I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[34182]
Open Access
Abstract: Chikungunya virus (CHIKV) outbreaks impose significant burdens on healthcare systems and raise an urgent need for effective antiviral therapies. So far there are no specific drugs against CHIKV infection. CHIKV-encoded macrodomain is critical for virulence and counteracts the host immune response, representing a promising antiviral drug target. Here, we describe small molecule inhibitors targeting the CHIKV macrodomain. Compound 1 (MDOLL-0273) was identified through a high-throughput screening using a fluorescence resonance energy transfer based assay, exhibiting an IC50 of 8.9 μM, and its inhibitory activity was validated through multiple orthogonal assays. The compound features a thiobarbiturate-indole scaffold and shows high selectivity over a panel of human and viral ADP-ribose binding and hydrolyzing proteins. X-ray crystallography revealed that the inhibitor occupies an adenine binding site of the macrodomain and extends into a novel cryptic pocket. Guided by structure-activity relationship studies, compound 11 (MDOLL-0591) of the developed series with similar IC50 of 10 μM but with increased lipophilicity was discovered to have antiviral activity against CHIKV in cell culture, demonstrating that macrodomain could be targeted in virus infections.
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Nov 2026
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[36130]
Open Access
Abstract: Nakaseomyces glabratus is an opportunistic pathogen of humans, causing invasive candidiasis (IC). Among the risk factors that favor IC are various host-specific factors. Although drugs are available to treat candidiasis, their clinical application remains limited. Therefore, it is necessary to identify therapeutic targets that will enable the development of new antifungals. In this regard, our research group has identified enzymes as potential therapeutic targets in N. glabratus, including fructose-1,6-bisphosphate aldolase (Fba1) and pyruvate kinase (Pk). Enzyme activity studies on these two enzymes have shown that they are important therapeutic targets against this pathogen. However, their three-dimensional structure has not yet been elucidated, an essential requirement for designating an enzyme as a therapeutic target. To propose Fba1 and Pk of N. glabratus as potential therapeutic targets, we investigated the solution structure and oligomeric state of N. glabratus Fba1 and Pk for the first time by combining Small-Angle X-ray Scattering (SAXS) with AlphaFold3 modeling. SAXS data were collected on the B21 beamline at Diamond Light Source (Didcot, UK), providing solution-scattering profiles, molecular-weight estimates, and low-resolution molecular envelopes. These data indicate that Pk is monomeric and Fba1 homodimeric in solution were used to evaluate and refine the corresponding AlphaFold3 atomic models by molecular dynamics. These structural findings for Fba1 and Pk from N. glabratus open the door to understanding these enzymes as potential therapeutic targets against this pathogen and, at the same time, a basis for future comparative studies.
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Sep 2026
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I03-Macromolecular Crystallography
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Open Access
Abstract: Carbapenem-resistant Enterobacterales (CRE), including Escherichia coli and Klebsiella pneumoniae are increasingly common pathogens. Resistance in CRE is often associated with production of K. pneumoniae (KPC) carbapenemases. We report a structurally modified meropenem derivative, JDB-1-200 (i.e., 8-epi-meropenem, in which the stereochemistry of the C8 hydroxyl is inverted), displaying enhanced activity relative to meropenem against KPC-producing CRE. JDB-1-200 alone displayed superior activity (MICs of 0.5 to 1 mg/L, compared with >8 mg/L for meropenem) against several clinically relevant Enterobacterales, including KPC-producing K. pneumoniae and Citrobacter freundii. Crystallographic analysis of JDB-1-200 complexes with the E166Q deacylation-deficient KPC-2 mutant, compared with an analogous meropenem (8R-hydroxyl) complex, indicates that the improvement arises from the JDB 8S-hydroxyl group acting as a hydrogen-bond donor to the hydrolytic water, slowing acyl-enzyme hydrolysis, with similar interactions observed in a CTX-M-15:JDB-1-200 complex. Kinetic inhibition data for KPC-2 and CTX-M-15 indicate that JDB-1-200 could be used alone or alongside a β-lactamase inhibitor for effective CRE treatment.
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Sep 2026
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I03-Macromolecular Crystallography
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Dylan
Kramer
,
Clarissa
Santos Rocha
,
Christopher A.
Gaulke
,
Marie
Nearing
,
Sumathi
Sankaran-Walters
,
Ikaika
Loque
,
Anugraha
Kidigannappa
,
Eric
Pham
,
Shuang
Hu
,
Patrawin
Wanakumjorn
,
Ramona
Abbattista
,
Abhaya
Dandekar
,
Roland
Faller
,
Satya
Dandekar
Open Access
Abstract: HIV infection disrupts gut epithelial barrier integrity and mucosal immunity, driving chronic inflammation and disease progression which are not fully resolved despite anti-retroviral therapy. Here we identify the microbiota-derived octadecanoid-hydroxy-fatty-acid metabolite 10-hydroxystearic acid (10-HSA), produced by Lactiplantibacillus plantarum, as a key mediator of gut epithelial barrier repair in human intestinal epithelial cells in vitro, ex vivo and in the non-human primate model of HIV/AIDS. X-ray crystallography and transcriptomics combined with functional analyses revealed that 10-HSA directly binds PPARα, inducing lipid metabolism, mitochondrial regeneration and subsequent epigenetic histone crotonylation, thereby promoting gut epithelial renewal. Co-administration of 10-HSA with anti-retroviral therapy in SIV-infected macaques accelerated viral suppression, resolved systemic inflammation, repaired gut epithelial integrity and recovered the gut microbiota. These findings identify a microbiota-derived lipid metabolite–PPARα–histone crotonylation axis that activates gut epithelial regeneration. This study defines a host–microbiome metabolic pathway that restores epithelial–immune homeostasis and enhances the efficacy of anti-retroviral therapy.
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Aug 2026
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I04-Macromolecular Crystallography
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Jemma K.
Betts
,
Cy M.
Jeffries
,
Tim C.
Passchier
,
Hermia C. Y.
Kung
,
Sarah P.
Graham
,
Mahmoud A. S.
Abdelhamid
,
Jamieson A. I.
Howard
,
Timothy D.
Craggs
,
Stephen C.
Graham
,
Ian
Brierley
,
Mark C.
Leake
,
Steven D.
Quinn
,
Chris H.
Hill
Diamond Proposal Number(s):
[32736]
Abstract: Programmed −1 ribosomal frameshifting (PRF) is a translational control mechanism used by RNA viruses to regulate the relative abundance of proteins encoded in different reading frames. Cardioviruses exhibit the highest known PRF efficiency, with ∼85% of ribosomes shifting into the −1 frame. This unusual event requires an interaction between the viral 2A protein and a stimulatory element in the RNA genome, but the basis for protein dependence is unclear. To address this, here we investigate the structure and dynamics of the PRF signal in Theiler’s murine encephalitis virus (TMEV). By combining X-ray crystallography, small-angle X-ray scattering (SAXS), and single-molecule fluorescence resonance energy transfer (smFRET), we show that 2A binding switches the RNA from a stem-loop conformation into a pseudoknot, and we demonstrate that pseudoknot formation is essential for efficient PRF in vitro and in cells. Together, these findings illustrate how the cardiovirus PRF element behaves as a protein-dependent riboswitch, defining the molecular mechanism by which frameshifting is conditionally activated.
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Aug 2026
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: Odorant-binding proteins (OBPs) in mosquitoes play central roles in chemosensory perception and are attractive targets for vector control strategies. However, some OBPs are also expressed in non-olfactory tissues, suggesting a pleiotropic function that has not been sufficiently investigated. The crystal structures of the pleiotropic AgamOBP9 of the Anopheles gambiae mosquito were determined in complex with three plant-derived bioactive molecules: the phenylpropanoids n-butyl cinnamate and methyl eugenol (ME), as well as the monoterpene p-menthane-3, 8-diol (PMD). Fluorescence competitive binding assays and binding free-energy calculations identified that n-butyl cinnamate and ME bind with Ki values in the micromolar range, in contrast to the weak affinity observed for PMD. Structural analysis revealed an extended internal cavity comprising two distinct ligand-binding regions. One region, located at the bottom of the cavity, accommodated all investigated ligands without undergoing significant conformational changes, suggesting a structurally preordered binding pocket. The second region, situated at the cavity entrance, binds MPD and PEG, both used as crystallization agents, and may therefore serve as a recognition site for molecules with diverse chemical features. In the AgamOBP9–butyl cinnamate–MPD complex, MPD binding to the entrance-site induces rearrangements of surrounding residues, including Arg8, Arg15, Tyr32, Lys33, and Trp35. These interactions appear to promote the convergence of helices α1, α2, and the α2-α3 connecting loop toward a more “closed” protein conformation. The conformational flexibility of AgamOBP9 at the entrance region was further supported by differential scanning calorimetry and molecular dynamics simulations, which suggested the presence of two independent thermodynamic domains within the protein. Structural superposition with the homologous AaegOBP22–linoleic acid complex indicated that the AgamOBP9 cavity could accommodate fatty acids or other long-chain molecules spanning both binding regions, implying potential functions beyond conventional olfactory signaling. These findings provide new insights into the molecular basis of ligand recognition by AgamOBP9, which may guide the discovery of novel OBP9-targeting ligands.
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Aug 2026
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I04-Macromolecular Crystallography
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Dustin A.
Ammendolia
,
Cara J.
Ellison
,
Yuelin
Zheng
,
Etienne
Coyaud
,
Estelle M. N.
Laurent
,
Bing-Ru
Yan
,
Bri
Manning
,
Anna K.
Waldmann
,
Joel M. J.
Tan
,
Scott
Frendo-Cumbo
,
Carina
Lyons
,
Ji-Young
Youn
,
Brian
Raught
,
Keith
Ireton
,
Darren E.
Higgins
,
Paul R.
Elliott
,
John H.
Brumell
Open Access
Abstract: Listeria monocytogenes (Lm) is an intracellular pathogen that can cause life-threatening systemic infections. Internalin C (InlC) is a secreted factor required for full Lm virulence in systemic models of infection by mechanisms that remain unclear. Here, we show that InlC binds to CYLD, a host deubiquitinase and regulator of innate immunity. Structurally, we reveal the LRR domain of InlC binds the CAP-Gly2 domain of CYLD, independently of InlC’s binding site for other host target proteins. Lm strains harboring amino acid substitutions in InlC that selectively disrupt binding to CYLD were examined. We demonstrate that after Lm accesses the host cytosol, InlC recruits CYLD to ubiquitin-positive bacteria. Recruitment of CYLD was dependent on the host E3 ligase RNF213, a major initiator of ubiquitin-mediated defenses. Furthermore, InlC-CYLD binding contributed to Lm virulence in mice. Together, these findings reveal how a secreted bacterial factor promotes the recruitment of a host deubiquitinase to cytosolic bacteria in response to ubiquitin-mediated defenses.
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Aug 2026
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I03-Macromolecular Crystallography
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Shibani
Biswas
,
Yogavel
Manickam
,
Choong Yeu
Khai
,
Ritu
Goswami
,
Sanskrita
Sukla
,
Rosicler
Barbosa
,
Sihyun
Sung
,
Rini
Chaturvedi
,
José Antonio
Márquez
,
Aditya
Chaki
,
Amit
Sharma
,
Ram
Das
,
J.
Sivaraman
,
Soumyananda
Chakraborti
Diamond Proposal Number(s):
[28534]
Abstract: Mosquito-borne diseases, particularly malaria, remain a major global health challenge. Among different mosquitoes, Anopheles are solely responsible for malaria. Insecticide-based interventions such as insecticide-treated nets (ITNs) and indoor residual spraying (IRS) are central to mosquito control. However, the widespread emergence of insecticide resistance, especially to pyrethroids, the most commonly used insecticides for mosquito worldwide, threatens their effectiveness. While genetic and metabolic mechanisms of resistance are well characterized, behavioral and sequestration-based mechanisms, particularly those involving chemosensory proteins (CSPs), are less understood. CSPs and odorant-binding proteins (OBPs) are integral components of the mosquito olfactory system, mediating host-seeking and other behavioral processes by transporting odorant molecules to olfactory receptors. Recent evidence suggests that CSPs may also bind insecticides like pyrethroids, contributing to resistance through sequestration. In this study, we investigated the structural and functional roles of CSPs in Anopheles mosquitoes. We determined three high-resolution X-ray crystal structures (apo-form) of CSPs (<2 Å) from Anopheles culicifacies and Anopheles gambiae, representing the first crystal structures of CSPs from any Anopheles species reported to date. Using structural, biophysical and computational approaches, we assessed CSP–ligand recognition of various insecticides, specifically pyrethroids and identified key residues potentially involved in pyrethroid interaction through site-directed mutagenesis. The moderate micromolar binding affinities of deltamethrin and permethrin, compared to other insecticides, support the role of CSPs in pyrethroid sequestration. Our findings offer mechanistic insights into pyrethroid sequestration and provide a foundation for developing novel vector control strategies that target CSP–insecticide interactions to combat pyrethroid resistance.
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Aug 2026
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[38144]
Open Access
Abstract: The PfPCRCR complex is essential for invasion of human erythrocytes by the deadliest malaria parasite, Plasmodium falciparum. Antibodies against each subunit of PfPCRCR prevent erythrocyte invasion and the PfRH5 component is currently the most advanced blood-stage malaria vaccine. Central within PfPCRCR is PfRIPR. This complex molecule contains a core and a flexible tail and allows PfPCRCR to bridge the parasite and erythrocyte during invasion. In this study, we generated a small panel of human monoclonal antibodies against PfRIPR. We structurally characterised four PfRIPR tail-binding antibodies in complex with PfRIPR fragments. We show that growth-inhibitory antibody RP.012 induces a kink in the PfRIPR tail while non-inhibitory antibodies do not. Furthermore, we show that these four antibodies modulate each other, either through antagonism or by acting synergistically. These studies have implications for the design of PfRIPR-based vaccine immunogens and indicate that the tail of PfRIPR undergoes essential conformational changes during erythrocyte invasion.
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Aug 2026
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B21-High Throughput SAXS
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Open Access
Abstract: C-type lectins (CTLs) play key roles in immunity and microbial carbohydrate recognition. In the vector-mosquito Aedes aegypti, the C-type lectin domain-single (CTLD-S) family comprises 34 soluble CTLs whose members are implicated in flavivirus dissemination and microbial homeostasis, yet their organization remains uncharacterized. We combine X-ray crystallography, small-angle X-ray scattering (SAXS), molecular dynamics, and machine learning-based structure prediction to characterize CTLs in Aedes aegypti. We determined the crystal structures of four representative CTLD-S proteins: mosGCTL-1, -3, -6, and -20. All crystals featured an identical homodimer arrangement, positioning both carbohydrate-binding sites on the same molecular face. Dimerization was confirmed in solution and AlphaFold predictions across the entire family indicated that dimer formation may be a unifying feature of CTLD-S proteins. For one mosGCTL structure, paucimannose glycans bound at a Ca2+-dependent site, demonstrating bidentate binding through one dimer. Machine learning-based predictions indicated hundreds of possible CTLD-S heterodimers may be viable, with wide-ranging implications for preferred glycan binding through one dimer. Our findings reveal a conserved dimeric arrangement among mosquito lectins that may underpin ligand recognition relevant to vector–pathogen interactions.
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Aug 2026
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