I24-Microfocus Macromolecular Crystallography
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Guido C.
Paesen
,
Nathaniel S.
Chapman
,
Jonna B.
Westover
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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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VMXi-Versatile Macromolecular Crystallography in situ
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Diamond Proposal Number(s):
[28534, 34263]
Open Access
Abstract: Schistosoma mansoni cathepsin D1 (SmCD1) has been shown to be an essential enzyme for helminth metabolism due to its role in haemoglobin degradation: a key amino-acid source for the developing parasite. Therefore, the enzyme is a potential target for the development of antischistosomal inhibitors. SmCD1 has significant sequence identity to cathepsin D-like proteases found in other schistosome species and homology to mammalian aspartic proteases. Here, we report the first crystal structures of a helminth cathepsin D, SmCD1, and have identified a single-domain antibody (nanobody) that specifically binds to SmCD1 with nanomolar affinity but does not recognize human cathepsin D. We have mapped the epitope of the nanobody by determining the crystal structure of the enzyme–nanobody complex, revealing the conformation of SmCD1 in the propeptide-bound state.
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Feb 2026
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Ingra M.
Claro
,
Erika R.
Manuli
,
Camila A. M.
Da Silva
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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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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[34221]
Open Access
Abstract: Point mutations in the exonuclease (ExoN) site of nonstructural protein 14 (NSP14) compromise the fitness of betacoronaviruses such as SARS-CoV-2, implicating NSP14 ExoN inhibition as an antiviral strategy. However, there are no advanced compounds that inhibit NSP14’s ExoN activity. Building upon the reported crystal structures of two fragments bound to NSP14’s ExoN site, we identified a series of 3,5-disubsituted pyrazoles that bound to and inhibited NSP14 ExoN. However, upon resynthesis, we discovered that these putative leads were false positives, perhaps due to contaminating divalent cations, which potently inhibit NSP14 ExoN. Our results provide a cautionary tale to the field about the sensitivity of NSP14 to divalent cations and illustrate the challenges associated with directly targeting the NSP14 ExoN site via fragment merging.
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Feb 2026
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[25587]
Open Access
Abstract: Nonstructural protein 3 (nsp3) is crucial for SARS-CoV-2 infection. It is the largest protein of the virus with roughly 2000 residues, and a major drug target. However, because of its size, disordered regions, and transmembrane domains, the atomic structure of the whole protein has not yet been established. Only 10 out of its 16 domains were individually determined in experiments. Here, we demonstrate how structural bioinformatics, AI-based fold prediction, and traditional experiments complement each other and can shed light on the makeup of this important protein, both in SARS-CoV-2 and in related viruses. Our method can be generalized for other multidomain proteins. Our prediction-based approach reveals a previously undescribed folded domain, which we could confirm experimentally. Our research also suggests a potential function of the domain Y1: this domain may be involved in the assembly of nsp3, nsp4, and nsp6 into the hexameric pore, which was discovered by electron tomography and exports RNA into the cytosol. The Y1 hexamer, however, could not be expressed on its own. We revise domain segmentation and nomenclature of nsp3 domains.
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Feb 2026
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[28402]
Open Access
Abstract: Despite the continual emergence of SARS-CoV-2 variants and increasing diversity within the receptor binding domain (RBD), some antibody responses that are directed to conserved regions can display cross-reactivity against variants. We previously isolated an RBD-directed monoclonal antibody (084-7D) from a Beta-infected donor that neutralized Beta and emerging Omicron variants. Here, we solved a high-resolution crystal structure of the 084-7D Fab in complex with the Beta RBD. These data revealed an epitope overlapping both the ACE2 binding site and those of other class 1 antibodies. Furthermore, the epitope includes highly conserved residues, Q409, D420, and Y489, that are present in recent Omicron variants. The N417 residue that emerged with Beta and has since persisted is tolerated within the epitope of 084-7D, explaining the preferential neutralization of contemporaneous N417-containing variants. These structural data defined the mechanism for cross-reactivity of a Beta-elicited neutralizing antibody, potentially informing the design of future broadly reactive SARS-CoV-2 therapeutics.
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Feb 2026
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Matthew R.
Singer
,
Zhen
Li
,
Juan S.
Rey
,
Joshua
Hope
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Florian
Chenavier
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Nicola J.
Cook
,
Emma
Punch
,
Jamie
Smith
,
Zhiyu
Zhou
,
Sarah
Maslen
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Laura
Masino
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Andrea
Nans
,
Mark
Skehel
,
Ian A.
Taylor
,
Giulia
Zanetti
,
Peijun
Zhang
,
Juan R.
Perilla
,
Alan N.
Engelman
,
Peter
Cherepanov
Open Access
Abstract: HIV-1 integrase (IN) promotes encapsulation of viral genomic RNA into mature viral cores, and this function is a target for ongoing antiretroviral drug development efforts1,2,3. Here we determined the cryogenic electron microscopy (cryo-EM) structure of a primate lentiviral IN in a complex with RNA, revealing a linear filament made of IN octamer repeat units, each comprising a pair of asymmetric homotetramers. The assembly is stabilized through IN–RNA interactions involving mainly the IN C-terminal domains and RNA backbone. The spacing and orientation of the IN filament repeat units closely matched those of consecutive capsid (CA) hexamers within the mature CA lattice. Using cryo-EM images of native purified HIV-1 cores, we refined the structure of the IN filament as it propagates along the luminal side of the CA lattice. Each IN tetramer within the filament nestled in a CA hexamer, engaging closely with the major homology regions. Substitutions of residues involved in IN–CA contacts yielded eccentric virions with RNA nucleoids located outside of the cores. Collectively, our results establish the structural basis for the HIV-1 IN–RNA interaction and reveal that IN forms an RNA-binding module on the luminal side of the mature CA lattice.
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Feb 2026
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B21-High Throughput SAXS
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Mohamad
Ahmad Najib
,
Anja
Winter
,
Khairul Mohd Fadzli
Mustaffa
,
Muhammad Hafiznur
Yunus
,
Eugene Boon Beng
Ong
,
Kasturi
Selvam
,
Muhammad Fazli
Khalid
,
Mohd Syafiq
Awang
,
Asrulnizam Abd
Manaf
,
Yazmin
Bustami
,
Habibah A.
Wahab
,
Ismail
Aziah
Diamond Proposal Number(s):
[34438]
Open Access
Abstract: Efficient and timely detection of biomarkers for typhoid fever is pivotal for improving treatment outcomes and preventing further transmission of the disease. This study addresses the pressing need for a rapid and accurate point-of-care test to detect Salmonella Typhi HlyE antigen, a vital biomarker for typhoid fever. Leveraging the growing prominence of computer-based simulations for aptamer-protein interactions modelling and electrochemical biosensors for their high sensitivity, this study presents the development of a novel electrochemical aptasensor designed to accurately and effectively detect Salmonella Typhi HlyE antigen in blood samples of typhoid patients. We previously identified and characterized selective aptamers against HlyE antigen, and identified AptHlyE97 as the aptamer with highest binding affinity and selectivity. In this study we showed that AptHlyE97 formed the most stable complex with HlyE in molecular docking and dynamics simulations and an unbound 5’ end to enable conjugation to the sensor surface. The aptasensor features AptHlyE97 conjugated via thiol immobilization to the surface of a screen-printed gold electrode, and utilizing potassium ferricyanide and potassium ferrocyanide for electrochemical detection. The aptasensor showed a significant (p < 0.001) square wave voltammetry (SWV) response to HlyE with a diagnostic performance of 100% sensitivity and 85.7% specificity against other bacterial communicable disease pathogens. Additionally, the aptasensor exhibited a strong linear response with a limit of detection (LoD) of 0.158 ng/mL. We present here a novel diagnostic aptasensor capable of efficiently and selectively detecting the HlyE antigen from Salmonella Typhi. This aptasensor provides a robust foundation for future research in typhoid diagnosis and also highlights the potential impact in advancing point-of-care detection strategies in community settings.
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Feb 2026
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B21-High Throughput SAXS
Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[24557, 39224]
Open Access
Abstract: Histone variants define distinct chromatin states by modulating the biophysical properties of nucleosomes. Variants play a particularly important role in the parasitic protist Trypanosoma brucei, which has unusual chromatin and lacks a canonical repressive heterochromatin system. Instead, T. brucei utilizes specialized divergent histone variants H3.V and H4.V. However, the biochemical basis of their repressive functions is unknown. Here, we determined the structure of the H3.V-H4.V nucleosome core particle and biochemically characterized variant-containing nucleosomes and nucleosome arrays, probing their unique properties. We discovered that surprisingly for repressive-state nucleosomes, H3.V promotes pronounced DNA splaying, largely via its N-terminal tail region, while retaining overall stability that is comparable to canonical nucleosomes. In contrast, H4.V exhibits near-identical binding to DNA but mediates a slight increase in histone octamer stability. The surface of the H3.V-H4.V nucleosome is altered and provides a differential platform for chromatin-binding proteins, linking the variants to parasite pathogenicity.
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Feb 2026
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23459]
Open Access
Abstract: Intestinal amoebiasis is caused by Entamoeba histolytica, one of the deadliest human-infective parasites. Central to its pathogenicity is its binding to mucosal carbohydrates, which precedes tissue damage by trogocytosis. Carbohydrate binding is mediated by a single adhesin, the galactose/N-acetylgalactosamine (Gal/GalNAc) lectin, which is the leading vaccine candidate for amoebiasis. We present the structure of the native heterodimeric lectin, revealing an ordered core containing the light chain and the N-terminal region of the heavy chain. Structures obtained in the presence of ligand show that the Gal/GalNAc binding site is in the light chain, which adopts a β-trefoil fold found in other lectins. An elongated arm emerges from the heavy chain, which adopts multiple positions and may be modulated by sugar binding. This study reveals the molecular basis for sugar binding by the Entamoeba histolytica Gal/GalNAc lectin, a prerequisite for parasite invasion and development of intestinal disease.
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Feb 2026
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