I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[25108, 18565]
Open Access
Abstract: The accessory secretion (aSec) system is a protein export pathway that is uniquely present in Gram-positive bacteria and is dedicated to the secretion of large, glycosylated cell wall-anchored adhesins called serine-rich repeat proteins (SRRPs). Strain-specific glycosylation of SRRPs has previously been reported in Limosilactobacillus reuteri and attributed to GtfC, a glycosyltransferase belonging to family 113, with LrGtfC100-23 from L. reuteri rat strain 100-23C showing specificity for UDP-Glc, while LrGtfC53608 from L. reuteri pig strain ATCC 53608, which differs at only ten amino-acid positions, shows a preference for UDP-GlcNAc. However, the structural basis underpinning GtfC sugar-donor specificity remains unclear. Here, we report X-ray crystal structures of the tetrameric LrGtfC100-23 in the apo form and its complexes with UDP and with the noncognate sugar donor UDP-N-acetylglucosamine (UDP-GlcNAc). Analysis of the LrGtfC100-23 structures identified candidate residues implicated in donor-sugar substrate specificity, which were supported by site-directed mutagenesis. Reciprocal swaps of candidate residues combined with thermal shift assays revealed that the W240C variant of LrGtfC100–23 could bind both UDP-sugar donors, while the P243S variant of LrGtfC53608 became specific for UDP-Glc, opening the door for glycoengineering approaches in bacteria.
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Dec 2025
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B18-Core EXAFS
I18-Microfocus Spectroscopy
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Diamond Proposal Number(s):
[33674, 35117, 35776, 40942]
Open Access
Abstract: Metal–organic frameworks (MOFs) are entering water technologies on the premise that abiotic stability predicts ecological safety. We overturn this assumption by showing that UiO-66 – often regarded as chemically and structurally robust – remains intact after 7-day aging in natural borehole water yet undergoes rapid in vivo transformation in Daphnia magna. Synchrotron Microfocus X-ray absorption spectroscopy (XAS) revealed collapse of the ordered Zr–carboxylate coordination into disordered Zr–O environments within the gut; Extended X-ray Absorption Fine Structure (EXAFS) showed loss of second-shell features, and Transmission Electron Microscopy (TEM) confirmed loss of crystallinity with nanoscale aggregates appearing within 24 h of ingestion. Although acute immobilization was limited (48 h EC50 ≈ 26.5 μg mL–1), a sublethal, environmentally relevant exposure (10 μg mL–1) caused pronounced chronic effects: brood initiation was delayed by 3–5 days and cumulative reproduction decreased by ∼74% without mortality. We attribute these outcomes to gut-level transformation and associated energetic/physiological burdens, not captured by standard acute tests. These results show that abiotic stability does not necessarily imply biological inertness and highlight the need to integrate in vivo transformation pathways with chronic end points in environmental risk assessment for water-sector materials. This perspective provides a mechanistic basis to inform Safe-and-Sustainable-by-Design (SSbD) MOFs before widespread deployment in water treatment.
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Dec 2025
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I24-Microfocus Macromolecular Crystallography
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Francesca
Coscia
,
Ioannis
Riziotis
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Antonina
Andreeva
,
Delhi
Kalwan
,
Jennifer
De Jong
,
Philip
Hinchliffe
,
Fabio
Parmeggiani
,
Paul R.
Race
,
Steven G.
Burston
,
Alex
Bateman
,
Rob
Barringer
Diamond Proposal Number(s):
[31440]
Open Access
Abstract: Many proteins harbor covalent intramolecular bonds that enhance their stability and resistance to thermal, mechanical, and proteolytic insults. Intramolecular isopeptide bonds represent one such covalent interaction, yet their distribution across protein domains and organisms has been largely unexplored. Here, we sought to address this by employing a large-scale prediction of intramolecular isopeptide bonds in the AlphaFold database using the structural template-based software Isopeptor. Our findings reveal an extensive phyletic distribution in bacterial and archaeal surface proteins resembling fibrillar adhesins and pilins. All identified intramolecular isopeptide bonds are found in two structurally distinct folds, CnaA-like or CnaB-like, from a relatively small set of related Pfam families, including 10 novel families that we predict to contain intramolecular isopeptide bonds. One CnaA-like domain of unknown function, DUF11 (renamed here to “CLIPPER”) is broadly distributed in cell-surface proteins from Gram-positive bacteria, Gram-negative bacteria, and archaea, and is structurally and biophysically characterized in this work. Using x-ray crystallography, we resolve a CLIPPER domain from a Gram-negative fibrillar adhesin that contains an intramolecular isopeptide bond and further demonstrate that it imparts thermostability and resistance to proteolysis. Our findings demonstrate the extensive distribution of intramolecular isopeptide bond-containing protein domains in nature and structurally resolve the previously cryptic CLIPPER domain.
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Dec 2025
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B18-Core EXAFS
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Diamond Proposal Number(s):
[29950]
Open Access
Abstract: Aims: To investigate the phase changes of bismuth oxide in contact with sodium hypochlorite responsible for tooth discolouration. Methodology: Bismuth oxide (monoclinic α−phase; C) was mixed with sodium hypochlorite at 20°C, 37°C, and 60°C (B20, B37, B60) for a period of 24 weeks with weekly refreshing of solutions. The products were imaged by scanning electron and optical microscopy and characterized by thermographic analysis (TGA), phase analysis by X-ray diffraction (XRD) using Bragg Brentano geometry and Pilatus detector, infrared spectroscopy (FT-IR), and X-ray absorption fine structure (XAFS). Results: The interaction of bismuth oxide with sodium hypochlorite resulted in a change in microstructure and colour. The thermographic assessment showed a change in mass (5%–10% weight change) and colour reversal to the yellow bismuth oxide at ~450°C. Phase changes dependent on temperature were demonstrated with δ-bismuth oxide, sodium bismuthate and bismuth oxychloride formed as by-products at the different temperatures. Conclusions: The δ-phase bismuth oxide formation led to the material darkening which will cause tooth discolouration in a clinical setting. Due to the phase changes, the material chemistry after the interaction is different from that of the material placed in the tooth. The by-products of the reaction have not been tested for use in patients. It is recommended to ban the use of bismuth oxide from dental materials and other clinical use due to its instability. The clinical guidance for endodontic treatment needs to be changed to reflect this.
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Dec 2025
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B21-High Throughput SAXS
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Sung Ryul
Choi
,
Thorsten B.
Blum
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Matteo
Giono
,
Bibhas
Roy
,
Ioannis
Vakonakis
,
Dominic
Schmid
,
Nicole
Oelgarth
,
Apisha
Ranganathan
,
Alvar D.
Gossert
,
G. V.
Shivashankar
,
Alfred
Zippelius
,
Michel O.
Steinmetz
Open Access
Abstract: Microtubules have long been recognized as upstream mediators of intracellular signaling, but the mechanisms underlying this fundamental function remain elusive. Here, we identify the structural basis by which microtubules regulate the guanine nucleotide exchange factor H1 (GEFH1), a key activator of the Ras homolog family member A (RhoA) pathway. We show that specific features of the microtubule lattice bind the C1 domain of GEFH1, leading to the sequestration and inactivation of this signaling protein. Targeted mutations in C1 residues disrupt this interaction, triggering GEFH1 release and activation of RhoA-dependent immune responses. Building on this sequestration-and-release mechanism, we identify microtubule-binding C1 domains in additional signaling proteins, including other guanine nucleotide exchange factors (GEFs), kinases, a GTPase-activating protein (GAP), and a tumor suppressor, and show that microtubule-mediated regulation via C1 domains is conserved in the Ras association domain-containing protein 1A (RASSF1A). Our findings establish a structural framework for understanding how microtubules can function as spatiotemporal signal sensors, integrating and processing diverse signaling pathways to control important cellular processes.
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Dec 2025
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I03-Macromolecular Crystallography
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Christopher
Lenz
,
Lewis
Elson
,
Johannes
Dopfer
,
Frederic
Farges
,
Andreas
Kramer
,
Frank
Löhr
,
Susanne
Müller
,
Stéphanie M.
Guéret
,
Herbert
Waldmann
,
Volker
Dötsch
,
Krishna
Saxena
,
Stefan
Knapp
Open Access
Abstract: Developing new E3 ligase ligands for the design of heterobivalent molecules, such as PROteolysis TArgeting Chimeras (PROTACs), requires careful evaluation of target engagement (TE). Characterizing protein–protein interactions (PPIs) is therefore essential in drug discovery, as it enables the assessment of ligand binding to sites that are often difficult to target. Degrons, peptide motifs recognized by E3 ligases, may serve as valuable starting points for designing E3 ligands. However, many degrons are highly polar and lack intrinsic membrane permeability, requiring alternative strategies for efficient cellular delivery. In this study, we used the SPRY domain-containing SOCS box protein 2 (SPSB2) E3 ligase as a model system to develop TE strategies in vitro and in cellulo using polar degron-based peptides. By conjugating various polycationic cell-penetrating peptides (CPPs) to the degron sequence, we present a study demonstrating cellular delivery. We obtained a high-resolution crystal structure and used various biophysical techniques to assess the influence of each modification, while confocal microscopy and BRET-based assays confirmed successful cellular delivery as well as potent TE.
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Dec 2025
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NONE-No attached Diamond beamline
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Deniz
Bicer
,
Rei
Matsuoka
,
Aurélien F. A.
Moumbock
,
Preethi
Sukumar
,
Albert
Suades
,
Harish
Cheruvara
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Andrew
Quigley
,
David
Drew
,
Els
Pardon
,
Jan
Steyaert
,
Peter J. F.
Henderson
,
Martin
Caffrey
,
Julia J.
Griese
,
Emmanuel
Nji
Open Access
Abstract: Under conditions of extreme acidity, the lysine-specific permease, LysP, not only mediates the import of L-lysine it also interacts with the transcriptional regulator, CadC, to activate expression of the cadAB operon. This operon encodes the lysine decarboxylase, CadA, which converts lysine to cadaverine while consuming a cytoplasmic proton, and the antiporter, CadB, which exports protonated cadaverine in exchange for extracellular lysine. Together, these processes contribute to cytoplasmic pH homeostasis and support bacterial acid resistance - a mechanism essential for the survival of pathogenic bacteria in acidic host environments. Here, we present the cryo-EM structure of LysP from Pseudomonas aeruginosa in an inward-occluded conformation (3.2–5.3 Å resolution), bound to L-lysine and a nanobody. L-Lysine is coordinated by hydrophobic contacts, cation–π interactions, and by hydrogen bonding mostly with polar uncharged residues. Reconstitution of LysP into proteoliposomes confirms specific L-lysine transport, which is competitively inhibited by L-4-thialysine. These findings provide a structural framework for understanding selective lysine recognition and inhibition, with implications for antibacterial drug design.
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Dec 2025
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I13-2-Diamond Manchester Imaging
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Diamond Proposal Number(s):
[21843]
Open Access
Abstract: Cortical bone is highly porous comprising of interconnecting network of vascular canals and osteocyte lacunae. Our understanding of the mechanisms coupling vascular: lacunar spatial organisation in cortical bone is poorly understood. Defining cellular cross talk mechanisms could be key in identification of reciprocal molecular signals driving increased cortical porosity with age.
Driven by the hypothesis that porosity within bone is heterogeneous and influenced by region-specific spatial cues, we utilised Synchrotron X-Ray computed tomography to characterise intracortical canal and osteocyte lacunae distribution, morphology and spatial arrangements in healthy and pathological murine bone.
We found that the posterior region of the tibiofibular (TFJ) junction exhibited the highest levels of cortical porosity and highest canal number density versus other regions. The volume of osteocyte lacunae volume positioned proximal to cortical vascular canals was highest in the posterior region. Following deletion of bone-derived VEGF the region-specific effects on lacunar: vascular arrangements described in the wild type TFJ were lost. Our results describe spatial diversity in osteocyte lacunae size within the bone cortex which associates with vascular canal arrangements which are maintained by VEGF.
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Dec 2025
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I02-Macromolecular Crystallography
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Martin
Orecchia
,
Katherine
Welbeck
,
Jason
Dexter
,
Laura
Hook
,
Chika
Akinseye
,
Marcin
Kot
,
Alan
Lewis
,
Don
Somers
,
Tejinder
Bhinder
,
Paul
Hamblin
,
Sarah
Elsey
,
David
Willé
,
Steven
Grant
Open Access
Abstract: This study describes the affinity maturation, molecular engineering, and preclinical assessment of depemokimab, an enhanced anti-interleukin-5 antagonist antibody. The molecular design objective for depemokimab was to generate a therapeutic antibody enabling a less frequent dosing regimen of once every 6 months compared with every 4 weeks for mepolizumab. Mepolizumab is a marketed monoclonal antibody used as an add-on prescription maintenance treatment for patients with severe asthma with an eosinophilic phenotype and other eosinophilic-associated disorders. A complementarity-determining region restricted affinity maturation strategy was used where affinity improved interleukin-5 binding antibody variants were subject to affinity driven selective pressure and identified using the Adimab yeast-based platform. Improved complementarity-determining region variants were combined with serum half-life extending amino acid mutations introduced into the fragment crystallizable region of the antibody. When compared with mepolizumab, depemokimab demonstrated improved in vitro interleukin-5 neutralization in a TF-1 (human erythroleukemia) functional cell assay. In vivo, depemokimab displayed significantly extended pharmacokinetic performance and pharmacodynamic duration determined via eosinophil suppression in cynomolgus monkey (Macaca fascicularis). These data provide compelling evidence that a less frequent dosing regimen for depemokimab in humans is possible and supported the advancement of depemokimab into a Phase I study in patients with asthma.
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Dec 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23459, 31353]
Open Access
Abstract: Traboulsi Syndrome is an autosomal recessive hereditary disease associated with developmental defects, in particular of the ocular system. Single nucleotide polymorphisms affecting the ASPH gene, which encodes for the 2-oxoglutarate (2OG)-dependent oxygenase aspartate/asparagine-β-hydroxylase (AspH), are associated with Traboulsi Syndrome. AspH catalyzes hydroxylations of conserved aspartate/asparagine residues in epidermal growth factor-like domain (EGFD) proteins. We report studies on the clinically-observed Traboulsi Syndrome-associated R688Q, R735Q, and R735W AspH variants. The results reveal that pathogenic active site substitutions substantially reduce, though do not ablate, EGFD hydroxylase activity compared to wildtype AspH. They imply that efficient AspH catalyzed EGFD hydroxylation is important during human development. Crystallographic studies reveal conservation of the overall AspH fold, but that the preferred conformations of 2OG in complex with the R735Q and R735W AspH variants differ from that with wildtype AspH. Screening of potential 2OG cosubstrate substitutes reveals certain 2-oxoacids, including naturally present metabolites, manifest enhanced catalytic efficiency of Traboulsi Syndrome-associated AspH variants compared to 2OG. The results thus provide proof-of-principle for a therapeutic strategy involving rescue of impaired activities of pathogenic active site AspH variants by use of 2-oxoacids, or 2-oxoacid precursors, other than 2OG.
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Dec 2025
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