Krios I-Titan Krios I at Diamond
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Open Access
Abstract: Double-stranded (ds)RNA viruses replicate and transcribe their genome within a proteinaceous viral capsid to evade host cell defenses. While Reovirales members use conservative transcription, most dsRNA viruses, including cystoviruses, utilize semi-conservative transcription, in which a newly synthesized positive strand replaces the parental positive strand, which is released as mRNA. Here, we visualize semi-conservative transcription activation in cystovirus ɸ6 double-layered particles using cryogenic electron microscopy. We observe nucleotide-triggered disassembly of the domain-swapped outer capsid layer, subsequent expansion of the inner capsid layer, and stepwise assembly of transcription complexes at the opposing poles of the spooled dsRNA genome. These complexes consist of the viral polymerases embedded into a triskelion formed by the minor protein P7, which we show as essential for continuous transcription. The packaging hexamers proximal to the transcription sites channel the viral mRNA exit. Our results define the complex molecular pathway from the quiescent state to activated semi-conservative transcription.
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Jan 2026
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Jingjing
Zhao
,
Chen
Huang
,
Ali
Mostaed
,
Amirafshar
Moshtaghpour
,
James M.
Parkhurst
,
Ivan
Lobato
,
Marcus
Gallagher-Jones
,
Judy S.
Kim
,
Mark
Boyce
,
David
Stuart
,
Elena A.
Andreeva
,
Jacques-Philippe
Colletier
,
Angus I.
Kirkland
Open Access
Abstract: Exit wavefunction reconstruction is important in transmission electron microscopy for structural studies. We describe electron Fourier ptychography and its application to phase reconstruction of both radiation-resistant and beam-sensitive materials. We demonstrate that the phase of the exit wave can be reconstructed to high resolution using a modified iterative phase retrieval algorithm from data collected in an alternative optical geometry. This method achieves a spatial resolution of 0.63 nm at a fluence of 4.5 × 102 e−/nm2, as validated on Cry11Aa protein crystals under cryogenic conditions. Notably, this method requires no instrumental modifications, is straightforward to implement, and can be seamlessly integrated with existing data collection software, providing a broadly accessible alternative approach for structural studies.
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Oct 2025
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Yanjun
Zhang
,
Yan
Wu
,
Meng-Qian
Zhang
,
Haiyue
Rao
,
Zhaoyong
Zhang
,
Xiangyue
He
,
Yiwen
Liang
,
Raoqing
Guo
,
Yaochang
Yuan
,
Jing
Sun
,
Helen M. E.
Duyvesteyn
,
Elizabeth E.
Fry
,
David I.
Stuart
,
Jingxian
Zhao
,
Xiaoyan
Pan
,
Shu-Lin
Liu
,
Jincun
Zhao
,
Jiandong
Huo
Open Access
Abstract: Current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are effective against severe disease and death, but do not prevent viral infections, probably due to the limited mucosal immunity induced by intramuscular administration of the vaccine. Fusion of SARS-CoV-2 subunit immunogens with a human IgG Fc backbone can be used as a mucosal vaccine but its effectiveness in delivery in animal models, and its immunogenicity and the vaccine-induced protection against viral infections requires further studies. Here we investigate a bivalent RBD-Fc vaccine that includes the spike receptor-binding domains (RBDs) of the ancestral and BQ.1.1 variant of SARS-CoV-2. Ex vivo fluorescent imaging demonstrates that this vaccine can be effectively delivered to the lungs of mice through intranasal administration, with enhancement of retention in the nasal cavity and lung parenchyma. In mice, the vaccine elicited potent and broad-spectrum antibody responses against different variants including KP.3 which could persist for at least 3 months after booster. Importantly, it was able to induce RBD-specific mucosal IgA responses. Further, heterologous intranasal immunisation with adeno-vectored Chadv1 and RBD-Fc elicited both potent neutralising antibody and T cell responses. Immunised BALB/c and K18-hACE2-transgenic mice were also protected against viral challenge of XBB.1 and viral transmission was effectively limited in hamsters through intranasal immunisation. This work thus demonstrates the potential of RBD-Fc antigens as mucosal vaccines for prevention of breakthrough infections and onward transmission. Moreover, Fc-fusion proteins can be used as an effective mucosal vaccine strategy which can be used either alone or in combination with other vaccine technology to constitute heterologous immunisations, enabling strong protection against SARS-CoV-2 and other respiratory viruses.
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May 2025
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[28534]
Open Access
Abstract: Influenza virus neuraminidase is a crucial target for protective antibodies, yet the development of recombinant neuraminidase protein as a vaccine has been held back by instability and variable expression. We have taken a pragmatic approach to improving expression and stability of neuraminidase by grafting antigenic surface loops from low-expressing neuraminidase proteins onto the scaffold of high-expressing counterparts. The resulting hybrid proteins retained the antigenic properties of the loop donor while benefiting from the high-yield expression, stability, and tetrameric structure of the loop recipient. These hybrid proteins were recognised by a broad set of human monoclonal antibodies elicited by influenza infection or vaccination, with X-ray structures validating the accurate structural conformation of the grafted loops and the enzymatic cavity. Immunisation of mice with neuraminidase hybrids induced inhibitory antibodies to the loop donor and protected against lethal influenza challenge. This pragmatic technique offers a robust solution for improving the expression and stability of influenza neuraminidase proteins for vaccine development.
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Apr 2025
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Krios I-Titan Krios I at Diamond
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Lee
Sherry
,
Keith
Grehan
,
Mohammad W.
Bahar
,
Jessica J.
Swanson
,
Helen
Fox
,
Sue
Matthews
,
Sarah
Carlyle
,
Ling
Qin
,
Claudine
Porta
,
Steven
Wilkinson
,
Suzanne
Robb
,
Naomi
Clark
,
John
Liddell
,
Elizabeth E.
Fry
,
David I.
Stuart
,
Andrew J.
Macadam
,
David J.
Rowlands
,
Nicola J.
Stonehouse
Diamond Proposal Number(s):
[28713]
Open Access
Abstract: The success of the poliovirus (PV) vaccines has enabled the near-eradication of wild PV, however, their continued use post-eradication poses concerns, due to the potential for virus escape during vaccine manufacture. Recombinant virus-like particles (VLPs) that lack the viral genome remove this risk. Here, we demonstrate the production of PV VLPs for all three serotypes by controlled fermentation using Pichia pastoris. We determined the cryo-EM structure of a new PV2 mutant, termed SC5a, in comparison to PV2-SC6b VLPs described previously and investigated the immunogenicity of PV2-SC5a VLPs. Finally, a trivalent immunogenicity trial using bioreactor-derived VLPs of all three serotypes in the presence of Alhydrogel adjuvant, showed that these VLPs outperform the current IPV vaccine in the standard vaccine potency assay, offering the potential for dose-sparing. Overall, these results provide further evidence that yeast-produced VLPs have the potential to be a next-generation polio vaccine in a post-eradication world.
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Mar 2025
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Krios I-Titan Krios I at Diamond
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Lee
Sherry
,
Mohammad W.
Bahar
,
Claudine
Porta
,
Helen
Fox
,
Keith
Grehan
,
Veronica
Nasta
,
Helen M. E.
Duyvesteyn
,
Luigi
De Colibus
,
Johanna
Marsian
,
Inga
Murdoch
,
Daniel
Ponndorf
,
Seong-Ryong
Kim
,
Sachin
Shah
,
Sarah
Carlyle
,
Jessica J.
Swanson
,
Sue
Matthews
,
Clare
Nicol
,
George P.
Lomonossoff
,
Andrew J.
Macadam
,
Elizabeth E.
Fry
,
David I.
Stuart
,
Nicola J.
Stonehouse
,
David J.
Rowlands
Diamond Proposal Number(s):
[14856, 20223]
Open Access
Abstract: Polioviruses have caused crippling disease in humans for centuries, prior to the successful development of vaccines in the mid-1900’s, which dramatically reduced disease prevalence. Continued use of these vaccines, however, threatens ultimate disease eradication and achievement of a polio-free world. Virus-like particles (VLPs) that lack a viral genome represent a safer potential vaccine, although they require particle stabilization. Using our previously established genetic techniques to stabilize the structural capsid proteins, we demonstrate production of poliovirus VLPs of all three serotypes, from four different recombinant expression systems. We compare the antigenicity, thermostability and immunogenicity of these stabilized VLPs against the current inactivated polio vaccine, demonstrating equivalent or superior immunogenicity in female Wistar rats. Structural analyses of these recombinant VLPs provide a rational understanding of the stabilizing mutations and the role of potential excipients. Collectively, we have established these poliovirus stabilized VLPs as viable next-generation vaccine candidates for the future.
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Jan 2025
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Abstract: A virus particle must work as a strongroom to protect its genome, but at the same time it must undergo dramatic conformational changes to infect the cell in order to replicate and assemble progeny. Thus, viruses are miniaturized wonders whose structural complexity requires investigation by a combination of different techniques that can tackle both static and dynamic processes. In this chapter, we will illustrate how major structural techniques such as X-ray crystallography and electron microscopy can be combined with other techniques to determine the structure of complex viruses. The power of these hybrid approaches is discussed through a number of examples.
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Dec 2024
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Roi
Asor
,
Anna
Olerinyova
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Sean A.
Burnap
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Manish S.
Kushwah
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Fabian
Soltermann
,
Lucas S. P.
Rudden
,
Mario
Hensen
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Mario
Hensen
,
Snežana
Vasiljevic
,
Juliane
Brun
,
Michelle
Hill
,
Liu
Chang
,
Wanwisa
Dejnirattisai
,
Piyada
Supasa
,
Juthathip
Mongkolsapaya
,
Daming
Zhou
,
David I.
Stuart
,
Gavin R.
Screaton
,
Matteo T.
Degiacomi
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Nicole
Zitzmann
,
Justin L. P.
Benesch
,
Weston B.
Struwe
,
Philipp
Kukura
Open Access
Abstract: Cellular processes are controlled by the thermodynamics of the underlying biomolecular interactions. Frequently, structural investigations use one monomeric binding partner, while ensemble measurements of binding affinities generally yield one affinity representative of a 1:1 interaction, despite the majority of the proteome consisting of oligomeric proteins. For example, viral entry and inhibition in SARS-CoV-2 involve a trimeric spike surface protein, a dimeric angiotensin-converting enzyme 2 (ACE2) cell-surface receptor and dimeric antibodies. Here, we reveal that cooperativity correlates with infectivity and inhibition as opposed to 1:1 binding strength. We show that ACE2 oligomerizes spike more strongly for more infectious variants, while exhibiting weaker 1:1 affinity. Furthermore, we find that antibodies use induced oligomerization both as a primary inhibition mechanism and to enhance the effects of receptor-site blocking. Our results suggest that naive affinity measurements are poor predictors of potency, and introduce an antibody-based inhibition mechanism for oligomeric targets. More generally, they point toward a much broader role of induced oligomerization in controlling biomolecular interactions.
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Oct 2024
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John D.
Clarke
,
Helen M. E.
Duyvesteyn
,
Eva
Perez-Martin
,
Undīne
Latišenko
,
Claudine
Porta
,
Kathleen V.
Humphreys
,
Abigail L.
Hay
,
Jingshan
Ren
,
Elizabeth E.
Fry
,
Erwin
Van Den Born
,
Bryan
Charleston
,
Marie
Bonnet-Di Placido
,
Raymond J.
Owens
,
David I.
Stuart
,
John A.
Hammond
Open Access
Abstract: Foot-and-mouth disease vaccination using inactivated virus is suboptimal, as the icosahedral viral capsids often disassemble into antigenically distinct pentameric units during long-term storage, or exposure to elevated temperature or lowered pH, and thus raise a response that is no longer protective. Furthermore, as foot-and-mouth disease virus (FMDV)’s seven serotypes are antigenically diverse, cross-protection from a single serotype vaccine is limited, and most existing mouse and bovine antibodies and camelid single-domain heavy chain-only antibodies are serotype-specific. For quality control purposes, there is a real need for pan-serotype antibodies that clearly distinguish between pentamer (12S) and protective intact FMDV capsid. To date, few cross-serotype bovine-derived antibodies have been reported in the literature. We identify a bovine antibody with an ultralong CDR-H3, Ab117, whose structural analysis reveals that it binds to a deep, hydrophobic pocket on the interior surface of the capsid via the CDR-H3. Main-chain and hydrophobic interactions provide broad serotype specificity. ELISA analysis confirms that Ab117 is a novel pan-serotype and conformational epitope-specific 12S reagent, suitable for assessing capsid integrity.
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Oct 2024
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Open Access
Abstract: The replication of RNA viruses relies on the activity of RNA-dependent RNA polymerases (RdRps). Despite large variations in their genomic sequences, viral RdRps share a common architecture generally known as a closed right hand. The P2 polymerase of cystovirus φ6 is currently among the best characterized viral RdRps. This polymerase is responsible for carrying out both replication and transcription of the viral double-stranded RNA genome using de novo initiation. Despite the extensive biochemical and structural studies conducted on φ6 P2, further structural information on other cystoviral RdRps is crucial to elucidate the structural and functional diversity of viral RdRps. Here, we have determined the atomic X-ray structure of the RdRp P2 from the φ6-related cystovirus φ8 at 3Å resolution. This structure completes the existing set of structural information on the φ8 polymerase complex and sheds light on the difference and similarities with related cystoviral RdRps.
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Oct 2024
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