I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[35587]
Open Access
Abstract: Canine and human anaplasmosis are tick-borne diseases caused by the obligate intracellular bacteria, Anaplasma phagocytophilum and Anaplasma platys, respectively. In veterinary medicine, peptides derived from the immunodominant P44 protein are used to detect antibodies in serological point-of-care tests for anaplasmosis. To inform efforts to develop improved chimeric diagnostic proteins for anaplasmosis, the structure of the predicted surface-exposed domain (residues 151–308) of P44 was determined at a resolution of 1.65 Å. This domain comprises six α-helices and six short β-strands connected by loop regions. A structural similarity search of the P44151–308 domain using the Distance Matrix Alignment server against the AlphaFold Database revealed that the domain represents a previously uncharacterized protein fold. The determined structure is consistent with AlphaFold v2 predictions, which further predict the core of P44 to be an eight-stranded β-barrel. Using P44 fragments, immunodominant epitopes were mapped within residues 151–308. Four chimeric genes (ADC1, ADC2, ADC3, and ADC4) were designed that incorporate the analogous immunodominant P44 domains from A. phagocytophilum and A. platys. The recombinant ADC chimerics were assessed for sensitivity and specificity by screening with serum from dogs diagnosed with anaplasmosis, Lyme disease, or ehrlichiosis, with sera from specific pathogen-free dogs serving as negative controls. Accuracy was further demonstrated by screening characterized serum samples from coyotes (Canis latrans; wild canids). The ADC1 chimeric provided 100% sensitivity and 98.04% specificity. The sensitivity and specificity of ADC1 support its potential use as a diagnostic Ag for anaplasmosis.
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Sep 2026
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Krios I-Titan Krios I at Diamond
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Ane
Martinez-Castillo
,
Andrea
Aebischer
,
Philippine
Toneatti
,
Lifei
Fu
,
Damien
Vitour
,
Corinne
Sailleau
,
Bernd
Hoffmann
,
Kati
Franzke
,
Michael
Eschbaumer
,
Saskia
Weber
,
Eva
Calvo Pinilla
,
Javier
Ortego
,
David
Gil-Carton
,
Emmanuel
Bréard
,
Stéphan
Zientara
,
Jeroen
Kortekaas
,
Martin
Beer
,
Nicola G. A.
Abrescia
Diamond Proposal Number(s):
[31586]
Open Access
Abstract: African horse sickness virus (AHSV) is a lethal equine pathogen with no licensed vaccine other than vaccines containing attenuated virus, which pose safety risks. Endemic to sub-Saharan Africa, AHSV has caused epizootics in Spain and Portugal, Cyprus, Morocco, the Middle East, India and Pakistan and, most recently, Thailand. Here, we resolve the 3.11 Å cryo-EM structure of full-length VP2 from AHSV serotype 4, adopting its native triskelion architecture and shedding light on an α-helical domain anchoring the triskelion core, which is absent in other structurally characterized orbiviruses. Structure-guided mapping identified a subdomain of VP2 as a key target of neutralizing antibodies. Displayed on nanoparticles using the SpyCatcher/SpyTag technology, the domain conferred complete protection from clinical disease after viral challenge infection in mice and elicited robust and long-lasting immune responses in horses, the target species of AHSV. These findings provide a structural blueprint for the next generation of recombinant vaccines against AHSV and related orbiviruses.
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Jul 2026
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[27314]
Open Access
Abstract: Marek’s disease (MD) is a highly contagious neoplastic disorder of poultry caused by MD virus (MDV; gallid alphaherpesvirus 2 [GaAHV2]). Infection results in profound immunosuppression, neurological dysfunction, and the development of malignant T-cell lymphomas. Continued viral evolution has produced increasingly virulent strains capable of partially or fully evading current vaccines, leaving few options for controlling emerging variants. This highlights the importance of identifying new antiviral targets, particularly those involved in the nuclear trafficking events essential for GaAHV2 replication. The UL36 large tegument protein of alphaherpesviruses contains N-terminal nuclear localization signals (NLSs) thought to guide capsid transport to the nuclear pore complex. However, the specific mechanism by which GaAHV2 UL36 engages the host nuclear import machinery remains unclear. In this work, we defined the NLS within the N-terminal region of GaAHV2 UL36 and characterized its interaction with importin proteins. Through high-resolution crystallography and quantitative binding assays, we pinpointed the residues and structural motifs within UL36 that mediate recognition by importin-α (IMPα) and compared their affinities across different IMPα isoforms. Our structural and biochemical data show that the predicted N-terminal NLS of GaAHV2 UL36 is essential for IMPα binding. These findings provide a detailed molecular framework for host–virus interactions during GaAHV2 nuclear entry and offer potential avenues for the development of targeted antiviral strategies.
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Jun 2026
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: Classical molecules encoded by the major histocompatibility complex (MHC) are central to immune responses. Compared to typical mammals, the chicken MHC is small and simple, determining life or death from economically important pathogens like avian influenza virus and Marek’s disease virus (MDV). Several genes within the tightly linked chicken MHC have been suggested to determine resistance and susceptibility to MDV, but it was a surprise to find that the dominantly expressed class II molecule from the resistant B2 haplotype employed a novel peptide-binding mode with a decamer core sequence compared to the susceptible B19 haplotype with a typical nonamer core. We examined the crystal structure of the dominantly expressed class II molecule from another resistant haplotype, B21, which is extremely frequent in commercial chicken flocks, to find that it bound the same MDV peptide with both nonamer and decamer cores, revealing an unexpected plasticity of binding that potentially increases the immune response to this devastating pathogen.
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Apr 2026
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I03-Macromolecular Crystallography
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Marie
Bonnet-Di Placido
,
Helen M. E.
Duyvesteyn
,
Angela W.
Steyn
,
Abigail L.
Hay
,
Claudine
Porta
,
Kristel Ramirez
Valdez
,
Elena
Lokhman
,
Sylvia
Crossley
,
Kevan
Hanson
,
William N.
Mwangi
,
Danish
Munir
,
Eva
Perez-Martin
,
Nick J.
Knowles
,
Alison
Burman
,
Abdelaziz A.
Yassin
,
Amin
Asfor
,
Cristina
Faralla
,
Katherine J.
Lam
,
Róisín
Mccomb
,
Carina
Leifeld
,
Kimberly
Pietersz
,
Donald P.
King
,
Erwin
Van Den Born
,
Sherie K.
Duncan
,
Bryan
Charleston
,
Elizabeth E.
Fry
,
Jingshan
Ren
,
David I.
Stuart
,
John A.
Hammond
Diamond Proposal Number(s):
[28534]
Open Access
Abstract: Foot-and-mouth disease virus (FMDV) causes a devastating disease that threatens global food security. Vaccination is hindered by antigenic diversity across serotypes. To identify cross-serotype neutralising epitopes, we isolated 24 FMDV-specific antibodies from cattle sequentially vaccinated with antigens from four serotypes, of which three neutralised three vaccine strains. These three antibodies neutralised 21 and bound 59 additional topotypes across O, A, Asia 1, and C serotypes. Cryo-EM complexes of Fabs with FMD virus-like particles indicated all three recognise a common flexible epitope at the VP1 C-terminus, confirmed by binding competition. Crystallography and structural modelling revealed that a normally inaccessible surface of the hydrophobic VP1 C-terminal peptides inserts into a similar groove in all three antibodies. Comparison of neutralisation activity and integrin receptor blocking by whole antibodies, F(ab’)2s, and Fabs suggests neutralisation is mediated by Fc steric hindrance of receptor binding. This cryptic, linear, and cross-serotype neutralising epitope may inform improved FMD vaccines.
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Apr 2026
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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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Krios I-Titan Krios I at Diamond
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Open Access
Abstract: Caliciviruses are important human and animal pathogens that cause varying clinical signs including gastroenteritis, respiratory illness, and hepatitis. Despite the availability of numerous calicivirus structures, relatively little is known about the mechanisms of capsid assembly and stability, or about genome packaging. Here we present the atomic structure of the RHDV virion and several related non-infectious virus-like particles, determined using cryo-EM at 2.5-3.3 Å resolution. The inherent molecular switch, responsible for the conformational flexibility of the capsid protein VP1, is located in its N-terminal arm (NTA). The NTA establishes an extensive network of interactions on the inner capsid surface that stabilizes the hexamers and pentamers. For this structural polymorphism, we show that the NTA must interact with the RNA viral genome, that is, the genomic RNA acts with the NTA as a molecular co-switch. The NTA-RNA interaction leads to specific conformational states that result in two types of VP1 dimers (the basic building blocks) necessary for T = 3 capsid assembly. In addition, we used atomic force microscopy (AFM) to assess whether differences in genomic RNA content influence viral properties such as capsid stiffness in physiological conditions. These analyses highlight the mechanical role of packed RNA genome in RHDV virions, as the virion capsid pentamers are strengthened by interactions of the NTA star-like structure promoted by the viral genome. These results indicate that the interactions between the NTA and the viral genome guide the conformational states of VP1 dimers, directing capsid assembly and modulating its mechanical properties. Through interference with intermediate assemblies, the NTA network promoted by the genome could be an attractive target in future antiviral strategies.
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Dec 2025
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Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[15997]
Open Access
Abstract: High-resolution structural studies have mainly focused on two out of the six adenovirus genera: mastadenoviruses and atadenoviruses. Here we report the high-resolution structure of an aviadenovirus, the poultry pathogen fowl adenovirus serotype 4 (FAdV-C4). FAdV-C4 virions are highly thermostable, despite lacking minor coat and core proteins shown to stabilize the mast- and atadenovirus particles, having no genus-specific cementing proteins, and packaging a 25% longer genome. Unique structural features of the FAdV-C4 hexon include a large insertion at the trimer equatorial region, and a long N-terminal tail. Protein IIIa conformation is closer to atadenoviruses than to mastadenoviruses, while protein VIII diverges from all previously reported structures. We interpret these differences in light of adenovirus evolution. Finally, we discuss the possible role of core composition in determining capsid stability properties. These results enlarge our view on the structural diversity of adenoviruses, and provide useful information to counteract fowl pathogens or use non-human adenoviruses as vectors.
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Oct 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[29835]
Open Access
Abstract: We present a comprehensive investigation into the catalytic mechanism of methylisocitrate lyase, a potential drug target candidate against the zoonotic pathogen Coxiella burnetii, the causative agent of Q fever and a federal select agent. Current treatment regimens are prolonged, often with incomplete clearance of the pathogen. We utilised a structure-based bioinformatics pipeline to identify methylisocitrate lyase as a candidate therapeutic target against C. burnetii from a list of essential genes. Wild-type C. burnetii methylisocitrate lyase has a kcat of 13.8 s-1 (compared to 105 s-1 for Salmonella enterica) and isocitrate inhibits with a KI of 11 mM. We have determined the previously uncharacterised substrate-bound structure of this enzyme family, alongside product and inhibitor-bound structures. These structures of wild-type enzyme reveal that in the active state the catalytic C118 is positioned 2.98 Å from O5 of methylisocitrate and Arg152 moves towards the substrate relative to the inhibitor bound structure. Analysis of structure-based mutants reveals that Arg152 and Glu110 are both essential for catalysis. We suggest that Arg152 acts as the catalytic base that initiates the methylisocitrate lyase reaction. These results deepen our understanding of the catalytic mechanism of methylisocitrate lyase and could aid the development of new therapeutics against C. burnetii.
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Apr 2025
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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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