–20 keV), which provide exceptionally intense, femtosecond x-ray pulses, marks a new frontier for time-resolved crystallography. The exploration of ultra-fast events becomes possible in high-resolution structural detail, on sub-picosecond time scales (Tenboer et al 2014 Science 346 1242–6, Barends et al 2015 Science 350 445–50, Pande et al 2016 Science 352 725–9). We review here state-of-the-art time-resolved crystallographic experiments both at synchrotrons and XFELs

Long-Wavelength X-Ray Diffraction and Its Applications in Macromolecular Crystallography.

PubMed

Weiss, Manfred S

2017-01-01

For many years, diffraction experiments in macromolecular crystallography at X-ray wavelengths longer than that of Cu-K α (1.54 Å) have been largely underappreciated. Effects caused by increased X-ray absorption result in the fact that these experiments are more difficult than the standard diffraction experiments at short wavelengths. However, due to the also increased anomalous scattering of many biologically relevant atoms, important additional structural information can be obtained. This information, in turn, can be used for phase determination, for substructure identification, in molecular replacement approaches, as well as in structure refinement. This chapter reviews the possibilities and the difficulties associated with such experiments, and it provides a short description of two macromolecular crystallography synchrotron beam lines dedicated to long-wavelength X-ray diffraction experiments.

Fine refinement of solid state structure of racemic form of phospho-tyrosine employing NMR Crystallography approach.

PubMed

Paluch, Piotr; Pawlak, Tomasz; Oszajca, Marcin; Lasocha, Wieslaw; Potrzebowski, Marek J

2015-02-01

We present step by step facets important in NMR Crystallography strategy employing O-phospho-dl-tyrosine as model sample. The significance of three major techniques being components of this approach: solid state NMR (SS NMR), X-ray diffraction of powdered sample (PXRD) and theoretical calculations (Gauge Invariant Projector Augmented Wave; GIPAW) is discussed. Each experimental technique provides different set of structural constraints. From the PXRD measurement the size of the unit cell, space group and roughly refined molecular structure are established. SS NMR provides information about content of crystallographic asymmetric unit, local geometry, molecular motion in the crystal lattice and hydrogen bonding pattern. GIPAW calculations are employed for validation of quality of elucidation and fine refinement of structure. Crystal and molecular structure of O-phospho-dl-tyrosine solved by NMR Crystallography is deposited at Cambridge Crystallographic Data Center under number CCDC 1005924. Copyright © 2014 Elsevier Inc. All rights reserved.

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

PubMed

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

2015-01-01

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

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

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

PubMed Central

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

2017-01-01

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

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

PubMed Central

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

2015-01-01

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

NMR and X-ray structural characterization and conformational aspects of fluorinated (5Z)-3-benzil-5-arylidenofuran-2(5H)-ones

NASA Astrophysics Data System (ADS)

Teixeira, R. R.; Barbosa, L. C. A.; Kabeshov, M. A.; Maltha, C. R. A.; Corrêa, R. S.; Doriguetto, A. C.

2014-10-01

Herein we describe structural insights of (5Z)-3-benzyl-5-(2-fluorobenzylidene)furan-2(5H)-one (6) and (5Z)-3-benzyl-5-(pentafluorobenzylidene)furan-2(5H)-one (7), γ-alkylidenebutenolides analogues of the natural products nostoclides. Their structures were investigated by NMR spectroscopy and X-ray crystallography. The stereochemistry of the exocyclic double bond of these fluorinated compounds was determined to be Z by NMR analysis and confirmed by X-ray data. Compounds 6 and 7 crystallized in the monoclinic crystal system P21/c group. A comparison between structural features of (6) and (7) and nostoclide derivatives previously published by us is described.

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.

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.

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.

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

NMR Crystallography of a Carbanionic Intermediate in Tryptophan Synthase: Chemical Structure, Tautomerization, and Reaction Specificity.

PubMed

Caulkins, Bethany G; Young, Robert P; Kudla, Ryan A; Yang, Chen; Bittbauer, Thomas J; Bastin, Baback; Hilario, Eduardo; Fan, Li; Marsella, Michael J; Dunn, Michael F; Mueller, Leonard J

2016-11-23

Carbanionic intermediates play a central role in the catalytic transformations of amino acids performed by pyridoxal-5'-phosphate (PLP)-dependent enzymes. Here, we make use of NMR crystallography-the synergistic combination of solid-state nuclear magnetic resonance, X-ray crystallography, and computational chemistry-to interrogate a carbanionic/quinonoid intermediate analogue in the β-subunit active site of the PLP-requiring enzyme tryptophan synthase. The solid-state NMR chemical shifts of the PLP pyridine ring nitrogen and additional sites, coupled with first-principles computational models, allow a detailed model of protonation states for ionizable groups on the cofactor, substrates, and nearby catalytic residues to be established. Most significantly, we find that a deprotonated pyridine nitrogen on PLP precludes formation of a true quinonoid species and that there is an equilibrium between the phenolic and protonated Schiff base tautomeric forms of this intermediate. Natural bond orbital analysis indicates that the latter builds up negative charge at the substrate C α and positive charge at C4' of the cofactor, consistent with its role as the catalytic tautomer. These findings support the hypothesis that the specificity for β-elimination/replacement versus transamination is dictated in part by the protonation states of ionizable groups on PLP and the reacting substrates and underscore the essential role that NMR crystallography can play in characterizing both chemical structure and dynamics within functioning enzyme active sites.

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.

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.

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.

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.

A convolutional neural network-based screening tool for X-ray serial crystallography

PubMed Central

Ke, Tsung-Wei; Brewster, Aaron S.; Yu, Stella X.; Ushizima, Daniela; Yang, Chao; Sauter, Nicholas K.

2018-01-01

A new tool is introduced for screening macromolecular X-ray crystallography diffraction images produced at an X-ray free-electron laser light source. Based on a data-driven deep learning approach, the proposed tool executes a convolutional neural network to detect Bragg spots. Automatic image processing algorithms described can enable the classification of large data sets, acquired under realistic conditions consisting of noisy data with experimental artifacts. Outcomes are compared for different data regimes, including samples from multiple instruments and differing amounts of training data for neural network optimization. PMID:29714177

A convolutional neural network-based screening tool for X-ray serial crystallography.

PubMed

Ke, Tsung Wei; Brewster, Aaron S; Yu, Stella X; Ushizima, Daniela; Yang, Chao; Sauter, Nicholas K

2018-05-01

A new tool is introduced for screening macromolecular X-ray crystallography diffraction images produced at an X-ray free-electron laser light source. Based on a data-driven deep learning approach, the proposed tool executes a convolutional neural network to detect Bragg spots. Automatic image processing algorithms described can enable the classification of large data sets, acquired under realistic conditions consisting of noisy data with experimental artifacts. Outcomes are compared for different data regimes, including samples from multiple instruments and differing amounts of training data for neural network optimization. open access.

A convolutional neural network-based screening tool for X-ray serial crystallography

DOE PAGES

Ke, Tsung-Wei; Brewster, Aaron S.; Yu, Stella X.; ...

2018-04-24

A new tool is introduced for screening macromolecular X-ray crystallography diffraction images produced at an X-ray free-electron laser light source. Based on a data-driven deep learning approach, the proposed tool executes a convolutional neural network to detect Bragg spots. Automatic image processing algorithms described can enable the classification of large data sets, acquired under realistic conditions consisting of noisy data with experimental artifacts. Outcomes are compared for different data regimes, including samples from multiple instruments and differing amounts of training data for neural network optimization.

A convolutional neural network-based screening tool for X-ray serial crystallography

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

Ke, Tsung-Wei; Brewster, Aaron S.; Yu, Stella X.

A new tool is introduced for screening macromolecular X-ray crystallography diffraction images produced at an X-ray free-electron laser light source. Based on a data-driven deep learning approach, the proposed tool executes a convolutional neural network to detect Bragg spots. Automatic image processing algorithms described can enable the classification of large data sets, acquired under realistic conditions consisting of noisy data with experimental artifacts. Outcomes are compared for different data regimes, including samples from multiple instruments and differing amounts of training data for neural network optimization.

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

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.

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

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.

Synthesis, spectroscopic investigations (X-ray, NMR and TD-DFT), antimicrobial activity and molecular docking of 2,6-bis(hydroxy(phenyl)methyl)cyclohexanone.

PubMed

Barakat, Assem; Ghabbour, Hazem A; Al-Majid, Abdullah Mohammed; Soliman, Saied M; Ali, M; Mabkhot, Yahia Nasser; Shaik, Mohammed Rafi; Fun, Hoong-Kun

2015-07-21

The synthesis of 2,6-bis(hydroxy(phenyl)methyl)cyclohexanone 1 is described. The molecular structure of the title compound 1 was confirmed by NMR, FT-IR, MS, CHN microanalysis, and X-ray crystallography. The molecular structure was also investigated by a set of computational studies and found to be in good agreement with the experimental data obtained from the various spectrophotometric techniques. The antimicrobial activity and molecular docking of the synthesized compound was investigated.

An NMR crystallography study of the hemihydrate of 2', 3'-O-isopropylidineguanosine.

PubMed

Reddy, G N Manjunatha; Cook, Daniel S; Iuga, Dinu; Walton, Richard I; Marsh, Andrew; Brown, Steven P

2015-02-01

An NMR crystallography study of the hemihydrate of 2', 3'-O-isopropylidineguanosine (Gace) is presented, together with powder X-ray diffraction and thermogravimetric analysis. (1)H double-quantum and (14)N-(1)H HMQC spectra recorded at 850MHz and 75kHz MAS (using a JEOL 1mm probe) are presented together with a (1)H-(13)C refocused INEPT spectrum recorded at 500MHz and 12.5kHz MAS using eDUMBO-122(1)H homonuclear decoupling. NMR chemical shieldings are calculated using the GIPAW (gauge-including projector augmented wave) method; good two-dimensional agreement between calculation and experiment is observed for (13)C and (1)H chemical shifts for directly bonded CH and CH3 peaks. There are two Gace molecules in the asymmetric unit cell: differences in specific (1)H chemical shifts are rationalised in terms of the strength of CH-π and intermolecular hydrogen bonding interactions. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

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.

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

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

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.

NMR Crystallography of a Carbanionic Intermediate in Tryptophan Synthase: Chemical Structure, Tautomerization, and Reaction Specificity

PubMed Central

2016-01-01

Carbanionic intermediates play a central role in the catalytic transformations of amino acids performed by pyridoxal-5′-phosphate (PLP)-dependent enzymes. Here, we make use of NMR crystallography—the synergistic combination of solid-state nuclear magnetic resonance, X-ray crystallography, and computational chemistry—to interrogate a carbanionic/quinonoid intermediate analogue in the β-subunit active site of the PLP-requiring enzyme tryptophan synthase. The solid-state NMR chemical shifts of the PLP pyridine ring nitrogen and additional sites, coupled with first-principles computational models, allow a detailed model of protonation states for ionizable groups on the cofactor, substrates, and nearby catalytic residues to be established. Most significantly, we find that a deprotonated pyridine nitrogen on PLP precludes formation of a true quinonoid species and that there is an equilibrium between the phenolic and protonated Schiff base tautomeric forms of this intermediate. Natural bond orbital analysis indicates that the latter builds up negative charge at the substrate Cα and positive charge at C4′ of the cofactor, consistent with its role as the catalytic tautomer. These findings support the hypothesis that the specificity for β-elimination/replacement versus transamination is dictated in part by the protonation states of ionizable groups on PLP and the reacting substrates and underscore the essential role that NMR crystallography can play in characterizing both chemical structure and dynamics within functioning enzyme active sites. PMID:27779384

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.

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.

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

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.

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.

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.

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.

Contrasting coordination behavior of Group 12 perchlorate salts with an acyclic N3O2 donor ligand by X-ray crystallography and (1)H NMR.

PubMed

Tice, Daniel B; Pike, Robert D; Bebout, Deborah C

2016-08-09

An unbranched N3O2 ligand 2,6-bis[((2-pyridinylmethyl)oxy)methyl]pyridine (L1) was used to prepare new mononuclear heteroleptic Group 12 perchlorate complexes characterized by IR, (1)H NMR and X-ray crystallography. Racemic complexes with pentadentate L1 and one to four oxygens from either water or perchlorate bound to a metal ion were structurally characterized. Octahedral [Zn(L1)(OH2)](ClO4)2 (1) and pentagonal bipyramidal [Cd(L1)(OH2)(OClO3)]ClO4 (2) structures were found with lighter congeners. The polymorphic forms of [Hg(L1)(ClO4)2] characterized (3 in P1[combining macron] and 4 in P21/c) had a mix of monodentate, anisobidentate and bidentate perchlorates, providing the first examples of a tricapped trigonal prismatic Hg(ii) coordination geometry, as well as additional examples of a rare square antiprismatic Hg(ii) coordination geometry. Solution state (1)H NMR characterization of the Group 12 complexes in CD3CN indicated intramolecular reorganization remained rapid under conditions where intermolecular M-L1 exchange was slow on the chemical shift time scale for Zn(ii) and on the J(M(1)H) time scale for Cd(ii) and Hg(ii). Solution studies with more than one equivalent of ligand also suggested that a complex with a 1 : 2 ratio of M : L1 contributed significantly to solution equilibria with Hg(ii) but not the other metal ions. The behavior of related linear pentadentate ligands with Group 12 perchlorate salts is discussed.

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.

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.

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

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.

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.

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.

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.

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.

Fine refinement of solid-state molecular structures of Leu- and Met-enkephalins by NMR crystallography.

PubMed

Pawlak, Tomasz; Potrzebowski, Marek J

2014-03-27

This paper presents a methodology that allows the fine refinement of the crystal and molecular structure for compounds for which the data deposited in the crystallographic bases are of poor quality. Such species belong to the group of samples with molecular disorder. In the Cambridge Crystallographic Data Center (CCDC), there are approximately 22,000 deposited structures with an R-factor over 10. The powerful methodology we present employs crystal data for Leu-enkephalin (two crystallographic forms) with R-factor values of 14.0 and 8.9 and for Met-enkephalin (one form) with an R-factor of 10.5. NMR crystallography was employed in testing the X-ray data and the quality of the structure refinement. The GIPAW (gauge invariant projector augmented wave) method was used to optimize the coordinates of the enkephalins and to compute NMR parameters. As we reveal, this complementary approach makes it possible to generate a reasonable set of new coordinates that better correlate to real samples. This methodology is general and can be employed in the study of each compound possessing magnetically active nuclei.

NMR crystallography of zeolites: How far can we go without diffraction data?

PubMed

Brouwer, Darren H; Van Huizen, Jared

2018-05-10

Nuclear magnetic resonance (NMR) crystallography-an approach to structure determination that seeks to integrate solid-state NMR spectroscopy, diffraction, and computation methods-has emerged as an effective strategy to determine structures of difficult-to-characterize materials, including zeolites and related network materials. This paper explores how far it is possible to go in determining the structure of a zeolite framework from a minimal amount of input information derived only from solid-state NMR spectroscopy. It is shown that the framework structure of the fluoride-containing and tetramethylammonium-templated octadecasil clathrasil material can be solved from the 1D 29 Si NMR spectrum and a single 2D 29 Si NMR correlation spectrum alone, without the space group and unit cell parameters normally obtained from diffraction data. The resulting NMR-solved structure is in excellent agreement with the structures determined previously by diffraction methods. It is anticipated that NMR crystallography strategies like this will be useful for structure determination of other materials, which cannot be solved from diffraction methods alone. Copyright © 2018 John Wiley & Sons, Ltd.

Synergic Investigation Of The Self-Assembly Structure And Mechanism Of Retroviral Capsid Proteins By Solid State NMR, Transmission Electron Microscopy And Multiscale simulation

DTIC Science & Technology

2017-03-29

310 helix. Green: this work. Cyans: solution NMR RSV CA structure in PDB entry 1D1D.[18] Magentas: X-ray crystallography structure of flat hexameric...to combine cryo-electron microscopy and X-ray crystallography , Methods, 49 (2009) 174-180. [8] K.Y. Chan, J. Gumbart, R. McGreevy, J.M. Watermeyer

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.

Molecular structure of actein: 13C CPMAS NMR, IR, X-ray diffraction studies and theoretical DFT-GIAO calculations

NASA Astrophysics Data System (ADS)

Jamróz, Marta K.; Bąk, Joanna; Gliński, Jan A.; Koczorowska, Agnieszka; Wawer, Iwona

2009-09-01

Actein is a prominent triterpene glycoside occurring in Actaea racemosa. The triterpene glycosides are believed to be responsible for the estrogenic activity of an extract prepared from this herb. We determined in the crystal structure of actein by X-ray crystallography to be monoclinic P2(1) chiral space group. Refining the disorder, we determined 70% and 30% of contributions of ( S)- and ( R)-actein, respectively. The IR and Raman spectra suggest that actein forms at least four different types of hydrogen bonds. The 13C NMR spectra of actein were recorded both in solution and solid state. The 13C CPMAS spectrum of actein displays multiplet signals, in agreement with the crystallographic data. The NMR shielding constants were calculated for actein using GIAO approach and a variety of basis sets: 6-31G**, 6-311G**, 6-31+G**, cc-pVDZ, cc-pVDZ-su1 and 6-31G**-su1, as well as IGLO approach combined with the IGLO II basis set. The best results (RMSD of 1.6 ppm and maximum error of 3.4 ppm) were obtained with the 6-31G**-su1 basis set. The calculations of the shielding constants are helpful in the interpretation of the 13C CPMAS NMR spectra of actein and actein's analogues.

Sterically Hindered Chiral Organometallic Complexes: AN X-Ray Crystallographic, NMR Spectroscopic and Ehmo Study.

NASA Astrophysics Data System (ADS)

Malisza, Krisztina Laura

results in elimination of water to yield 2-methylcyclopentene derivatives of which the dimolybdenum cluster has been characterized by x-ray crystallography. The sterically crowded complexes rm(C _6H_5)_3SiOH[ Cr(CO)_3 ]_{n}, where n = 1,2,3, have also been characterized X-ray crystallographically and all three have propellor-type geometries. The question of whether the phenyl ring rotations are correlated has been studied by variable-temperature NMR spectroscopy. These systems do not yield stable silicenium cations, but the analogous Cr(CO)_3 complex of triphenylcarbinol not only shows fluxional behaviour but also yields a metal -stabilized cation. The pathway for phenyl rotations in such systems can be followed via a Dunitz-type trajectory approach in which a number of x-ray structures were analyzed.

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.

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.

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.

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

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.

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

PubMed

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

2017-05-10

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

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

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

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.

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.

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.

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.…

NMR crystallography: structure and properties of materials from solid-state nuclear magnetic resonance observables

PubMed Central

Bryce, David L.

2017-01-01

This topical review provides a brief overview of recent developments in NMR crystallography and related NMR approaches to studying the properties of molecular and ionic solids. Areas of complementarity with diffraction-based methods are underscored. These include the study of disordered systems, of dynamic systems, and other selected examples where NMR can provide unique insights. Highlights from the literature as well as recent work from my own group are discussed. PMID:28875022

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.

NMR contributions to structural dynamics studies of intrinsically disordered proteins☆

PubMed Central

Konrat, Robert

2014-01-01

Intrinsically disordered proteins (IDPs) are characterized by substantial conformational plasticity. Given their inherent structural flexibility X-ray crystallography is not applicable to study these proteins. In contrast, NMR spectroscopy offers unique opportunities for structural and dynamic studies of IDPs. The past two decades have witnessed significant development of NMR spectroscopy that couples advances in spin physics and chemistry with a broad range of applications. This article will summarize key advances in basic physical-chemistry and NMR methodology, outline their limitations and envision future R&D directions. PMID:24656082

Structural studies of homoisoflavonoids: NMR spectroscopy, X-ray diffraction, and theoretical calculations

NASA Astrophysics Data System (ADS)

Sievänen, Elina; Toušek, Jaromír; Lunerová, Kamila; Marek, Jaromír; Jankovská, Dagmar; Dvorská, Margita; Marek, Radek

2010-08-01

In this article we present a detailed structural investigation for five homoisoflavonoids, molecules important from the pharmacological point of view. For studying the electron distribution as well as its influence on the physicochemical properties, NMR spectroscopy, X-ray diffraction, and theoretical calculations have been used. Nuclear magnetic shieldings obtained by using DFT calculations for optimized molecular geometries are correlated with the experimentally determined chemical shifts. The theoretical data are well in agreement with the experimental values. The single crystal X-ray structures of homoisoflavonoid derivatives 1, 3, and 4 have been solved. The molecular geometries and crystal packing determined by X-ray diffraction are used for characterizing the intermolecular interactions. Electron distribution is crucial for the stability of radicals and hence the antioxidant efficiency of flavonoid structures. The hydrogen bonding governs the formation of complexes of homoisoflavonoids with biological targets.

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

Structural features of cytochromes P450 and ligands that affect drug metabolism as revealed by X-ray crystallography and NMR.

PubMed

Gay, Sean C; Roberts, Arthur G; Halpert, James R

2010-09-01

Cytochromes P450 (P450s) play a major role in the clearance of drugs, toxins, and environmental pollutants. Additionally, metabolism by P450s can result in toxic or carcinogenic products. The metabolism of pharmaceuticals by P450s is a major concern during the design of new drug candidates. Determining the interactions between P450s and compounds of very diverse structures is complicated by the variability in P450-ligand interactions. Understanding the protein structural elements and the chemical attributes of ligands that dictate their orientation in the P450 active site will aid in the development of effective and safe therapeutic agents. The goal of this review is to describe P450-ligand interactions from two perspectives. The first is the various structural elements that microsomal P450s have at their disposal to assume the different conformations observed in X-ray crystal structures. The second is P450-ligand dynamics analyzed by NMR relaxation studies.

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.

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

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

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.

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.

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.

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

Magic Angle Spinning NMR of Viruses

PubMed Central

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

2015-01-01

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

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

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

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.

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

Simultaneous use of solution NMR and X-ray data in REFMAC5 for joint refinement/detection of structural differences.

PubMed

Rinaldelli, Mauro; Ravera, Enrico; Calderone, Vito; Parigi, Giacomo; Murshudov, Garib N; Luchinat, Claudio

2014-04-01

The program REFMAC5 from CCP4 was modified to allow the simultaneous use of X-ray crystallographic data and paramagnetic NMR data (pseudocontact shifts and self-orientation residual dipolar couplings) and/or diamagnetic residual dipolar couplings. Incorporation of these long-range NMR restraints in REFMAC5 can reveal differences between solid-state and solution conformations of molecules or, in their absence, can be used together with X-ray crystallographic data for structural refinement. Since NMR and X-ray data are complementary, when a single structure is consistent with both sets of data and still maintains reasonably `ideal' geometries, the reliability of the derived atomic model is expected to increase. The program was tested on five different proteins: the catalytic domain of matrix metalloproteinase 1, GB3, ubiquitin, free calmodulin and calmodulin complexed with a peptide. In some cases the joint refinement produced a single model consistent with both sets of observations, while in other cases it indicated, outside the experimental uncertainty, the presence of different protein conformations in solution and in the solid state.

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

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

Membrane lateral compressibility determined by NMR and x-ray diffraction: effect of acyl chain polyunsaturation.

PubMed Central

Koenig, B W; Strey, H H; Gawrisch, K

1997-01-01

The elastic area compressibility modulus, Ka, of lamellar liquid crystalline bilayers was determined by a new experimental approach using 2H-NMR order parameters of lipid hydrocarbon chains together with lamellar repeat spacings measured by x-ray diffraction. The combination of NMR and x-ray techniques yields accurate determination of lateral area per lipid molecule. Samples of saturated, monounsaturated, and polyunsaturated phospholipids were equilibrated with polyethylene glycol (PEG) 20,000 solutions in water at concentrations from 0 to 55 wt % PEG at 30 degrees C. This procedure is equivalent to applying 0 to 8 dyn/cm lateral pressure to the bilayers. The resulting reductions in area per lipid were measured with a resolution of +/-0.2 A2 and the fractional area decrease was proportional to applied lateral pressure. For 1,2-dimyristoyl(d54)-sn-glycero-3-phosphocholine, 1-stearoyl(d35)-2-oleoyl-sn-glycero-3-phosphocholine (SOPC-d35), and 1-stearoyl(d35)-2-docosahexaenoyl-sn-glycero-3-phosphocholine (SDPC-d35) cross-sectional areas per molecule in excess water of 59.5, 61.4, and 69.2 A2 and bilayer elastic area compressibility moduli of 141, 221, and 121 dyn/cm were determined, respectively. Combining NMR and x-ray results enables the determination of compressibility differences between saturated and unsaturated hydrocarbon chains. In mixed-chain SOPC-d35 both chains have similar compressibility moduli; however, in mixed-chain polyunsaturated SDPC-d35, the saturated stearic acid chain appears to be far less compressible than the polyunsaturated docosahexaenoic acid chain. Images FIGURE 3 FIGURE 5 PMID:9336191

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

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

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

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.

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

DTIC Science & Technology

2017-08-01

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

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.

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.

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.

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.

Structure of indazole N1-oxide derivatives studied by X-ray, theoretical methods, 1H, 13C, 15N NMR and EI/MS

NASA Astrophysics Data System (ADS)

Gerpe, Alejandra; Piro, Oscar E.; Cerecetto, Hugo; González, Mercedes

2007-12-01

A series of indazole N1-oxide derivatives has been spectroscopically studied in solution using 1H, 13C, and 15N NMR based on pulsed field gradient selected PFG 1H sbnd X (X = 13C and 15N) gHMQC and gHMBC experiments. Some indazoles were prepared using a new methodology to compare its spectral and structural data with the indazole N1-oxide parent compounds. The 13C resonances of the indazole N1-oxide carbon 3 and 7a demonstrate the N-oxide push-electron capability. The 15N resonances of the indazole N-oxide, nitrogen 1, are near to 30 ppm more shielded than the corresponding values in the indazole heterocycle (deoxygenated form). Moreover, the structures of one indazole and one indazole N-oxide were unambiguously confirmed by X-ray crystallography. The solid state structures were contrasted with the theoretical ones obtained in vacuo at different calculus level. The aromaticity of the derivatives was studied analyzing the H sbnd H coupling constants of indazole's aromatic hydrogens and measuring C sbnd C distances in the solid state. The fragmentation that takes place in EI/MS was gathered for all the indazole N-oxide derivatives and the general fragmentation pattern analyzed.

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.

Synthesis, high-resolution NMR spectroscopic analysis, and single-crystal X-ray diffraction of isoxazoline tetracycles.

PubMed

Fascio, Mirta L; Alvarez-Larena, Angel; D'Accorso, Norma B

2002-11-29

Three isoxazoline tetracycles were obtained enantiomerically pure by intramolecular 1,3-dipolar cycloaddition. The characterization of the new compounds was performed by high-resolution 1H and 13C NMR spectroscopy. The relative configuration of the new chiral centers was determined by NOESY experiments and confirmed by single-crystal X-ray structural analysis.

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.

Structure of dimethylphenyl betaine hydrochloride studied by X-ray diffraction, DFT calculation, NMR and FTIR spectra

NASA Astrophysics Data System (ADS)

Szafran, M.; Katrusiak, A.; Dega-Szafran, Z.; Kowalczyk, I.

2013-01-01

The structure of dimethylphenyl betaine hydrochloride (1) has been studied by X-ray diffraction, DFT calculations, NMR and FTIR spectra. The crystals are monoclinic, space group P21/c. In the crystal, the Cl- anion is connected with protonated betaine through the O-H⋯Cl- hydrogen bond of 2.943(2) Å. The structures in the gas phase (2) and water solution (3) have been optimized by the B3LYP/6-311++G(d,p) approach and the geometrical results have been compared with the X-ray data of 1. The FTIR spectrum of the solid compound is consistent with the X-ray results. The probable assignments of the anharmonic experimental vibrational frequencies of the investigated chloride (1) based on the calculated harmonic frequencies in water solution (3) are proposed. The correlations between the experimental 1H and 13C NMR chemical shifts (δexp) of 1 in D2O and the magnetic isotropic shielding constants (σcalc) calculated by the GIAO/B3LYP/6-311G++(d,p) approach, using the screening solvation model (COSMO), δexp = a + b σcalc, for optimized molecule 3 in water solution are linear and correctly reproduce the experimental chemical shifts.

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.

Chalcogen analogues of nicotine lactam studied by NMR, FTIR, DFT and X-ray methods

NASA Astrophysics Data System (ADS)

Jasiewicz, Beata; Malczewska-Jaskóła, Karolina; Kowalczyk, Iwona; Warżajtis, Beata; Rychlewska, Urszula

2014-07-01

The selenoanalogue of nicotine has been synthesized and characterized by spectroscopic and X-ray diffraction methods. The crystals of selenonicotine are isomorphic with the thionicotine homologue and consist of molecules engaged in columnar π⋯π stacking interactions between antiparallely arranged pyridine moieties. These interactions, absent in other crystals containing nicotine fragments, seem to be induced by the presence of a lactam group. The molecular structures in the vacuum of the oxo-, thio- and selenonicotine homologues have been calculated by the DFT method and compared with the available X-ray data. The delocalized structure of thionicotine is stabilized by intramolecular Csbnd H⋯S hydrogen bond, which becomes weaker in the partial zwitterionic resonance structure of selenonicotine in favor of multiple Csbnd H⋯Se intermolecular hydrogen-bonds. The calculated data allow a complete assignment of vibration modes in the solid state FTIR spectra. The 1H and 13C NMR chemical shifts were calculated by the GIAO method with B3LYP/6-311G(3df) level. A comparison between experimental and calculated theoretical results indicates that the density functional B3LYP method provided satisfactory results for predicting FTIR, 1H, 13C NMR spectra properties.

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

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.

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

Deformation and Failure of Protein Materials in Physiologically Extreme Conditions and Disease

DTIC Science & Technology

2009-03-01

resonance (NMR) spectroscopy and X- ray crystallography have advanced our ability to identify 3D protein structures57. Site-specific studies using NMR, a... ray crystallography, providing structural and temporal information about mechanisms of deformation and assembly (for example in intermediate...tens of thousands of 3D atomistic protein structures, identifying the structure of numerous proteins from varying species sources60. X- ray

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

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

Automation of NMR structure determination of proteins.

PubMed

Altieri, Amanda S; Byrd, R Andrew

2004-10-01

The automation of protein structure determination using NMR is coming of age. The tedious processes of resonance assignment, followed by assignment of NOE (nuclear Overhauser enhancement) interactions (now intertwined with structure calculation), assembly of input files for structure calculation, intermediate analyses of incorrect assignments and bad input data, and finally structure validation are all being automated with sophisticated software tools. The robustness of the different approaches continues to deal with problems of completeness and uniqueness; nevertheless, the future is very bright for automation of NMR structure generation to approach the levels found in X-ray crystallography. Currently, near completely automated structure determination is possible for small proteins, and the prospect for medium-sized and large proteins is good. Copyright 2004 Elsevier Ltd.

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.

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

Quantum-chemical, NMR and X-ray diffraction studies on (+/-)-1-[3,4-(methylenedioxy)phenyl]-2-methylaminopropane.

PubMed

Zapata-Torres, Gerald; Cassels, Bruce K; Parra-Mouchet, Julia; Mascarenhas, Yvonne P; Ellena, Javier; De Araujo, A S

2008-06-01

Time-averaged conformations of (+/-)-1-[3,4-(methylenedioxy)phenyl]-2-methylaminopropane hydrochloride (MDMA, "ecstasy") in D(2)O, and of its free base and trifluoroacetate in CDCl(3), were deduced from their (1)H NMR spectra and used to calculate their conformer distribution. Their rotational potential energy surface (PES) was calculated at the RHF/6-31G(d,p), B3LYP/6-31G(d,p), B3LYP/cc-pVDZ and AM1 levels. Solvent effects were evaluated using the polarizable continuum model. The NMR and theoretical studies showed that, in the free base, the N-methyl group and the ring are preferentially trans. This preference is stronger in the salts and corresponds to the X-ray structure of the hydrochloride. However, the energy barriers separating these forms are very low. The X-ray diffraction crystal structures of the anhydrous salt and its monohydrate differed mainly in the trans or cis relationship of the N-methyl group to the alpha-methyl, although these two forms interconvert freely in solution.

Structural investigations of Pu{sup III} phosphate by X-ray diffraction, MAS-NMR and XANES spectroscopy

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

Popa, Karin; Raison, Philippe E., E-mail: philippe.raison@ec.europa.eu; Martel, Laura

2015-10-15

PuPO{sub 4} was prepared by a solid state reaction method and its crystal structure at room temperature was solved by powder X-ray diffraction combined with Rietveld refinement. High resolution XANES measurements confirm the +III valence state of plutonium, in agreement with valence bond derivation. The presence of the americium (as β{sup −} decay product of plutonium) in the +III oxidation state was determined based on XANES spectroscopy. High resolution solid state {sup 31}P NMR agrees with the XANES results and the presence of a solid-solution. - Graphical abstract: A full structural analysis of PuPO{sub 4} based on Rietveld analysis ofmore » room temperature X-ray diffraction data, XANES and MAS NMR measurements was performed. - Highlights: • The crystal structure of PuPO{sub 4} monazite is solved. • In PuPO{sub 4} plutonium is strictly trivalent. • The presence of a minute amount of Am{sup III} is highlighted. • We propose PuPO{sub 4} as a potential reference material for spectroscopic and microscopic studies.« less

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.

(sup 6)Li and (sup 7)MAS NMR and In Situ X-Ray Diffraction Studies of Lithium Manganate Cathode Materials

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

Lee, Young Joo; Wang, Francis; Grey, Clare P.

{sup 6}Li MAS NMR spectra of lithium manganese oxides with differing manganese oxidation states (LiMn{sub 2}O{sub 4}, Li{sub 4}Mn{sub 5}O{sub 12}, Li{sub 2}Mn{sub 4}O{sub 9}, and Li{sub 2}Mn{sub 2}O{sub 4}) are presented. Improved understanding of the lithium NMR spectra of these model compounds is used to interpret the local structure of the Li{sub x}Mn{sub 2}O{sub 4} cathode materials following electrochemical Li{sup +} deintercalation to various charging levels. In situ x-ray diffraction patterns of the same material during charging are also reported for comparison. Evidence for two-phase behavior for x <0.4 (Li{sub x}Mn{sub 2}O{sub 4}) is seen by both NMR andmore » diffraction.« less

NMR and X-ray studies of isomeric 22,23-dihydroxy stigmastanes

NASA Astrophysics Data System (ADS)

Khripach, Vladimir A.; Zhabinskii, Vladimir N.; Ivanova, Galina V.; Fando, Galina P.; Tsavlovskii, Dmitrii V.; Khripach, Natalya B.; Lyakhov, Alexander S.; Misharin, Alexander Yu.

2010-06-01

A comparative conformational study of steroidal side chain of (22 R,23 R)- and (22 S,23 S)-dihydroxy stigmastane derivatives was performed using single crystal X-ray diffraction and NMR spectroscopy. The preferred conformation in solution was shown to be close to that in the crystal. (22 R,23 R)-Isomers typical for natural plant steroid hormones brassinosteroids adopt a conformation in which both hydroxyl groups are pointed toward unhindered α-side of the steroidal plane and can thus participate in biochemical processes. Unnatural (22 S,23 S)-counterparts exhibit a conformation with the two hydroxyl groups oriented in the opposite direction and sterically hindered by 21-methyl group and terminal side chain fragment.

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.

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

PubMed

Ubarretxena-Belandia, Iban; Stokes, David L

2010-01-01

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

Present and future of membrane protein structure determination by electron crystallography

PubMed Central

Ubarretxena-Belandia, Iban; Stokes, David L.

2011-01-01

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

Solid-state acid-base interactions in complexes of heterocyclic bases with dicarboxylic acids: crystallography, hydrogen bond analysis, and 15N NMR spectroscopy.

PubMed

Li, Z Jane; Abramov, Yuriy; Bordner, Jon; Leonard, Jason; Medek, Ales; Trask, Andrew V

2006-06-28

A cancer candidate, compound 1, is a weak base with two heterocyclic basic nitrogens and five hydrogen-bonding functional groups, and is sparingly soluble in water rendering it unsuitable for pharmaceutical development. The crystalline acid-base pairs of 1, collectively termed solid acid-base complexes, provide significant increases in the solubility and bioavailability compared to the free base, 1. Three dicarboxylic acid-base complexes, sesquisuccinate 2, dimalonate 3, and dimaleate 4, show the most favorable physicochemical profiles and are studied in greater detail. The structural analyses of the three complexes using crystal structure and solid-state NMR reveal that the proton-transfer behavior in these organic acid-base complexes vary successively correlating with Delta pKa. As a result, 2 is a neutral complex, 3 is a mixed ionic and zwitterionic complex and 4 is an ionic salt. The addition of the acidic components leads to maximized hydrogen bond interactions forming extended three-dimensional networks. Although structurally similar, the packing arrangements of the three complexes are considerably different due to the presence of multiple functional groups and the flexible backbone of 1. The findings in this study provide insight into the structural characteristics of complexes involving heterocyclic bases and carboxylic acids, and demonstrate that X-ray crystallography and 15N solid-state NMR are truly complementary in elucidating hydrogen bonding interactions and the degree of proton transfer of these complexes.

Combined Approach for the Structural Characterization of Alkali Fluoroscandates: Solid-State NMR, Powder X-ray Diffraction, and Density Functional Theory Calculations.

PubMed

Rakhmatullin, Aydar; Polovov, Ilya B; Maltsev, Dmitry; Allix, Mathieu; Volkovich, Vladimir; Chukin, Andrey V; Boča, Miroslav; Bessada, Catherine

2018-02-05

The structures of several fluoroscandate compounds are presented here using a characterization approach combining powder X-ray diffraction and solid-state NMR. The structure of K 5 Sc 3 F 14 was fully determined from Rietveld refinement performed on powder X-ray diffraction data. Moreover, the local structures of NaScF 4 , Li 3 ScF 6 , KSc 2 F 7 , and Na 3 ScF 6 compounds were studied in detail from solid-state 19 F and 45 Sc NMR experiments. The 45 Sc chemical shift ranges for six- and seven-coordinated scandium environments were defined. The 19 F chemical shift ranges for bridging and terminal fluorine atoms were also determined. First-principles calculations of the 19 F and 45 Sc NMR parameters were carried out using plane-wave basis sets and periodic boundary conditions (CASTEP), and the results were compared with the experimental data. A good agreement between the calculated shielding constants and experimental chemical shifts was obtained. This demonstrates the good potential of computational methods in spectroscopic assignments of solid-state 45 Sc NMR spectroscopy.

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.

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.

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.

Experimental (13C NMR, 1H NMR, FT-IR, single-crystal X-ray diffraction) and DFT studies on 3,4-bis(isoproylamino)cyclobut-3-ene-1,2-dione.

PubMed

Süleymanoğlu, Nevin; Ustabaş, Reşat; Alpaslan, Yelda Bingöl; Eyduran, Fatih; Ozyürek, Cengiz; Iskeleli, Nazan Ocak

2011-12-01

In this work, 3,4-bis(isoproylamino)cyclobut-3-ene-1,2-dione C(10)H(16)N(2)O(2) (I), was synthesized and characterized by (13)C NMR, (1)H NMR, FT-IR, UV-vis spectroscopy and single-crystal X-ray diffraction. DFT method with 6-31G(d,p) basis set has been used to calculate the optimized geometrical parameters, atomic charges, vibrational frequencies and chemical shift values. The calculated vibrational frequencies and chemical shift values are compared with experimental FT-IR and NMR spectra. The results of the calculation shows good agreement between experimental and calculated values of the compound I. The existence of N-H⋯O type intermolecular ve C-H⋯O type intramolecular hydrogen bonds can be deduced from differences between experimental and calculated results of FT-IR and NMR. In addition, the molecular electrostatic potential map and frontier molecular orbitals and electronic absorption spectra were performed at B3LYP/6-31G(d,p) level of theory. HOMO-LUMO electronic transition of 4.90 eV are derived from the contribution of the bands π→π* and n→π* The spectral results obtained from FT-IR, NMR and X-ray of I revealed that the compound I is in predominantly enamine tautomeric form, which was supported by DFT calculations. Copyright © 2011 Elsevier B.V. All rights reserved.

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

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.

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

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

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.

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.

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

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

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

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

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.

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.

Unique Structure and Dynamics of the EphA5 Ligand Binding Domain Mediate Its Binding Specificity as Revealed by X-ray Crystallography, NMR and MD Simulations

PubMed Central

Mitra, Sayantan; Zhu, Wanlong; Qin, Haina; Pasquale, Elena B.; Song, Jianxing

2013-01-01

The 16 EphA and EphB receptors represent the largest family of receptor tyrosine kinases, and their interactions with 9 ephrin-A and ephrin-B ligands initiate bidirectional signals controlling many physiological and pathological processes. Most interactions occur between receptor and ephrins of the same class, and only EphA4 can bind all A and B ephrins. To understand the structural and dynamic principles that enable Eph receptors to utilize the same jellyroll β-sandwich fold to bind ephrins, the VAPB-MSP domain, peptides and small molecules, we have used crystallography, NMR and molecular dynamics (MD) simulations to determine the first structure and dynamics of the EphA5 ligand-binding domain (LBD), which only binds ephrin-A ligands. Unexpectedly, despite being unbound, the high affinity ephrin-binding pocket of EphA5 resembles that of other Eph receptors bound to ephrins, with a helical conformation over the J–K loop and an open pocket. The openness of the pocket is further supported by NMR hydrogen/deuterium exchange data and MD simulations. Additionally, the EphA5 LBD undergoes significant picosecond-nanosecond conformational exchanges over the loops, as revealed by NMR and MD simulations, but lacks global conformational exchanges on the microsecond-millisecond time scale. This is markedly different from the EphA4 LBD, which shares 74% sequence identity and 87% homology. Consequently, the unbound EphA5 LBD appears to comprise an ensemble of open conformations that have only small variations over the loops and appear ready to bind ephrin-A ligands. These findings show how two proteins with high sequence homology and structural similarity are still able to achieve distinctive binding specificities through different dynamics, which may represent a general mechanism whereby the same protein fold can serve for different functions. Our findings also suggest that a promising strategy to design agonists/antagonists with high affinity and selectivity might be to

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.

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.

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

A neutron-X-ray, NMR and calorimetric study of glassy Probucol synthesized using containerless techniques

NASA Astrophysics Data System (ADS)

Weber, J. K. R.; Benmore, C. J.; Tailor, A. N.; Tumber, S. K.; Neuefeind, J.; Cherry, B.; Yarger, J. L.; Mou, Q.; Weber, W.; Byrn, S. R.

2013-10-01

Acoustic levitation was used to trap 1-3 mm diameter drops of Probucol and other pharmaceutical materials in containerless conditions. Samples were studied in situ using X-ray diffraction and ex situ using neutron diffraction, NMR and DSC techniques. The materials were brought into non-equilibrium states by supersaturating solutions or by supercooling melts. The glass transition and crystallization temperatures of glassy Probucol were 29 ± 1 and 71 ± 1 °C respectively. The glassy form was stable with a shelf life of at least 8 months. A neutron/X-ray difference function of the glass showed that while molecular sub-groups remain rigid, many of the hydrogen correlations observed in the crystal become smeared out in the disordered material. The glass is principally comprised of slightly distorted Form I Probucol molecules with disordered packing rather than large changes in the individual molecular structure. Avoiding surface contact-induced nucleation provided access to highly non-equilibrium phases and enabled synthesis of phase-pure glasses.

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

Solution and solid-state effects on NMR chemical shifts in sesquiterpene lactones: NMR, X-ray, and theoretical methods.

PubMed

Dračínský, Martin; Buděšínský, Miloš; Warżajtis, Beata; Rychlewska, Urszula

2012-01-12

Selected guaianolide type sesquiterpene lactones were studied combining solution and solid-state NMR spectroscopy with theoretical calculations of the chemical shifts in both environments and with the X-ray data. The experimental (1)H and (13)C chemical shifts in solution were successfully reproduced by theoretical calculations (with the GIAO method and DFT B3LYP 6-31++G**) after geometry optimization (DFT B3LYP 6-31 G**) in vacuum. The GIPAW method was used for calculations of solid-state (13)C chemical shifts. The studied cases involved two polymorphs of helenalin, two pseudopolymorphs of 6α-hydroxydihydro-aromaticin and two cases of multiple asymmetric units in crystals: one in which the symmetry-independent molecules were connected by a series of hydrogen bonds (geigerinin) and the other in which the symmetry-independent molecules, deprived of any specific intermolecular interactions, differed in the conformation of the side chain (badkhysin). Geometrically different molecules present in the crystal lattices could be easily distinguished in the solid-state NMR spectra. Moreover, the experimental differences in the (13)C chemical shifts corresponding to nuclei in different polymorphs or in geometrically different molecules were nicely reproduced with the GIPAW calculations.

Computational approach to integrate 3D X-ray microtomography and NMR data.

PubMed

Lucas-Oliveira, Everton; Araujo-Ferreira, Arthur G; Trevizan, Willian A; Fortulan, Carlos A; Bonagamba, Tito J

2018-05-04

Nowadays, most of the efforts in NMR applied to porous media are dedicated to studying the molecular fluid dynamics within and among the pores. These analyses have a higher complexity due to morphology and chemical composition of rocks, besides dynamic effects as restricted diffusion, diffusional coupling, and exchange processes. Since the translational nuclear spin diffusion in a confined geometry (e.g. pores and fractures) requires specific boundary conditions, the theoretical solutions are restricted to some special problems and, in many cases, computational methods are required. The Random Walk Method is a classic way to simulate self-diffusion along a Digital Porous Medium. Bergman model considers the magnetic relaxation process of the fluid molecules by including a probability rate of magnetization survival under surface interactions. Here we propose a statistical approach to correlate surface magnetic relaxivity with the computational method applied to the NMR relaxation in order to elucidate the relationship between simulated relaxation time and pore size of the Digital Porous Medium. The proposed computational method simulates one- and two-dimensional NMR techniques reproducing, for example, longitudinal and transverse relaxation times (T 1 and T 2 , respectively), diffusion coefficients (D), as well as their correlations. For a good approximation between the numerical and experimental results, it is necessary to preserve the complexity of translational diffusion through the microstructures in the digital rocks. Therefore, we use Digital Porous Media obtained by 3D X-ray microtomography. To validate the method, relaxation times of ideal spherical pores were obtained and compared with the previous determinations by the Brownstein-Tarr model, as well as the computational approach proposed by Bergman. Furthermore, simulated and experimental results of synthetic porous media are compared. These results make evident the potential of computational physics in the

Computational approach to integrate 3D X-ray microtomography and NMR data

NASA Astrophysics Data System (ADS)

Lucas-Oliveira, Everton; Araujo-Ferreira, Arthur G.; Trevizan, Willian A.; Fortulan, Carlos A.; Bonagamba, Tito J.

2018-07-01

Nowadays, most of the efforts in NMR applied to porous media are dedicated to studying the molecular fluid dynamics within and among the pores. These analyses have a higher complexity due to morphology and chemical composition of rocks, besides dynamic effects as restricted diffusion, diffusional coupling, and exchange processes. Since the translational nuclear spin diffusion in a confined geometry (e.g. pores and fractures) requires specific boundary conditions, the theoretical solutions are restricted to some special problems and, in many cases, computational methods are required. The Random Walk Method is a classic way to simulate self-diffusion along a Digital Porous Medium. Bergman model considers the magnetic relaxation process of the fluid molecules by including a probability rate of magnetization survival under surface interactions. Here we propose a statistical approach to correlate surface magnetic relaxivity with the computational method applied to the NMR relaxation in order to elucidate the relationship between simulated relaxation time and pore size of the Digital Porous Medium. The proposed computational method simulates one- and two-dimensional NMR techniques reproducing, for example, longitudinal and transverse relaxation times (T1 and T2, respectively), diffusion coefficients (D), as well as their correlations. For a good approximation between the numerical and experimental results, it is necessary to preserve the complexity of translational diffusion through the microstructures in the digital rocks. Therefore, we use Digital Porous Media obtained by 3D X-ray microtomography. To validate the method, relaxation times of ideal spherical pores were obtained and compared with the previous determinations by the Brownstein-Tarr model, as well as the computational approach proposed by Bergman. Furthermore, simulated and experimental results of synthetic porous media are compared. These results make evident the potential of computational physics in the

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

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

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

Solid-state 11B and 13C NMR, IR, and X-ray crystallographic characterization of selected arylboronic acids and their catechol cyclic esters.

PubMed

Oh, Se-Woung; Weiss, Joseph W E; Kerneghan, Phillip A; Korobkov, Ilia; Maly, Kenneth E; Bryce, David L

2012-05-01

Nine arylboronic acids, seven arylboronic catechol cyclic esters, and two trimeric arylboronic anhydrides (boroxines) are investigated using (11)B solid-state NMR spectroscopy at three different magnetic field strengths (9.4, 11.7, and 21.1 T). Through the analysis of spectra of static and magic-angle spinning samples, the (11)B electric field gradient and chemical shift tensors are determined. The effects of relaxation anisotropy and nutation field strength on the (11)B NMR line shapes are investigated. Infrared spectroscopy was also used to help identify peaks in the NMR spectra as being due to the anhydride form in some of the arylboronic acid samples. Seven new X-ray crystallographic structures are reported. Calculations of the (11)B NMR parameters are performed using cluster model and periodic gauge-including projector-augmented wave (GIPAW) density functional theory (DFT) approaches, and the results are compared with the experimental values. Carbon-13 solid-state NMR experiments and spectral simulations are applied to determine the chemical shifts of the ipso carbons of the samples. One bond indirect (13)C-(11)B spin-spin (J) coupling constants are also measured experimentally and compared with calculated values. The (11)B/(10)B isotope effect on the (13)C chemical shift of the ipso carbons of arylboronic acids and their catechol esters, as well as residual dipolar coupling, is discussed. Overall, this combined X-ray, NMR, IR, and computational study provides valuable new insights into the relationship between NMR parameters and the structure of boronic acids and esters. Copyright © 2012 John Wiley & Sons, Ltd.

Structural and Molecular Modeling Features of P2X Receptors

PubMed Central

Alves, Luiz Anastacio; da Silva, João Herminio Martins; Ferreira, Dinarte Neto Moreira; Fidalgo-Neto, Antonio Augusto; Teixeira, Pedro Celso Nogueira; de Souza, Cristina Alves Magalhães; Caffarena, Ernesto Raúl; de Freitas, Mônica Santos

2014-01-01

Currently, adenosine 5′-triphosphate (ATP) is recognized as the extracellular messenger that acts through P2 receptors. P2 receptors are divided into two subtypes: P2Y metabotropic receptors and P2X ionotropic receptors, both of which are found in virtually all mammalian cell types studied. Due to the difficulty in studying membrane protein structures by X-ray crystallography or NMR techniques, there is little information about these structures available in the literature. Two structures of the P2X4 receptor in truncated form have been solved by crystallography. Molecular modeling has proven to be an excellent tool for studying ionotropic receptors. Recently, modeling studies carried out on P2X receptors have advanced our knowledge of the P2X receptor structure-function relationships. This review presents a brief history of ion channel structural studies and shows how modeling approaches can be used to address relevant questions about P2X receptors. PMID:24637936

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

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.

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.

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

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.

N-acetylglyoxylic amide bearing a nitrophenyl group as anion receptors: NMR and X-ray investigations on anion binding and selectivity

NASA Astrophysics Data System (ADS)

Suryanti, Venty; Bhadbhade, Mohan; Black, David StC; Kumar, Naresh

2017-10-01

N-Nitrophenylglyoxylic amides 1 and 2 in presence of tetrabutylammonium cation (TBA) act as receptors for anions HSO4-, Cl-, Br- and NO3- as investigated by NMR studies. The receptors formed 1:1 host-guest complexes in solution. X-ray structure of 1 along with TBA that bind a chloride anion is reported. Molecule 1 showed the highest selectivity for HSO4- anion over others measured. X-ray structure of the bound Cl- revealed a pocket containing the anion making strong (Nsbnd H⋯Cl) and weak hydrogen bonds (Csbnd H⋯Cl) that contribute to the recognition of the chloride anion. Nsbnd H and Csbnd H hydrogen bonds resulted in a relatively strong binding for chloride ions.

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

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.

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

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

The application of tailor-made force fields and molecular dynamics for NMR crystallography: a case study of free base cocaine

PubMed Central

Neumann, Marcus A.

2017-01-01

Motional averaging has been proven to be significant in predicting the chemical shifts in ab initio solid-state NMR calculations, and the applicability of motional averaging with molecular dynamics has been shown to depend on the accuracy of the molecular mechanical force field. The performance of a fully automatically generated tailor-made force field (TMFF) for the dynamic aspects of NMR crystallography is evaluated and compared with existing benchmarks, including static dispersion-corrected density functional theory calculations and the COMPASS force field. The crystal structure of free base cocaine is used as an example. The results reveal that, even though the TMFF outperforms the COMPASS force field for representing the energies and conformations of predicted structures, it does not give significant improvement in the accuracy of NMR calculations. Further studies should direct more attention to anisotropic chemical shifts and development of the method of solid-state NMR calculations. PMID:28250956

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.

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

Dinuclear NHC-palladium complexes containing phosphine spacers: synthesis, X-ray structures and their catalytic activities towards the Hiyama coupling reaction.

PubMed

Yang, Jin; Li, Pinhua; Zhang, Yicheng; Wang, Lei

2014-05-21

Six dinuclear N-heterocyclic carbene (NHC) palladium complexes, [PdCl2(IMes)]2(μ-dppe) (1), [PdCl2(IPr)]2(μ-dppe) (2), [PdCl2(IMes)]2(μ-dppb) (3), [PdCl2(IPr)]2(μ-dppb) (4), [PdCl2(IMes)]2(μ-dpph) (5), and [PdCl2(IPr)]2(μ-dpph) (6) [IMes = N,N'-bis-(2,4,6-trimethylphenyl)imidazol-2-ylidene; IPr = N,N'-bis-(2,6-di(iso-propyl)phenyl)imidazol-2-ylidene; dppe = 1,2-bis(diphenylphosphino)ethane, dppb = 1,4-bis(diphenylphosphino)butane; and dpph = 1,6-bis(diphenylphosphino)hexane], have been synthesized through bridge-cleavage reactions of chloro-bridged dimeric compounds, [Pd(μ-Cl)(Cl)(NHC)]2, with the corresponding diphosphine ligands. The obtained compounds were fully characterized by (1)H NMR, (13)C NMR and (31)P NMR spectroscopy, FT-IR, elemental analysis and single-crystal X-ray crystallography. Moreover, further explorations of the catalytic potential of the dinuclear carbene palladium complexes as catalysts for the Pd-catalyzed transformations have been performed under microwave irradiation conditions, and the complexes exhibited moderate to good catalytic activity in the Hiyama coupling reaction of trimethoxyphenylsilane with aryl chlorides.

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)

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

The structure investigations of dehydroacetic acid and 1,8-diaminonaphthalene condensation product by NMR, MS, and X-ray measurements

NASA Astrophysics Data System (ADS)

Kołodziej, B.; Morawiak, M.; Kamieński, B.; Schilf, W.

2016-05-01

A new unexpected product of condensation reaction of 1,8-diaminonaphthalene (DAN) and carbonyl compound (here: dehydroacetic acid (dha)) was synthesized. Discussion about the molecular structure of possible products of this reaction was done on the base of NMR studies. The structure of the titled product in both DMSO solution and in the solid state was resolved by analysis of its spectral data (X-ray structure analysis, multinuclear NMR in solution and solid state spectra) and MS measurements. The presented studies provided clear evidence that the titled product exists in diluted DMSO solution as the mixture of two kinetic free ionic species whereas in concentrated DMSO solution as well as in the solid state this system forms associated ionic pairs bonded together by hydrogen bonds.

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

A multicomponent CuAAC "click" approach to a library of hybrid polydentate 2-pyridyl-1,2,3-triazole ligands: new building blocks for the generation of metallosupramolecular architectures.

PubMed

Crowley, James D; Bandeen, Pauline H

2010-01-14

A one pot, multicomponent CuAAC reaction has been exploited for the safe generation of alkyl, benzyl or aryl linked polydentate pyridyl-1,2,3-triazole ligands from their corresponding halides, sodium azide and alkynes in excellent yields. The ligands have been fully characterised by elemental analysis, HR-ESMS, IR, (1)H and (13)C NMR and in two cases the structures were confirmed by X-ray crystallography. Additionally, we have examined the Ag(I) coordination chemistry of these ligands and found, using HR-ESMS, (1)H NMR, and X-ray crystallography, that both discrete and polymeric metallosupramolecular architectures can be formed.

First X-ray crystal structure and internal reference diffusion-ordered NMR spectroscopy study of the prototypical Posner reagent, MeCu(SPh)Li(THF)3.

PubMed

Bertz, Steven H; Hardin, Richard A; Heavey, Thomas J; Jones, Daniel S; Monroe, T Blake; Murphy, Michael D; Ogle, Craig A; Whaley, Tara N

2013-07-29

Grow slow: The usual direct treatment of MeLi and CuSPh did not yield X-ray quality crystals of MeCu(SPh)Li. An indirect method starting from Me2CuLi⋅LiSPh and chalcone afforded the desired crystals by the slow reaction of the intermediate π-complex (see scheme). This strategy produced the first X-ray crystal structure of a Posner cuprate. A complementary NMR study showed that the contact ion pair was also the main species in solution. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Study of the gel films of Acetobacter Xylinum cellulose and its modified samples by {sup 1}H NMR cryoporometry and small-angle X-ray scattering

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

Babushkina, T. A.; Klimova, T. P.; Shtykova, E. V.

2010-03-15

Gel films of Acetobacter Xylinum cellulose and its modified samples have been investigated by 1H nuclear magnetic resonance (NMR) cryoporometry and small-angle X-ray scattering. The joint use of these two methods made it possible to characterize the sizes of aqueous pores in gel films and estimate the sizes of structural inhomogeneities before and after the sorption of polyvinylpyrrolidone and Se{sub 0} nanoparticles (stabilized by polyvinylpyrrolidone) into the films. According to small-angle X-ray scattering data, the sizes of inhomogeneities in a gel film change only slightly upon the sorption of polyvinylpyrrolidone and nanoparticles. The impregnated material is sorbed into water-filled cavitiesmore » that are present in the gel film. {sup 1}H NMR cryoporometry allowed us to reveal the details of changes in the sizes of small aqueous pores during modifications.« less

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

Protein folding on the ribosome studied using NMR spectroscopy

PubMed Central

Waudby, Christopher A.; Launay, Hélène; Cabrita, Lisa D.; Christodoulou, John

2013-01-01

NMR spectroscopy is a powerful tool for the investigation of protein folding and misfolding, providing a characterization of molecular structure, dynamics and exchange processes, across a very wide range of timescales and with near atomic resolution. In recent years NMR methods have also been developed to study protein folding as it might occur within the cell, in a de novo manner, by observing the folding of nascent polypeptides in the process of emerging from the ribosome during synthesis. Despite the 2.3 MDa molecular weight of the bacterial 70S ribosome, many nascent polypeptides, and some ribosomal proteins, have sufficient local flexibility that sharp resonances may be observed in solution-state NMR spectra. In providing information on dynamic regions of the structure, NMR spectroscopy is therefore highly complementary to alternative methods such as X-ray crystallography and cryo-electron microscopy, which have successfully characterized the rigid core of the ribosome particle. However, the low working concentrations and limited sample stability associated with ribosome–nascent chain complexes means that such studies still present significant technical challenges to the NMR spectroscopist. This review will discuss the progress that has been made in this area, surveying all NMR studies that have been published to date, and with a particular focus on strategies for improving experimental sensitivity. PMID:24083462

Synthesis of 24-phenyl-24-oxo steroids derived from bile acids by palladium-catalyzed cross coupling with phenylboronic acid. NMR characterization and X-ray structures.

PubMed

Mayorquín-Torres, Martha C; Romero-Ávila, Margarita; Flores-Álamo, Marcos; Iglesias-Arteaga, Martin A

2013-11-01

Palladium-catalyzed cross coupling of phenyboronic acid with acetylated bile acids in which the carboxyl functions have been activated by formation of a mixed anhydride with pivalic anhydride afforded moderate to good yield of 24-phenyl-24-oxo-steroids. Unambiguous assignments of the NMR signals were made with the aid of combined 1D and 2D NMR techniques. X-ray diffraction studies confirmed the obtained structures. Copyright © 2013 Elsevier Inc. All rights reserved.

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.

Programming A Molecular Relay for Ultrasensitive Biodetection through 129 Xe NMR

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

Wang, Yanfei; Roose, Benjamin W.; Philbin, John P.

2015-12-21

We reported a supramolecular strategy for detecting specific proteins in complex media by using hyperpolarized 129Xe NMR. A cucurbit[6]uril (CB[6])-based molecular relay was programmed for three sequential equilibrium conditions by designing a two-faced guest (TFG) that initially binds CB[6] and blocks the CB[6]–Xe interaction. Moreover, the protein analyte recruits the TFG and frees CB[6] for Xe binding. TFGs containing CB[6]- and carbonic anhydrase II (CAII)-binding domains were synthesized in one or two steps. X-ray crystallography confirmed TFG binding to Zn 2+ in the deep CAII active-site cleft, which precludes simultaneous CB[6] binding. The molecular relay was reprogrammed to detect avidinmore » by using a different TFG. Finally, Xe binding by CB[6] was detected in buffer and in E. coli cultures expressing CAII through ultrasensitive 129Xe NMR spectroscopy.« less

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.

Challenges in NMR-based structural genomics

NASA Astrophysics Data System (ADS)

Sue, Shih-Che; Chang, Chi-Fon; Huang, Yao-Te; Chou, Ching-Yu; Huang, Tai-huang

2005-05-01

Understanding the functions of the vast number of proteins encoded in many genomes that have been completely sequenced recently is the main challenge for biologists in the post-genomics era. Since the function of a protein is determined by its exact three-dimensional structure it is paramount to determine the 3D structures of all proteins. This need has driven structural biologists to undertake the structural genomics project aimed at determining the structures of all known proteins. Several centers for structural genomics studies have been established throughout the world. Nuclear magnetic resonance (NMR) spectroscopy has played a major role in determining protein structures in atomic details and in a physiologically relevant solution state. Since the number of new genes being discovered daily far exceeds the number of structures determined by both NMR and X-ray crystallography, a high-throughput method for speeding up the process of protein structure determination is essential for the success of the structural genomics effort. In this article we will describe NMR methods currently being employed for protein structure determination. We will also describe methods under development which may drastically increase the throughput, as well as point out areas where opportunities exist for biophysicists to make significant contribution in this important field.

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.…

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

Structure of Oxide Glass and Melts at High-Pressure: A View from Inelastic X-ray Scattering and 2D Solid-State NMR

NASA Astrophysics Data System (ADS)

Lee, S.; Mysen, B. O.; Fei, Y.; Cody, G. D.; Mao, H.; Eng, P.

2006-12-01

Full understanding of the atomic arrangement of oxides glasses and melts both at ambient and high-pressure has long been one of the fundamental and yet difficult problems in earth sciences, condensed matter physics as well as glass sciences. The structures of archetypal oxide glasses (e.g. SiO2 and B2O3) as well as complex silicate glasses (ternary and quaternary aluminosilicate glasses) at high pressure are essential to elucidate origins of anomalous macroscopic properties of melts and global geophysical processes in the Earth's interior. Recent progress in inelastic x-ray scattering (IXS) with high brilliance 3rd generation synchrotron x-rays combined with DAC techniques allows us to explore pressure-induced changes in the bonding nature of archetypal amorphous oxide, illustrating a new opportunity to study amorphous oxides with IXS (Lee SK et al. Nature Materials 2005, 4, p851). 2 dimensional solid-state NMR have offered much improved resolution over conventional 1D NMR, unveiling previously unknown structural details of amorphous silicates at high pressure (Lee SK. Geochim. Cosmochim. Acta 2005, 69, p3695; J. Phys. Chem. B. 2006, 110, p16408) Here, we report the synchrotron inelastic x-ray scattering results (oxygen and boron K-edge) for divers oxide glasses at pressure up to 40 GPa, revealing the nature of pressure-induced bonding changes and the structure. Direct in-situ measurements provide evidence for a continuous transformation with multiple densification mechanisms. 2D solid-state NMR spectra for silicate and germinate glasses shows detailed information about extent of disorder among framework units at high pressure. The chemical ordering among framework units leads to the formation of ^{[5,6]}Si-O-^{[4]}Si in silicates and ^{[5,6]}Al-O-^{[4]}Si in aluminosilicates. Whereas the densification mechanism can be dependent on the chemical composition and the fraction of non-bridging oxygen, the pressure dependence of both simple and complex multi

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

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.

Protein-Carbohydrate Interactions Studied by NMR: From Molecular Recognition to Drug Design

PubMed Central

Fernández-Alonso, María del Carmen; Díaz, Dolores; Berbis, Manuel Álvaro; Marcelo, Filipa; Cañada, Javier; Jiménez-Barbero, Jesús

2012-01-01

Diseases that result from infection are, in general, a consequence of specific interactions between a pathogenic organism and the cells. The study of host-pathogen interactions has provided insights for the design of drugs with therapeutic properties. One area that has proved to be promising for such studies is the constituted by carbohydrates which participate in biological processes of paramount importance. On the one hand, carbohydrates have shown to be information carriers with similar, if not higher, importance than traditionally considered carriers as amino acids and nucleic acids. On the other hand, the knowledge on molecular recognition of sugars by lectins and other carbohydrate-binding proteins has been employed for the development of new biomedical strategies. Biophysical techniques such as X-Ray crystallography and NMR spectroscopy lead currently the investigation on this field. In this review, a description of traditional and novel NMR methodologies employed in the study of sugar-protein interactions is briefly presented in combination with a palette of NMR-based studies related to biologically and/or pharmaceutically relevant applications. PMID:23305367

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.

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

Toward a structure determination method for biomineral-associated protein using combined solid- state NMR and computational structure prediction.

PubMed

Masica, David L; Ash, Jason T; Ndao, Moise; Drobny, Gary P; Gray, Jeffrey J

2010-12-08

Protein-biomineral interactions are paramount to materials production in biology, including the mineral phase of hard tissue. Unfortunately, the structure of biomineral-associated proteins cannot be determined by X-ray crystallography or solution nuclear magnetic resonance (NMR). Here we report a method for determining the structure of biomineral-associated proteins. The method combines solid-state NMR (ssNMR) and ssNMR-biased computational structure prediction. In addition, the algorithm is able to identify lattice geometries most compatible with ssNMR constraints, representing a quantitative, novel method for investigating crystal-face binding specificity. We use this method to determine most of the structure of human salivary statherin interacting with the mineral phase of tooth enamel. Computation and experiment converge on an ensemble of related structures and identify preferential binding at three crystal surfaces. The work represents a significant advance toward determining structure of biomineral-adsorbed protein using experimentally biased structure prediction. This method is generally applicable to proteins that can be chemically synthesized. Copyright © 2010 Elsevier Ltd. All rights reserved.

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

Synthesis, spectra and X-ray crystallography of dipyridin-2-ylmethanone oxime and its CuX2(oxime)2 complexes: Thermal, Hirshfeld surface and DFT analysis

NASA Astrophysics Data System (ADS)

Warad, Ismail; Abdoh, Muneer; Al Ali, Anas; Shivalingegowda, Naveen; Kumara, Karthik; Zarrouk, Abdelkader; Lokanath, Neartur Krishnappagowda

2018-02-01

Dipyridin-2-ylmethanone oxime (C11H9N3O), was prepared using di-2-pyridyl ketone. The oxime ligand and its neutral CuX2 (oxime)2 (X = Cl or Br) complexes have been identified with the aid of several spectroscopic techniques such as: IR, EI-MS, EA, UV-visible, TG, 1H-NMR and finally the structure of the free oxime ligand was confirmed by X-ray diffraction studies. The oxime crystallizes in the monoclinic space group P21/c, with cell parameters a = 8.8811 (8) Å, b = 10.6362 (8) Å, c = 11.2050 (8) Å, β = 109.085 (4) º, V = 1000.26 (14) Å3 and Z = 4. The molecular conformation is stabilized by a strong intramolecular Osbnd H⋯N hydrogen bonding between the hydroxyl group of the oxime moiety and the nitrogen of the pyridine ring. Since the oxime structure was solved by XRD, the ligand structure parameters like bond length and angles were compared to the DFT computed one, the UV-visible to TD-SCF and Hirshfeld surface to MEP analysis.

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

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

DFT calculations in the assignment of solid-state NMR and crystal structure elucidation of a lanthanum(iii) complex with dithiocarbamate and phenanthroline.

PubMed

Gowda, Vasantha; Laitinen, Risto S; Telkki, Ville-Veikko; Larsson, Anna-Carin; Antzutkin, Oleg N; Lantto, Perttu

2016-12-06

The molecular, crystal, and electronic structures as well as spectroscopic properties of a mononuclear heteroleptic lanthanum(iii) complex with diethyldithiocarbamate and 1,10-phenanthroline ligands (3 : 1) were studied by solid-state 13 C and 15 N cross-polarisation (CP) magic-angle-spinning (MAS) NMR, X-ray diffraction (XRD), and first principles density functional theory (DFT) calculations. A substantially different powder XRD pattern and 13 C and 15 N CP-MAS NMR spectra indicated that the title compound is not isostructural to the previously reported analogous rare earth complexes with the space group P2 1 /n. Both 13 C and 15 N CP-MAS NMR revealed the presence of six structurally different dithiocarbamate groups in the asymmetric unit cell, implying a non-centrosymmetric packing arrangement of molecules. This was supported by single-crystal X-ray crystallography showing that the title compound crystallised in the triclinic space group P1[combining macron]. In addition, the crystal structure also revealed that one of the dithiocarbamate ligands has a conformational disorder. NMR chemical shift calculations employing the periodic gauge including projector augmented wave (GIPAW) approach supported the assignment of the experimental 13 C and 15 N NMR spectra. However, the best correspondences were obtained with the structure where the atomic positions in the X-ray unit cell were optimised at the DFT level. The roles of the scalar and spin-orbit relativistic effects on NMR shielding were investigated using the zeroth-order regular approximation (ZORA) method with the outcome that already the scalar relativistic level qualitatively reproduces the experimental chemical shifts. The electronic properties of the complex were evaluated based on the results of the natural bond orbital (NBO) and topology of the electron density analyses. Overall, we apply a multidisciplinary approach acquiring comprehensive information about the solid-state structure and the metal

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

Exploiting the Synergy of Powder X-ray Diffraction and Solid-State NMR Spectroscopy in Structure Determination of Organic Molecular Solids

PubMed Central

2013-01-01

We report a strategy for structure determination of organic materials in which complete solid-state nuclear magnetic resonance (NMR) spectral data is utilized within the context of structure determination from powder X-ray diffraction (XRD) data. Following determination of the crystal structure from powder XRD data, first-principles density functional theory-based techniques within the GIPAW approach are exploited to calculate the solid-state NMR data for the structure, followed by careful scrutiny of the agreement with experimental solid-state NMR data. The successful application of this approach is demonstrated by structure determination of the 1:1 cocrystal of indomethacin and nicotinamide. The 1H and 13C chemical shifts calculated for the crystal structure determined from the powder XRD data are in excellent agreement with those measured experimentally, notably including the two-dimensional correlation of 1H and 13C chemical shifts for directly bonded 13C–1H moieties. The key feature of this combined approach is that the quality of the structure determined is assessed both against experimental powder XRD data and against experimental solid-state NMR data, thus providing a very robust validation of the veracity of the structure. PMID:24386493

Exploiting the Synergy of Powder X-ray Diffraction and Solid-State NMR Spectroscopy in Structure Determination of Organic Molecular Solids.

PubMed

Dudenko, Dmytro V; Williams, P Andrew; Hughes, Colan E; Antzutkin, Oleg N; Velaga, Sitaram P; Brown, Steven P; Harris, Kenneth D M

2013-06-13

We report a strategy for structure determination of organic materials in which complete solid-state nuclear magnetic resonance (NMR) spectral data is utilized within the context of structure determination from powder X-ray diffraction (XRD) data. Following determination of the crystal structure from powder XRD data, first-principles density functional theory-based techniques within the GIPAW approach are exploited to calculate the solid-state NMR data for the structure, followed by careful scrutiny of the agreement with experimental solid-state NMR data. The successful application of this approach is demonstrated by structure determination of the 1:1 cocrystal of indomethacin and nicotinamide. The 1 H and 13 C chemical shifts calculated for the crystal structure determined from the powder XRD data are in excellent agreement with those measured experimentally, notably including the two-dimensional correlation of 1 H and 13 C chemical shifts for directly bonded 13 C- 1 H moieties. The key feature of this combined approach is that the quality of the structure determined is assessed both against experimental powder XRD data and against experimental solid-state NMR data, thus providing a very robust validation of the veracity of the structure.

Structural study of the membrane protein MscL using cell-free expression and solid-state NMR

NASA Astrophysics Data System (ADS)

Abdine, Alaa; Verhoeven, Michiel A.; Park, Kyu-Ho; Ghazi, Alexandre; Guittet, Eric; Berrier, Catherine; Van Heijenoort, Carine; Warschawski, Dror E.

2010-05-01

High-resolution structures of membrane proteins have so far been obtained mostly by X-ray crystallography, on samples where the protein is surrounded by detergent. Recent developments of solid-state NMR have opened the way to a new approach for the study of integral membrane proteins inside a membrane. At the same time, the extension of cell-free expression to the production of membrane proteins allows for the production of proteins tailor made for NMR. We present here an in situ solid-state NMR study of a membrane protein selectively labeled through the use of cell-free expression. The sample consists of MscL (mechano-sensitive channel of large conductance), a 75 kDa pentameric α-helical ion channel from Escherichia coli, reconstituted in a hydrated lipid bilayer. Compared to a uniformly labeled protein sample, the spectral crowding is greatly reduced in the cell-free expressed protein sample. This approach may be a decisive step required for spectral assignment and structure determination of membrane proteins by solid-state NMR.

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.

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.

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

Detergent/Nanodisc Screening for High-Resolution NMR Studies of an Integral Membrane Protein Containing a Cytoplasmic Domain

PubMed Central

Maslennikov, Innokentiy; Choe, Senyon; Riek, Roland

2013-01-01

Because membrane proteins need to be extracted from their natural environment and reconstituted in artificial milieus for the 3D structure determination by X-ray crystallography or NMR, the search for membrane mimetic that conserve the native structure and functional activities remains challenging. We demonstrate here a detergent/nanodisc screening study by NMR of the bacterial α-helical membrane protein YgaP containing a cytoplasmic rhodanese domain. The analysis of 2D [15N,1H]-TROSY spectra shows that only a careful usage of low amounts of mixed detergents did not perturb the cytoplasmic domain while solubilizing in parallel the transmembrane segments with good spectral quality. In contrast, the incorporation of YgaP into nanodiscs appeared to be straightforward and yielded a surprisingly high quality [15N,1H]-TROSY spectrum opening an avenue for the structural studies of a helical membrane protein in a bilayer system by solution state NMR. PMID:23349867

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

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.

The structure and dipole moment of globular proteins in solution and crystalline states: use of NMR and X-ray databases for the numerical calculation of dipole moment.

PubMed

Takashima, S

2001-04-05

The large dipole moment of globular proteins has been well known because of the detailed studies using dielectric relaxation and electro-optical methods. The search for the origin of these dipolemoments, however, must be based on the detailed knowledge on protein structure with atomic resolutions. At present, we have two sources of information on the structure of protein molecules: (1) x-ray databases obtained in crystalline state; (2) NMR databases obtained in solution state. While x-ray databases consist of only one model, NMR databases, because of the fluctuation of the protein folding in solution, consist of a number of models, thus enabling the computation of dipole moment repeated for all these models. The aim of this work, using these databases, is the detailed investigation on the interdependence between the structure and dipole moment of protein molecules. The dipole moment of protein molecules has roughly two components: one dipole moment is due to surface charges and the other, core dipole moment, is due to polar groups such as N--H and C==O bonds. The computation of surface charge dipole moment consists of two steps: (A) calculation of the pK shifts of charged groups for electrostatic interactions and (B) calculation of the dipole moment using the pK corrected for electrostatic shifts. The dipole moments of several proteins were computed using both NMR and x-ray databases. The dipole moments of these two sets of calculations are, with a few exceptions, in good agreement with one another and also with measured dipole moments.

X-Ray Structure determination of the Glycine Cleavage System Protein H of Mycobacterium tuberculosis Using An Inverse Compton Synchrotron X-Ray Source

PubMed Central

Abendroth, Jan; McCormick, Michael S.; Edwards, Thomas E.; Staker, Bart; Loewen, Roderick; Gifford, Martin; Rifkin, Jeff; Mayer, Chad; Guo, Wenjin; Zhang, Yang; Myler, Peter; Kelley, Angela; Analau, Erwin; Hewitt, Stephen Nakazawa; Napuli, Alberto J.; Kuhn, Peter; Ruth, Ronald D.; Stewart, Lance J.

2010-01-01

Structural genomics discovery projects require ready access to both X-ray and NMR instrumentation which support the collection of experimental data needed to solve large numbers of novel protein structures. The most productive X-ray crystal structure determination laboratories make extensive frequent use of tunable synchrotron X-ray light to solve novel structures by anomalous diffraction methods. This requires that frozen cryo-protected crystals be shipped to large government-run synchrotron facilities for data collection. In an effort to eliminate the need to ship crystals for data collection, we have developed the first laboratory-scale synchrotron light source capable of performing many of the state-of-the-art synchrotron applications in X-ray science. This Compact Light Source is a first-in-class device that uses inverse Compton scattering to generate X-rays of sufficient flux, tunable wavelength and beam size to allow high-resolution X-ray diffraction data collection from protein crystals. We report on benchmarking tests of X-ray diffraction data collection with hen egg white lysozyme, and the successful high-resolution X-ray structure determination of the Glycine cleavage system protein H from Mycobacterium tuberculosis using diffraction data collected with the Compact Light Source X-ray beam. PMID:20364333

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

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.

Electron paramagnetic resonance, scanning electron microscopy with energy dispersion X-ray spectrometry, X-ray powder diffraction, and NMR characterization of iron-rich fired clays.

PubMed

Presciutti, Federica; Capitani, Donatella; Sgamellotti, Antonio; Brunetti, Brunetto Giovanni; Costantino, Ferdinando; Viel, Stéphane; Segre, Annalaura

2005-12-01

The aim of this study is to clarify the structure of an iron-rich clay and the structural changes involved in the firing process as a preliminary step to get information on ancient ceramic technology. To this purpose, illite-rich clay samples fired at different temperatures were characterized using a multitechnique approach, i.e., by electron paramagnetic resonance, scanning electron microscopy with electron dispersion X-ray spectrometry, X-ray powder diffraction, magic angle spinning and multiple quantum magic angle spinning NMR. During firing, four main reaction processes occur: dehydration, dehydroxylation, structural breakdown, and recrystallization. When the results are combined from all characterization methods, the following conclusions could be obtained. Interlayer H2O is located close to aluminum in octahedral sites and is driven off at temperatures lower than 600 degrees C. Between 600 and 700 degrees C dehydroxylation occurs whereas, between 800 and 900 degrees C, the aluminum in octahedral sites disappears, due to the breakdown of the illite structure, and all iron present is oxidized to Fe3+. In samples fired at 1000 and 1100 degrees C iron clustering was observed as well as large single crystals of iron with the occurrence of ferro- or ferrimagnetic effects. Below 900 degrees C the aluminum in octahedral sites presents a continuous distribution of chemical shift, suggesting the presence of slightly distorted sites. Finally, over the whole temperature range, the presence of at least two tetrahedral aluminum sites was revealed, characterized by different values of the quadrupolar coupling constant.

Synthesis, X-ray diffraction method, spectroscopic characterization (FT-IR, 1H and 13C NMR), antimicrobial activity, Hirshfeld surface analysis and DFT computations of novel sulfonamide derivatives

NASA Astrophysics Data System (ADS)

Demircioğlu, Zeynep; Özdemir, Fethi Ahmet; Dayan, Osman; Şerbetçi, Zafer; Özdemir, Namık

2018-06-01

Synthesized compounds of N-(2-aminophenyl)benzenesulfonamide 1 and (Z)-N-(2-((2-nitrobenzylidene)amino)phenyl)benzenesulfonamide 2 were characterized by antimicrobial activity, FT-IR, 1H and 13C NMR. Two new Schiff base ligands containing aromatic sulfonamide fragment of (Z)-N-(2-((3-nitrobenzylidene)amino)phenyl)benzenesulfonamide 3 and (Z)-N-(2-((4-nitrobenzylidene)amino)phenyl)benzenesulfonamide 4 were synthesized and investigated by spectroscopic techniques including 1H and 13C NMR, FT-IR, single crystal X-ray diffraction, Hirshfeld surface, theoretical method analyses and by antimicrobial activity. The molecular geometry obtained from the X-ray structure determination was optimized Density Functional Theory (DFT/B3LYP) method with the 6-311++G(d,p) basis set in ground state. From the optimized geometry of the molecules of 3 and 4, the geometric parameters, vibrational wavenumbers and chemical shifts were computed. The optimized geometry results, which were well represented the X-ray data, were shown that the chosen of DFT/B3LYP 6-311G++(d,p) was a successful choice. After a successful optimization, frontier molecular orbitals, chemical activity, non-linear optical properties (NLO), molecular electrostatic mep (MEP), Mulliken population method, natural population analysis (NPA) and natural bond orbital analysis (NBO), which cannot be obtained experimentally, were calculated and investigated.

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.

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.

Cation Ordering in Li[NixMnxCo(1-2x)]O2-Layered Cathode Materials: A Nuclear Magnetic Resonance (NMR), Pair Distribution Function, X-ray Absorption Spectroscopy, and Electrochemical Study

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

Zeng,D.; Cabana, J.; Breger, J.

2007-01-01

Several members of the compositional series Li[NixMnxCo(1-2x)]O2 (0.01 = x = 1/3) were synthesized and characterized. X-ray diffraction results confirm the presence of the layered a-NaFeO2-type structure, while X-ray absorption near-edge spectroscopy experiments verify the presence of Ni2+, Mn4+, and Co3+. Their local environment and short-range ordering were investigated by using a combination of 6Li magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy and neutron pair distribution function (PDF) analysis, associated with reverse Monte Carlo (RMC) calculations. The 6Li MAS NMR spectra of compounds with low Ni/Mn contents (x = 0.10) show several well-resolved resonances, which start to mergemore » when the amount of Ni and Mn increases, finally forming a broad resonance at high Ni/Mn contents. Analysis of the 6Li MAS NMR 6Li[Ni0.02Mn0.02Co0.96]O2 spectrum, is consistent with the formation of Ni2+ and Mn4+ clusters within the transition-metal layers, even at these low-doping levels. The oxidation state of Ni in this high Co content sample strongly depends upon the Li/transition metal ratio of the starting materials. Neutron PDF analysis of the highest Ni/Mn content sample Li[Ni1/3Mn1/3Co1/3]O2 shows a tendency for Ni cations to be close to Mn cations in the first coordination shell; however, the Co3+ ions are randomly distributed. Analysis of the intensity of the 'LiCoO2' resonance, arising from Li surrounded by Co3+ in its first two cation coordination shells, for the whole series provides further evidence for a nonrandom distribution of the transition-metal cations. The presence of the insulator-to-metal transition seen in the electrochemical profiles of these materials upon charging correlates strongly with the concentration of the 'LiCoO2' resonance.« less

Synthesis and structural characterization by NMR and X-ray of new Morita-Baylis-Hillman adducts derived from 7-chloroquinoline

NASA Astrophysics Data System (ADS)

da Silva Caleffi, Guilherme; de Oliveira, João Paulo Gomes; da Paz Silva, Everton; Olegário, Tayná Rodrigues; Mendes, Rhuan Karlos Santos; Lima-Junior, Cláudio Gabriel; Silva, Fábio Pedrosa Lins; Martins, Felipe Terra; Vasconcellos, Mário Luiz Araújo de Almeida

2017-04-01

Herein we describe the design, synthesis and structural characterization by NMR and X-ray of new molecular hybrids 4a-4c containing the nitroaromatic MBHA moiety and the 7-chloroquinoline pharmacophores, linked by an aliphatic spacer as a model for antiprotozoal drugs. Firstly, the 4,7-dichloroquinoline (7) reacted with excess of ethylene glycol, through a SNAr promoted by t-BuOK, to prepare the 2-(7-chloroquinolin-4-yloxy)ethanol (6) in 98% isolated yield. Then, the novel 2-(7-chloroquinolin-4-yloxy) ethyl acrylate (5) was synthesized in 78% isolated yield by reacting the alcohol (6) with acryloyl chloride and TEA in CH2Cl2. Finally, this acrylate (5) reacted with o-nitrobenzaldehyde, m-nitrobenzaldehyde and p-nitrobenzaldehyde through Morita-Baylis-Hillman reactions promoted by DABCO at room temperature, using t-BuOH:Water (9:1) as solvent, to afford the new hybrids 4a-4c in 73-76% isolated yields. Three-dimensional structures were characterized in both monomeric and intermolecular packing by X-Ray experiments.

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

From single-site tantalum complexes to nanoparticles of Ta x N y and TaO x N y supported on silica: elucidation of synthesis chemistry by dynamic nuclear polarization surface enhanced NMR spectroscopy and X-ray absorption spectroscopy.

PubMed

Mohandas, Janet C; Abou-Hamad, Edy; Callens, Emmanuel; Samantaray, Manoja K; Gajan, David; Gurinov, Andrei; Ma, Tao; Ould-Chikh, Samy; Hoffman, Adam S; Gates, Bruce C; Basset, Jean-Marie

2017-08-01

Air-stable catalysts consisting of tantalum nitride nanoparticles represented as a mixture of Ta x N y and TaO x N y with diameters in the range of 0.5 to 3 nm supported on highly dehydroxylated silica were synthesized from TaMe 5 (Me = methyl) and dimeric Ta 2 (OMe) 10 with guidance by the principles of surface organometallic chemistry (SOMC). Characterization of the supported precursors and the supported nanoparticles formed from them was carried out by IR, NMR, UV-Vis, extended X-ray absorption fine structure, and X-ray photoelectron spectroscopies complemented with XRD and high-resolution TEM, with dynamic nuclear polarization surface enhanced NMR spectroscopy being especially helpful by providing enhanced intensities of the signals of 1 H, 13 C, 29 Si, and 15 N at their natural abundances. The characterization data provide details of the synthesis chemistry, including evidence of (a) O 2 insertion into Ta-CH 3 species on the support and (b) a binuclear to mononuclear transformation of species formed from Ta 2 (OMe) 10 on the support. A catalytic test reaction, cyclooctene epoxidation, was used to probe the supported nanoparticles, with 30% H 2 O 2 serving as the oxidant. The catalysts gave selectivities up to 98% for the epoxide at conversions as high as 99% with a 3.4 wt% loading of Ta present as Ta x N y /TaO x N y .

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.

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.

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

Proton transfer and hydrogen bonding in the organic solid state: a combined XRD/XPS/ssNMR study of 17 organic acid-base complexes.

PubMed

Stevens, Joanna S; Byard, Stephen J; Seaton, Colin C; Sadiq, Ghazala; Davey, Roger J; Schroeder, Sven L M

2014-01-21

The properties of nitrogen centres acting either as hydrogen-bond or Brønsted acceptors in solid molecular acid-base complexes have been probed by N 1s X-ray photoelectron spectroscopy (XPS) as well as (15)N solid-state nuclear magnetic resonance (ssNMR) spectroscopy and are interpreted with reference to local crystallographic structure information provided by X-ray diffraction (XRD). We have previously shown that the strong chemical shift of the N 1s binding energy associated with the protonation of nitrogen centres unequivocally distinguishes protonated (salt) from hydrogen-bonded (co-crystal) nitrogen species. This result is further supported by significant ssNMR shifts to low frequency, which occur with proton transfer from the acid to the base component. Generally, only minor chemical shifts occur upon co-crystal formation, unless a strong hydrogen bond is formed. CASTEP density functional theory (DFT) calculations of (15)N ssNMR isotropic chemical shifts correlate well with the experimental data, confirming that computational predictions of H-bond strengths and associated ssNMR chemical shifts allow the identification of salt and co-crystal structures (NMR crystallography). The excellent agreement between the conclusions drawn by XPS and the combined CASTEP/ssNMR investigations opens up a reliable avenue for local structure characterization in molecular systems even in the absence of crystal structure information, for example for non-crystalline or amorphous matter. The range of 17 different systems investigated in this study demonstrates the generic nature of this approach, which will be applicable to many other molecular materials in organic, physical, and materials chemistry.

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.

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.

Hydrogenation of Carbon Dioxide Catalyzed by Ruthenium Trimethylphosphine Complexes: A Mechanistic Investigation using High-Pressure NMR Spectroscopy

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

Getty, April D.; Tai, Chih-Cheng; Linehan, John C.

2009-08-26

The previously reported complex, cis-(PMe3)4RuCl(OAc) (1) acts as a catalyst for CO2 hydrogenation into formic acid in the presence of a base and an alcohol co-catalyst. NMR spectroscopy has revealed that 1 exists in solution in equilibrium with fac-(PMe3)3RuCl(h2-OAc) (2), [(PMe3)4Ru(h2-OAc)]Cl (3a), and free PMe3. Complex 2 has been isolated and characterized by elemental analysis, NMR spectroscopy, and X-ray crystallography. 2 has been tested as a CO2 hydrogenation catalyst, however, it performed poorly under the conditions of catalysis used for 1. Complex 3a can be prepared by adding certain alcohols, such as MeOH, EtOH, or o-C6H5OH, to a solution ofmore » 1 in CDCl3. The chloride ion of 3a has been exchanged for the non-coordinating anions BPh4 or B(ArF )4 (B(ArF)4 = tetrakis(3,5-bis(trifluoromethyl)phenyl)borate) to produce [(PMe3)4Ru(h2-OAc)]BPh4 (3b) and [(PMe3)4Ru(h2-OAc)]B(ArF)4 (3c). Both of these complexes have been isolated and characterized by elemental analysis, NMR spectroscopy, and in the case of 3b, X-ray crystallography. Complexes 3b and 3c perform just as well as 1 for CO2 hydrogenation to formic acid in the presence of an alcohol co-catalyst; however, 3b,c perform equally well without the added alcohol. High-pressure NMR has been used to investigate the mechanism of CO2 hydrogenation via 3a,b in the presence of base. Two of the intermediates involved have been identified as cis-(PMe3)4RuH2 (5) and cis-(PMe3)4Ru(H)O2CH (6), and the role of the base includes not only trapping of the formic acid product, but also initiation of the catalysis by aiding the conversion of 3b,c to 5.« less

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.

A Sterically Biased Unsymmetrical Azobenzene Derivative: Synthesis, Molecular Structure, and 15N NMR Spectroscopic Analysis of (E)-1-(2,6-Diisopropylphenyl)-2-phenyldiazine

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

Baumann, David O.; Erickson, Karla A.; Scott, Brian L.

We synthesized and characterized the title compound, (E)-1-(2,6-diisopropylphenyl)-2-phenyldiazine (I) using a combination of 1H, 13C, and 15N NMR spectroscopy, infrared and UV/Vis spectroscopy, X-ray crystallography, and GC mass spectrometry. The solid-state structure is also reported. The unsymmetric azobenzene crystallizes in the space group P2 1/c with unit cell parameters a = 8.001(7) Å, b = 17.827(16) Å, c = 11.129(10) Å, β = 101.960(10)°, V = 1553(2) Å 3, Z = 4, D calc = 1.139 g/cm 3.

A Sterically Biased Unsymmetrical Azobenzene Derivative: Synthesis, Molecular Structure, and 15N NMR Spectroscopic Analysis of (E)-1-(2,6-Diisopropylphenyl)-2-phenyldiazine

DOE PAGES

Baumann, David O.; Erickson, Karla A.; Scott, Brian L.; ...

2017-10-24

We synthesized and characterized the title compound, (E)-1-(2,6-diisopropylphenyl)-2-phenyldiazine (I) using a combination of 1H, 13C, and 15N NMR spectroscopy, infrared and UV/Vis spectroscopy, X-ray crystallography, and GC mass spectrometry. The solid-state structure is also reported. The unsymmetric azobenzene crystallizes in the space group P2 1/c with unit cell parameters a = 8.001(7) Å, b = 17.827(16) Å, c = 11.129(10) Å, β = 101.960(10)°, V = 1553(2) Å 3, Z = 4, D calc = 1.139 g/cm 3.

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

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

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.

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.

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.

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

Racemic crystals of trolox derivatives compared to their chiral counterparts: Structural studies using solid-state NMR, DFT calculations and X-ray diffraction

NASA Astrophysics Data System (ADS)

Wałejko, P.; Paradowska, K.; Szeleszczuk, Ł.; Wojtulewski, S.; Baj, A.

2018-03-01

Trolox C (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) is a water-soluble vitamin E analogue that is available in enantiomeric forms R or S. Enantiomerically pure Trolox 1, its derivatives 2, 3 (R and S enantiomers) and racemic forms 1-3 were studied using solid-state 13C cross-polarisation (CP) magic angle spinning (MAS) NMR (13C CPMAS NMR). Gauge-including projector-augmented wave density functional theory (GIPAW DFT) calculations of the shielding constants supported the assignment of 13C resonances in the solid-state NMR spectra. For the 13C CPMAS NMR spectra of 1, resonances of pure enantiomers were significantly broader than those of the racemic R/S form. In order to explain these effects, five of the available crystal structures were analysed (1R/S, 3R/S, 2S and the newly measured 2R/S and 3S). Cyclic dimers with one R and one S enantiomer linked by two OHsbnd Odbnd C2b hydrogen bonds were formed in 1R/S. Similar hydrogen-bonded dimers were present in 3S but not in 3R/S, in which interactions are water-mediated. A comparison of X-ray diffraction, CPMAS NMR data and the DFT GIPAW calculations of racemic forms and pure enantiomers was conducted for the first time. Our results, particularly the solid-state NMR data, were discussed in relation to Wallach's rule, that the racemic crystal appears as more ordered than its chiral counterpart.

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.

Distinguishing multiple chemotaxis Y protein conformations with laser-polarized 129Xe NMR

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

Lowery, Thomas J.; Doucleff, Michealeen; Ruiz, E. Janette

2005-02-01

The chemical shift of the {sup 129}Xe NMR signal has been shown to be extremely sensitive to the local environment around the atom and has been used to follow processes such as ligand binding by bacterial periplasmic binding proteins (Rubin et al. 2000; Lowery et al. 2004). Here we show that the {sup 129}Xe shift can sense more subtle changes: magnesium binding, BeF{sub 3}{sup -} activation, and peptide binding by the E. coli chemotaxis Y protein. {sup 1}H-{sup 15}N correlation spectroscopy and x-ray crystallography were used to identify two xenon-binding cavities in CheY that are primarily responsible for the shiftmore » changes. One site is near the active site, and the other is near the peptide binding site.« less

Synthesis, X-ray crystallography characterization, vibrational spectroscopic, molecular electrostatic potential maps, thermodynamic properties studies of N,N'-di(p-thiazole)formamidine.

PubMed

Rofouei, M K; Fereyduni, E; Sohrabi, N; Shamsipur, M; Attar Gharamaleki, J; Sundaraganesan, N

2011-01-01

In this work, we will report a combined experimental and theoretical study on molecular and vibrational structure of N,N'-di(p-thiazole)formamidine (DpTF). DpTF has been synthesized and characterized by elemental analysis, FT-IR, FT-Raman, 1H NMR, 13C NMR spectroscopy and X-ray single crystal diffraction. The FT-IR and FT-Raman spectra of DpTF were recorded in the solid phase. The optimized geometry was calculated by HF and B3LYP methods using 6-31G(d) basis set. The FT-IR and FT-Raman spectra of DpTF was calculated at the HF/B3LYP/6-31G(d) level and were interpreted in terms of potential energy distribution (PED) analysis. The scaled theoretical wavenumber showed very good agreement with the experimental values. A detailed interpretation of the infrared and Raman spectra of DpTF was reported. On the basis of vibrational analyses, the thermodynamic properties of the title compound at different temperatures have been calculated, revealing the correlations between Cp,m°, Sm°, Hm° and temperatures. Furthermore, molecular electrostatic potential maps (MESP) and total dipole moment properties of the compound have been calculated. Copyright © 2010 Elsevier B.V. All rights reserved.

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

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

Ruthenium-Nitrosyl Complexes with Glycine, l-Alanine, l-Valine, l-Proline, d-Proline, l-Serine, l-Threonine, and l-Tyrosine: Synthesis, X-ray Diffraction Structures, Spectroscopic and Electrochemical Properties, and Antiproliferative Activity

PubMed Central

2014-01-01

The reactions of [Ru(NO)Cl5]2– with glycine (Gly), l-alanine (l-Ala), l-valine (l-Val), l-proline (l-Pro), d-proline (d-Pro), l-serine (l-Ser), l-threonine (l-Thr), and l-tyrosine (l-Tyr) in n-butanol or n-propanol afforded eight new complexes (1–8) of the general formula [RuCl3(AA–H)(NO)]−, where AA = Gly, l-Ala, l-Val, l-Pro, d-Pro, l-Ser, l-Thr, and l-Tyr, respectively. The compounds were characterized by elemental analysis, electrospray ionization mass spectrometry (ESI-MS), 1H NMR, UV–visible and ATR IR spectroscopy, cyclic voltammetry, and X-ray crystallography. X-ray crystallography studies have revealed that in all cases the same isomer type (from three theoretically possible) was isolated, namely mer(Cl),trans(NO,O)-[RuCl3(AA–H)(NO)], as was also recently reported for osmium analogues with Gly, l-Pro, and d-Pro (see Z. Anorg. Allg. Chem.2013, 639, 1590–1597). Compounds 1, 4, 5, and 8 were investigated by ESI-MS with regard to their stability in aqueous solution and reactivity toward sodium ascorbate. In addition, cell culture experiments in three human cancer cell lines, namely, A549 (nonsmall cell lung carcinoma), CH1 (ovarian carcinoma), and SW480 (colon carcinoma), were performed, and the results are discussed in conjunction with the lipophilicity of compounds. PMID:24555845

Probing the calcium and sodium local environment in bones and teeth using multinuclear solid state NMR and X-ray absorption spectroscopy.

PubMed

Laurencin, Danielle; Wong, Alan; Chrzanowski, Wojciech; Knowles, Jonathan C; Qiu, Dong; Pickup, David M; Newport, Robert J; Gan, Zhehong; Duer, Melinda J; Smith, Mark E

2010-02-07

Despite the numerous studies of bone mineral, there are still many questions regarding the exact structure and composition of the mineral phase, and how the mineral crystals become organised with respect to each other and the collagen matrix. Bone mineral is commonly formulated as hydroxyapatite, albeit with numerous substitutions, and has previously been studied by (31)P and (1)H NMR, which has given considerable insight into the complexity of the mineral structure. However, to date, there has been no report of an NMR investigation of the other major component of bone mineral, calcium, nor of common minority cations like sodium. Here, direct analysis of the local environment of calcium in two biological apatites, equine bone (HB) and bovine tooth (CT), was carried out using both (43)Ca solid state NMR and Ca K-edge X-ray absorption spectroscopy, revealing important structural information about the calcium coordination shell. The (43)Ca delta(iso) in HB and CT is found to correlate with the average Ca-O bond distance measured by Ca K-edge EXAFS, and the (43)Ca NMR linewidths show that there is a greater distribution in chemical bonding around calcium in HB and CT, compared to synthetic apatites. In the case of sodium, (23)Na MAS NMR, high resolution 3Q-MAS NMR, as well as (23)Na{(31)P} REDOR and (1)H{(23)Na} R(3)-HMQC correlation experiments give the first direct evidence that some sodium is located inside the apatite phase in HB and CT, but with a greater distribution of environments compared to a synthetic sodium substituted apatite (Na-HA).

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.

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.

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.

Recent developments in structural proteomics for protein structure determination.

PubMed

Liu, Hsuan-Liang; Hsu, Jyh-Ping

2005-05-01

The major challenges in structural proteomics include identifying all the proteins on the genome-wide scale, determining their structure-function relationships, and outlining the precise three-dimensional structures of the proteins. Protein structures are typically determined by experimental approaches such as X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. However, the knowledge of three-dimensional space by these techniques is still limited. Thus, computational methods such as comparative and de novo approaches and molecular dynamic simulations are intensively used as alternative tools to predict the three-dimensional structures and dynamic behavior of proteins. This review summarizes recent developments in structural proteomics for protein structure determination; including instrumental methods such as X-ray crystallography and NMR spectroscopy, and computational methods such as comparative and de novo structure prediction and molecular dynamics simulations.

Difference in the structures of alanine tri- and tetra-peptides with antiparallel β-sheet assessed by X-ray diffraction, solid-state NMR and chemical shift calculations by GIPAW.

PubMed

Asakura, Tetsuo; Yazawa, Koji; Horiguchi, Kumiko; Suzuki, Furitsu; Nishiyama, Yusuke; Nishimura, Katsuyuki; Kaji, Hironori

2014-01-01

Alanine oligomers provide a key structure for silk fibers from spider and wild silkworms.We report on structural analysis of L-alanyl-L-alanyl-L-alanyl-L-alanine (Ala)4 with anti-parallel (AP) β-structures using X-ray and solid-state NMR. All of the Ala residues in the (Ala)4 are in equivalent positions, whereas for alanine trimer (Ala)3 there are two alternative locations in a unit cell as reported previously (Fawcett and Camerman, Acta Cryst., 1975, 31, 658-665). (Ala)4 with AP β-structure is more stable than AP-(Ala)3 due to formation of the stronger hydrogen bonds. The intermolecular structure of (Ala)4 is also different from polyalanine fiber structure, indicating that the interchain arrangement of AP β-structure changes with increasing alanine sequencelength. Furthermore the precise (1)H positions, which are usually inaccesible by X-ray diffraction method, are determined by high resolution (1)H solid state NMR combined with the chemical shift calculations by the gauge-including projector augmented wave method. Copyright © 2013 Wiley Periodicals, Inc.

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.

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

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.

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

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.

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.

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.

High-Throughput Screening by Nuclear Magnetic Resonance (HTS by NMR) for the Identification of PPIs Antagonists.

PubMed

Wu, Bainan; Barile, Elisa; De, Surya K; Wei, Jun; Purves, Angela; Pellecchia, Maurizio

2015-01-01

In recent years the ever so complex field of drug discovery has embraced novel design strategies based on biophysical fragment screening (fragment-based drug design; FBDD) using nuclear magnetic resonance spectroscopy (NMR) and/or structure-guided approaches, most often using X-ray crystallography and computer modeling. Experience from recent years unveiled that these methods are more effective and less prone to artifacts compared to biochemical high-throughput screening (HTS) of large collection of compounds in designing protein inhibitors. Hence these strategies are increasingly becoming the most utilized in the modern pharmaceutical industry. Nonetheless, there is still an impending need to develop innovative and effective strategies to tackle other more challenging targets such as those involving protein-protein interactions (PPIs). While HTS strategies notoriously fail to identify viable hits against such targets, few successful examples of PPIs antagonists derived by FBDD strategies exist. Recently, we reported on a new strategy that combines some of the basic principles of fragment-based screening with combinatorial chemistry and NMR-based screening. The approach, termed HTS by NMR, combines the advantages of combinatorial chemistry and NMR-based screening to rapidly and unambiguously identify bona fide inhibitors of PPIs. This review will reiterate the critical aspects of the approach with examples of possible applications.

High-throughput screening by Nuclear Magnetic Resonance (HTS by NMR) for the identification of PPIs antagonists

PubMed Central

Wu, Bainan; Barile, Elisa; De, Surya K.; Wei, Jun; Purves, Angela; Pellecchia, Maurizio

2015-01-01

In recent years the ever so complex field of drug discovery has embraced novel design strategies based on biophysical fragment screening (fragment-based drug design; FBDD) using nuclear magnetic resonance spectroscopy (NMR) and/or structure-guided approaches, most often using X-ray crystallography and computer modeling. Experience from recent years unveiled that these methods are more effective and less prone to artifacts compared to biochemical high-throughput screening (HTS) of large collection of compounds in designing protein inhibitors. Hence these strategies are increasingly becoming the most utilized in the modern pharmaceutical industry. Nonetheless, there is still an impending need to develop innovative and effective strategies to tackle other more challenging targets such as those involving protein-protein interactions (PPIs). While HTS strategies notoriously fail to identify viable hits against such targets, few successful examples of PPIs antagonists derived by FBDD strategies exist. Recently, we reported on a new strategy that combines some of the basic principles of fragment-based screening with combinatorial chemistry and NMR-based screening. The approach, termed HTS by NMR, combines the advantages of combinatorial chemistry and NMR-based screening to rapidly and unambiguously identify bona fide inhibitors of PPIs. This review will reiterate the critical aspects of the approach with examples of possible applications. PMID:25986689

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.

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

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

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.

Characterizing phosphorus speciation of Chesapeake Bay sediments using chemical extraction, 31P NMR, and X-ray absorption fine structure spectroscopy.

PubMed

Li, Wei; Joshi, Sunendra R; Hou, Guangjin; Burdige, David J; Sparks, Donald L; Jaisi, Deb P

2015-01-06

Nutrient contamination has been one of the lingering issues in the Chesapeake Bay because the bay restoration is complicated by temporally and seasonally variable nutrient sources and complex interaction between imported and regenerated nutrients. Differential reactivity of sedimentary phosphorus (P) pools in response to imposed biogeochemical conditions can record past sediment history and therefore a detailed sediment P speciation may provide information on P cycling particularly the stability of a P pool and the formation of one pool at the expense of another. This study examined sediment P speciation from three sites in the Chesapeake Bay: (i) a North site in the upstream bay, (ii) a middle site in the central bay dominated by seasonally hypoxic bottom water, and (iii) a South site at the bay-ocean boundary using a combination of sequential P extraction (SEDEX) and spectroscopic techniques, including (31)P NMR, P X-ray absorption near edge structure spectroscopy (XANES), and Fe extended X-ray absorption fine structure (EXAFS). Results from sequential P extraction reveal that sediment P is composed predominantly of ferric Fe-bound P and authigenic P, which was further confirmed by solid-state (31)P NMR, XANES, and EXAFS analyses. Additionally, solution (31)P NMR results show that the sediments from the middle site contain high amounts of organic P such as monoesters and diesters, compared to the other two sites, but that these compounds rapidly decrease with sediment depth indicating remineralized P could have precipitated as authigenic P. Fe EXAFS enabled to identify the changes in Fe mineral composition and P sinks in response to imposed redox condition in the middle site sediments. The presence of lepidocrocite, vermiculite, and Fe smectite in the middle site sediments indicates that some ferric Fe minerals can still be present along with pyrite and vivianite, and that ferric Fe-bound P pool can be a major P sink in anoxic sediments. These results provide

Host-Guest Interaction of Cucurbit[8]uril with N-(3-Aminopropyl)cyclohexylamine: Cyclohexyl Encapsulation Triggered Ternary Complex.

PubMed

Xia, Yu; Wang, Chuan-Zeng; Tian, Mengkui; Tao, Zhu; Ni, Xin-Long; Prior, Timothy J; Redshaw, Carl

2018-01-15

The host-guest interaction of a series of cyclohexyl-appended guests with cucurbit[8]uril (Q[8]) was studied by ¹H NMR spectroscopy, isothermal titration calorimetry (ITC), and X-ray crystallography. The X-ray structure revealed that two cycloalkane moieties can be simultaneously encapsulated in the hydrophobic cavity of the Q[8] host to form a ternary complex for the first time.

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

Chevrel-phase solid solution Mo 6Se 8- xTe x. Study of its superconducting, magnetic and NMR properties

NASA Astrophysics Data System (ADS)

Hamard1a, C.; Auffret, V.; Peña, O.; Le Floch, M.; Nowak, B.; Wojakowski, A.

2000-09-01

The Chevrel-phase solid solution Mo 6Se 8-Mo 6Te 8 was studied by X-ray diffraction, AC and DC magnetic susceptibility and 77Se and 125Te NMR spectroscopy. From the smooth evolution of the lattice parameters and superconducting critical temperatures, a progressive substitution of selenium atoms by tellurium is shown, on the whole range of composition 0⩽ x⩽8, in the formulation Mo 6Se 8- xTe x: the unit-cell volume increases linearly because of the larger ionic size of tellurium, while Tc decreases rapidly (from 6.45 down to 0 K) because of the different formal oxidation states of the anions and a probable evolution of the Fermi level in the density of states. Results of magnetic susceptibility support this model and suggest the inhibition of the intrinsic metallic behavior with increasing x. The NMR spectra of the binaries Mo 6Se 8 and Mo 6Te 8 reveal two significant features, attributed to two different chalcogen positions in the R 3¯ symmetry. At low Se contents in Mo 6Se 8- xTe x ( x=7.5, 7 and 6), selenium first fills the two X(2) sites along the three-fold axis (2c positions), and then it becomes statistically distributed over the general 6f positions, leading to broad 77Se NMR lines. On the other hand, substitution of Te atoms in Mo 6Se 8 seems to occur in a random way, creating large perturbations on the 125Te NMR spectra, over the whole range of x. Theoretical analysis based on the presence of two anisotropic lines (of axial and non-axial symmetries, respectively) allowed us to estimate their anisotropy factors and to perfectly simulate the frequency response of both Mo 6Se 8 and Mo 6Te 8 binaries. Analysis of the Knight shift anisotropy leads us to conclude about the importance of the molybdenum z 2 molecular orbital contribution which controls the Mo-X dipolar interactions.

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

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.

Exo-pi-bonding to an ortho-carborane hypercarbon atom: systematic icosahedral cage distortions reflected in the structures of the fluoro-, hydroxy- and amino-carboranes, 1-X-2-Ph-1,2-C2B10H10 (X=F, OH or NH2) and related anions.

PubMed

Boyd, Lynn A; Clegg, William; Copley, Royston C B; Davidson, Matthew G; Fox, Mark A; Hibbert, Thomas G; Howard, Judith A K; Mackinnon, Angus; Peace, Richard J; Wade, Kenneth

2004-09-07

The structures of derivatives of phenyl-ortho-carborane bearing on the second cage hypercarbon atom a pi-donor substituent (F, OH, O-, NH2, NH- and CH2-) were investigated by NMR, X-ray crystallography and computational studies. The molecular structures of these compounds, notably their cage C1-C2 distances and the orientations of their pi-donor substituents (OH, NH2, NH- and CH2-) show remarkable and systematic variations with the degree of exo pi-bonding, which varies as expected with the pi-donor characteristics of the substituent.

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

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

Efficient ultrasound-assisted synthesis, spectroscopic, crystallographic and biological investigations of pyrazole-appended quinolinyl chalcones

NASA Astrophysics Data System (ADS)

Prasath, R.; Bhavana, P.; Sarveswari, S.; Ng, Seik Weng; Tiekink, Edward R. T.

2015-02-01

Two series of new quinolinyl chalcones containing a pyrazole group, 3a-f and 4a-r, have been synthesized by Claisen-Schmidt condensation of the derivatives of 2-methyl-3-acetylquinoline with either substituted 1,3-diphenyl-1H-pyrazole-4-carbaldehyde or 5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde in 76-93% yield under ultrasonic method. The compounds were characterized using IR, 1H NMR and ESI-MS spectroscopic methods and, for representative compounds, by X-ray crystallography. An E-configuration about the Cdbnd C ethylene bond has been established via 1H NMR spectroscopy and X-ray crystallography. These compounds show promising anti-microbial properties, with 4a and 3e being the most potent against bacterial and fungal strains, respectively and the methoxy substituted compounds showed moderate anti-oxidant activity.

Integrative, Dynamic Structural Biology at Atomic Resolution—It’s About Time

PubMed Central

van den Bedem, Henry; Fraser, James S.

2015-01-01

Biomolecules adopt a dynamic ensemble of conformations, each with the potential to interact with binding partners or perform the chemical reactions required for a multitude of cellular functions. Recent advances in X-ray crystallography, Nuclear Magnetic Resonance (NMR) spectroscopy, and other techniques are helping us realize the dream of seeing—in atomic detail—how different parts of biomolecules exchange between functional sub-states using concerted motions. Integrative structural biology has advanced our understanding of the formation of large macromolecular complexes and how their components interact in assemblies by leveraging data from many low-resolution methods. Here, we review the growing opportunities for integrative, dynamic structural biology at the atomic scale, contending there is increasing synergistic potential between X-ray crystallography, NMR, and computer simulations to reveal a structural basis for protein conformational dynamics at high resolution. PMID:25825836

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

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

Measuring the lactate-to-creatine ratio via 1H NMR spectroscopy can be used to noninvasively evaluate apoptosis in glioma cells after X-ray irradiation.

PubMed

Li, Hongxia; Cui, Yi; Li, Fuyan; Shi, Wenqi; Gao, Wenjing; Wang, Xiao; Zeng, Qingshi

2018-01-01

Radiotherapy is among the commonly applied treatment options for glioma, which is one of the most common types of primary brain tumor. To evaluate the effect of radiotherapy noninvasively, it is vital for oncologists to monitor the effects of X-ray irradiation on glioma cells. Preliminary research had showed that PKC-ι expression correlates with tumor cell apoptosis induced by X-ray irradiation. It is also believed that the lactate-to-creatine (Lac/Cr) ratio can be used as a biomarker to evaluate apoptosis in glioma cells after X-ray irradiation. In this study, we evaluated the relationships between the Lac/Cr ratio, apoptotic rate, and protein kinase C iota (PKC-ι) expression in glioma cells. Cells of the glioma cell lines C6 and U251 were randomly divided into 4 groups, with every group exposed to X-ray irradiation at 0, 1, 5, 10 and 15 Gy. Single cell gel electrophoresis (SCGE) was conducted to evaluate the DNA damage. Flow cytometry was performed to measure the cell cycle blockage and apoptotic rates. Western blot analysis was used to detect the phosphorylated PKC-ι (p-PKC-ι) level. 1 H NMR spectroscopy was employed to determine the Lac/Cr ratio. The DNA damage increased in a radiation dose-dependent manner ( p  < 0.05). With the increase in X-ray irradiation, the apoptotic rate also increased (C6, p  < 0.01; U251, p  < 0.05), and the p-PKC-ι level decreased (C6, p  < 0.01; U251, p  < 0.05). The p-PKC-ι level negatively correlated with apoptosis, whereas the Lac/Cr ratio positively correlated with the p-PKC-ι level. The Lac/Cr ratio decreases with an increase in X-ray irradiation and thus can be used as a biomarker to reflect the effects of X-ray irradiation in glioma cells.

NMR crystallography to probe the breathing effect of the MIL-53(Al) metal-organic framework using solid-state NMR measurements of 13C-27Al distances.

PubMed

Giovine, Raynald; Volkringer, Christophe; Trébosc, Julien; Amoureux, Jean Paul; Loiseau, Thierry; Lafon, Olivier; Pourpoint, Frédérique

2017-03-01

The metal-organic framework MIL-53(Al) (aluminium terephthalate) exhibits a structural transition between two porous structures with large pore (lp) or narrow pore (np) configurations. This transition, called the breathing effect, is observed upon changes in temperature or external pressure, as well as with the adsorption of guest molecules, such as H 2 O, within the pores. We show here how these different pore openings can be detected by observing the dephasing of 13 C magnetization under 13 C- 27 Al dipolar couplings using Rotational-Echo Saturation-Pulse Double-Resonance (RESPDOR) solid-state NMR experiments with Simultaneous Frequency and Amplitude Modulation (SFAM) recoupling. These double-resonance NMR experiments between 13 C and 27 Al nuclei, which have close Larmor frequencies, are feasible thanks to the use of a frequency splitter. The experimental SFAM-RESPDOR signal fractions agree well with those simulated from the MIL-53(Al)-lp and -np crystal structures obtained from powder X-ray diffraction analysis. Hence, these 13 C- 27 Al solid-state NMR experiments validate these structures and confirm their rigidity. A similar agreement is reported for the framework ligands in the as-synthesized (as) MIL-53(Al), in which the pores contain free ligands. Furthermore, in this case, 13 C-{ 27 Al} SFAM-RESPDOR experiments allow an estimation of the average distance between the free ligands and the 27 Al nuclei of the framework.

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.

Detection and characterization of serine and threonine hydroxyl protons in Bacillus circulans xylanase by NMR spectroscopy.

PubMed

Brockerman, Jacob A; Okon, Mark; McIntosh, Lawrence P

2014-01-01

Hydroxyl protons on serine and threonine residues are not well characterized in protein structures determined by both NMR spectroscopy and X-ray crystallography. In the case of NMR spectroscopy, this is in large part because hydroxyl proton signals are usually hidden under crowded regions of (1)H-NMR spectra and remain undetected by conventional heteronuclear correlation approaches that rely on strong one-bond (1)H-(15)N or (1)H-(13)C couplings. However, by filtering against protons directly bonded to (13)C or (15)N nuclei, signals from slowly-exchanging hydroxyls can be observed in the (1)H-NMR spectrum of a uniformly (13)C/(15)N-labeled protein. Here we demonstrate the use of a simple selective labeling scheme in combination with long-range heteronuclear scalar correlation experiments as an easy and relatively inexpensive way to detect and assign these hydroxyl proton signals. Using auxtrophic Escherichia coli strains, we produced Bacillus circulans xylanase (BcX) labeled with (13)C/(15)N-serine or (13)C/(15)N-threonine. Signals from two serine and three threonine hydroxyls in these protein samples were readily observed via (3)JC-OH couplings in long-range (13)C-HSQC spectra. These scalar couplings (~5-7 Hz) were measured in a sample of uniformly (13)C/(15)N-labeled BcX using a quantitative (13)C/(15)N-filtered spin-echo difference experiment. In a similar approach, the threonine and serine hydroxyl hydrogen exchange kinetics were measured using a (13)C/(15)N-filtered CLEANEX-PM pulse sequence. Collectively, these experiments provide insights into the structural and dynamic properties of several serine and threonine hydroxyls within this model protein.

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

Does Z' equal 1 or 2? Enhanced powder NMR crystallography verification of a disordered room temperature crystal structure of a p38 inhibitor for chronic obstructive pulmonary disease.

PubMed

Widdifield, Cory M; Nilsson Lill, Sten O; Broo, Anders; Lindkvist, Maria; Pettersen, Anna; Svensk Ankarberg, Anna; Aldred, Peter; Schantz, Staffan; Emsley, Lyndon

2017-06-28

The crystal structure of the Form A polymorph of N-cyclopropyl-3-fluoro-4-methyl-5-[3-[[1-[2-[2-(methylamino)ethoxy]phenyl]cyclopropyl]amino]-2-oxo-pyrazin-1-yl]benzamide (i.e., AZD7624), determined using single-crystal X-ray diffraction (scXRD) at 100 K, contains two molecules in the asymmetric unit (Z' = 2) and has regions of local static disorder. This substance has been in phase IIa drug development trials for the treatment of chronic obstructive pulmonary disease, a disease which affects over 300 million people and contributes to nearly 3 million deaths annually. While attempting to verify the crystal structure using nuclear magnetic resonance crystallography (NMRX), we measured 13 C solid-state NMR (SSNMR) spectra at 295 K that appeared consistent with Z' = 1 rather than Z' = 2. To understand this surprising observation, we used multinuclear SSNMR ( 1 H, 13 C, 15 N), gauge-including projector augmented-wave density functional theory (GIPAW DFT) calculations, crystal structure prediction (CSP), and powder XRD (pXRD) to determine the room temperature crystal structure. Due to the large size of AZD7624 (ca. 500 amu, 54 distinct 13 C environments for Z' = 2), static disorder at 100 K, and (as we show) dynamic disorder at ambient temperatures, NMR spectral assignment was a challenge. We introduce a method to enhance confidence in NMR assignments by comparing experimental 13 C isotropic chemical shifts against site-specific DFT-calculated shift distributions established using CSP-generated crystal structures. The assignment and room temperature NMRX structure determination process also included measurements of 13 C shift tensors and the observation of residual dipolar coupling between 13 C and 14 N. CSP generated ca. 90 reasonable candidate structures (Z' = 1 and Z' = 2), which when coupled with GIPAW DFT results, room temperature pXRD, and the assigned SSNMR data, establish Z' = 2 at room temperature. We find that the polymorphic Form A of AZD7624 is maintained at

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.

Reliable resonance assignments of selected residues of proteins with known structure based on empirical NMR chemical shift prediction

NASA Astrophysics Data System (ADS)

Li, Da-Wei; Meng, Dan; Brüschweiler, Rafael

2015-05-01

A robust NMR resonance assignment method is introduced for proteins whose 3D structure has previously been determined by X-ray crystallography. The goal of the method is to obtain a subset of correct assignments from a parsimonious set of 3D NMR experiments of 15N, 13C labeled proteins. Chemical shifts of sequential residue pairs are predicted from static protein structures using PPM_One, which are then compared with the corresponding experimental shifts. Globally optimized weighted matching identifies the assignments that are robust with respect to small changes in NMR cross-peak positions. The method, termed PASSPORT, is demonstrated for 4 proteins with 100-250 amino acids using 3D NHCA and a 3D CBCA(CO)NH experiments as input producing correct assignments with high reliability for 22% of the residues. The method, which works best for Gly, Ala, Ser, and Thr residues, provides assignments that serve as anchor points for additional assignments by both manual and semi-automated methods or they can be directly used for further studies, e.g. on ligand binding, protein dynamics, or post-translational modification, such as phosphorylation.

Reliable Resonance Assignments of Selected Residues of Proteins with Known Structure Based on Empirical NMR Chemical Shift Prediction

PubMed Central

Li, Da-Wei; Meng, Dan; Brüschweiler, Rafael

2015-01-01

A robust NMR resonance assignment method is introduced for proteins whose 3D structure has previously been determined by X-ray crystallography. The goal of the method is to obtain a subset of correct assignments from a parsimonious set of 3D NMR experiments of 15N, 13C labeled proteins. Chemical shifts of sequential residue pairs are predicted from static protein structures using PPM_One, which are then compared with the corresponding experimental shifts. Globally optimized weighted matching identifies the assignments that are robust with respect to small changes in NMR cross-peak positions. The method, termed PASSPORT, is demonstrated for 4 proteins with 100 – 250 amino acids using 3D NHCA and a 3D CBCA(CO)NH experiments as input producing correct assignments with high reliability for 22% of the residues. The method, which works best for Gly, Ala, Ser, and Thr residues, provides assignments that serve as anchor points for additional assignments by both manual and semi-automated methods or they can be directly used for further studies, e.g. on ligand binding, protein dynamics, or post-translational modification, such as phosphorylation. PMID:25863893

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

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

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

A Study of the Structure-Activity Relationship of GABAA-Benzodiazepine Receptor Bivalent Ligands by Conformational Analysis with Low Temperature NMR and X-ray Analysis

PubMed Central

Han, Dongmei; Försterling, F. Holger; Li, Xiaoyan; Deschamps, Jeffrey R.; Parrish, Damon; Cao, Hui; Rallapalli, Sundari; Clayton, Terry; Teng, Yun; Majumder, Samarpan; Sankar, Subramaniam; Roth, Bryan L.; Sieghart, Werner; Furtmuller, Roman; Rowlett, James; Weed, Mike R.; Cook, James M.

2013-01-01

The stable conformations of GABAA-benzodiazepine receptor bivalent ligands were determined by low temperature NMR spectroscopy and confirmed by single crystal X-ray analysis. The stable conformations in solution correlated well with those in the solid state. The linear conformation was important for these dimers to access the binding site and exhibit potent in vitro affinity and was illustrated for α5 subtype selective ligands. Bivalent ligands with an oxygen-containing linker folded back upon themselves both in solution and the solid state. Dimers which are folded do not bind to Bz receptors. PMID:18790643

Organobase catalyzed 1,4-conjugate addition of 4-hydroxycoumarin on chalcones: Synthesis, NMR and single-crystal X-ray diffraction studies of novel warfarin analogues

NASA Astrophysics Data System (ADS)

Talhi, Oualid; Fernandes, José A.; Pinto, Diana C. G. A.; Almeida Paz, Filipe A.; Silva, Artur M. S.

2015-08-01

The synthesis of a new series of warfarin analogues by convenient organobase catalyzed 1,4-conjugate addition of 4-hydroxycoumarin to chalcone derivatives is described. 1H NMR spectroscopy evidenced the presence of a predominant acyclic open-form together with the cyclic hemiketal tautomers of the resulting Michael adducts. The acyclic open-form has been unequivocally proved by single-crystal X-ray diffraction analysis. The use of the B ring ortho-hydroxychalcone synthons in this reaction has led to a diastereoselective synthesis of warfarin bicyclo[3.3.1]nonane ketal derivatives.

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

Structure of shock compressed model basaltic glass: Insights from O K-edge X-ray Raman scattering and high-resolution 27Al NMR spectroscopy

NASA Astrophysics Data System (ADS)

Lee, Sung Keun; Park, Sun Young; Kim, Hyo-Im; Tschauner, Oliver; Asimow, Paul; Bai, Ligang; Xiao, Yuming; Chow, Paul

2012-03-01

The detailed atomic structures of shock compressed basaltic glasses are not well understood. Here, we explore the structures of shock compressed silicate glass with a diopside-anorthite eutectic composition (Di64An36), a common Fe-free model basaltic composition, using oxygen K-edge X-ray Raman scattering and high- resolution 27Al solid-state NMR spectroscopy and report previously unknown details of shock-induced changes in the atomic configurations. A topologically driven densification of the Di64An36 glass is indicated by the increase in oxygen K-edge energy for the glass upon shock compression. The first experimental evidence of the increase in the fraction of highly coordinated Al in shock compressed glass is found in the 27Al NMR spectra. This unambiguous evidence of shock-induced changes in Al coordination environments provides atomistic insights into shock compression in basaltic glasses and allows us to microscopically constrain the magnitude of impact events or relevant processes involving natural basalts on Earth and planetary surfaces.

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.

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.

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

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.

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.

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.

Water Structure and Dynamics in Smectites: X-ray Diffraction and 2 H NMR Spectroscopy of Mg–, Ca–, Sr–, Na–, Cs–, and Pb–Hectorite

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

Reddy, U. Venkateswara; Bowers, Geoffrey M.; Loganathan, Narasimhan

2016-04-06

Variable-temperature X-ray diffraction and 2H NMR spectroscopy of the smectite mineral, hectorite, containing interlayer Na +, K +, Cs +, Mg 2+, Ca 2+, Sr 2+, and Pb 2+ equilibrated at 43% relative humidity (RH) and mixed with 2H 2O to form a paste provide a comprehensive picture of the structural environments and dynamics of interlayer 2H 2O and the relationships of these properties to interlayer hydration state, the hydration energy and polarizability of the cation, temperature, and the formation of ice-1h in the interparticle pores. The variation in basal spacing shown by the XRD data correlates well with themore » 2H NMR behavior, and the XRD data show for the first time in hectorites that crystallization of interparticle ice-1h causes a decrease in the interlayer spacing, likely due to removal of interlayer 2H 2O. The variation of the 2H NMR behavior of all the samples with decreasing temperature reflects decreasing frequencies of motion for the rotation of the 2H 2O molecules around their dipoles, reorientation of the 2H 2O molecules, and exchange of the 2H 2O molecules between interlayer sites coordinated to and not coordinated to the cations.« less

Cadmium (II) macrocyclic Schiff-base complexes containing piperazine moiety: Synthesis, spectroscopic, X-ray structure, theoretical and antibacterial studies

NASA Astrophysics Data System (ADS)

Keypour, Hassan; Mahmoudabadi, Masoumeh; Shooshtari, Amir; Bayat, Mehdi; Mohsenzadeh, Fariba; Gable, Robert William

2018-03-01

The new Cd(II) macrocyclic Schiff-base complexes were prepared via the metal templated [1 + 1] cyclocondensation of 2,2'-(piperazine-1,4-diylbis (methylene))dianiline (A) and 2,6-pyridinedicarbaldehyde or 2,6-diacetylpyridine. The products were characterized by elemental analysis, mass spectrometry and spectroscopic methods such as: FT-IR, 1H and 13C-NMR, the crystal structure of [CdL1(ClO4)2](CH3CN) (1) complex was also obtained by single-crystal X-ray crystallography. The complexes were tested for in vitro antibacterial properties against some bacteria. The complexes had antibacterial properties and in some cases were active even more than standards. The geometries of the [CdLn (ClO4)2], (n = 1,2) complexes have been optimized at the BP86/def2-SVP level of theory. Also the nature of Cd←Ln (n = 1, 2) bonds in [CdLn (ClO4)2], (n = 1,2) complexes are studied with the help of NBO and Energy decomposition analysis (EDA). Results showed that the nature of metal-ligand bond in the complexes is slightly more electrostatic with a contribution of about 52% in total interaction energy.

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.

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).

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

Obtaining aluminas from the thermal decomposition of their different precursors: An {sup 27}Al MAS NMR and X-ray powder diffraction studies

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

Chagas, L.H.; De Carvalho, G.S.G.; San Gil, R.A.S.

2014-01-01

Graphical abstract: - Highlights: • We synthesized three precursors of alumina from different methods. • The calcination of the precursors generated several alumina polymorphs. • XRD and NMR were used for structural investigation of the polymorphs. • The synthesis route determines the structural and textural properties of the solids. - Abstract: A commercial sample of Boehmite was used as precursor of alumina polymorphs. For comparison, three other precursors were synthesized from different methods. Particularly, the use of excess of urea promoted a very crystalline form of basic aluminum carbonate. The characteristics of the four precursors were investigated by thermal, vibrationalmore » and X-ray powder diffraction (XRD) analysis. Additionally, the nuclear magnetic resonance, with magic angle spinning ({sup 27}Al MAS NMR), was used to verify the coordination of aluminum cations. Each precursor was calcined at various temperatures generating alumina polymorphs, which were structurally analyzed by XRD and {sup 27}Al MAS NMR. Due to interest in catalysis supports, special attention was given to the γ-Al{sub 2}O{sub 3} phase, which in addition to structural investigation was subjected to textural analysis. The results showed that, from different synthesis procedures and common route of calcination, one can obtain materials with the same composition but with different structural and textural properties, which in turn can significantly influence the performance of a supported catalyst.« less

"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

Synthesis, NMR, FT-IR, X-ray structural characterization, DFT analysis and isomerism aspects of 5-(2,6-dichlorobenzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione.

PubMed

Barakat, Assem; Al-Najjar, Hany J; Al-Majid, Abdullah Mohammed; Soliman, Saied M; Mabkhot, Yahia Nasser; Shaik, Mohammed Rafi; Ghabbour, Hazem A; Fun, Hoong-Kun

2015-08-05

The synthesis and spectral characterization of the 5-(2,6-dichlorobenzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione;3 was reported. The solid state molecular structure of 3 was studied using X-ray crystallography. The relative stabilities of the seven possible isomers of 3 were calculated by DFT/B3LYP method using 6-311 G(d,p) basis set. The calculated total energies and thermodynamic parameters were used to predict the relative stabilities of these isomers. The effect of solvent polarity on the relative stability of these isomers was studied at the same level of theory using PCM. It was found that the keto form, (T0), is the most stable isomer both in the gaseous state and solution. In solution, the calculated total energies of all isomers are decreased indicating that all isomers are stabilized by the solvent effect. The vibrational spectra of the most stable isomer, 3(T0) are calculated using the same level of theory and the results are compared with the experimentally measured FTIR spectra. Good correlation was obtained between the experimental and calculated vibrational frequencies (R(2)=0.9992). The electronic spectra of 3(T0) in gas phase as well as in solutions were calculated using the TD-DFT method. All the predicted electronic transitions showed very little spectral shifts and increase in the intensity of absorption due to solvent effect. Also the (1)H- and (13)C-NMR chemical shifts of the stable isomer were calculated and the results were correlated with the experimental data. Good correlations between the experimental and calculated chemical shifts were obtained. Copyright © 2015 Elsevier B.V. All rights reserved.

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 ...

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

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.

Determination of NMR chemical shifts for cholesterol crystals from first-principles

NASA Astrophysics Data System (ADS)

Kucukbenli, Emine; de Gironcoli, Stefano

2011-03-01

Solid State Nuclear Magnetic Resonance (NMR) is a powerful tool in crystallography when combined with theoretical predictions. So far, empirical calculations of spectra have been employed for an unambiguous identification. However, many complex systems are outside the scope of these methods. Our implementation of ultrasoft and projector augmented wave pseudopotentials within ab initio gauge including projector augmented plane wave (GIPAW) method in Quantum Espresso simulation package allows affordable calculations of NMR spectra for systems of thousands of electrons. We report here the first ab initio determination of NMR spectra for several crystal structures of cholesterol. Cholesterol crystals, the main component of human gallstones, are of interest to medical research as their structural properties can shed light on the pathologies of gallbladder. With our application we show that ab initio calculations can be employed to aid NMR crystallography.

Micro-scale NMR Experiments for Monitoring the Optimization of Membrane Protein Solutions for Structural Biology.

PubMed

Horst, Reto; Wüthrich, Kurt

2015-07-20

Reconstitution of integral membrane proteins (IMP) in aqueous solutions of detergent micelles has been extensively used in structural biology, using either X-ray crystallography or NMR in solution. Further progress could be achieved by establishing a rational basis for the selection of detergent and buffer conditions, since the stringent bottleneck that slows down the structural biology of IMPs is the preparation of diffracting crystals or concentrated solutions of stable isotope labeled IMPs. Here, we describe procedures to monitor the quality of aqueous solutions of [ 2 H, 15 N]-labeled IMPs reconstituted in detergent micelles. This approach has been developed for studies of β-barrel IMPs, where it was successfully applied for numerous NMR structure determinations, and it has also been adapted for use with α-helical IMPs, in particular GPCRs, in guiding crystallization trials and optimizing samples for NMR studies (Horst et al ., 2013). 2D [ 15 N, 1 H]-correlation maps are used as "fingerprints" to assess the foldedness of the IMP in solution. For promising samples, these "inexpensive" data are then supplemented with measurements of the translational and rotational diffusion coefficients, which give information on the shape and size of the IMP/detergent mixed micelles. Using microcoil equipment for these NMR experiments enables data collection with only micrograms of protein and detergent. This makes serial screens of variable solution conditions viable, enabling the optimization of parameters such as the detergent concentration, sample temperature, pH and the composition of the buffer.

Cd(II) and Zn(II) complexes of two new hexadentate Schiff base ligands derived from different aldehydes and ethanol amine; X-ray crystal structure, IR and NMR spectroscopy studies

NASA Astrophysics Data System (ADS)

Golbedaghi, Reza; Rezaeivala, Majid; Albeheshti, Leila

2014-11-01

Four new [Cd(H2L1)(NO3)]ClO4 (1), [Zn(H2L1)](ClO4)2 (2), [Cd(H2L2)(NO3)]ClO4 (3), and [Zn(H2L2)](ClO4)2 (4), complexes were prepared by the reaction of two new Schiff base ligands and Cd(II) and Zn(II) metal ions in equimolar ratios. The ligands H2L1 and H2L2 were synthesized by reaction of 2-[2-(2-formyl phenoxy)ethoxy]benzaldehyde and/or 2-[2-(3-formyl phenoxy)propoxy]benzaldehyde and ethanol amine and characterized by IR, 1H and 13C NMR spectroscopy. All complexes were characterized by IR, 1H and 13C NMR, COSY, and elemental analysis. Also, the complex 1 was characterized by X-ray in addition to the above methods. The X-ray crystal structure of compound 1 showed that all nitrogen and oxygen atoms of ligand (N2O4) and a molecule of nitrate with two donor oxygen atom have been coordinated to the metal ion and the Cd(II) ion is in an eight-coordinate environment that is best described as a distorted dodecahedron geometry.

NMR-based platform for fragment-based lead discovery used in screening BRD4-targeted compounds

PubMed Central

Yu, Jun-lan; Chen, Tian-tian; Zhou, Chen; Lian, Fu-lin; Tang, Xu-long; Wen, Yi; Shen, Jing-kang; Xu, Ye-chun; Xiong, Bing; Zhang, Nai-xia

2016-01-01

Aim: Fragment-based lead discovery (FBLD) is a complementary approach in drug research and development. In this study, we established an NMR-based FBLD platform that was used to screen novel scaffolds targeting human bromodomain of BRD4, and investigated the binding interactions between hit compounds and the target protein. Methods: 1D NMR techniques were primarily used to generate the fragment library and to screen compounds. The inhibitory activity of hits on the first bromodomain of BRD4 [BRD4(I)] was examined using fluorescence anisotropy binding assay. 2D NMR and X-ray crystallography were applied to characterize the binding interactions between hit compounds and the target protein. Results: An NMR-based fragment library containing 539 compounds was established, which were clustered into 56 groups (8–10 compounds in each group). Eight hits with new scaffolds were found to inhibit BRD4(I). Four out of the 8 hits (compounds 1, 2, 8 and 9) had IC50 values of 100–260 μmol/L, demonstrating their potential for further BRD4-targeted hit-to-lead optimization. Analysis of the binding interactions revealed that compounds 1 and 2 shared a common quinazolin core structure and bound to BRD4(I) in a non-acetylated lysine mimetic mode. Conclusion: An NMR-based platform for FBLD was established and used in discovery of BRD4-targeted compounds. Four potential hit-to-lead optimization candidates have been found, two of them bound to BRD4(I) in a non-acetylated lysine mimetic mode, being selective BRD4(I) inhibitors. PMID:27238211

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.

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).

A discrete three-layer stack aggregate of a linear porphyrin tetramer: solution-phase structure elucidation by NMR and X-ray scattering.

PubMed

Hutin, Marie; Sprafke, Johannes K; Odell, Barbara; Anderson, Harry L; Claridge, Tim D W

2013-08-28

Formation of stacked aggregates can dramatically alter the properties of aromatic π-systems, yet the solution-phase structure elucidation of these aggregates is often impossible because broad distributions of species are formed, giving uninformative spectroscopic data. Here, we show that a butadiyne-linked zinc porphyrin tetramer forms a remarkably well-defined aggregate, consisting of exactly three molecules, in a parallel stacked arrangement (in chloroform at room temperature; concentration 1 mM-0.1 μM). The aggregate has a mass of 14.7 kDa. Unlike most previously reported aggregates, it gives sharp NMR resonances and aggregation is in slow exchange on the NMR time scale. The structure was elucidated using a range of NMR techniques, including diffusion-editing, (1)H-(29)Si HMBC, (1)H-(1)H COSY, TOCSY and NOESY, and (1)H-(13)C edited HSQC spectroscopy. Surprisingly, the (1)H-(1)H COSY spectrum revealed many long-range residual dipolar couplings (RDCs), and detailed analysis of magnetic field-induced (1)H-(13)C RDCs provided further evidence for the structural model. The size and shape of the aggregate is supported by small-angle X-ray scattering (SAXS) data. It adopts a geometry that maximizes van der Waals contact between the porphyrins, while avoiding clashes between side chains. The need for interdigitation of the side chains prevents formation of stacks consisting of more than three layers. Although a detailed analysis has only been carried out for one compound (the tetramer), comparison with the NMR spectra of other oligomers indicates that they form similar three-layer stacks. In all cases, aggregation can be prevented by addition of pyridine, although at low pyridine concentrations, disaggregation takes many hours to reach equilibrium.