I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[34263]
Open Access
Abstract: Deoxyuridine triphosphatase (dUTPase) is essential for DNA replication fidelity and embryonic development in metazoa, yet whether a protein inhibitor can acutely disrupt dUTPase function in a living vertebrate has not been tested. The Staphylococcus aureus Stl protein inhibits diverse dUTPases in vitro, but its efficacy in a eukaryotic organism was unknown. We determined the crystal structure of zebrafish dUTPase in complex with the N-terminal Stl fragment to 2.26 Å resolution, revealing conserved active-site engagement and displacement of the C-terminal arm. Biolayer interferometry confirmed low-nanomolar binding affinity with KD = 0.7 nM, and enzyme assays demonstrated ~50% inhibition of zebrafish dUTPase activity at saturating Stl concentrations. These structural and biophysical data establish that Stl forms a tight inhibitory complex with zebrafish dUTPase, prompting us to test whether this interaction can perturb enzyme function in vivo. Microinjection of Stl or its point mutant, retaining dUTPase-inhibitory activity but unable to bind DNA, into fertilized zebrafish oocytes reduces embryo survival to ~60% within 48 h compared to ~90% in controls, demonstrating that lethality depends on dUTPase targeting rather than DNA binding. Accelerated mortality relative to genetic dUTPase knockout indicates that maternally deposited dUTPase supports viability during early cleavage. Stl thus acts as an immediate, proteinaceous dUTPase inhibitor in a living vertebrate, providing a tool to dissect dUTPase function and regulation during embryonic development.
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Oct 2026
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I03-Macromolecular Crystallography
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Luise
Pallasdies
,
Helen
Barber
,
Mihwa
Lee
,
Laura
Burchill
,
Ho N. N.
Ho
,
Adam W. E.
Stewart
,
Artur
Nastasovici
,
Hanna
Sanne-Wander
,
Dilushini T.
Rathnayake
,
Vinzenz
Hofferek
,
Leila
Jebeli
,
Taylor A.
Mcdaniels
,
Malcolm J.
Mcconville
,
Robert A.
Field
,
Sacha
Pidot
,
Nichollas E.
Scott
,
Yi
Jin
,
Spencer J.
Williams
Diamond Proposal Number(s):
[31850]
Abstract: Small aminoorganosulfonates are reservoirs of carbon, nitrogen, and sulfur in marine and host-associated ecosystems. Homotaurine, a C3-aminoorganosulfonate produced by marine algae and present in algal-derived foods, is comparatively well documented, but its complete microbial catabolism has remained unresolved. Here we identify a complete bacterial pathway for homotaurine catabolism in Caballeronia jiangsuensis strain McCoy, isolated by metabolic enrichment from soil by growth on homotaurine. Growth, proteomic, metabolomic, biochemical, structural, and heterologous-expression analyses show that homotaurine is imported and oxidatively deaminated to sulfopropanoate, then converted to sulfolactate through E-sulfoacrylate by SpuABCD and SpuIJ, enzymes related to succinate dehydrogenase and fumarase. Sulfolactate is cleaved by redundant SuyAB sulfolyases to release pyruvate and sulfite, which is oxidized and excreted as sulfate, completing biomineralization. Crystal structures of the HtaA homotaurine-binding protein and SuyAB2 sulfolyase reveal ligand recognition and provide a structural framework for sulfolactate cleavage. Hidden Markov model searches and Tara Oceans and human gut metagenomes show that the core sulfopropanoate-to-sulfolactate module is widespread in marine Proteobacteria, especially Roseobacter-lineage bacteria, and occurs more narrowly in human gut-associated sulfur-metabolizing taxa such as Bilophila wadsworthia. This pathway connects algal, dietary, and xenobiotic C3-aminoorganosulfonates to microbial carbon, nitrogen, and sulfur cycling, and identifies sulfopropanoate turnover as a conserved module in organosulfonate metabolism.
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Sep 2026
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I03-Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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Open Access
Abstract: In vertebrates, a nuclear gene encoding mitochondrial cytochrome c (CYC) was duplicated and diverged into one with ubiquitous expression (somatic-type) and the other with testis-preferred expression (testis-type). In this study, synteny based phylogenetic analyses suggest that cyc ohnologs originated through whole genome duplication (WGD) events in the common ancestor of vertebrates and have been conserved for approximately 450–540 million years. Molecular evolutionary analyses revealed pervasive purifying selection and an approximate Phe/Tyr dichotomy at position 46 between somatic- and testis-type CYC proteins. This was the case in two closely related amphibians, Buergeria buergeri and its hot-spring-dwelling species, Buergeria japonica. The recombinant testis-type CYC proteins from B. buergeri (Bb-CYCT) and B. japonica (Bj-CYCT) exhibited significantly higher resistance to hydrogen peroxide (H2O2) than somatic-type counterpart proteins (Bb-CYCS and Bj-CYCS). Structural analysis showed that the Tyr-46 residues in the testis-type CYC proteins face the heme, suggesting a protective role against H2O2 through an oxidation of its hydroxyl group compared to the spatially corresponding Phe-46 residue in the somatic-type counterparts. When the Tyr-46 residue in Bj-CYCT was replaced with the Phe residue, the H2O2 resistance was significantly reduced, confirming the functional importance of Tyr-46. These results demonstrate that functional divergence between somatic- and testis-type Buergeria CYC proteins is at least partly attributable to the Phe-to-Tyr substitution, suggesting that ancient nuclear genome duplication contributed to tissue-specific divergence of mitochondrial function.
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Sep 2026
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[32728]
Open Access
Abstract: Plant intracellular immune receptors are widely deployed in breeding to protect crops from disease. In addition to nucleotide-binding leucine-rich repeat receptors (NLRs), tandem kinase proteins (TKPs) have recently emerged as an important family of immune receptors within staple cereal food crops, but how TKPs recognize effectors and whether they are amenable to engineering is essentially unknown. Here, we show that the barley and wheat TKPs Rmo2 and Rwt7 recognize different blast fungus effectors via their integrated HMA domains using different protein interfaces with nanomolar binding affinity. Structural analysis pinpointed interface residues that dictate effector recognition and enabled engineering of dual-specificity TKPs. These results establish integrated HMA domains as programmable modules within TKPs for designing new specificities in plant immunity for diseases relevant to global agriculture.
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Sep 2026
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I04-Macromolecular Crystallography
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Abstract: ELAV/Hu RNA-binding proteins (RBPs) are key regulators of neuronal alternative splicing and polyadenylation programs across animals. How ELAV/Hu RBPs achieve gene-specific regulation by recognizing spaced U-rich motifs through multimerization, remains uncertain. We determined X-ray crystal structures of ELAV RNA recognition motif 3 (RRM3) to reveal that multimerization is mediated by two evolutionarily conserved interfaces in non-RNA-binding parts of the RRM to form a tetramer and RNA binding is not required for multimerization. Mutational probing of these two interfaces in Drosophila photoreceptor neurons shows that both interfaces contribute to ELAV function in development. Notably, multimerization defective Drosophila elav mutants are embryonic lethal. Genomic profiling demonstrates that multimerization is required to direct neuronal alternative splicing and polyadenylation programs of some, but not all ELAV target genes. Our study provides a structural basis for a mechanistic understanding how ELAV/Hu proteins can extract gene-specific regulation from a landscape of redundant sequence motifs.
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Aug 2026
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B21-High Throughput SAXS
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Antonin
Galien
,
Stephanie
Hutin
,
Sarah
Le Hir
,
Michel
Paul
,
Pascal
Albanese
,
Sylvie
Kieffer-Jaquinod
,
Audrey
Guillotin
,
Nozomu
Iwabuchi
,
Kensaku
Maejima
,
Max H.
Nanao
,
Mark D.
Tully
,
Veronique
Hugouvieux
,
Chloe
Zubieta
Open Access
Abstract: Phytoplasmas are obligate plant pathogens that include species causing severe developmental abnormalities in diverse plant families. Phytoplasma infections may result in dwarfism, witches’ broom, virescence and phyllody, the conversion of reproductive organs into leaf-like structures. Phyllody is due to the activity of phytoplasma effector proteins, called phyllogens, that interact with the host MADS transcription factor (MTF) network. This interaction was believed to be specific for members of the AP1/SEP/AGL6 superclade of tetrameric MTFs, key regulators of floral meristem identity and organogenesis. Here, we determined the molecular and atomic basis for the interaction between the phyllogen from Onions Yellow phytoplasma (PHYLOY) and the floral MTFs, APETALA1, SEPALLATA and AGAMOUS using biochemical, structural and genetic experiments. The DNA-binding patterns of homo and heteromeric MTFs with PHYLOY are shown using electrophoretic mobility shift assays, revealing broader interaction patterns between PHYLOY and MTFs that depend on highly conserved hydrophobic residues. Point mutations of these residues allowed gain or loss of interaction between MTFs and PHYLOY. Expressing PHYLOY under the control of the SEP3 and AP1 promoter in Arabidopsis further demonstrated whorl specific phenotypes, recapitulating the in vitro interaction and DNA-binding patterns. These findings reveal how PHYLOY targets specific MTFs through structural mimicry, resulting in impaired MTF tetramer formation and homeotic conversion of floral organs due to loss of active MTF tetramers.
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Aug 2026
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B21-High Throughput SAXS
B23-Circular Dichroism
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Diamond Proposal Number(s):
[39005, 42397, 40578, 42398]
Open Access
Abstract: Aspergillus fumigatus infections are a major yet often neglected global health challenge magnified by a growing at-risk population, limited treatment options, and the emergence of drug-resistant strains. Regulation of the DNA damage response (DDR) by ADP-ribosylation signalling has recently emerged as an important feature of fungal pathogenesis, but the underlying mechanisms remain largely elusive. Here we present a comprehensive phylogenetic and functional characterisation of Af-PARP1, the A. fumigatus PARP homologue. Our data reveal Af-PARP1 as a DNA-dependent poly(ADP-ribosyl)transferase with unique domain architecture, DNA damage selectivity, and activation dynamics distinct from its mammalian and plant homologues. We show that the fungal specific BRCT domain plays a crucial role in both damage recognition and ADP-ribosylation signal establishment. Collectively, our findings reveal a divergence in DDR-associated ADP-ribosylation specific to fungi, highlighting the potential of this signalling pathway as target for antifungal therapy.
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Jul 2026
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Roger B.
Dodd
,
Masahiro
Enomoto
,
Ye
Zhou
,
Kanayo
Satoh
,
Yalun
Zhang
,
Fusheng
Chen
,
Beatrice
Acheson
,
Deniz
Ghaffari
,
Elizabeth
Sayn-Wittgenstein
,
Jamie J.
Manning
,
George V.
Dukas
,
Ronak
Patel
,
Alondra Schweizer
Burguete
,
Mason J.
Kralovec
,
Mamunur
Rashid
,
Jennifer L.
Hall
,
Kirstin A.
Tamucci
,
Zena
Chatila
,
Minghua
Liu
,
Annie J.
Lee
,
Badri N.
Vardarajan
,
Mariko F.
Taga
,
Sirkku
Pollari
,
Alon
Rabinovitch
,
Cory D.
Rillahan
,
Andrey A.
Bobkov
,
Eduard
Sergienko
,
William
Meadows
,
Seema
Qamar
,
Suzanne J.
Randle
,
Christopher M.
Johnson
,
Jean
Sevalle
,
Jennifer
Griffin
,
Christopher
Bohm
,
Mitsuhiko
Ikura
,
Xunde
Xian
,
Joachim
Herz
,
Megan A.
Kelly
,
Jennifer
West
,
Sandeep
Satapathy
,
Mark R.
Wilson
,
Jonathan A.
Javitch
,
Paul E.
Fraser
,
David A.
Bennett
,
Philip L.
De Jager
,
Zvi
Fishelson
,
Dan
Frenkel
,
Wesley B.
Asher
,
Elizabeth M.
Bradshaw
,
Peter
St George-Hyslop
Diamond Proposal Number(s):
[8547]
Open Access
Abstract: Mechanisms linking CD33 variants to Alzheimer Disease (AD) are poorly defined. Here, we combine structural, cellular, and genetic analyses to delineate how the CD33M splice isoform, upregulated in carriers of CD33 risk alleles, modulates microglial function. We show that CD33M ectodomain dimerizes, enabling binding of large multi-sialylated molecules. We demonstrate that another AD risk protein - clusterin (CLU) ± Aβ oligomers (but not ApoE) binds with nanomolar avidity to CD33M, but not CD33m. We show that in human monocytes CD33M:CLU binding induces CD33M ITIM phosphorylation, recruits SHP-1, suppresses Aβ phagocytosis, and impairs clearance of amyloid plaques. We identify a soluble CD33M ectodomain fragment (sCD33M) - absent from CD33m-expressing cells - which could contribute to the role of CD33M in AD. Genetic analyses confirm that CD33:CLU interaction modulates amyloid burden, cognition, and disease risk. These findings define a mechanistic CLU:Aβ:CD33M axis, highlighting CD33M dimerization and ligand-binding sites as potential therapeutic targets.
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Jul 2026
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[35754]
Open Access
Abstract: Proteins with intrinsically disordered regions (IDRs) perform essential cellular functions despite lacking stable structures, challenging the traditional structure–function paradigm. Neurofilament-light (NFL) proteins self-assemble into bottlebrush filaments, whose disordered tail domains mediate nematic hydrogel formation critical for neuronal integrity. Mutations in NFL are linked to Charcot–Marie–Tooth (CMT) disease, yet their molecular effects remain unclear. Here, aiming to gain insight into these molecular mechanisms, we combine small-angle X-ray scattering, microscopy, and deep-learning conformational analysis to investigate CMT-associated NFL tail mutations. We find that these mutations compact the hydrogel, disrupt filament nematic order by generating microdomains, and alter water retention dynamics by shifting sequence-dependent conformational ensembles, leading to macroscopic network rearrangements. These findings demonstrate how subtle sequence changes in IDRs modulate protein network organization and function, offering structural insights into IDR-related pathologies.
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Jul 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, 21005]
Open Access
Abstract: RNA polymerase II (RNAPII) drives gene expression through iterative nucleotide addition cycles (NACs) comprising translocation, substrate binding, and catalysis. The lack of pre-catalysis and post-catalysis intermediates has precluded a complete mechanistic understanding of the NAC. Here we present 31 Cryo-electron Microscopy structures (with 43 maps) capturing distinct stages of Saccharomyces cerevisiae RNAPII elongation complex (EC) NAC, including previously intractable transition intermediates. We establish a continuous spectrum of RNAPII EC structural dynamics during the NAC, which can be divided into two coordinated phases: a substrate-induced EC tightening phase and a post-catalysis EC relaxation phase. For the substrate-induced EC tightening phase, the substrate binding initiates allosteric conformational changes across the entire RNAPII EC, including Trigger Loop folding, funnel closure, clamp closure, transcription bubble ordering, and precise alignment of the RNA 3′-end with substrate to form a catalysis-competent configuration. For the post-catalysis EC relaxation phase, we capture the long-sought, short-lived post-catalysis product state and identify a series of intermediates that reveal a reverse conformational transition that facilitates rapid translocation. Together, our findings define a comprehensive structural and dynamic framework for RNAPII NAC, yielding a “molecular movie” of RNAPII in action and revealing a fundamental principle by which the enzyme balances speed and fidelity through coordinated conformational dynamics.
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Jul 2026
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