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48 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4, 5 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I02-Macromolecular Crystallography I23-Long wavelength MX	Native de novo structural determinations of non-canonical nucleic acid motifs by X-ray crystallography at long wavelengths Yashu Zhang , Kamel El Omari , Ramona Duman , Sisi Liu , Shozeb Haider , Armin Wagner , Gary N. Parkinson , Dengguo Wei Nucleic Acids Research 45 DOI: 10.1093/nar/gkaa439 Diamond Proposal Number(s): [12305] Open Access Show Abstract ▼ Abstract: Obtaining phase information remains a formidable challenge for nucleic acid structure determination. The introduction of an X-ray synchrotron beamline designed to be tunable to long wavelengths at Diamond Light Source has opened the possibility to native de novo structure determinations by the use of intrinsic scattering elements. This provides opportunities to overcome the limitations of introducing modifying nucleotides, often required to derive phasing information. In this paper, we build on established methods to generate new tools for nucleic acid structure determinations. We report on the use of (i) native intrinsic potassium single-wavelength anomalous dispersion methods (K-SAD), (ii) use of anomalous scattering elements integral to the crystallization buffer (extrinsic cobalt and intrinsic potassium ions), (iii) extrinsic bromine and intrinsic phosphorus SAD to solve complex nucleic acid structures. Using the reported methods we solved the structures of (i) Pseudorabies virus (PRV) RNA G-quadruplex and ligand complex, (ii) PRV DNA G-quadruplex, and (iii) an i-motif of human telomeric sequence. Our results highlight the utility of using intrinsic scattering as a pathway to solve and determine non-canonical nucleic acid motifs and reveal the variability of topology, influence of ligand binding, and glycosidic angle rearrangements seen between RNA and DNA G-quadruplexes of the same sequence.	May 2020
I03-Macromolecular Crystallography I23-Long wavelength MX Krios III-Titan Krios III at Diamond	The architecture of EMC reveals a path for membrane protein insertion John P. O'donnell , Ben P. Phillips , Yuichi Yagita , Szymon Juszkiewicz , Armin Wagner , Duccio Malinverni , Robert J Keenan , Elizabeth Miller , Ramanujan S. Hegde Elife 9 DOI: 10.7554/eLife.57887 Diamond Proposal Number(s): [21426] Open Access Show Abstract ▼ Abstract: Approximately 25% of eukaryotic genes code for integral membrane proteins that are assembled at the endoplasmic reticulum. An abundant and widely conserved multi-protein complex termed EMC has been implicated in membrane protein biogenesis, but its mechanism of action is poorly understood. Here, we define the composition and architecture of human EMC using biochemical assays, crystallography of individual subunits, site-specific photocrosslinking, and cryo-EM reconstruction. Our results suggest that EMC's cytosolic domain contains a large, moderately hydrophobic vestibule that can bind a substrate's transmembrane domain (TMD). The cytosolic vestibule leads into a lumenally-sealed, lipid-exposed intramembrane groove large enough to accommodate a single substrate TMD. A gap between the cytosolic vestibule and intramembrane groove provides a potential path for substrate egress from EMC. These findings suggest how EMC facilitates energy-independent membrane insertion of TMDs, explain why only short lumenal domains are translocated by EMC, and constrain models of EMC's proposed chaperone function.	May 2020
I23-Long wavelength MX	Structure and characterization of Crimean-Congo hemorrhagic fever virus GP38 Akaash K. Mishra , Crystal L. Moyer , Dafna M. Abelson , Daniel J. Deer , Kamel El Omari , Ramona Duman , Leslie Lobel , Julius J. Lutwama , John M. Dye , Armin Wagner , Kartik Chandran , Robert W. Cross , Thomas W. Geisbert , Larry Zeitlin , Zachary A. Bornholdt , Jason S. Mclellan Journal Of Virology DOI: 10.1128/JVI.02005-19 Open Access Show Abstract ▼ Abstract: Crimean–Congo hemorrhagic fever virus (CCHFV) is the causative agent of the most widespread tick-borne viral infection in humans. CCHFV encodes a secreted glycoprotein (GP38) of unknown function that is the target of a protective antibody. Here we present the crystal structure of GP38 at a resolution of 2.5 Å, which revealed a novel fold primarily consisting of a 3-helix bundle and a β-sandwich. Sequence alignment and homology modeling showed distant homology between GP38 and the ectodomain of Gn (a structural glycoprotein in CCHFV), suggestive of a gene duplication event. Analysis of convalescent sera showed high titers of GP38 antibodies indicating immunogenicity in humans during natural CCHFV infection. The only protective antibody for CCHFV in an adult mouse model reported to date, 13G8, bound GP38 with sub-nanomolar affinity and protected against heterologous CCHFV challenge in a STAT1-knockout mouse model. Our data strongly suggests that GP38 should be evaluated as a vaccine antigen and its structure provides a foundation to investigate functions of this protein in the viral life cycle.	Jan 2020
I23-Long wavelength MX	Identifying dynamic, partially occupied residues using anomalous scattering Serena Rocchio , Ramona Duman , Kamel El Omari , Vitaliy Mykhaylyk , Christian Orr , Zhen Yan , Loïc Salmon , Armin Wagner , James C. A. Bardwell , Scott Horowitz Acta Crystallographica Section D Structural Biology 75, 1084 - 1095 DOI: 10.1107/S2059798319014475 Diamond Proposal Number(s): [18691] Open Access Show Abstract ▼ Abstract: Although often presented as taking single `snapshots' of the conformation of a protein, X-ray crystallography provides an averaged structure over time and space within the crystal. The important but difficult task of characterizing structural ensembles in crystals is typically limited to small conformational changes, such as multiple side-chain conformations. A crystallographic method was recently introduced that utilizes residual electron and anomalous density (READ) to characterize structural ensembles encompassing large-scale structural changes. Key to this method is an ability to accurately measure anomalous signals and distinguish them from noise or other anomalous scatterers. This report presents an optimized data-collection and analysis strategy for partially occupied iodine anomalous signals. Using the long-wavelength-optimized beamline I23 at Diamond Light Source, the ability to accurately distinguish the positions of anomalous scatterers with occupancies as low as ∼12% is demonstrated. The number and positions of these anomalous scatterers are consistent with previous biophysical, kinetic and structural data that suggest that the protein Im7 binds to the chaperone Spy in multiple partially occupied conformations. Finally, READ selections demonstrate that re-measured data using the new protocols are consistent with the previously characterized structural ensemble of the chaperone Spy with its client Im7. This study shows that a long-wavelength beamline results in easily validated anomalous signals that are strong enough to be used to detect and characterize highly disordered sections of crystal structures.	Dec 2019
I03-Macromolecular Crystallography I23-Long wavelength MX I24-Microfocus Macromolecular Crystallography	Desolvation of the substrate binding protein TauA dictates ligand specificity for the alkanesulfonate ABC importer TauABC Feng Qu , Kamel El Omari , Armin Wagner , Alfonso De Simone , Konstantinos Beis Biochemical Journal DOI: 10.1042/BCJ20190779 Diamond Proposal Number(s): [12579] Open Access Show Abstract ▼ Abstract: Under limiting sulfur availability, bacteria can assimilate sulfur from alkanesulfonates. Bacteria utilize ATP-binding cassette (ABC) transporters to internalise them for further processing to release sulfur. In gram-negative bacteria the TauABC and SsuABC ensure internalization, although, these two systems have common substrates, the former has been characterized as a taurine specific system. TauA and SsuA are substrate-binding proteins (SBPs) that bind and bring the alkanesulfonates to the ABC importer for transport. Here, we have determined the crystal structure of TauA and have characterized its thermodynamic binding parameters by isothermal titration calorimetry in complex with taurine and different alkanesulfonates. Our structures revealed that the coordination of the alkanesulfonates is conserved, with the exception of Asp205 that is absent from SsuA, but the thermodynamic parameters revealed a very high enthalpic penalty cost for binding of the other alkanesulfonates relative to taurine. Our molecular dynamic simulations indicated that the different levels of hydration of the binding site contributed to the selectivity for taurine over the other alkanesulfonates. Such selectivity mechanism is very likely to be employed by other SBPs of ABC transporters.	Nov 2019
I23-Long wavelength MX	The morphogen Sonic hedgehog inhibits its receptor Patched by a pincer grasp mechanism Amalie F. Rudolf , Maia Kinnebrew , Christiane Kowatsch , T. Bertie Ansell , Kamel El Omari , Benjamin Bishop , Els Pardon , Rebekka A. Schwab , Tomas Malinauskas , Mingxing Qian , Ramona Duman , Douglas F. Covey , Jan Steyaert , Armin Wagner , Mark S. P. Sansom , Rajat Rohatgi , Christian Siebold Nature Chemical Biology 15, 975 - 982 DOI: 10.1038/s41589-019-0370-y Show Abstract ▼ Abstract: Hedgehog (HH) ligands, classical morphogens that pattern embryonic tissues in all animals, are covalently coupled to two lipids—a palmitoyl group at the N terminus and a cholesteroyl group at the C terminus. While the palmitoyl group binds and inactivates Patched 1 (PTCH1), the main receptor for HH ligands, the function of the cholesterol modification has remained mysterious. Using structural and biochemical studies, along with reassessment of previous cryo-electron microscopy structures, we find that the C-terminal cholesterol attached to Sonic hedgehog (Shh) binds the first extracellular domain of PTCH1 and promotes its inactivation, thus triggering HH signaling. Molecular dynamics simulations show that this interaction leads to the closure of a tunnel through PTCH1 that serves as the putative conduit for sterol transport. Thus, Shh inactivates PTCH1 by grasping its extracellular domain with two lipidic pincers, the N-terminal palmitate and the C-terminal cholesterol, which are both inserted into the PTCH1 protein core.	Oct 2019
I23-Long wavelength MX	Importance of potassium ions for ribosome structure and function revealed by long-wavelength X-ray diffraction Alexey Rozov , Iskander Khusainov , Kamel El Omari , Ramona Duman , Vitaliy Mykhaylyk , Marat Yusupov , Eric Westhof , Armin Wagner , Gulnara Yusupova Nature Communications DOI: 10.1038/s41467-019-10409-4 Open Access Show Abstract ▼ Abstract: The ribosome, the largest RNA-containing macromolecular machinery in cells, requires metal ions not only to maintain its three-dimensional fold but also to perform protein synthesis. Despite the vast biochemical data regarding the importance of metal ions for efficient protein synthesis and the increasing number of ribosome structures solved by X-ray crystallography or cryo-electron microscopy, the assignment of metal ions within the ribosome remains elusive due to methodological limitations. Here we present extensive experimental data on the potassium composition and environment in two structures of functional ribosome complexes obtained by measurement of the potassium anomalous signal at the K-edge, derived from long-wavelength X-ray diffraction data. We elucidate the role of potassium ions in protein synthesis at the three-dimensional level, most notably, in the environment of the ribosome functional decoding and peptidyl transferase centers. Our data expand the fundamental knowledge of the mechanism of ribosome function and structural integrity.	Jun 2019
I23-Long wavelength MX	The long-wavelength macromolecular crystallography I23 at Diamond Light Source Armin Wagner , Ramona Duman , Kamel El Omari , Vinay Grama , Vitaliy Mykhaylyk Nihon Kessho Gakkaishi 60, 233 - 239 DOI: 10.5940/jcrsj.60.233 Show Abstract ▼ Abstract: Long-wavelength macromolecular crystallography（MX）has been proposed for a long time as a tool for phasing novel macromolecular crystal structures without additional heavy atom labelling. Making use of anomalous diffraction from atoms natively present in the crystal, such as sulphur and phosphorus, has become increasingly popular over the past years. Nevertheless, the full potential of this technique, has not been fully exploited due to lack of dedicated experimental setups able to easily access wavelengths longer than 2 Å. Since the wavelengths for the absorption edges of sulphur and phosphorus are significantly longer, standard beamline setups are not suitable to provide high-quality, high-resolution data, as the experiments are limited by the increasing absorption effects and the diffraction angles for longer wavelengths. Currently only two synchrotron beamlines offer access to optimised sample environments using wavelengths longer than 2.7 Å：BL1A at Photon Factory, Japan and I23 at Diamond Light Source, UK. Here, we describe the challenges and solutions implemented at the in-vacuum long-wavelength MX beamline I23 and present first results.	Dec 2018
I23-Long wavelength MX	Anomalous X-ray diffraction studies of ion transport in K+ channels Patricia S. Langan , Venu Gopal Vandavasi , Kevin L. Weiss , Pavel V. Afonine , Kamel El Omari , Ramona Duman , Armin Wagner , Leighton Coates Nature Communications 9 DOI: 10.1038/s41467-018-06957-w Diamond Proposal Number(s): [14493] Open Access Show Abstract ▼ Abstract: Potassium ion channels utilize a highly selective filter to rapidly transport K+ ions across cellular membranes. This selectivity filter is composed of four binding sites which display almost equal electron density in crystal structures with high potassium ion concentrations. This electron density can be interpreted to reflect a superposition of alternating potassium ion and water occupied states or as adjacent potassium ions. Here, we use single wavelength anomalous dispersion (SAD) X-ray diffraction data collected near the potassium absorption edge to show experimentally that all ion binding sites within the selectivity filter are fully occupied by K+ ions. These data support the hypothesis that potassium ion transport occurs by direct Coulomb knock-on, and provide an example of solving the phase problem by K-SAD.	Oct 2018
B16-Test Beamline	Characterisation of tungstate and molybdate crystals ABO4 (A=Ca, Sr, Zn, Cd; B=W, Mo) for luminescence lifetime cryothermometry N. Ahmed , H. Kraus , H. J. Kim , V. Mokina , V. Tsiumra , A. Wagner , Y. Zhydachevskyy , V. B. Mykhaylyk Materialia DOI: 10.1016/j.mtla.2018.09.039 Diamond Proposal Number(s): [18454] Open Access Show Abstract ▼ Abstract: Luminescence lifetime thermometry for remote temperature monitoring of cryogenic objects requires materials that exhibit a suitably large change of the luminescence kinetics at low temperatures. Results of systematic studies of the temperature-induced changes in the luminescence of tungstates and molybdates with the general formula ABO4 (A=Ca, Sr, Zn, Cd; B=W, Mo) over the 4.5-300 K temperature range are summarized. It is shown through analysing changes of the emission and excitation spectra, as well as the decay kinetics, that in these materials the luminescence is due to the emission by self-trapped excitons, a process that exhibits strong temperature dependence. The main emphasis of the study is on establishing the factors that determine the character of the temperature dependence of the luminescence decay time constant. We discuss our findings in terms of a model that analyses the dynamics of radiative and non-radiative transitions between the excited and ground states of the emission center. Two thermally activated processes drive the observed changes. The first is the non-radiative decay of excited states, resulting in a decrease of the luminescence decay time constant at high temperatures. Additionally it is demonstrated that in molybdates and tungstates the fine splitting of the excited state facilitates a second mechanism for thermally activated exchange of charged carriers between the two split levels. This has a noticeable effect on the dynamics of the radiative decay at low temperatures. We established that the sensitivity of the luminescence lifetime to temperature changes can be estimated by using information on the energy structure of materials. It is concluded that within tungstates and molybdates under study SrWO4 is the most promising material for application in luminescence lifetime cryothermometry.	Oct 2018

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