I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[10627]
Abstract: A large number of Food and Drug Administration (FDA) approved drugs have been found to inhibit cell entry of Ebola virus (EBOV). However, since these drugs have various primary pharmacological targets their mechanisms of action against EBOV remain largely unknown. We have previously shown that six FDA approved drugs inhibit EBOV infection by interacting with and destabilizing the viral glycoprotein (GP). Here we show that the antidepressants imipramine and clomipramine, and antipsychotic drug thioridazine also directly interact with EBOV GP, and determine the mode of interaction by crystallographic analysis of the complexes. The compounds bind within the same pocket as observed for other, chemically divergent, complexes but with different binding modes. These details should be of value for the development of potent EBOV inhibitors.
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May 2018
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M01-Polara at OPIC (Oxford)
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Open Access
Abstract: Foot-and-mouth disease virus (FMDV) belongs to the aphthovirus genus of the Picornaviridae, a family of small, icosahedral, non-enveloped, single-stranded RNA viruses. It is a highly infectious pathogen and is one of the biggest hindrances to the international trade of animals and animal products. FMDV capsids (which are unstable below pH6.5) release their genome into the host cell from an acidic compartment, such as that of an endosome, and in the process dissociate into pentamers. Whilst other members of the family (enteroviruses) have been visualized to form an expanded intermediate capsid with holes from which inner capsid proteins (VP4), N-termini (VP1) and RNA can be released, there has been no visualization of any such state for an aphthovirus, instead the capsid appears to simply dissociate into pentamers. Here we present the 8-Å resolution structure of isolated dissociated pentamers of FMDV, lacking VP4. We also found these pentamers to re-associate into a rigid, icosahedrally symmetric assembly, which enabled their structure to be solved at higher resolution (5.2 Å). In this assembly, the pentamers unexpectedly associate ‘inside out’, but still with their exposed hydrophobic edges buried. Stabilizing interactions occur between the HI loop of VP2 and its symmetry related partners at the icosahedral 3-fold axes, and between the BC and EF loops of VP3 with the VP2 βB-strand and the CD loop at the 2-fold axes. A relatively extensive but subtle structural rearrangement towards the periphery of the dissociated pentamer compared to that in the mature virus provides insight into the mechanism of dissociation of FMDV and the marked difference in antigenicity.
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Sep 2017
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Krios I-Titan Krios I at Diamond
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Open Access
Abstract: Poliovirus (PV) is the causative agent of poliomyelitis, a crippling human disease known since antiquity. PV occurs in two distinct antigenic forms, D and C, of which only the D form elicits a robust neutralizing response. Developing a synthetically produced stabilized virus-like particle (sVLP)-based vaccine with D antigenicity, without the drawbacks of current vaccines, will be a major step towards the final eradication of poliovirus. Such a sVLP would retain the native antigenic conformation and the repetitive structure of the original virus particle, but lack infectious genomic material. In this study, we report the production of synthetically stabilized PV VLPs in plants. Mice carrying the gene for the human PV receptor are protected from wild-type PV when immunized with the plant-made PV sVLPs. Structural analysis of the stabilized mutant at 3.6 Å resolution by cryo-electron microscopy and single-particle reconstruction reveals a structure almost indistinguishable from wild-type PV3.
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Aug 2017
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Abstract: Viruses have impacted the biosphere in numerous ways since the dawn of life. However, the evolution, genetic, structural, and taxonomic diversity of viruses remain poorly understood, in part because sparse sampling of the virosphere has concentrated mostly on exploring the abundance and diversity of dsDNA viruses. Furthermore, viral genomes are highly diverse, and using only the current sequence-based methods for classifying viruses and studying their phylogeny is complicated. Here we describe a virus, FLiP (Flavobacterium-infecting, lipid-containing phage), with a circular ssDNA genome and an internal lipid membrane enclosed in the icosahedral capsid. The 9,174-nt-long genome showed limited sequence similarity to other known viruses. The genetic data imply that this virus might use replication mechanisms similar to those found in other ssDNA replicons. However, the structure of the viral major capsid protein, elucidated at near-atomic resolution using cryo-electron microscopy, is strikingly similar to that observed in dsDNA viruses of the PRD1–adenovirus lineage, characterized by a major capsid protein bearing two β-barrels. The strong similarity between FLiP and another member of the structural lineage, bacteriophage PM2, extends to the capsid organization (pseudo T = 21 dextro) despite the difference in the genetic material packaged and the lack of significant sequence similarity.
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Jul 2017
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Philip
Roedig
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Helen M.
Ginn
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Tim
Pakendorf
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Geoff
Sutton
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Karl
Harlos
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Thomas S.
Walter
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Jan
Meyer
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Pontus
Fischer
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Ramona
Duman
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Ismo
Vartiainen
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Bernd
Reime
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Martin
Warmer
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Aaron S.
Brewster
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Iris D.
Young
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Tara
Michels-clark
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Nicholas K.
Sauter
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Abhay
Kotecha
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James
Kelly
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David J.
Rowlands
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Marcin
Sikorsky
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Silke
Nelson
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Daniel S.
Damiani
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Roberto
Alonso-mori
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Jingshan
Ren
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Elizabeth E.
Fry
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Christian
David
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David I. Stuart
Stuart
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Armin
Wagner
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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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Krios I-Titan Krios I at Diamond
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Daniel K.
Clare
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C. Alistair
Siebert
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Corey
Hecksel
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Christoph
Hagen
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Valerie
Mordhorst
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Michael
Grange
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Alun W.
Ashton
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Martin A.
Walsh
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Kay
Grunewald
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Helen R.
Saibil
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Dave I.
Stuart
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Peijun
Zhang
Open Access
Abstract: The recent resolution revolution in cryo-EM has led to a massive increase in demand for both time on high-end cryo-electron microscopes and access to cryo-electron microscopy expertise. In anticipation of this demand, eBIC was set up at Diamond Light Source in collaboration with Birkbeck College London and the University of Oxford, and funded by the Wellcome Trust, the UK Medical Research Council (MRC) and the Biotechnology and Biological Sciences Research Council (BBSRC) to provide access to high-end equipment through peer review. eBIC is currently in its start-up phase and began by offering time on a single FEI Titan Krios microscope equipped with the latest generation of direct electron detectors from two manufacturers. Here, the current status and modes of access for potential users of eBIC are outlined. In the first year of operation, 222 d of microscope time were delivered to external research groups, with 95 visits in total, of which 53 were from unique groups. The data collected have generated multiple high- to intermediate-resolution structures (2.8–8 Å), ten of which have been published. A second Krios microscope is now in operation, with two more due to come online in 2017. In the next phase of growth of eBIC, in addition to more microscope time, new data-collection strategies and sample-preparation techniques will be made available to external user groups. Finally, all raw data are archived, and a metadata catalogue and automated pipelines for data analysis are being developed.
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Jun 2017
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I24-Microfocus Macromolecular Crystallography
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Abhay
Kotecha
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Quan
Wang
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Xianchi
Dong
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Serban L.
Ilca
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Marina
Ondiviela
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Rao
Zihe
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Julian
Seago
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Bryan
Charleston
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Elizabeth E.
Fry
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Nicola G. A.
Abrescia
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Timothy A.
Springer
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Juha T.
Huiskonen
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David I.
Stuart
Open Access
Abstract: Foot-and-mouth disease virus (FMDV) mediates cell entry by attachment to an integrin receptor, generally αvβ6, via a conserved arginine–glycine–aspartic acid (RGD) motif in the exposed, antigenic, GH loop of capsid protein VP1. Infection can also occur in tissue culture adapted virus in the absence of integrin via acquired basic mutations interacting with heparin sulphate (HS); this virus is attenuated in natural infections. HS interaction has been visualized at a conserved site in two serotypes suggesting a propensity for sulfated-sugar binding. Here we determined the interaction between αvβ6 and two tissue culture adapted FMDV strains by cryo-electron microscopy. In the preferred mode of engagement, the fully open form of the integrin, hitherto unseen at high resolution, attaches to an extended GH loop via interactions with the RGD motif plus downstream hydrophobic residues. In addition, an N-linked sugar of the integrin attaches to the previously identified HS binding site, suggesting a functional role.
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May 2017
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Abstract: Foot-and-mouth disease virus (FMDV) is notoriously unstable, particularly the O and SAT serotypes. Consequently, vaccines derived from heat-labile SAT viruses have been linked to the induction of poor duration immunity and hence require more frequent vaccinations to ensure protection. In-silico calculations predicted residue substitutions that would increase interactions at the inter-pentameric interface supporting increased stability. We assessed the stability of the 18 recombinant mutant viruses for their growth kinetics; antigenicity; plaque morphology; genetic stability; temperature, ionic and pH stability using the thermofluor and inactivation assays, in order to evaluate potential SAT2 vaccine candidates with improved stability. The most stable mutation was the single mutant S2093Y for temperature and pH stability, whilst other promising single mutants were E3198A, L2094V,S2093H and the triple mutant F2062Y-H2087M-H3143V. Although the S2093Y mutant had the greatest stability it exhibited smaller plaques; a reduced growth rate; a change in a monoclonal antibody footprint, and poor genetic stability properties compared to the wild-type virus. However, these factors affecting production can be overcome. The addition of 1M NaCl salt was found to further increase the stability of the SAT2 panel of viruses. The S2093Y and S2093H mutants were selected for future use in stabilising SAT2 vaccines.
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Mar 2017
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I03-Macromolecular Crystallography
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Xiangxi
Wang
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Ling
Zhu
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Minghao
Dang
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Zhongyu
Hu
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Qiang
Gao
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Shuai
Yuan
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Yao
Sun
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Bo
Zhang
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Jingshan
Ren
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Abhay
Kotecha
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Thomas S.
Walter
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Junzhi
Wang
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Elizabeth
Fry
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David I.
Stuart
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Zihe
Rao
Abstract: Hepatitis A virus (HAV) infects ∼1.4 million people annually and, although there is a vaccine, there are no licensed therapeutic drugs. HAV is unusually stable (making disinfection problematic) and little is known of how it enters cells and releases its RNA. Here we report a potent HAV-specific monoclonal antibody, R10, which neutralizes HAV infection by blocking attachment to the host cell. High-resolution cryo-EM structures of HAV full and empty particles and of the complex of HAV with R10 Fab reveal the atomic details of antibody binding and point to a receptor recognition site at the pentamer interface. These results, together with our observation that the R10 Fab destabilizes the capsid, suggest the use of a receptor mimic mechanism to neutralize virus infection, providing new opportunities for therapeutic intervention.
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Jan 2017
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Ling
Zhu
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Xiangxi
Wang
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Jingshan
Ren
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Abhay
Kotecha
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Thomas S.
Walter
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Shuai
Yuan
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Teruo
Yamashita
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Tobias J.
Tuthill
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Elizabeth E.
Fry
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Zihe
Rao
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David I.
Stuart
Abstract: Aichi virus (AiV), an unusual and poorly characterized picornavirus, classified in the genus Kobuvirus, can cause severe gastroenteritis and deaths in children below the age of five years, especially in developing countries1,2. The seroprevalence of AiV is approximately 60% in children under the age of ten years and reaches 90% later in life3,4. There is no available vaccine or effective antiviral treatment. Here, we describe the structure of AiV at 3.7 Å. This first high-resolution structure for a kobuvirus is intermediate between those of the enteroviruses and cardioviruses, with a shallow, narrow depression bounded by the prominent VP0 CD loops (linking the C and D strands of the β-barrel), replacing the depression known as the canyon, frequently the site of receptor attachment in enteroviruses. VP0 is not cleaved to form VP2 and VP4, so the ‘VP2’ β-barrel structure is complemented with a unique extended structure on the inside of the capsid. On the outer surface, a polyproline helix structure, not seen previously in picornaviruses is present at the C terminus of VP1, a position where integrin binding motifs are found in some other picornaviruses. A peptide corresponding to this polyproline motif somewhat attenuates virus infectivity, presumably blocking host-cell attachment. This may guide cellular receptor identification.
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Sep 2016
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