I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[26793]
Open Access
Abstract: Bacteria coexist in polymicrobial communities where they engage in complex interactions, including interbacterial antagonism. The environmental bacterial pathogen Chromobacterium violaceum possesses an active type VI secretion system (T6SS), which relies mainly on VgrG3 for its activity and role in interbacterial competition. However, the arsenal of toxic effectors delivered by this T6SS remains unknown. Here, we identify the repertoire of C. violaceum T6SS effectors and characterize a novel antibacterial Rhs-family effector, RhsF (Rhs with a FIX domain), and its cognate immunity protein, RhsFi. Using mass spectrometry analyses of secreted proteins and proteins co-immunoprecipitated with VgrG3, we identified six novel effector candidates, namely four phospholipases, a protein of unknown function, and the previously-uncharacterized Rhs protein, RhsF (CV_1431). RhsF contains an N-terminal FIX domain and can intoxicate susceptible bacteria in a T6SS-dependent manner. The action of the C-terminal toxin domain of RhsF (RhsF-CT) is prevented by RhsFi (CV_1430), confirming that RhsF-RhsFi comprises an effector-immunity pair. The structure of the RhsF-CT/RhsFi complex determined by X-ray crystallography (1.85 Å resolution) revealed that RhsF-CT shares structural similarity with ADP-ribosyltransferase toxins and that RhsFi inhibits toxicity via direct occlusion of the RhsF-CT catalytic site. Functional assays indicated that RhsF-CT ADP-ribosylates RNA in vitro and that RhsF toxicity requires a catalytic triad composed of R1403, Y1456, and E1497 residues. Overall, our findings reveal effectors secreted by the T6SS of C. violaceum, establish RhsF as a potent antibacterial toxin, and confirm T6SS-dependent delivery of a FIX-containing Rhs protein, expanding the known repertoire of bacterial arms involved in microbial competition.
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Jun 2026
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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María
Conde-Giménez
,
Sandra
Salillas
,
María
Galiana-Cameo
,
Juan E.
Martínez-Oliván
,
Alejandro
Mahía
,
Manuel
Ledesma
,
Juan José
Galano-Frutos
,
Ritwik
Maity
,
Adrián
Velázquez-Campoy
,
María D.
Díaz-De-Villegas
,
Ramon
Hurtado-Guerrero
,
Javier
Sancho
Diamond Proposal Number(s):
[14739]
Open Access
Abstract: henylketonuria (PKU) is an inherited metabolic disorder caused by pathogenic variants in phenylalanine hydroxylase (PAH), leading to toxic phenylalanine accumulation and severe neurological complications if untreated. Current pharmacological treatment relies on tetrahydrobiopterin (BH4), which benefits only a subset of patients, highlighting a major unmet need for alternative therapies. Here, we combined high-throughput screening, computational modelling, and drug repurposing to identify pharmacological chaperones capable of rescuing PAH function. We evaluated 26 structurally diverse small molecules in HEK293T cells expressing wild-type PAH or one of eight PKU-associated variants spanning phenotypes from mild to classical disease. Chaperoning efficacy was strongly variant-dependent, and for every variant tested at least one compound produced a greater activity increase than BH4 under identical assay conditions. Notably, belinostat, a clinically approved histone deacetylase inhibitor, emerged as the most effective compound for several clinically severe variants. Mechanistically, functional rescue consistently correlated with an increased population of tetrameric, catalytically competent PAH, as quantified by mass photometry. The crystal structure of the PAH–belinostat complex (PDB ID: 9T1O), together with structural models for all compounds, provide a framework for rational optimization. These results establish a preclinical proof-of-concept for genotype-guided pharmacological chaperone therapy in PKU and support the feasibility of personalized, variant-specific treatment strategies.
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Jun 2026
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Krios III-Titan Krios III at Diamond
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Ho Fong
Leong
,
Giovanni
Consoli
,
Geoffry A.
Davis
,
Ben
Hancox-Lachman
,
Kenta
Renard
,
Fiazall
Tufail
,
Lauren E.
Lee
,
Lucas
Gautier
,
James W.
Murray
,
Andrea
Fantuzzi
,
A. William
Rutherford
Diamond Proposal Number(s):
[33230]
Open Access
Abstract: Far-red light photoacclimation enables some cyanobacteria to survive in white-light-depleted environments by extending the red limit of photosynthesis. In far-red Photosystem II, paralogous subunits replace their canonical counterparts, allowing the incorporation of some chlorophyll f molecules and one chlorophyll d that are red-shifted and spectrally distinct from the chlorophyll a manifold, and from each other. Here, we present a comparative study of far-red Photosystem II from Chroococcidiopsis thermalis PCC 7203 and Calothrix sp. NIES-3974. In C. thermalis, the cryo-electron microscopy structure reveals the far-red-exclusive subunit, PsbH2’, which forms part of a chlorophyll f binding site. We also assign four chlorophyll f sites using sequence comparisons and electrostatic potential analyses. In Calothrix, psbH2’ is absent, and the same analyses show that only two of these chlorophyll f sites are present. Comparative phylogenetic, structural, and spectroscopic analyses allow the assignment of specific wavelengths to all the red-shifted chlorophylls. This provides the framework needed to model excitation energy transfer in far-red Photosystem II, and to understand the conserved features that allow survival under far-red light.
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Jun 2026
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I13-2-Diamond Manchester Imaging
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Karo
De Rycke
,
Marina
Horvat
,
Lisa
Caboor
,
Petra
Vermassen
,
Griet
De Smet
,
Sophie
Lobbestael
,
Marta
Santana Silva
,
Wouter
Steyaert
,
Matthias
Van Impe
,
Patrick
Segers
,
Julie
De Backer
,
Patrick
Sips
Diamond Proposal Number(s):
[32919]
Open Access
Abstract: Fibrillin defects lead to severe cardiovascular complications in Marfan syndrome (MFS), including aortic dilation, dissection, and rupture. To model MFS, zebrafish mutants lacking various fibrillin genes were generated. Among these mutant lines, only fibrillin-3–deficient zebrafish exhibited cardiovascular phenotypes mimicking human disease. Multimodal imaging revealed early cardiac defects, bulbus arteriosus dilation, and valve abnormalities. Transcriptomic analysis identified altered regulation of pathways related to extracellular matrix homeostasis and immune system activation. This zebrafish model, recapitulating key cardiovascular features of MFS, provides a valuable platform to investigate disease mechanisms and identify novel treatment strategies.
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May 2026
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[23268]
Open Access
Abstract: Localization of mRNAs is a widespread mechanism for dictating where proteins operate in cells and underpins many fundamental processes, from embryonic patterning to synaptic plasticity. This spatial control is mediated by the interaction of ‘localization signals’ in target mRNAs with RNA-binding proteins (RBPs). These signals frequently lack overt sequence or structural patterns, raising the question of how specificity is achieved. Here we investigate this issue using the Drosophila RBP Egalitarian (Egl), which couples mRNAs to microtubule-based transport through Bicaudal D (BicD) and the dynein motor. We present cryo-electron microscopy structures of Egl–BicD bound to six different RNAs. Egl uses multiple noncanonical double-stranded RNA-binding domains to cooperatively form a recognition pocket around localization signals. Despite substantial variation in length and sequence, each signal adopts a bent stem-loop conformation that, together with base-pair identities at two defined sites, drives Egl engagement. We further demonstrate that Egl dimers couple RNA binding to transport initiation through coincident detection of two RNA elements within the same transcript. Thus, localizing mRNAs are recognized through a combination of shape, positional sequence features and number of structured RNA elements. This work reveals a molecular strategy by which diverse mRNAs can be selectively engaged by a single RBP.
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May 2026
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I03-Macromolecular Crystallography
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Julian
Blagg
,
Philippe
Riou
,
Alexia
Hervieu
,
Eleonora
Piumatti
,
Maria T.
Rodriguez-Plata
,
Paolo
Battuello
,
Adam
Peall
,
Vito
Amodio
,
Pietro Paolo
Vitiello
,
Daniel J. H.
Nightingale
,
Ruzica
Bago
,
Paige
Tongue
,
Tessa
Slater
,
Kalpesh
Parmar
,
Pradip
Patel
,
Javier
Rodríguez González
,
David E.
Clark
,
Gareth W.
Langley
,
Charles
Nichols
,
Alba
Guarne
,
Paul C. M.
Winship
,
Matthew
Baker
,
Martin
Drysdale
,
Giovanni
Germano
,
Alberto
Bardelli
Diamond Proposal Number(s):
[29364]
Open Access
Abstract: DNA mismatch repair (MMR) detects and corrects post-replicative DNA alterations; it is deregulated in up to 20% of human cancers. MMR-deficient (MMR-d) cancers display increased tumour mutational burden (TMB), microsatellite instability (MSI) and are eligible for checkpoint inhibitor (CPI) immunotherapy which commonly elicits durable responses. We reasoned that pharmacological blockade of MMR could broaden the patient population eligible for immunotherapy. Here we reveal MMR protein PMS2 as a druggable target and describe the discovery and characterisation of first-in-class small molecule MMR pathway modulator NP1867. In vitro treatment of murine cancer cells abrogates MMR function and elicits an MMR-d genotype including increased TMB, MMR-d mutational signatures, and MSI-High (MSI- H) status. Inoculation of syngeneic immunocompetent mice with cancer cells pretreated with NP1867 leads to CPI sensitivity, tumour growth delay, and complete responses. For the first time, we demonstrate pharmacological targeting of MMR to proactively rewire the tumour-host relationship for therapeutic purposes.
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Apr 2026
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Krios I-Titan Krios I at Diamond
Krios II-Titan Krios II at Diamond
Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[29812, 34631, 21005]
Open Access
Abstract: RNA polymerase II (RNA Pol II) is central to gene expression, but its catalytic mechanism remains elusive due to the absence of high-resolution structural data. The role of water molecules in RNA Pol II catalysis is unknown. Here, we present 3 high-resolution cryo-electron microscopy structures of active Saccharomyces cerevisiae RNA Pol II elongation complexes in distinct catalytic states: two pre-catalysis states at 1.96 Å and 2.26 Å resolution and a post-catalysis state at 2.33 Å resolution. Each structure contains over 700–1,350 ordered water molecules, many located at functionally critical positions. Comparative analysis shows that these waters play essential roles in proton-transfer steps during RNA Pol II catalysis, facilitating substrate recognition and trigger-loop folding during nucleotide addition. Strikingly, these waters are conserved between prokaryotic and eukaryotic transcription machineries (see Mueller and Darst). These findings provide unprecedented mechanistic insights into RNA Pol II catalysis and reveal vital and evolutionarily conserved roles of water molecules in transcription.
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Apr 2026
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[34349]
Open Access
Abstract: Recently, there has been growing interest in the function and physiological importance of human TANGO2 (transport and Golgi organization 2 homolog), particularly whether it acts as a heme-trafficking protein. To address this question, we experimentally determined the three-dimensional structure of TANGO2. Our crystallographic analysis indicates that interactions between heme and TANGO2 are nonspecific. Structural comparison of the TANGO2 crystal structure with known cysteine Ntn-hydrolases allowed us to identify a putative active site, catalytic residues and a substrate-binding cavity that correspond to residues that are mutated in pathogenic TANGO2 variants. Based on these features, we propose that TANGO2 may utilize fatty-acid derivatives as substrates, suggesting a potential role in lipid metabolism. Mutations in the human TANGO2 gene cause TANGO2 deficiency disorder, a multisystem, life-threatening disease with onset in early childhood. Together, our results provide new insights into the molecular function of TANGO2 and help to resolve the ongoing debate regarding whether it functions as a heme-trafficking protein.
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Apr 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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I13-2-Diamond Manchester Imaging
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Md Al Amin
Sheikh
,
Kenichi
Kimura
,
Eri
Motoyama
,
Keiichi
Asano
,
Violette
Deleeuw
,
Patrick
Sips
,
Chiho
Tokunaga
,
Hiroko
Matsunaga
,
Sachiko
Kanki
,
Shigeki
Koizumi
,
Kaori
Sugiyama
,
Julie
De Backer
,
Lynn Y.
Sakai
,
Haruko
Takeyama
,
Yuji
Hiramatsu
,
Haruka
Ozaki
,
Hiromi
Yanagisawa
Diamond Proposal Number(s):
[32919]
Open Access
Abstract: Aortic dissection (AD) is characterized by separation within the medial layers of the aortic wall. Pathogenic variants in the fibrillin-1 gene (FBN1), which cause Marfan syndrome, represent a major genetic cause of AD. In a recently established Fbn1G234D/G234D mouse model, intimomedial tears develop at 3 weeks of age, and 50% of mice die by 5 weeks from aortic rupture. Despite this severe phenotype, the magnitude and expansion of AD lesions, as well as the molecular alterations within the medial layers remain incompletely understood. In this study, we used three-dimensional propagation-based X-ray phase-contrast synchrotron imaging for reconstruction of the ascending aortas, together with single-cell RNA sequencing (scRNA-seq) analysis in Fbn1G234D/G234D mice. Synchrotron imaging revealed 1–2 elastic lamellar breaks evolved into widespread disruptions spanning the entire elastic lamellae, accompanied by localized adventitial thickening. scRNA-seq analysis followed by immunofluorescence staining showed upregulation of fibronectin (Fn1) in Fbn1G234D/G234D smooth muscle cells (SMCs). Consistently, increased FN1 expression was observed in human non-heritable AD samples. Furthermore, enhanced expression of fibronectin receptors and activation of focal adhesion kinase signaling suggested augmented extracellular matrix–SMC interactions during disease progression. These findings indicate that AD progression involves coordinated medial structural failure, adventitial remodeling, and fibronectin-associated SMC dysfunction.
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Apr 2026
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