I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[36097]
Open Access
Abstract: The tRNA m1G37 methyltransferase (TrmD) is considered essential in various bacteria, including Staphylococcus aureus, a pathogen responsible for a wide range of diseases. Here, we have performed a high-throughput nanomole-scale synthesis campaign (nanoSAR) by late-stage copper(I)-catalyzed alkyne–azide cycloaddition (CuAAC)-functionalizing a library of structurally diverse azides (N = 320) to a pyrrolopyrimidone alkyne. We have identified selective S. aureus TrmD inhibitors with inhibitory activity in the nanomolar to low micromolar range using a direct-to-biology assay read-out. A carbamate-masked guanidine intermediate of the lead structure selectively inhibited S. aureus growth at low micromolar concentrations in cell-based assays, while Gram-negative bacteria and an off-target panel of methyltransferases were not affected. Subsequent cocrystallization resulted in a crystal structure of S. aureus TrmD bound to an inhibitor, providing detailed insights into its binding mode and enabling future structure-guided optimization.
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Dec 2025
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B24-Cryo Soft X-ray Tomography
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Diamond Proposal Number(s):
[18925, 19958, 21485, 23508, 25247, 26657, 30442]
Open Access
Abstract: Numerous viral genes are involved in the assembly of herpes simplex virus-1 (HSV-1), but their relative importance and function remain poorly characterised. Transmission electron microscopy has been used to study viral protein function in cells infected with HSV-1 mutants; however, these studies were usually conducted without correlative light microscopy to identify specific viral components. In this study, fluorescent capsid (eYFP-VP26) and envelope (gM-mCherry) proteins were imaged by structured illumination microscopy under cryogenic conditions (cryoSIM) and cellular ultrastructure was captured from the same infected cells using cryo-soft-X-ray tomography (cryoSXT). Nine fluorescent HSV-1 mutants, each lacking a different viral protein, were compared to assess the importance of viral proteins in different stages of HSV-1 morphogenesis. The relative importance of five viral proteins to nuclear egress were ranked (pUL34 >pUL21>VP16>pUL16>pUS3) according to the levels of attenuation observed for each virus. Correlative imaging also revealed the roles of five viral proteins in cytoplasmic envelopment. VP16 was found to be important in capsid delivery to envelopment compartments, while cytoplasmic clusters of virus particles plus features of stalled envelopment not previously described were observed in the absence of pUL11, pUL51, gK, and gE. Finally, this 3D imaging approach was used to capture different assembly stages during cytoplasmic envelopment and to determine that envelopment occurs by particle budding rather than wrapping. The findings demonstrate that tomographic 3D correlative imaging is an emerging technology that sheds new light on viral protein functions and virion morphogenesis.
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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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I04-Macromolecular Crystallography
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Xue
Han
,
Izaak N.
Beck
,
Moise
Mansour
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Tom J.
Arrowsmith
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Roland
Barriot
,
Paul
Chansigaud
,
Carine
Pagès
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Hussein
Hamze
,
Hatice
Akarsu
,
Laurent
Falquet
,
Peter
Redder
,
Xibing
Xu
,
Tim R.
Blower
,
Pierre
Genevaux
Diamond Proposal Number(s):
[24948]
Open Access
Abstract: Toxin–antitoxin (TA) systems are central to bacterial immunity, genome maintenance, and pathogenicity. Toxins of TA systems use diverse strategies to control bacterial growth and represent attractive therapeutic targets to fight pathogens. In this work, we have investigated the toxic mechanism of the three RelE toxins of Mycobacterium tuberculosis, the bacterium responsible for tuberculosis in humans. Structural studies showed that RelBE1, RelBE2, and RelBE3 TA complexes share conserved structural motifs distinct from the RelBE complex of Escherichia coli. Although RelE homologs have previously been reported to perform ribosome-dependent messenger RNA (mRNA) cleavage, detection of cleavage products by nEMOTE demonstrated that only RelE3 targets mRNA. In contrast, in vitro and in vivo analyses using Mycobacterium smegmatis and M. tuberculosis revealed that RelE1 is a site-specific RNase, able to cleave 16S rRNA from free 30S and formed 70S ribosomes, to release the anti-Shine–Dalgarno region and prevent translation. This stunning mode of action, which is likely shared with RelE2, demonstrates that there is broader diversity for toxic mechanisms within the widespread RelE family.
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Nov 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[38168]
Open Access
Abstract: Cyclophilins (Cyps) are ubiquitous cytosolic proteins with peptidyl-prolyl cis-trans isomerase (PPIase) activity and the ability to bind the immunosuppressant cyclosporin A (CsA). The genome of Toxoplasma gondii, the parasite responsible for toxoplasmosis, encodes multiple putative Cyps, whose specific functions remain largely unexplored.
Here, we characterize TgCyp21, a predicted Cyp from T. gondii. TgCyp21 displays PPIase activity and is inhibited by CsA in vitro. Importantly, its activity decreases markedly under oxidizing conditions but is partially restored by reducing agents, including dithiothreitol (DTT) and the parasite endogenous thioredoxin (TgTrx). TgCyp21 contains four cysteines, with Cys87 and Cys141 predicted to be spatially close based on structural modeling. Substitution of both residues significantly reduced PPIase activity, with Cys87 emerging as the main contributor to this loss. Structural modeling further indicates that Cys87 and Cys141 are suitably oriented to interact with the conserved active-site cysteines of TgTrx. This interaction is supported experimentally by mixed disulfide trapping, which identifies a stable disulfide-linked intermediate between TgCyp21 and TgTrx, consistent with a thiol-disulfide exchange mechanism. Small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy further confirm the formation of the complex.
Taken together, our data indicate that TgCyp21 behaves in vitro as a redox-responsive Cyp and a substrate for Trx, suggesting a potential involvement in Trx-mediated redox processes in T. gondii.
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Nov 2025
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Aquilos-CryoFIB at Diamond
Scios-Scios at Diamond
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Diamond Proposal Number(s):
[31607]
Open Access
Abstract: Hepatitis C virus (HCV) infection induces extensive rearrangements of host cytoplasmic membranes, leading to the formation of multiple membranous structures that facilitate RNA replication. Current knowledge of these membranous structures has largely relied on correlative light and electron microscopy techniques using chemical fixation and resin embedding. To overcome these limitations, cryo-preserved cells were prepared using cryo-focused ion beam (cryo-FIB) milling and cryo-ultramicrotomy. For the first time, the contents within the membranous structures have been observed in situ using cryo-electron tomography (cryo-ET) performed on lamellae (prepared via cryo-FIB) and on ultrathin sections (prepared via cryo-ultramicrotomy) from HCV subgenomic replicon-harbouring cells. Observations from 112 cryo-electron tomograms of cryo-FIB-derived samples revealed the presence of densities within the inner vesicles of a subset of single- and double-membrane vesicles, as well as within multi-vesicular bodies, which are consistent with the presence of the viral genome replication machinery. Notably, this study also presents the first direct visualization of densities within a multi-membrane vesicle observed by cryo-electron microscopy of vitreous sections. The cryo-ET methodologies established here lay the groundwork for future investigations into the architecture of the HCV replication complex, leveraging advanced computational tools for deeper structural and functional analyses.
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Nov 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[33176, 35926]
Open Access
Abstract: Mycobacterial infections, including tuberculosis, remain a major global health challenge, causing millions of deaths annually. Their treatment is increasingly hindered by limited therapeutic options and rising antimicrobial resistance, highlighting the urgent need for alternative strategies. Mycobacteriophage LysA endolysins are complex multi-domain peptidoglycan hydrolases emerging as potential tools to treat mycobacterial infections. However, despite the therapeutic prospects of LysAs, our understanding of their mechanism of action remains limited. This study provides a comprehensive structural-functional analysis of the catalytic domains of D29LysA and DS6ALysA endolysins (D29N4/D29GH19 and DS6AGH19/DS6AAmi2B), characterised alone and in complex with PG analogues, using protein engineering, X-ray crystallography, small-angle X-ray scattering, and in silico tools. Our results reveal precise details of the substrate-binding site and the catalytic platforms at each domain, including information about substrate-binding mode and conformational changes associated with peptidoglycan recognition and hydrolysis. Moreover, these findings also suggest a coordinated mechanism of action of both catalytic domains in DS6ALysA lysin. These insights represent a significant advance in understanding the structural basis of mycobacterial cell-wall degradation by mycobacteriophage endolysins. Information that may aid in further exploring these endolysins as therapeutic antimicrobial tools in the future.
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Nov 2025
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Krios I-Titan Krios I at Diamond
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Andreas
Schedlbauer
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Xu
Han
,
Wouter
Van Bakel
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Tatsuya
Kaminishi
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Borja
Ochoa-Lizarralde
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Idoia
Iturrioz
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Retina
Çapuni
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Ransford
Parry
,
Ronny
Zegarra
,
David
Gil-Carton
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Jorge P.
López-Alonso
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Kristina
Barragan Sanz
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Letizia
Brandi
,
Claudio O.
Gualerzi
,
Paola
Fucini
,
Sean R.
Connell
Diamond Proposal Number(s):
[17171, 31586]
Open Access
Abstract: The initiation phase is the rate-limiting step of protein synthesis (translation) and is finely regulated, making it an important drug target. In bacteria, initiation is guided by three initiation factors and involves positioning the start site on the messenger RNA within the P-site on the small ribosomal subunit (30S), where it is decoded by the initiator fMet–tRNA. This process can be efficiently inhibited by GE81112, a natural hydrophilic, noncyclic, nonribosomal tetrapeptide. It is found in nature in three structural variants (A, B, and B1 with molecular masses of 643–658 Da). Previous biochemical and structural characterization of GE81112 indicates that the primary mechanism of action of this antibiotic is to (i) prevent the initiator fMet–tRNA from binding correctly to the P-site and (ii) block conformational rearrangements in initiation factor IF3, resulting in an unlocked 30S preIC state. In this study, using cryo-EM, we have determined the binding site of GE81112 in initiation complexes (3.2–3.7 Å) and on empty ribosomes (2.09 Å). This binding site is within the mRNA channel but remote from the binding site of the initiation factors and initiator fMet–tRNA. This suggests that it acts allosterically to prevent the initiator fMet–tRNA from being locked into place. The binding mode is consistent with previous biochemical studies and recent work identifying the key pharmacophores of GE81112.
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Oct 2025
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Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[34172]
Open Access
Abstract: Plasmodium falciparum is a eukaryotic pathogen responsible for the majority of malaria-related fatalities. Plasmodium belongs to the phylum Apicomplexa and, like most members of this phylum, contains a non-photosynthetic plastid called the apicoplast. The apicoplast has its own genome, replicated by a dedicated replisome. Unlike other cellular replisomes, the apicoplast replisome uses a single DNA polymerase (apPol). This suggests that apPol can multitask and catalyse both replicative and lesion bypass synthesis. Replicative synthesis relies on a restrictive active site for high accuracy while lesion bypass typically requires an open active site. This raises the question: how does apPol combine the structural features of multiple DNA polymerases in a single protein? Using single-particle electron cryomicroscopy (cryoEM), we have solved the structures of apPol bound to its undamaged DNA and nucleotide substrates in five pre-chemistry conformational states. We found that apPol can accommodate a nascent base pair with the fingers in an open configuration, which might facilitate the lesion bypass activity. In the fingers-open state, we identified a nascent base pair checkpoint that preferentially selects Watson–Crick base pairs, an essential requirement for replicative synthesis. Taken together, these structural features might explain how apPol balances replicative and lesion bypass synthesis.
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Oct 2025
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B23-Circular Dichroism
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Open Access
Abstract: The emergence of multidrug-resistant (MDR) bacterial pathogens is an alarming global health threat that demands new therapeutic strategies beyond conventional antibiotics. Here, we present a rationally designed antimicrobial peptide (AMP) derived from mammalian cathelicidins and defensins that selectively targets bacterial membranes with low cytotoxicity toward mammalian cells. Circular dichroism spectroscopy revealed that the peptide adopts an α-helical conformation upon membrane interaction, a key feature of its mechanism. Surface plasmon resonance and isothermal titration calorimetry demonstrated high-affinity and selective binding to bacterial lipid membranes. Functionally, the peptide was strongly bactericidal against clinical MDR Escherichia coli (E. coli) and clinically important ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.). Compared with the parent peptide LL-37, our AMP exhibited lower minimum inhibitory concentrations (MICs) and faster bactericidal kinetics across both Gram-negative and Gram-positive strains. Calcein leakage assays, showing effective membrane disruption. Importantly, cytotoxicity experiments with human epithelial (Caco-2) and immune (THP-1) cells indicated low cytotoxicity at concentrations exceeding bactericidal levels, supporting a favorable therapeutic window. ELISA quantifications of cytokines (IL-6, TNF-α) further suggested immunomodulatory effects at bactericidal concentrations. Transcriptomic profiling of E. coli treated with sub-lethal concentrations of the peptide exhibited upregulation of bacterial stress response pathways and downregulation of vital metabolic processes, reflecting the complex antimicrobial action of the peptide. Collectively, these findings highlight this LL-37-derived AMP as a promising candidate for treating MDR bacterial infections caused by E. coli and ESKAPE pathogens and for guiding the development of next-generation antimicrobial agents.
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Oct 2025
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