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57 items found (0 items not approved), displaying 41 to 50.[First/Prev] 1, 2, 3, 4, 5, 6 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Identification of a tyrosine switch in copper-haem nitrite reductases Jianshu Dong , Daisuke Sasaki , Robert R. Eady , Svetlana V. Antonyuk , S. Samar Hasnain Iucrj 5, 510 - 518 DOI: 10.1107/S2052252518008242 Open Access Show Abstract ▼ Abstract: There are few cases where tyrosine has been shown to be involved in catalysis or the control of catalysis despite its ability to carry out chemistry at much higher potentials (1 V versus NHE). Here, it is shown that a tyrosine that blocks the hydrophobic substrate-entry channel in copper-haem nitrite reductases can be activated like a switch by the treatment of crystals of Ralstonia pickettii nitrite reductase (RpNiR) with nitric oxide (NO) (−0.8 ± 0.2 V). Treatment with NO results in an opening of the channel originating from the rotation of Tyr323 away from AspCAT97. Remarkably, the structure of a catalytic copper-deficient enzyme also shows Tyr323 in the closed position despite the absence of type 2 copper (T2Cu), clearly demonstrating that the status of Tyr323 is not controlled by T2Cu or its redox chemistry. It is also shown that the activation by NO is not through binding to haem. It is proposed that activation of the Tyr323 switch is controlled by NO through proton abstraction from tyrosine and the formation of HNO. The insight gained here for the use of tyrosine as a switch in catalysis has wider implications for catalysis in biology.	Jul 2018
I24-Microfocus Macromolecular Crystallography	Enzyme catalysis captured using multiple structures from one crystal at varying temperatures Sam Horrell , Demet Kekilli , Kakali Sen , Robin L. Owen , Florian S. N. Dworkowski , Svetlana V. Antonyuk , Thomas W. Keal , Chin W. Yong , Robert R. Eady , S. Samar Hasnain , Richard W. Strange , Mike Hough Iucrj 5 DOI: 10.1107/S205225251800386X Diamond Proposal Number(s): [11175] Open Access Show Abstract ▼ Abstract: High-resolution crystal structures of enzymes in relevant redox states have transformed our understanding of enzyme catalysis. Recent developments have demonstrated that X-rays can be used, via the generation of solvated electrons, to drive reactions in crystals at cryogenic temperatures (100 K) to generate `structural movies' of enzyme reactions. However, a serious limitation at these temperatures is that protein conformational motion can be significantly supressed. Here, the recently developed MSOX (multiple serial structures from one crystal) approach has been applied to nitrite-bound copper nitrite reductase at room temperature and at 190 K, close to the glass transition. During both series of multiple structures, nitrite was initially observed in a `top-hat' geometry, which was rapidly transformed to a `side-on' configuration before conversion to side-on NO, followed by dissociation of NO and substitution by water to reform the resting state. Density functional theory calculations indicate that the top-hat orientation corresponds to the oxidized type 2 copper site, while the side-on orientation is consistent with the reduced state. It is demonstrated that substrate-to-product conversion within the crystal occurs at a lower radiation dose at 190 K, allowing more of the enzyme catalytic cycle to be captured at high resolution than in the previous 100 K experiment. At room temperature the reaction was very rapid, but it remained possible to generate and characterize several structural states. These experiments open up the possibility of obtaining MSOX structural movies at multiple temperatures (MSOX-VT), providing an unparallelled level of structural information during catalysis for redox enzymes.	May 2018
I03-Macromolecular Crystallography	X-ray and cryo-EM structures of inhibitor-bound cytochrome bc 1 complexes for structure-based drug discovery Kangsa Amporndanai , Rachel M. Johnson , Paul M. O'Neill , Colin W. G. Fishwick , Alexander H. Jamson , Shaun Rawson , Stephen Muench , S. Samar Hasnain , Svetlana V. Antonyuk Iucrj 5 DOI: 10.1107/S2052252518001616 Diamond Proposal Number(s): [11740] Open Access Show Abstract ▼ Abstract: Cytochrome bc1, a dimeric multi-subunit electron-transport protein embedded in the inner mitochondrial membrane, is a major drug target for the treatment and prevention of malaria and toxoplasmosis. Structural studies of cytochrome bc1 from mammalian homologues co-crystallized with lead compounds have underpinned structure-based drug design to develop compounds with higher potency and selectivity. However, owing to the limited amount of cytochrome bc1 that may be available from parasites, all efforts have been focused on homologous cytochrome bc1 complexes from mammalian species, which has resulted in the failure of some drug candidates owing to toxicity in the host. Crystallographic studies of the native parasite proteins are not feasible owing to limited availability of the proteins. Here, it is demonstrated that cytochrome bc1 is highly amenable to single-particle cryo-EM (which uses significantly less protein) by solving the apo and two inhibitor-bound structures to ∼4.1 Å resolution, revealing clear inhibitor density at the binding site. Therefore, cryo-EM is proposed as a viable alternative method for structure-based drug discovery using both host and parasite enzymes.	Mar 2018
	Background modelling of diffraction data in the presence of ice rings James M. Parkhurst , Andrea Thorn , Melanie Vollmar , Graeme Winter , David G. Waterman , Luis Fuentes-Montero , Richard J. Gildea , Garib N. Murshudov , Gwyndaf Evans Iucrj 4 DOI: 10.1107/S2052252517010259 Open Access Show Abstract ▼ Abstract: An algorithm for modelling the background for each Bragg reflection in a series of X-ray diffraction images containing Debye–Scherrer diffraction from ice in the sample is presented. The method involves the use of a global background model which is generated from the complete X-ray diffraction data set. Fitting of this model to the background pixels is then performed for each reflection independently. The algorithm uses a static background model that does not vary over the course of the scan. The greatest improvement can be expected for data where ice rings are present throughout the data set and the local background shape at the size of a spot on the detector does not exhibit large time-dependent variation. However, the algorithm has been applied to data sets whose background showed large pixel variations (variance/mean > 2) and has been shown to improve the results of processing for these data sets. It is shown that the use of a simple flat-background model as in traditional integration programs causes systematic bias in the background determination at ice-ring resolutions, resulting in an overestimation of reflection intensities at the peaks of the ice rings and an underestimation of reflection intensities either side of the ice ring. The new global background-model algorithm presented here corrects for this bias, resulting in a noticeable improvement in R factors following refinement.	Sep 2017
I02-Macromolecular Crystallography	Active-site protein dynamics and solvent accessibility in native Achromobacter cycloclastes copper nitrite reductase Kakali Sen , Sam Horrell , Demet Kekilli , Chin W. Yong , Thomas W. Keal , Hakan Atakisi , David W. Moreau , Robert E. Thorne , Michael A. Hough , Richard W. Strange Iucrj 4 DOI: 10.1107/S2052252517007527 Diamond Proposal Number(s): [13467] Open Access Show Abstract ▼ Abstract: Microbial nitrite reductases are denitrifying enzymes that are a major component of the global nitrogen cycle. Multiple structures measured from one crystal (MSOX data) of copper nitrite reductase at 240 K, together with molecular-dynamics simulations, have revealed protein dynamics at the type 2 copper site that are significant for its catalytic properties and for the entry and exit of solvent or ligands to and from the active site. Molecular-dynamics simulations were performed using different protonation states of the key catalytic residues (AspCAT and HisCAT) involved in the nitrite-reduction mechanism of this enzyme. Taken together, the crystal structures and simulations show that the AspCAT protonation state strongly influences the active-site solvent accessibility, while the dynamics of the active-site `capping residue' (IleCAT), a determinant of ligand binding, are influenced both by temperature and by the protonation state of AspCAT. A previously unobserved conformation of IleCAT is seen in the elevated temperature series compared with 100 K structures. DFT calculations also show that the loss of a bound water ligand at the active site during the MSOX series is consistent with reduction of the type 2 Cu atom.	Jul 2017
I04-Macromolecular Crystallography	New leads for fragment-based design of rhenium/technetium radiopharmaceutical agents Alice Brink , John R. Helliwell Iucrj 4 DOI: 10.1107/S2052252517003475 Diamond Proposal Number(s): [8997] Open Access Show Abstract ▼ Abstract: Multiple possibilities for the coordination of fac-[Re(CO)3(H2O)3]+ to a protein have been determined and include binding to Asp, Glu, Arg and His amino-acid residues as well as to the C-terminal carboxylate in the vicinity of Leu and Pro. The large number of rhenium metal complex binding sites that have been identified on specific residues thereby allow increased target identification for the design of future radiopharmaceuticals. The core experimental concept involved the use of state-of-art tuneable synchrotron radiation at the Diamond Light Source to optimize the rhenium anomalous dispersion signal to a large value (f′′ of 12.1 electrons) at its LI absorption edge with a selected X-ray wavelength of 0.9763 Å. At the Cu Kα X-ray wavelength (1.5418 Å) the f′′ for rhenium is 5.9 electrons. The expected peak-height increase owing to the optimization of the Re f′′ was therefore 2.1. This X-ray wavelength tuning methodology thereby showed the lower occupancy rhenium binding sites as well as the occupancies of the higher occupancy rhenium binding sites.	May 2017
I13-1-Coherence	Procedures for cryogenic X-ray ptychographic imaging of biological samples M. Yusuf , F. Zhang , B. Chen , A. Bhartiya , K. Cunnea , U. Wagner , F. Cacho-Nerin , J. Schwenke , I. K. Robinson Iucrj 4 DOI: 10.1107/S2052252516020029 Open Access Show Abstract ▼ Abstract: Biological sample-preparation procedures have been developed for imaging human chromosomes under cryogenic conditions. A new experimental setup, developed for imaging frozen samples using beamline I13 at Diamond Light Source, is described. This manuscript describes the equipment and experimental procedures as well as the authors' first ptychographic reconstructions using X-rays.	Mar 2017
I19-Small Molecule Single Crystal Diffraction	Observation of a re-entrant phase transition in the molecular complex tris¬(μ2-3,5-diiso¬propyl-1,2,4-triazolato-κ2N1:N2)trigold(I) under high pressure Christopher H. Woodall , Jeppe Christensen , Jonathan M. Skelton , Lauren E. Hatcher , Andrew Parlett , Paul R. Raithby , Aron Walsh , Stephen C. Parker , Christine M. Beavers , Simon J. Teat , Mourad Intissar , Christian Reber , David R. Allan Iucrj 3, 367 - 376 DOI: 10.1107/S2052252516013129 Open Access Show Abstract ▼ Abstract: We report a molecular crystal that exhibits four successive phase transitions under hydrostatic pressure, driven by aurophilic interactions, with the ground-state structure re-emerging at high pressure. The effect of pressure on two polytypes of tris(μ2-3,5-diisopropyl-1,2,4-triazolato-κ2N1:N2)trigold(I) (denoted Form-I and Form-II) has been analysed using luminescence spectroscopy, single-crystal X-ray diffraction and first-principles computation. A unique phase behaviour was observed in Form-I, with a complex sequence of phase transitions between 1 and 3.5 GPa. The ambient C2/c mother cell transforms to a P21/n phase above 1 GPa, followed by a P21/a phase above 2 GPa and a large-volume C2/c supercell at 2.70 GPa, with the previously observed P21/n phase then reappearing at higher pressure. The observation of crystallographically identical low- and high-pressure P21/n phases makes this a rare example of a re-entrant phase transformation. The phase behaviour has been characterized using detailed crystallographic theory and modelling, and rationalized in terms of molecular structural distortions. The dramatic changes in conformation are correlated with shifts of the luminescence maxima, from a band maximum at 14040 cm−1 at 2.40 GPa, decreasing steeply to 13550 cm−1 at 3 GPa. A similar study of Form-II displays more conventional crystallographic behaviour, indicating that the complex behaviour observed in Form-I is likely to be a direct consequence of the differences in crystal packing between the two polytypes.	Sep 2016
I04-Macromolecular Crystallography	Serial crystallography captures enzyme catalysis in copper nitrite reductase at atomic resolution from one crystal Sam Horrell , Svetlana V. Antonyuk , Robert R. Eady , S. Samar Hasnain , Michael A. Hough , Richard W. Strange Iucrj 3, 271 - 281 DOI: 10.1107/S205225251600823X Diamond Proposal Number(s): [11740] Open Access Show Abstract ▼ Abstract: 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.	Jul 2016
Data acquisition	Native SAD is maturing John P. Rose , Bi-Cheng Wang , Manfred S. Weiss Iucrj 2, 431 - 440 DOI: 10.1107/S2052252515008337 PMID: 26175902 Open Access Show Abstract ▼ Abstract: Native SAD phasing uses the anomalous scattering signal of light atoms in the crystalline, native samples of macromolecules collected from single-wavelength X-ray diffraction experiments. These atoms include sodium, magnesium, phosphorus, sulfur, chlorine, potassium and calcium. Native SAD phasing is challenging and is critically dependent on the collection of accurate data. Over the past five years, advances in diffraction hardware, crystallographic software, data-collection methods and strategies, and the use of data statistics have been witnessed which allow `highly accurate data' to be routinely collected. Today, native SAD sits on the verge of becoming a `first-choice' method for both de novo and molecular-replacement structure determination. This article will focus on advances that have caught the attention of the community over the past five years. It will also highlight both de novo native SAD structures and recent structures that were key to methods development.	Jul 2015

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