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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Hui
Sun
,
Yanan
Jiang
,
Miaolin
Lan
,
Ming
Zhou
,
Gangshun
Yi
,
Juan
Shen
,
Tingting
Deng
,
Liqin
Liu
,
Yang
Huang
,
Yu
Li
,
Jinfu
Su
,
Yanling
Lin
,
Zhenqin
Chen
,
Lizhi
Zhou
,
Tingting
Li
,
Hai
Yu
,
Tong
Cheng
,
Yali
Zhang
,
Lunzhi
Yuan
,
Shaowei
Li
,
Ying
Gu
,
Peijun
Zhang
,
Ningshao
Xia
,
Qingbing
Zheng
Open Access
Abstract: The rapid evolution of SARS-CoV-2 and the subsequent emergence of Omicron subvariants pose significant challenges to the efficacy of existing vaccines and therapeutics, including those previously reported most broad neutralizing antibodies (bnAbs). Here, we investigated the molecular basis of the altered neutralization profile of a bnAb, 1C4, against recent variants. 1C4 is effective against early variants from Alpha to Omicron BQ.1, but is circumvented by BQ.1.1, XBB and thereafter variants, primarily due to an additional R346T mutation that diminishes its binding affinity. Cryo-electron microscopy analysis revealed that despite the loss of neutralizing potency, 1C4 retained residual binding to the spike protein of immune-evasive variants such as XBB, which harbor altered receptor-binding domain (RBD). Furthermore, 1C4 exhibited a diminished capacity to inhibit ACE2 engagement with Omicron variants, amplifying the intricacies of viral immune evasion tactics. To address this, we employed the mi3-SpyCatcher-based nanoparticle to polymerize 1C4 (mi3-1C4), which reestablished the neutralization potency against recent variants by enhancing avidity via multivalent binding. Such multivalent binding can promote efficient spike aggregation as well as viral cross-linking, thereby providing enhanced protection against both the infection of Beta and XBB variants in a hamster model. Together, our findings delineate the molecular landscape of immune evasion by neutralizing antibodies and provide strategic insight for the adaptation of antibody engineering to keep pace with viral evolution.
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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
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Nutpakal
Ketprasit
,
Chia-Wei
Tai
,
Vivek Kumar
Sharma
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Yogavel
Manickam
,
Yogesh
Khandokar
,
Xi
Ye
,
Con
Dogovski
,
David H.
Hilko
,
Craig J.
Morton
,
Anne-Sophie C.
Braun
,
Michael G.
Leeming
,
Bagale
Siddharam
,
Gerald J.
Shami
,
Pushpangadan Indira
Pradeepkumar
,
Santosh
Panjikar
,
Sally-Ann
Poulsen
,
Michael D. W.
Griffin
,
Amit
Sharma
,
Leann
Tilley
,
Stanley C.
Xie
Diamond Proposal Number(s):
[28534]
Open Access
Abstract: Malaria poses an enormous threat to human health. With ever-increasing resistance to currently deployed antimalarials, new targets and starting point compounds with novel mechanisms of action need to be identified. Here, we explore the antimalarial activity of the Streptomyces sp natural product, 5′-O-sulfamoyl-2-chloroadenosine (dealanylascamycin, DACM) and compare it with the synthetic adenosine monophosphate (AMP) mimic, 5-O-sulfamoyladenosine (AMS). These nucleoside sulfamates exhibit potent inhibition of P. falciparum growth with an efficacy comparable to that of the current front-line antimalarial, dihydroartemisinin. Exposure of P. falciparum to DACM leads to inhibition of protein translation, driven by eIF2α phosphorylation. We show that DACM targets multiple aminoacyl-tRNA synthetases (aaRSs), including the cytoplasmic aspartyl tRNA synthetase (AspRS). The mechanism involves hijacking of the reaction product, leading to the formation of a tightly bound inhibitory amino acid-sulfamate conjugate. We show that recombinant P. falciparum and P. vivax AspRS are susceptible to hijacking by DACM and AMS, generating Asp-DACM and Asp-AMS adducts that stabilize these proteins. By contrast, human AspRS appears less susceptible to hijacking. X-ray crystallography reveals that apo P. vivax AspRS exhibits a stabilized flipping loop over the active site that is poised to bind substrates. By contrast, human AspRS exhibits disorder in an extended region around the flexible flipping loop as well as in a loop in motif II. These structural differences may underpin the decreased susceptibility of human AspRS to reaction-hijacking by DACM and AMS. Our work reveals Plasmodium AspRS as a promising antimalarial target and highlights structural features that underpin differences in the susceptibility of aaRSs to reaction hijacking inhibition.
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Jul 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[34289]
Open Access
Abstract: Pseudomonas aeruginosa employs the Type VI secretion system (T6SS) to outcompete other bacteria in its environment. Among the effectors secreted by the T6SS of P. aeruginosa PAO1, Tse4 is known for its potent antibacterial activity. This study elucidates the molecular function of Tse4, which promotes cell depolarization in competing bacteria. Our results show that Tse4 spontaneously incorporates into lipid monolayers and forms multiionic channels in planar bilayers, with either ohmic conduction or diode-like rectifying currents and a preference for cations over anions. These observations allow us to propose a model of action whereby Tse4 channels couple cell depolarization with K+ efflux. These insights into Tse4’s pore-forming activity enhance our understanding of bacterial competition and exemplify a finely tuned antibacterial strategy, coupling its ability to cause membrane depolarization with potassium efflux that synergises with other T6SS effectors. These results highlight the sophistication of Pseudomonas aeruginosa’s competitive arsenal.
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Jun 2025
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B21-High Throughput SAXS
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Subash
Chapagain
,
Nicolas
Salcedo-Porras
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Amir
Abdolahzadeh
,
Yaohua
Zhang
,
Higor Sette
Pereira
,
Stephane
Flibotte
,
Kevin
Low
,
Christina
Young
,
Yuhang
Wu
,
Shao
Wang
,
Soh
Ishiguro
,
Nozomu
Yachie
,
Trushar
Patel
,
Artem
Babaian
,
Eric
Jan
Diamond Proposal Number(s):
[36363]
Open Access
Abstract: All viruses must co-opt the host translational machinery for viral protein synthesis. The dicistrovirus intergenic region internal ribosome entry site (IGR-IRES) utilizes the most streamlined translation mechanism by adopting a triple pseudoknot structure that directly recruits and binds within the intersubunit space of the ribosome and initiates translation from a non-AUG codon. The origin of this unprecedented mechanism is not known. Using a bioinformatics pipeline to examine the diversity and function of IRESs across RNA viromes, we searched for IRES-like RNA structures using RNA covariance models for multiple IRES sub-types, and tested functional IRES by using a dual-fluorescent lentiviral library reporter screen. We identified over >4,700 dicistro-like genomes with ~32% containing putative IRES structures, including novel viral genome arrangements with multiple IRESs and IRESs embedded within open-reading frames (ORFs). Predicted IRESs bound directly to purified ribosomes and supported internal ribosome entry activity in vitro and in vivo. Moreover, internal IRESs embedded within an ORF of monocistronic genomes were functional and operated simultaneously to produce the downstream ORF. We also identified IRES-like structures within non-dicistrovirus viral genomes, including in the families Tombusviridae and Narnaviridae that bound to ribosomes directly and a subset can direct internal ribosome entry. This study provides a framework to map the origin of factorless IRES mechanisms and study the diverse viral strategies utilizing RNA-based mechanisms.
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Jun 2025
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B21-High Throughput SAXS
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Anuradha
De Silva
,
Kihun
Kim
,
John
Weiland
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Jihyun
Hwang
,
Jacky
Chung
,
Higor S.
Pereira
,
Trushar R.
Patel
,
Joan
Teyra
,
Ankoor
Patel
,
Mohammed M.
Mira
,
Mazdak
Khajehpour
,
Melvin
Bolton
,
Claudio
Stasolla
,
Sachdev S.
Sidhu
,
Brian L.
Mark
Diamond Proposal Number(s):
[26855]
Open Access
Abstract: RNA viruses have evolved numerous strategies to overcome host resistance and immunity, including the use of multifunctional proteases that not only cleave viral polyproteins during virus replication but also deubiquitinate cellular proteins to suppress ubiquitin (Ub)-mediated antiviral mechanisms. Here, we report an approach to attenuate the infection of Arabidopsis thaliana by Turnip Yellow Mosaic Virus (TYMV) by suppressing the polyprotein cleavage and deubiquitination activities of the TYMV protease (PRO). Performing selections using a library of phage-displayed Ub variants (UbVs) for binding to recombinant PRO yielded several UbVs that bound the viral protease with nanomolar affinities and blocked its function. The strongest binding UbV (UbV3) candidate had a EC50 of 0.3 nM and inhibited both polyprotein cleavage and DUB activity of PRO in vitro. X-ray crystal structures of UbV3 alone and in complex with PRO reveal that the inhibitor exists as a dimer that binds two copies of PRO. Consistent with our biochemical and structural findings, transgenic expression of UbV3 in the cytosol of A. thaliana suppressed TYMV replication in planta, with the reduction in viral load being correlated to UbV3 expression level. Our results demonstrate the potential of using UbVs to protect plants from tymovirus infection, a family of viruses that contain numerous members of significant agricultural concern, as well as other plant viruses that express functionally related proteases with deubiquitinating activity.
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Jan 2025
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B24-Cryo Soft X-ray Tomography
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Simon
Leclerc
,
Alka
Gupta
,
Visa
Ruokolainen
,
Jian-Hua
Chen
,
Kari
Kunnas
,
Axel A.
Ekman
,
Henri
Niskanen
,
Ilya
Belevich
,
Helena
Vihinen
,
Paula
Turkki
,
Ana J.
Perez-Berna
,
Sergey
Kapishnikov
,
Elina
Mäntylä
,
Maria
Harkiolaki
,
Eric
Dufour
,
Vesa
Hytönen
,
Eva
Pereiro
,
Tony
Mcenroe
,
Kenneth
Fahy
,
Minna U.
Kaikkonen
,
Eija
Jokitalo
,
Carolyn A.
Larabell
,
Venera
Weinhardt
,
Salla
Mattola
,
Vesa
Aho
,
Maija
Vihinen-Ranta
Open Access
Abstract: iruses target mitochondria to promote their replication, and infection-induced stress during the progression of infection leads to the regulation of antiviral defenses and mitochondrial metabolism which are opposed by counteracting viral factors. The precise structural and functional changes that underlie how mitochondria react to the infection remain largely unclear. Here we show extensive transcriptional remodeling of protein-encoding host genes involved in the respiratory chain, apoptosis, and structural organization of mitochondria as herpes simplex virus type 1 lytic infection proceeds from early to late stages of infection. High-resolution microscopy and interaction analyses unveiled infection-induced emergence of rough, thin, and elongated mitochondria relocalized to the perinuclear area, a significant increase in the number and clustering of endoplasmic reticulum-mitochondria contact sites, and thickening and shortening of mitochondrial cristae. Finally, metabolic analyses demonstrated that reactivation of ATP production is accompanied by increased mitochondrial Ca2+ content and proton leakage as the infection proceeds. Overall, the significant structural and functional changes in the mitochondria triggered by the viral invasion are tightly connected to the progression of the virus infection.
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Apr 2024
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I24-Microfocus Macromolecular Crystallography
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Michael J.
Ormsby
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Filipa
Vaz
,
Joseph A.
Kirk
,
Anna
Barwinska-Sendra
,
Jennifer C.
Hallam
,
Paola
Lanzoni-Mangutchi
,
John
Cole
,
Roy R.
Chaudhuri
,
Paula S.
Salgado
,
Robert P.
Fagan
,
Gillian R.
Douce
Diamond Proposal Number(s):
[24948]
Open Access
Abstract: Clostridioides difficile is responsible for substantial morbidity and mortality in antibiotically-treated, hospitalised, elderly patients, in which toxin production correlates with diarrhoeal disease. While the function of these toxins has been studied in detail, the contribution of other factors, including the paracrystalline surface layer (S-layer), to disease is less well understood. Here, we highlight the essentiality of the S-layer in vivo by reporting the recovery of S-layer variants, following infection with the S-layer-null strain, FM2.5. These variants carry either correction of the original point mutation, or sequence modifications which restored the reading frame, and translation of slpA. Selection of these variant clones was rapid in vivo, and independent of toxin production, with up to 90% of the recovered C. difficile population encoding modified slpA sequence within 24 h post infection.
Two variants, subsequently named FM2.5varA and FM2.5varB, were selected for study in greater detail. Structural determination of SlpA from FM2.5varB indicated an alteration in the orientation of protein domains, resulting in a reorganisation of the lattice assembly, and changes in interacting interfaces, which might alter function. Interestingly, variant FM2.5varB displayed an attenuated, FM2.5-like phenotype in vivo compared to FM2.5varA, which caused disease severity more comparable to that of R20291. Comparative RNA sequencing (RNA-Seq) analysis of in vitro grown isolates revealed large changes in gene expression between R20291 and FM2.5. Downregulation of tcdA/tcdB and several genes associated with sporulation and cell wall integrity may account for the reported attenuated phenotype of FM2.5 in vivo. RNA-seq data correlated well with disease severity with the more virulent variant, FM2.5varA, showing s similar profile of gene expression to R20291 in vitro, while the attenuated FM2.5varB showed downregulation of many of the same virulence associated traits as FM2.5. Cumulatively, these data add to a growing body of evidence that the S-layer contributes to C. difficile pathogenesis and disease severity.
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Jun 2023
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14744]
Open Access
Abstract: The prolyl-tRNA synthetase (PRS) is a validated drug target for febrifugine and its synthetic analog halofuginone (HFG) against multiple apicomplexan parasites including Plasmodium falciparum and Toxoplasma gondii. Here, a novel ATP-mimetic centered on 1-(pyridin-4-yl) pyrrolidin-2-one (PPL) scaffold has been validated to bind to Toxoplasma gondii PRS and kill toxoplasma parasites. PPL series exhibited potent inhibition at the cellular (T. gondii parasites) and enzymatic (TgPRS) levels compared to the human counterparts. Cell-based chemical mutagenesis was employed to determine the mechanism of action via a forward genetic screen. Tg-resistant parasites were analyzed with wild-type strain by RNA-seq to identify mutations in the coding sequence conferring drug resistance by computational analysis of variants. DNA sequencing established two mutations, T477A and T592S, proximal to terminals of the PPL scaffold and not directly in the ATP, tRNA, or L-pro sites, as supported by the structural data from high-resolution crystal structures of drug-bound enzyme complexes. These data provide an avenue for structure-based activity enhancement of this chemical series as anti-infectives.
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Feb 2023
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