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

Instruments / Technical Area	Publication Details	Published
I23-Long wavelength MX	Directional regulation of cytosolic PEPCK catalysis is mediated by competitive binding of anions Sarah Barwell , Ramona Duman , Armin Wagner , Todd Holyoak Biochemical And Biophysical Research Communications 2 DOI: 10.1016/j.bbrc.2022.11.025 Diamond Proposal Number(s): [442] Show Abstract ▼ Abstract: Phosphoenolpyruvate carboxykinase (PEPCK) is a well-characterized enzyme involved in primary glucose metabolism, responsible for catalyzing one of the key steps of gluconeogenesis. It is well demonstrated that PEPCK can efficiently catalyze the reversible interconversion of oxaloacetic acid (OAA) to phosphoenolpyruvate (PEP) in vitro, but the enzyme is typically ascribed a metabolic role that requires preferential catalysis in the direction of PEP synthesis in vivo. Here we present structural and functional data that demonstrate the preferential synthesis of PEP from OAA catalyzed by PEPCK in vivo is facilitated by anion-mediated enzyme inhibition that reduces enzyme activity more significantly in the direction of OAA synthesis than in the direction of PEP synthesis. From our studies we conclude that the specific binding of small, ubiquitous anions like chloride, present in millimolar concentrations under normal cellular conditions allows for metabolic control by restricting PEPCK to function in the direction of PEP synthesis.	Nov 2022
I23-Long wavelength MX	High-resolution mapping of metal ions reveals principles of surface layer assembly in Caulobacter crescentus cells Matthew Herdman , Andriko Von Kugelgen , Danguole Kureisaite-Ciziene , Ramona Duman , Kamel El Omari , Elspeth F. Garman , Andreas Kjaer , Dimitrios Kolokouris , Jan Lowe , Armin Wagner , Phillip J. Stansfeld , Tanmay A. M. Bharat Structure 55 DOI: 10.1016/j.str.2021.10.012 Open Access Show Abstract ▼ Abstract: Surface layers (S-layers) are proteinaceous crystalline coats that constitute the outermost component of most prokaryotic cell envelopes. In this study, we have investigated the role of metal ions in the formation of the Caulobacter crescentus S-layer using high-resolution structural and cell biology techniques, as well as molecular simulations. Utilizing optical microscopy of fluorescently tagged S-layers, we show that calcium ions facilitate S-layer lattice formation and cell-surface binding. We report all-atom molecular dynamics simulations of the S-layer lattice, revealing the importance of bound metal ions. Finally, using electron cryomicroscopy and long-wavelength X-ray diffraction experiments, we mapped the positions of metal ions in the S-layer at near-atomic resolution, supporting our insights from the cellular and simulations data. Our findings contribute to the understanding of how C. crescentus cells form a regularly arranged S-layer on their surface, with implications on fundamental S-layer biology and the synthetic biology of self-assembling biomaterials.	Nov 2021
I04-Macromolecular Crystallography I23-Long wavelength MX	Phosphorus and sulfur SAD phasing of the nucleic acid-bound DNA-binding domain of interferon regulatory factor 4 Alessandro Agnarelli , Kamel El Omari , Ramona Duman , Armin Wagner , Erika J. Mancini Acta Crystallographica Section F Structural Biology Communications 77 DOI: 10.1107/S2053230X21006506 Diamond Proposal Number(s): [20145] Open Access Show Abstract ▼ Abstract: Pivotal to the regulation of key cellular processes such as the transcription, replication and repair of DNA, DNA-binding proteins play vital roles in all aspects of genetic activity. The determination of high-quality structures of DNA-binding proteins, particularly those in complexes with DNA, provides crucial insights into the understanding of these processes. The presence in such complexes of phosphate-rich oligonucleotides offers the choice of a rapid method for the routine solution of DNA-binding proteins through the use of long-wavelength beamlines such as I23 at Diamond Light Source. This article reports the use of native intrinsic phosphorus and sulfur single-wavelength anomalous dispersion methods to solve the complex of the DNA-binding domain (DBD) of interferon regulatory factor 4 (IRF4) bound to its interferon-stimulated response element (ISRE). The structure unexpectedly shows three molecules of the IRF4 DBD bound to one ISRE. The sole reliance on native intrinsic anomalous scattering elements that belong to DNA–protein complexes renders the method of general applicability to a large number of such protein complexes that cannot be solved by molecular replacement or by other phasing methods.	Jul 2021
I23-Long wavelength MX	X-ray tomographic reconstruction and segmentation pipeline for the long-wavelength macromolecular crystallography beamline at Diamond Light Source Daniil Kazantsev , Ramona Duman , Armin Wagner , Vitaliy Mykhaylyk , Kazimir Wanelik , Mark Basham , Nicola Wadeson Journal Of Synchrotron Radiation 28 DOI: 10.1107/S1600577521003453 Open Access Show Abstract ▼ Abstract: In this paper a practical solution for the reconstruction and segmentation of low-contrast X-ray tomographic data of protein crystals from the long-wavelength macromolecular crystallography beamline I23 at Diamond Light Source is provided. The resulting segmented data will provide the path lengths through both diffracting and non-diffracting materials as basis for analytical absorption corrections for X-ray diffraction data taken in the same sample environment ahead of the tomography experiment. X-ray tomography data from protein crystals can be difficult to analyse due to very low or absent contrast between the different materials: the crystal, the sample holder and the surrounding mother liquor. The proposed data processing pipeline consists of two major sequential operations: model-based iterative reconstruction to improve contrast and minimize the influence of noise and artefacts, followed by segmentation. The segmentation aims to partition the reconstructed data into four phases: the crystal, mother liquor, loop and vacuum. In this study three different semi-automated segmentation methods are experimented with by using Gaussian mixture models, geodesic distance thresholding and a novel morphological method, RegionGrow, implemented specifically for the task. The complete reconstruction-segmentation pipeline is integrated into the MPI-based data analysis and reconstruction framework Savu, which is used to reduce computation time through parallelization across a computing cluster and makes the developed methods easily accessible.	May 2021
I23-Long wavelength MX	Sample preparation and transfer protocol for in-vacuum long-wavelength crystallography on beamline I23 at Diamond Light Source Ramona Duman , Christian Orr , Vitaliy Mykhaylyk , Kamel El Omari , Robert Pocock , Vinay Grama , Armin Wagner Journal Of Visualized Experiments DOI: 10.3791/62364 Open Access Show Abstract ▼ Abstract: Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, chlorine, or calcium, that are often natively present in macromolecules. This enables the direct structure solution of proteins and nucleic acids via experimental phasing without the need of additional labelling. To eliminate the significant air absorption of X-rays in this wavelength regime, these experiments are performed in a vacuum environment. Beamline I23 at Diamond Light Source, UK, is the first synchrotron instrument of its kind, designed and optimized for MX experiments in the long wavelength range towards 5 Å. To make this possible, a large vacuum vessel encloses all endstation components of the sample environment. The necessity to maintain samples at cryogenic temperatures during storage and data collection in vacuum requires the use of thermally conductive sample holders. This facilitates efficient heat removal to ensure sample cooling to approximately 50 K. The current protocol describes the procedures used for sample preparation and transfer of samples into vacuum on beamline I23. Ensuring uniformity in practices and methods already established within the macromolecular crystallography community, sample cooling to liquid nitrogen temperature can be performed in any laboratory setting equipped with standard MX tools. Cryogenic storage and transport of samples only require standard commercially available equipment. Specialized equipment is required for the transfer of cryogenically cooled crystals from liquid nitrogen into the vacuum endstation. Bespoke sample handling tools and a dedicated Cryogenic Transfer System (CTS) have been developed in house. Diffraction data collected on samples prepared using this protocol show excellent merging statistics, indicating that the quality of samples is unaltered during the procedure. This opens unique opportunities for in-vacuum MX in a wavelength range beyond standard synchrotron beamlines.	Apr 2021
I23-Long wavelength MX	Experimental phasing with vanadium and application to nucleotide-binding membrane proteins Kamel El Omari , Nada Mohamad , Kiran Bountra , Ramona Duman , Maria Romano , Katja Schlegel , Hok-Sau Kwong , Vitaliy Mykhaylyk , Claus Olesen , Jesper Vuust Moller , Maike Bublitz , Konstantinos Beis , Armin Wagner Iucrj 7 DOI: 10.1107/S2052252520012312 Open Access Show Abstract ▼ Abstract: The structure determination of soluble and membrane proteins can be hindered by the crystallographic phase problem, especially in the absence of a suitable homologous structure. Experimental phasing is the method of choice for novel structures; however, it often requires heavy-atom derivatization, which can be difficult and time-consuming. Here, a novel and rapid method to obtain experimental phases for protein structure determination by vanadium phasing is reported. Vanadate is a transition-state mimic of phosphoryl-transfer reactions and it has the advantage of binding specifically to the active site of numerous enzymes catalyzing this reaction. The applicability of vanadium phasing has been validated by determining the structures of three different protein–vanadium complexes, two of which are integral membrane proteins: the rabbit sarcoplasmic reticulum Ca2+-ATPase, the antibacterial peptide ATP-binding cassette transporter McjD from Escherichia coli and the soluble enzyme RNAse A from Bos taurus. Vanadium phasing was successful even at low resolution and despite severe anisotropy in the data. This method is principally applicable to a large number of proteins, representing six of the seven Enzyme Commission classes. It relies exclusively on the specific chemistry of the protein and it does not require any modifications, making it a very powerful addition to the phasing toolkit. In addition to the phasing power of this technique, the protein–vanadium complexes also provide detailed insights into the reaction mechanisms of the studied proteins.	Nov 2020
I23-Long wavelength MX	K2P channel C-type gating involves asymmetric selectivity filter order-disorder transitions Marco Lolicato , Andrew M. Natale , Fayal Abderemane-Ali , David Crottès , Sara Capponi , Ramona Duman , Armin Wagner , John M. Rosenberg , Michael Grabe , Daniel L. Minor Science Advances 6 DOI: 10.1126/sciadv.abc9174 Open Access Show Abstract ▼ Abstract: K2P potassium channels regulate cellular excitability using their selectivity filter (C-type) gate. C-type gating mechanisms, best characterized in homotetrameric potassium channels, remain controversial and are attributed to selectivity filter pinching, dilation, or subtle structural changes. The extent to which such mechanisms control C-type gating of innately heterodimeric K2Ps is unknown. Here, combining K2P2.1 (TREK-1) x-ray crystallography in different potassium concentrations, potassium anomalous scattering, molecular dynamics, and electrophysiology, we uncover unprecedented, asymmetric, potassium-dependent conformational changes that underlie K2P C-type gating. These asymmetric order-disorder transitions, enabled by the K2P heterodimeric architecture, encompass pinching and dilation, disrupt the S1 and S2 ion binding sites, require the uniquely long K2P SF2-M4 loop and conserved “M3 glutamate network,” and are suppressed by the K2P C-type gate activator ML335. These findings demonstrate that two distinct C-type gating mechanisms can operate in one channel and underscore the SF2-M4 loop as a target for K2P channel modulator development.	Oct 2020
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
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

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