I24-Microfocus Macromolecular Crystallography
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Guido C.
Paesen
,
Nathaniel S.
Chapman
,
Jonna B.
Westover
,
Cynthia M.
Mcmillen
,
Natalia A.
Kuzmina
,
Emmett A.
Dews
,
Luke
Myers
,
Robert
Stass
,
Joel M.
Montgomery
,
Alexander
Bukreyev
,
Amy L.
Hartman
,
Brian B.
Gowen
,
James E.
Crowe
,
Thomas A.
Bowden
Diamond Proposal Number(s):
[28534]
Open Access
Abstract: Rift Valley fever virus (RVFV) poses a continued threat to human health and animal husbandry. Two neutralizing and protective human monoclonal antibodies (mAbs), RVFV-268 and RVFV-379, exhibit similar affinities and epitope footprints on the Gn glycoprotein component of the RVFV Gn-Gc capsomeric lattice. Here, we define fine details of the biophysical determinants of Gn recognition used by RVFV human monoclonal antibodies through studying an antibody encoded by a set of recombined genes not previously identified in RVFV antibodies. We find that RVFV-379 exhibits a larger footprint than that observed for RVFV-268 and other antibodies targeting the same region, which involves major contributions of both the light and heavy chains. RVFV-379 also uses an oblique angle of approach towards the virion surface that contrasts with the perpendicular angle of engagement observed for some other potently neutralizing human mAbs. Further, consistent with amino acid sequence variation within and proximal to the RVFV-379 epitope, in vitro neutralization screening reveals a limited degree of neutralization breadth across prevalent RVFV strains, suggesting that RVFV has fewer functional constraints at this region of the virus envelope. By dissecting the molecular determinants of mAb recognition of Gn, this integrated analysis refines strategies needed for the rational design of vaccines that can elicit a potent and species-wide protective antibody immune response to this important re-emerging pathogen.
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Feb 2026
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Ingra M.
Claro
,
Erika R.
Manuli
,
Camila A. M.
Da Silva
,
Thaís M.
Coletti
,
Philippe
Lemey
,
Ana Catharina
Nastri
,
Luciana Vilas Boas
Casadio
,
Amaro Nunes
Duarte-Neto
,
Joshua
Quick
,
Camila M.
Romano
,
Charles
Whittaker
,
Sarah C.
Hill
,
Carlos A.
Prete
,
Darlan S.
Candido
,
Filipe R. R.
Moreira
,
Mariana S.
Ramundo
,
Ian Nunes
Valença
,
Jaqueline G.
De Jesus
,
Flavia C. S.
Sales
,
Mariana S.
Cunha
,
Juliana M.
Guerra
,
Maria Cassia
Mendes-Correa
,
Tania R.
Tozetto-Mendoza
,
Marcilio Jorge
Fumagalli
,
Yeh-Li
Ho
,
Peter
Simmonds
,
Weng M.
Ng
,
Thomas A.
Bowden
,
William M.
De Souza
,
Oliver G.
Pybus
,
Anna S.
Levin
,
Nicholas
Loman
,
Ester C.
Sabino
,
Nuno R.
Faria
Open Access
Abstract: Between December 2019 and January 2020, two patients suspected of having severe yellow fever were admitted to a tertiary healthcare facility in São Paulo, Brazil, presenting with acute hemorrhagic syndrome and neurological alterations; both cases had fatal outcomes. Upon admission, both tested negative for yellow fever viral RNA, and Sabiá virus (SABV), a New World arenavirus, was identified as the causative pathogen. To date, only four humans naturally acquired SABV infections have been confirmed, all fatal and linked to rural settings. We applied next-generation sequencing to generate complete and near-complete genomes from two patients (SP17 and SP19). Existing molecular diagnostics failed to detect SABV; therefore, new molecular tests were developed. Genetic analyses of SP17 and SP19 genomes along with other arenaviruses, revealed that the new cases were genetically diverse, showing 93-98.2% amino acid identity at the NP level between SP17, SP19, and the 1990 reference strain (SPH114202). Time-scaled phylogenetic analyses confirmed that SP17 and SP19 were not epidemiologically linked and suggested that SABV has been circulating undetected in Brazil for over a century. Additionally, homology modeling and structure-based mapping provided insights into SABV receptor-binding sequence conservation, suggesting that SABV shares similar receptor binding structure compared to other clade B arenaviruses, despite some amino acid variation around receptor binding site. Our findings underscore the need for retrospective and prospective surveillance of undiagnosed hemorrhagic fever cases to assess the public health impact of SABV in Brazil.
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Feb 2026
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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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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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Victoria A.
Avanzato
,
Trenton
Bushmaker
,
Kasopefoluwa Y.
Oguntuyo
,
Claude Kwe
Yinda
,
Helen M. E.
Duyvesteyn
,
Robert
Stass
,
Kimberly
Meade-White
,
Rebecca
Rosenke
,
Tina
Thomas
,
Neeltje
Van Doremalen
,
Greg
Saturday
,
Katie J.
Doores
,
Benhur
Lee
,
Thomas A.
Bowden
,
Vincent J.
Munster
Diamond Proposal Number(s):
[20223]
Abstract: Nipah virus (NiV) is a highly pathogenic paramyxovirus capable of causing severe respiratory and neurologic disease in humans. Currently, there are no licensed vaccines or therapeutics against NiV, underscoring the urgent need for the development of countermeasures. The NiV surface-displayed glycoproteins, NiV-G and NiV-F, mediate host cell attachment and fusion, respectively, and are heavily targeted by host antibodies. Here, we describe a vaccination-derived neutralizing monoclonal antibody, mAb92, that targets NiV-F. Structural characterization of the Fab region bound to NiV-F (NiV-F–Fab92) by cryo-electron microscopy analysis reveals an epitope in the DIII domain at the membrane distal apex of NiV-F, an established site of vulnerability on the NiV surface. Further, prophylactic treatment of hamsters with mAb92 offered complete protection from NiV disease, demonstrating beneficial activity of mAb92 in vivo. This work provides support for targeting NiV-F in the development of vaccines and therapeutics against NiV.
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Sep 2024
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I03-Macromolecular Crystallography
I23-Long wavelength MX
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Kamel
El Omari
,
Ramona
Duman
,
Vitaliy
Mykhaylyk
,
Christian M.
Orr
,
Merlyn
Latimer-Smith
,
Graeme
Winter
,
Vinay
Grama
,
Feng
Qu
,
Kiran
Bountra
,
Hok Sau
Kwong
,
Maria
Romano
,
Rosana
Reis
,
Lutz
Vogeley
,
Luca
Vecchia
,
C. David
Owen
,
Sina
Wittmann
,
Max
Renner
,
Miki
Senda
,
Naohiro
Matsugaki
,
Yoshiaki
Kawano
,
Thomas A.
Bowden
,
Isabel
Moraes
,
Jonathan M.
Grimes
,
Erika J.
Mancini
,
Martin A.
Walsh
,
Cristiane R.
Guzzo
,
Raymond J.
Owens
,
E. Yvonne
Jones
,
David G.
Brown
,
Dave I.
Stuart
,
Konstantinos
Beis
,
Armin
Wagner
Open Access
Abstract: Despite recent advances in cryo-electron microscopy and artificial intelligence-based model predictions, a significant fraction of structure determinations by macromolecular crystallography still requires experimental phasing, usually by means of single-wavelength anomalous diffraction (SAD) techniques. Most synchrotron beamlines provide highly brilliant beams of X-rays of between 0.7 and 2 Å wavelength. Use of longer wavelengths to access the absorption edges of biologically important lighter atoms such as calcium, potassium, chlorine, sulfur and phosphorus for native-SAD phasing is attractive but technically highly challenging. The long-wavelength beamline I23 at Diamond Light Source overcomes these limitations and extends the accessible wavelength range to λ = 5.9 Å. Here we report 22 macromolecular structures solved in this extended wavelength range, using anomalous scattering from a range of elements which demonstrate the routine feasibility of lighter atom phasing. We suggest that, in light of its advantages, long-wavelength crystallography is a compelling option for experimental phasing.
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Oct 2023
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B21-High Throughput SAXS
I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[10627, 14744]
Open Access
Abstract: Increased viral surveillance has led to the isolation and identification of numerous uncharacterized paramyxoviruses, rapidly expanding our understanding of paramyxoviral diversity beyond the bounds of known genera. Despite this diversity, a key feature that unites paramyxoviruses is the presence of a receptor-binding protein (RBP), which facilitates host-cell attachment and plays a fundamental role in determining host range. Here, we study the RBP presented on the surface of rodent-borne paramyxoviruses Mossman and Nariva (MosV and NarV, respectively), viruses that constitute founding members of the recently defined Narmovirus genus within the Paramyxoviridae family. Crystallographic analysis of the C-terminal head region of the dimeric MosV and NarV RBPs demonstrates that while these glycoproteins retain the canonical six-bladed β-propeller fold found in other paramyxoviral RBPs, they lack the structural motifs associated with established paramyxovirus host-cell receptor entry pathways. Consistent with MosV-RBP and NarV-RBP undergoing a distinct entry pathway from other characterized paramyxoviruses, structure-based phylogenetic analysis demonstrates that these six-bladed β-propeller head domains form a singular structural class that is distinct from other paramyxoviral RBPs. Additionally, using an integrated crystallographic and small-angle X-ray scattering analysis, we confirm that MosV-RBP and NarV-RBP form homodimeric arrangements that are distinct from those adopted by other paramyxovirus RBPs. Altogether, this investigation provides a molecular-level blueprint of the narmovirus RBP that broadens our understanding of the structural space and functional diversity available to paramyxovirus RBPs.
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Sep 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Nathaniel S.
Chapman
,
Ruben J.g.
Hulswit
,
Jonna L. B.
Westover
,
Robert
Stass
,
Guido C.
Paesen
,
Elad
Binshtein
,
Joseph X.
Reidy
,
Taylor B.
Engdahl
,
Laura S.
Handal
,
Alejandra
Flores
,
Brian B.
Gowen
,
Thomas A.
Bowden
,
James E.
Crowe
Diamond Proposal Number(s):
[28534]
Open Access
Abstract: The zoonotic Rift Valley fever virus (RVFV) can cause severe disease in humans and has pandemic potential, yet no approved vaccine or therapy exists. Here we describe a dual-mechanism human monoclonal antibody (mAb) combination against RVFV that is effective at minimal doses in a lethal mouse model of infection. We structurally analyze and characterize the binding mode of a prototypical potent Gn domain-A-binding antibody that blocks attachment and of an antibody that inhibits infection by abrogating the fusion process as previously determined. Surprisingly, the Gn domain-A antibody does not directly block RVFV Gn interaction with the host receptor low density lipoprotein receptor-related protein 1 (LRP1) as determined by a competitive assay. This study identifies a rationally designed combination of human mAbs deserving of future investigation for use in humans against RVFV infection. Using a two-pronged mechanistic approach, we demonstrate the potent efficacy of a rationally designed combination mAb therapeutic.
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Sep 2023
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[28534]
Open Access
Abstract: Rodent-borne hantaviruses are prevalent worldwide and upon spillover to human populations, cause severe disease for which no specific treatment is available. A potent antibody response is key for recovery from hantavirus infection. Here we study a highly neutralizing human monoclonal antibody, termed SNV-42, which was derived from a memory B cell isolated from an individual with previous Sin Nombre virus (SNV) infection. Crystallographic analysis demonstrates that SNV-42 targets the Gn subcomponent of the tetrameric (Gn−Gc)4 glycoprotein assembly that is relevant for viral entry. Integration of our 1.8 Å structure with the (Gn−Gc)4 ultrastructure arrangement indicates that SNV-42 targets the membrane-distal region of the virus envelope. Comparison of the SNV-42 paratope encoding variable genes with inferred germline gene segments reveals high sequence conservation, suggesting that germline-encoded antibodies inhibit SNV. Furthermore, mechanistic assays reveal that SNV-42 interferes with both receptor recognition and fusion during host-cell entry. This work provides a molecular-level blueprint for understanding the human neutralizing antibody response to hantavirus infection.
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Jun 2023
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