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Philip
Roedig
,
Helen M.
Ginn
,
Tim
Pakendorf
,
Geoff
Sutton
,
Karl
Harlos
,
Thomas S.
Walter
,
Jan
Meyer
,
Pontus
Fischer
,
Ramona
Duman
,
Ismo
Vartiainen
,
Bernd
Reime
,
Martin
Warmer
,
Aaron S.
Brewster
,
Iris D.
Young
,
Tara
Michels-clark
,
Nicholas K.
Sauter
,
Abhay
Kotecha
,
James
Kelly
,
David J.
Rowlands
,
Marcin
Sikorsky
,
Silke
Nelson
,
Daniel S.
Damiani
,
Roberto
Alonso-mori
,
Jingshan
Ren
,
Elizabeth E.
Fry
,
Christian
David
,
David I. Stuart
Stuart
,
Armin
Wagner
,
Alke
Meents
Abstract: 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 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 most efficient use of the limited beam time available at XFELs and should enable a substantial increase in sample throughput at these facilities.
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Jun 2017
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Detectors
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Saeed
Oghbaey
,
Antoine
Sarracini
,
Helen M.
Ginn
,
Olivier
Pare-labrosse
,
Anling
Kuo
,
Alexander
Marx
,
Sascha W.
Epp
,
Darren A.
Sherrell
,
Bryan T.
Eger
,
Yinpeng
Zhong
,
Rolf
Loch
,
Valerio
Mariani
,
Roberto
Alonso-mori
,
Silke
Nelson
,
Henrik T.
Lemke
,
Robin L.
Owen
,
Arwen R.
Pearson
,
David I.
Stuart
,
Oliver P.
Ernst
,
Henrike
Mueller-werkmeister
,
R. J. Dwayne
Miller
Open Access
Abstract: The advent of ultrafast highly brilliant coherent X-ray free-electron laser sources has driven the development of novel structure-determination approaches for proteins, and promises visualization of protein dynamics on sub-picosecond timescales with full atomic resolution. Significant efforts are being applied to the development of sample-delivery systems that allow these unique sources to be most efficiently exploited for high-throughput serial femtosecond crystallography. Here, the next iteration of a fixed-target crystallography chip designed for rapid and reliable delivery of up to 11 259 protein crystals with high spatial precision is presented. An experimental scheme for predetermining the positions of crystals in the chip by means of in situ spectroscopy using a fiducial system for rapid, precise alignment and registration of the crystal positions is presented. This delivers unprecedented performance in serial crystallography experiments at room temperature under atmospheric pressure, giving a raw hit rate approaching 100% with an effective indexing rate of approximately 50%, increasing the efficiency of beam usage and allowing the method to be applied to systems where the number of crystals is limited.
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Aug 2016
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Open Access
Abstract: The indexing methods currently used for serial femtosecond crystallography were originally developed for experiments in which crystals are rotated in the X-ray beam, providing significant three-dimensional information. On the other hand, shots from both X-ray free-electron lasers and serial synchrotron crystallography experiments are still images, in which the few three-dimensional data available arise only from the curvature of the Ewald sphere. Traditional synchrotron crystallography methods are thus less well suited to still image data processing. Here, a new indexing method is presented with the aim of maximizing information use from a still image given the known unit-cell dimensions and space group. Efficacy for cubic, hexagonal and orthorhombic space groups is shown, and for those showing some evidence of diffraction the indexing rate ranged from 90% (hexagonal space group) to 151% (cubic space group). Here, the indexing rate refers to the number of lattices indexed per image.
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Aug 2016
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Open Access
Abstract: As serial femtosecond crystallography expands towards a variety of delivery methods, including chip-based methods, and smaller collected data sets, the requirement to optimize the data analysis to produce maximum structure quality is becoming increasingly pressing. Here cppxfel, a software package primarily written in C++, which showcases several data analysis techniques, is released. This software package presently indexes images using DIALS (diffraction integration for advanced light sources) and performs an initial orientation matrix refinement, followed by post-refinement of individual images against a reference data set. Cppxfel is released with the hope that the unique and useful elements of this package can be repurposed for existing software packages. However, as released, it produces high-quality crystal structures and is therefore likely to be also useful to experienced users of X-ray free-electron laser (XFEL) software who wish to maximize the information extracted from a limited number of XFEL images.
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Jun 2016
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I24-Microfocus Macromolecular Crystallography
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Abhay
Kotecha
,
Julian
Seago
,
Katherine
Scott
,
Alison
Burman
,
Silvia
Loureiro
,
Jingshan
Ren
,
Claudine
Porta
,
Helen M
Ginn
,
Terry
Jackson
,
Eva
Perez-martin
,
C Alistair
Siebert
,
Guntram
Paul
,
Juha T
Huiskonen
,
Ian M
Jones
,
Robert
Esnouf
,
Elizabeth
Fry
,
Francois F
Maree
,
Bryan
Charleston
,
Dave
Stuart
Diamond Proposal Number(s):
[10627]
Open Access
Abstract: Virus capsids are primed for disassembly, yet capsid integrity is key to generating a protective immune response. Foot-and-mouth disease virus (FMDV) capsids comprise identical pentameric protein subunits held together by tenuous noncovalent interactions and are often unstable. Chemically inactivated or recombinant empty capsids, which could form the basis of future vaccines, are even less stable than live virus. Here we devised a computational method to assess the relative stability of protein-protein interfaces and used it to design improved candidate vaccines for two poorly stable, but globally important, serotypes of FMDV: O and SAT2. We used a restrained molecular dynamics strategy to rank mutations predicted to strengthen the pentamer interfaces and applied the results to produce stabilized capsids. Structural analyses and stability assays confirmed the predictions, and vaccinated animals generated improved neutralizing-antibody responses to stabilized particles compared to parental viruses and wild-type capsids.
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Sep 2015
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[6387]
Open Access
Abstract: Polyhedra represent an ancient system used by a number of insect viruses to protect virions during long periods of environmental exposure. We present high resolution crystal structures of polyhedra for seven previously uncharacterised types of cypoviruses, four using ab initio selenomethionine phasing (two of these required over 100 selenomethionine crystals each). Approximately 80% of residues are structurally equivalent between all polyhedrins (pairwise rmsd ⩽1.5Å), whilst pairwise sequence identities, based on structural alignment, are as little as 12%. These illustrate the effect of 400 million years of evolution on a system where the crystal lattice is the functionally conserved feature in the face of massive sequence variability. The conservation of crystal contacts is maintained across most of the molecular surface, except for a dispensable virus recognition domain. By spreading the contacts over so much of the protein surface the lattice remains robust in the face of many individual changes. Overall these unusual structural constraints seem to have skewed the molecule’s evolution so that surface residues are almost as conserved as the internal residues.
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Aug 2015
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Open Access
Abstract: Research towards using X-ray free-electron laser (XFEL) data to solve structures using experimental phasing methods such as sulfur single-wavelength anomalous dispersion (SAD) has been hampered by shortcomings in the diffraction models for X-ray diffraction from FELs. Owing to errors in the orientation matrix and overly simple partiality models, researchers have required large numbers of images to converge to reliable estimates for the structure-factor amplitudes, which may not be feasible for all biological systems. Here, data for cytoplasmic polyhedrosis virus type 17 (CPV17) collected at 1.3 Å wavelength at the Linac Coherent Light Source (LCLS) are revisited. A previously published definition of a partiality model for reflections illuminated by self-amplified spontaneous emission (SASE) pulses is built upon, which defines a fraction between 0 and 1 based on the intersection of a reflection with a spread of Ewald spheres modelled by a super-Gaussian wavelength distribution in the X-ray beam. A method of post-refinement to refine the parameters of this model is suggested. This has generated a merged data set with an overall discrepancy (by calculating the Rsplit value) of 3.15% to 1.46 Å resolution from a 7225-image data set. The atomic numbers of C, N and O atoms in the structure are distinguishable in the electron-density map. There are 13 S atoms within the 237 residues of CPV17, excluding the initial disordered methionine. These only possess 0.42 anomalous scattering electrons each at 1.3 Å wavelength, but the 12 that have single predominant positions are easily detectable in the anomalous difference Fourier map. It is hoped that these improvements will lead towards XFEL experimental phase determination and structure determination by sulfur SAD and will generally increase the utility of the method for difficult cases.
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Jun 2015
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I24-Microfocus Macromolecular Crystallography
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Helen M
Ginn
,
Marc
Messerschmidt
,
Xiaoyun
Ji
,
Hanwen
Zhang
,
Danny
Axford
,
Richard J
Gildea
,
Graeme
Winter
,
Aaron S.
Brewster
,
Johan
Hattne
,
Armin
Wagner
,
Jonathan M
Grimes
,
Gwyndaf
Evans
,
Nicholas K.
Sauter
,
Geoff
Sutton
,
David I
Stuart
Open Access
Abstract: The X-ray free-electron laser (XFEL) allows the analysis of small weakly diffracting protein crystals, but has required very many crystals to obtain good data. Here we use an XFEL to determine the room temperature atomic structure for the smallest cytoplasmic polyhedrosis virus polyhedra yet characterized, which we failed to solve at a synchrotron. These protein microcrystals, roughly a micron across, accrue within infected cells. We use a new physical model for XFEL diffraction, which better estimates the experimental signal, delivering a high-resolution XFEL structure (1.75 Å), using fewer crystals than previously required for this resolution. The crystal lattice and protein core are conserved compared with a polyhedrin with less than 10% sequence identity. We explain how the conserved biological phenotype, the crystal lattice, is maintained in the face of extreme environmental challenge and massive evolutionary divergence. Our improved methods should open up more challenging biological samples to XFEL analysis.
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Mar 2015
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Data acquisition
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Open Access
Abstract: The macromolecular crystallography (MX) user experience at synchrotron radiation facilities continues to evolve, with the impact of developments in X-ray detectors, computer hardware and automation methods making it possible for complete data sets to be collected on timescales of tens of seconds. Data can be reduced in a couple of minutes and in favourable cases structures solved and refined shortly after. The information-rich database ISPyB, automatically populated by data acquisition software, data processing and structure solution pipelines at the Diamond Light Source beamlines, allows users to automatically track MX experiments in real time. In order to improve the synchrotron users' experience, efficient access to the data contained in ISPyB is now provided via an iOS 6.0+ app for iPhones and iPads. This provides users, both local and remote, with a succinct summary of data collection, visualization of diffraction images and crystals, and key metrics for data quality in real time.
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Oct 2014
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I24-Microfocus Macromolecular Crystallography
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Abstract: T-box proteins; transcription factors; protein-DNA interactions; crystal structure; DNA binding; Patient mutations
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Dec 2011
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