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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I24-Microfocus Macromolecular Crystallography
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Francesca
Coscia
,
Ioannis
Riziotis
,
Antonina
Andreeva
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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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B21-High Throughput SAXS
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Sung Ryul
Choi
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Thorsten B.
Blum
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Matteo
Giono
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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
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Johannes
Dopfer
,
Frederic
Farges
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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
,
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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I02-Macromolecular Crystallography
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Martin
Orecchia
,
Katherine
Welbeck
,
Jason
Dexter
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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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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[38313]
Open Access
Abstract: Radical enzymes, including glycyl radical enzymes (GREs) and B12-dependent enzymes, catalyze a wide range of biochemical transformations through radical-based mechanisms. An unusual property—conditional resistance to chymotrypsin digestion—has previously been reported for two GREs. However, whether this feature is broadly conserved among related radical enzymes and what factors trigger it has remained unclear. In this study, we investigated five radical enzymes: four GREs and one B12-dependent diol dehydratase. Proteolytic assays demonstrated that substrate binding significantly enhances resistance to chymotrypsin degradation, suggesting a conserved conformational shift from an open, protease-sensitive state to a closed, protease-resistant form. X-ray crystallographic analysis of a GRE-type 1,2-propanediol dehydratase from Raoultella planticola confirmed that active site occupancy correlates with increased protease resistance. Importantly, non-substrate analogs such as 1,3-propanediol and β-methylcholine failed to induce protection, underscoring the specificity of ligand-induced stabilization. These findings reveal a broadly conserved mechanism of substrate-induced conformational stabilization in GREs and B12-dependent radical enzymes and offer a scalable strategy for ligand identification with potential applications in enzyme engineering.
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Dec 2025
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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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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[36130]
Open Access
Abstract: The initiation of allergic responses critically depends on the recognition of an allergenic epitope by the paratope of IgE antibodies. While previous structural studies have focused on recombinant fragments or engineered forms of IgE, the structure of full-length IgE in its native state remains poorly understood. In this study, we investigate the conformational changes of a native murine IgE (2F5), both in its free form and upon binding to the Hevea brasiliensis allergen profilin (Hev b 8). Small-angle X-ray scattering (SAXS) data reveal that unbound IgE adopts an extended conformation with open Fab arms. However, when it binds to profilin, it transitions to a more compact arrangement characterized by closer proximity of the arms. Molecular dynamics (MD) simulations of the Fab region further identified conformational rearrangements upon allergen binding, including a twisting motion and partial disruption of interactions between the naturally paired heavy and light chains. These findings indicate that there may be allosteric communication between Fab and Fc regions, even in the absence of a hinge region, which is not present in IgE. Overall, this study provides valuable insights into the dynamic structural properties of native IgE and enhances our understanding of the molecular mechanisms underlying allergen recognition.
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Nov 2025
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