I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[10627, 14744]
Open Access
Abstract: Paramyxoviral transmission between hosts may be, in part, attributed to the ability of the viral envelope-displayed receptor-binding protein (RBP) to bind to cell surface receptors of different host species. We sought to elucidate the architecture of the receptor-binding head region of the RBPs presented by jeilongviruses, a group of emerging and genetically unique paramyxoviruses belonging to the genus Jeilongvirus, family Paramyxoviridae. Structure determination of J and Beilong jeilongvirus RBPs reveals that the proteins exhibit a prototypical six-bladed β-propeller fold, present a binding site with residues associated with sialic acid recognition and hydrolysis, and bear a close structural relationship with sialic acid binding hemagglutinin-neuraminidase (HN)-type paramyxoviral RBPs. Additionally, unlike other paramyxoviruses, jeilongviruses encode an RBP with an unusually long C-terminal extension. In our dimeric Beilong virus RBP structure, we find that the C-terminal extension exchanges a hat-like domain with the central region of the β-propeller of the opposing protomer through domain-swapping. The hat-like domain occludes residues putatively associated with sialic acid binding and hydrolysis, providing a structural rationale for the absence of observed hemadsorption and neuraminidase activity. The insights gleaned from this analysis expand our appreciation of the structural palette available to the plastic paramyxoviral RBP and how their architectures may be adapted to regulate host-cell interactions at the cell surface.
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Nov 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946, 28534]
Open Access
Abstract: The spillover of New World (NW) arenaviruses from rodent reservoirs into human populations poses a continued risk to human health. NW arenaviruses present a glycoprotein (GP) complex on the envelope surface of the virion, which orchestrates host cell entry and is a key target of the immune response arising from infection and immunization. Each protomer of the trimeric GP is composed of a stable signal peptide, a GP1 attachment glycoprotein, and a GP2 fusion glycoprotein. To glean insights into the architecture of this key therapeutic target, we determined the crystal structures of NW GP1−GP2 heterodimeric complexes from Junín virus and Machupo virus. Due to the metastability of the interaction between GP1 and GP2, structural elucidation required the introduction of a disulfide bond at the GP1−GP2 complex interface, but no other stabilizing modifications were required. While the overall assembly of NW GP1−GP2 is conserved with that presented by Old World (OW) arenaviruses, including Lassa virus and lymphocytic choriomeningitis virus, NW GP1−GP2 complexes are structurally distinct. Indeed, we note that when compared to the OW GP1−GP2 complex, the globular portion of NW GP1 undergoes limited structural alterations upon detachment from its cognate GP2. We further demonstrate that our engineered GP1−GP2 heterodimers are antigenically relevant and recognized by neutralizing antibodies. These data provide insights into the distinct assemblies presented by NW and OW arenaviruses, as well as provide molecular-level blueprints that may guide vaccine development.
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Jun 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8423, 10627]
Open Access
Abstract: The unenveloped Bluetongue virus capsid comprises several structural layers, the inner two comprising a core, which assembles before addition of the outer proteins, VP2 and VP5. Two symmetric trimers of VP5 fit like pegs into two distinct pits on the core and undergo pH conformational changes in the context of the virus, associated with cell entry. Here we show that in isolation VP5 alone undergoes essentially the same changes with pH and confirm a helical transition, indicating that VP5 is a motor during cell entry. In the absence of VP5 the two pits on the core differ from each other, presumably due to the asymmetric underlying structure of VP3, the innermost capsid protein. On insertion of VP5 these pits become closely similar and remain similar at low pH whilst VP5 is present. This natural asymmetry presumably destabilises the attachment of VP5, facilitating ejection upon low pH, membrane penetration and cell entry.
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Aug 2024
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Open Access
Abstract: Many bioimaging research projects require objects of interest to be identified, located, and then traced to allow quantitative measurement. Depending on the complexity of the system and imaging, instance segmentation is often done manually, and automated approaches still require weeks to months of an individual’s time to acquire the necessary training data for AI models. As such, there is a strong need to develop approaches for instance segmentation that minimize the use of expert annotation while maintaining quality on challenging image analysis problems.
Herein, we present our work on a citizen science project we ran called Science Scribbler: Virus Factory on the Zooniverse platform, in which citizen scientists annotated a cryo-electron tomography volume by locating and categorising viruses using point-based annotations instead of manually drawing outlines. One crowdsourcing workflow produced a database of virus locations, and the other workflow produced a set of classifications of those locations. Together, this allowed mask annotation to be generated for training a deep learning–based segmentation model. From this model, segmentations were produced that allowed for measurements such as counts of the viruses by virus class.
The application of citizen science–driven crowdsourcing to the generation of instance segmentations of volumetric bioimages is a step towards developing annotation-efficient segmentation workflows for bioimaging data. This approach aligns with the growing interest in citizen science initiatives that combine the collective intelligence of volunteers with AI to tackle complex problems while involving the public with research that is being undertaken in these important areas of science.
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Jan 2024
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Krios III-Titan Krios III at Diamond
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Pranav N. M.
Shah
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James B.
Gilchrist
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Björn O.
Forsberg
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Alister
Burt
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Andrew
Howe
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Shyamal
Mosalaganti
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William
Wan
,
Julika
Radecke
,
Yuriy
Chaban
,
Geoff
Sutton
,
David I.
Stuart
,
Mark
Boyce
Diamond Proposal Number(s):
[21004]
Open Access
Abstract: Rotavirus assembly is a complex process that involves the stepwise acquisition of protein layers in distinct intracellular locations to form the fully assembled particle. Understanding and visualization of the assembly process has been hampered by the inaccessibility of unstable intermediates. We characterize the assembly pathway of group A rotaviruses observed in situ within cryo-preserved infected cells through the use of cryoelectron tomography of cellular lamellae. Our findings demonstrate that the viral polymerase VP1 recruits viral genomes during particle assembly, as revealed by infecting with a conditionally lethal mutant. Additionally, pharmacological inhibition to arrest the transiently enveloped stage uncovered a unique conformation of the VP4 spike. Subtomogram averaging provided atomic models of four intermediate states, including a pre-packaging single-layered intermediate, the double-layered particle, the transiently enveloped double-layered particle, and the fully assembled triple-layered virus particle. In summary, these complementary approaches enable us to elucidate the discrete steps involved in forming an intracellular rotavirus particle.
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Mar 2023
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Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[18477]
Open Access
Abstract: Traditionally, molecular assembly pathways for viruses are inferred from high resolution structures of purified stable intermediates, low resolution images of cell sections and genetic approaches. Here, we directly visualise an unsuspected ‘single shelled’ intermediate for a mammalian orthoreovirus in cryo-preserved infected cells, by cryo-electron tomography of cellular lamellae. Particle classification and averaging yields structures to 5.6 Å resolution, sufficient to identify secondary structural elements and produce an atomic model of the intermediate, comprising 120 copies each of protein λ1 and σ2. This λ1 shell is ‘collapsed’ compared to the mature virions, with molecules pushed inwards at the icosahedral fivefolds by ~100 Å, reminiscent of the first assembly intermediate of certain prokaryotic dsRNA viruses. This supports the supposition that these viruses share a common ancestor, and suggests mechanisms for the assembly of viruses of the Reoviridae. Such methodology holds promise for dissecting the replication cycle of many viruses.
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Sep 2020
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Developing methods to determine high-resolution structures from micrometre- or even submicrometre-sized protein crystals has become increasingly important in recent years. This applies to both large protein complexes and membrane proteins, where protein production and the subsequent growth of large homogeneous crystals is often challenging, and to samples which yield only micro- or nanocrystals such as amyloid or viral polyhedrin proteins. The versatile macromolecular crystallography microfocus (VMXm) beamline at Diamond Light Source specializes in X-ray diffraction measurements from micro- and nanocrystals. Because of the possibility of measuring data from crystalline samples that approach the resolution limit of visible-light microscopy, the beamline design includes a scanning electron microscope (SEM) to visualize, locate and accurately centre crystals for X-ray diffraction experiments. To ensure that scanning electron microscopy is an appropriate method for sample visualization, tests were carried out to assess the effect of SEM radiation on diffraction quality. Cytoplasmic polyhedrosis virus polyhedrin protein crystals cryocooled on electron-microscopy grids were exposed to SEM radiation before X-ray diffraction data were collected. After processing the data with DIALS, no statistically significant difference in data quality was found between datasets collected from crystals exposed and not exposed to SEM radiation. This study supports the use of an SEM as a tool for the visualization of protein crystals and as an integrated visualization tool on the VMXm beamline.
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May 2020
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Helen M. E.
Duyvesteyn
,
Helen M.
Ginn
,
Maija K.
Pietila
,
Armin
Wagner
,
Johan
Hattne
,
Jonathan M.
Grimes
,
Elina
Hirvonen
,
Gwyndaf
Evans
,
Marie-Laure
Parsy
,
Nicholas K.
Sauter
,
Aaron S.
Brewster
,
Juha
Huiskonen
,
David I.
Stuart
,
Geoff
Sutton
,
Dennis H.
Bamford
Open Access
Abstract: Viruses are a significant threat to both human health and the economy, and there is an urgent need for novel anti-viral drugs and vaccines. High-resolution viral structures inform our understanding of the virosphere, and inspire novel therapies. Here we present a method of obtaining such structural information that avoids potentially disruptive handling, by collecting diffraction data from intact infected cells. We identify a suitable combination of cell type and virus to accumulate particles in the cells, establish a suitable time point where most cells contain virus condensates and use electron microscopy to demonstrate that these are ordered crystalline arrays of empty capsids. We then use an X-ray free electron laser to provide extremely bright illumination of sub-micron intracellular condensates of bacteriophage phiX174 inside living Escherichia coli at room temperature. We have been able to collect low resolution diffraction data. Despite the limited resolution and completeness of these initial data, due to a far from optimal experimental setup, we have used novel methodology to determine a putative space group, unit cell dimensions, particle packing and likely maturation state of the particles.
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Feb 2018
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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
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Nicholas K.
Sauter
,
Abhay
Kotecha
,
James
Kelly
,
David J.
Rowlands
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Marcin
Sikorsky
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Silke
Nelson
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Daniel S.
Damiani
,
Roberto
Alonso-Mori
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Jingshan
Ren
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Elizabeth E.
Fry
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Christian
David
,
David I.
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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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Fringes and speckles occur within diffraction spots when a crystal is illuminated
with coherent radiation during X-ray diffraction. The additional information in
these features provides insight into the imperfections in the crystal at the submicrometre
scale. In addition, these features can provide more accurate
intensity measurements (e.g. by model-based profile fitting), detwinning (by
distinguishing the various components), phasing (by exploiting sampling of the
molecular transform) and refinement (by distinguishing regions with different
unit-cell parameters). In order to exploit these potential benefits, the features
due to coherent diffraction have to be recorded and any change due to radiation
damage properly modelled. Initial results from recording coherent diffraction at
cryotemperatures from polyhedrin crystals of approximately 2 mm in size are
described. These measurements allowed information about the type of crystal
imperfections to be obtained at the sub-micrometre level, together with the
changes due to radiation damage
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Jan 2016
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