I04-Macromolecular Crystallography
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Stéphane
Bourg
,
Matthieu
Place
,
Chloé
Copin
,
Apirat
Chaikuad
,
Thomas
Robert
,
Hanna
Holzmann
,
Susanne
Müller
,
Stéphane
Bach
,
Sandrine
Ruchaud
,
Stefan
Knapp
,
Frédéric
Buron
,
Sylvain
Routier
,
Pascal
Bonnet
Open Access
Abstract: CLK1 is one of the four human isoforms of the cdc2-like (CLK) kinases that has been suggested as a therapeutic target in diverse diseases based on its important role regulating mRNA splicing. For example, CLKs and closely related kinases such as DYRK1A have been targeted in Alzheimer’s disease and other diseases in which splice site selection contributes to the disease development. Here we have developed an efficient in silico fragment-based ligand design approach to identify novel CLK1 inhibitors with excellent ligand efficiency based on an imidazo[2,1-b][1,3,4]thiadiazole fragment. More than one million docking poses were generated from 26,225 unique virtual compounds, and after applying several filtering steps, 11 compounds were selected, synthesized and their CLK1 inhibition and cellular potency were evaluated. Gratifyingly, inhibitor potencies were in excellent agreement with predicted values and crystallographic data of an inhibitor bound to CLK1 confirmed the unusual binding mode of the compounds.
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Oct 2026
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[28402, 35088]
Open Access
Abstract: Keratin is an abundant structural fibrous protein and extremely recalcitrant biopolymer. β-Keratin is the major constituent of feathers, which, due to the widespread poultry industry, has become a major waste product. Biotechnological upcycling of feather waste has gained interest as various bacteria and fungi capable of degrading keratin have been isolated. These microorganisms produce proteases, termed keratinases, responsible for the enzymatic hydrolysis of keratin. The structural properties that confer keratinolytic activity to proteases are, however, not well understood. Here, we investigated the structure-function relationship of a subtilisin-like S8 endopeptidase (FerB) from the thermophile Fervidobacterium pennivorans strain T. FerB was crystallized and its structure solved to 1.5 Å resolution, revealing an auto-processed state where the pro-peptide domain is non-covalently attached to the catalytic domain. The carboxyl group of the scissile peptide bond is coordinated in the active site within hydrogen bonding distance of the catalytic triad’s serine residue. Unlike fervidolysin, no β-sandwich domains are present. However, a tyrosine-rich β-hairpin structure is found in the corresponding position within the FerB structure. Deletion of the β-hairpin reduced the protein’s integrity and keratinase activity.
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Aug 2026
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[35585]
Open Access
Abstract: The self-assembly in aqueous solutions, cytocompatibility, and collagen production of lipopeptide C16–KTTKS and a variant with arginine substitution for lysine, C16–RTTRS, are investigated. C16–KTTKS is known commercially as Matrixyl and is used in cosmetic formulations as it can stimulate collagen production. The self-assembly and conformations and collagen-stimulating effects of the two lipopeptides in two salt forms, trifluoroacetate (TFA) and acetate, are compared. Lipopeptide C16-KTTKS self-assembles into nanotapes based on a multi-bilayer stacking across a pH range pH 4–7 and micelles at pH 2, with little influence of the counterion. In contrast, C16-RTTRS forms a substantial population of spherical micelles for the acetate salt for pH 2–7, but mainly nanotapes for the TFA salt for pH 4–7. Conditions for hydrogel formation by C16-KTTKS were identified. Both lipopeptides show good cytocompatibility to fibroblasts at sufficiently low concentration. The two lipopeptides also stimulate collagen production in Human Dermal Fibroblasts (HDFa) at low concentration (0.0062 wt%). No significant effect of the counterion was noted on cell viability or collagen production. Our results suggest that the peptide sequence influences the pH-dependent self-assembly properties and that this can be modulated for certain lipopeptides by the nature of the counterions.
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Aug 2026
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[21005]
Open Access
Abstract: The dynamic nature of protein and macromolecular complexes means that the capture of multiple sequential states along a reaction pathway can provide much greater insight into function than that obtained from a single static structure. We present a set of modular, easy-to-implement tools and workflows for optical excitation, on-grid characterization and tightly coupled rapid vitrification, establishing a proof-of-principle framework for time-resolved cryoEM and cryo-electron tomography (cryoET). We apply this framework to E. coli chemotaxis, in which serine-sensitive chemoreceptors initiate signalling upon ligand binding and undergo critical conformational changes within the chemosensory arrays. Using DMNB-caged serine [O-(4,5-dimethoxy-2-nitrobenzyl)-L-serine] as a model trigger, we quantified its photophysical properties and uncaging efficiency using UV–Vis spectroscopy and two-dimensional gas chromatography mass spectrometry (GC×GC-MS). Coupling a femtosecond-pulsed laser to a Vitrobot enabled reproducible reaction-to-vitrification delays of ∼150 ms, yielding intact E. coli minicells with well-preserved chemotaxis arrays suitable for in situ structural analysis by cryoET. This integrated approach provides a robust and generalisable framework for millisecond time-resolved cryoET, laying the groundwork for capturing transient conformational states in their native cellular context.
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Jul 2026
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B21-High Throughput SAXS
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Bridget
Tang
,
Philip
Kitchen
,
Luke M.
Broadbent
,
Steven D.
Quinn
,
Nawal
Hassan
,
Siriporn
Chaimueangchuen
,
Barbara
Gerbelli
,
Katsuaki
Inoue
,
Nathan
Cowieson
,
Fátima
Herranz-Trillo
,
Alice J.
Rothnie
,
Roslyn M.
Bill
,
Paul D.
Topham
,
Alan D.
Goddard
,
Jacob J. K.
Kirkensgaard
,
Matthew J.
Derry
,
Andreas Haahr
Larsen
Diamond Proposal Number(s):
[39511]
Open Access
Abstract: Amphiphilic copolymers have emerged as powerful, detergent-free tools for solubilizing biological membranes, enabling the extraction and stabilization of membrane proteins within native-like lipid environments. We report a comprehensive, multi-technique elucidation of how polymer:lipid stoichiometry governs the formation, size, and stability of styrene-maleic acid lipid particles (SMALPs). Using commercial SMA2000, a styrene-maleic acid copolymer made using free radical polymerization and 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) as a model phospholipid, we prepared SMALPs across a wide range of polymer-to-lipid weight ratios and employed an integrated suite of orthogonal characterization methods, including size exclusion chromatography (SEC), dynamic light scattering (DLS), flow-induced dispersion analysis (FIDA), mass photometry, ensemble and time-resolved Förster resonance energy transfer (FRET), and small-angle X-ray scattering (SAXS), to establish the structural consequences of varying polymer content. Our data reveal that efficient lipid solubilization into nanodiscs requires a minimum amount of polymer. Above ~1% (w/v) SMA2000: 1% DMPC, well-defined nanodiscs of ~10 nm diameter are formed that, on average, exhibit a consistent stoichiometry of ~130 lipids encircled by ~11 polymer chains. Through a new molecularly-constrained SAXS model, we show that these nanodiscs possess a stable bilayer height across variations in polymer to lipid ratio and a narrow polymer belt, and that their structural parameters remain invariant once excess polymer is used. In contrast, insufficient polymer (<1% w/v) generates bimodal populations including substantially larger discs. Notably, nanodiscs formed at optimal polymer:lipid ratios remain structurally stable for at least two months. Together, these results provide a rigorous quantification of SMALP composition and preferred size, enhancing our understanding of polymer-lipid nanodisc formation and offering critical design rules for detergent-free membrane protein extraction.
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Jul 2026
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I03-Macromolecular Crystallography
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Elisa
Peirano
,
Laura
O'Regan
,
Philip
Harrer
,
Ivana
Dzinovic
,
Magda S.
Chegkazi
,
Petra
Havrankova
,
Theresa
Brunet
,
Rossella
Capolino
,
Claudia
Cesario
,
Stefania
Ferro
,
Eva
Hammar
,
Russia
Hà-Vinh Leuchter
,
Elisabetta
Indelicato
,
Maureen
Jacob
,
Lukas
Kunc
,
Henri
Margot
,
Oriano
Marin
,
Maria
Mazurkiewicz-Bełdzińska
,
Niccolò E.
Mencacci
,
Antonio
Novelli
,
Laura
Orec
,
Michael
Poschmann
,
Alexandra
Sitzberger
,
Ugo
Sorrentino
,
Melanie
Spanjaard
,
Matias
Wagner
,
Magdalena
Krygier
,
Sylvia
Boesch
,
Jan
Necpal
,
Matej
Skorvanek
,
Donald L.
Gilbert
,
Robert
Jech
,
Mark
Dodding
,
Roberto A.
Steiner
,
Michael
Zech
Open Access
Abstract: Background: Although de novo causation in dystonia is widely acknowledged, there have been only a few trio-sequencing analyses in this field. We sought to prioritise de novo variants in dystonia and characterise the clinical and molecular features associated with the top gene candidate identified after genomic matchmaking.
Methods: We (re)assessed exome-sequencing data for de novo variants in genes with strong mutational constraint in a sample of 257 dystonia trios. Via data sharing, we collected information on individuals with variants in KLC1, encoding a subunit of the axonal-transport motor protein kinesin-1. Biophysical, biochemical, and functional studies, including differential scanning fluorimetry, X-ray crystallography, fluorescence-polarisation measurements, and immunoprecipitation from cells were performed for representative KLC1 variants. Findings: Missense and loss-of-function de novo variants in constrained genes without implication in autosomal dominant or X-linked conditions were found in 11.7% (30/257) of cases with dystonia. We then ascertained 7 unrelated patients with movement and neurodevelopmental disorders who harboured distinct, predicted deleterious de novo KLC1 missense variants. These variants clustered within the cargo adaptor-binding tetratricopeptide repeat domain and 3 variants mapped to an identical amino-acid position. Highly similar infantile-onset dystonic-spastic phenotypes were observed in the subjects with the recurrently affected residue. For all functionally tested variants, we observed changes in KLC1 stability and/or altered binding behaviour to known kinesin-1 interactors, such as JIP3, previously associated with dystonia and neurodevelopmental impairment. Interpretation: Our research supports the existence of a kinesinopathy linked to KLC1, featuring phenotypic overlap with diseases related to mutational defects of key interactors of KLC1. The full dystonia de-novo variant compendium is reported as a resource for additional disease-gene discovery.
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Jul 2026
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I13-2-Diamond Manchester Imaging
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Atal
Pande
,
Lucille
Rose
,
Sam J.
England
,
Imran
Rahman
,
Ricardo
Pérez-De La Fuente
,
Andrew J.
Bodey
,
Kaz
Wanelik
,
Christian M.
Schlepütz
,
Jonathan
Günther
,
Christoph
Rau
,
Alexander
Blanke
,
Lauren
Sumner-Rooney
Diamond Proposal Number(s):
[20721, 32952, 17205]
Open Access
Abstract: Vision is one of the most important senses used by animals and contributes to fundamental behaviors, including foraging, navigation, and mate detection and selection.1 Although much is known about how eye position and orientation correlate to ecology in the context of binocularity,2 animals with multipartite visual systems (more than two eyes) remain comparatively neglected. Spiders are highly successful predators that occupy a range of ecological niches and usually possess eight eyes. Here, we use three-dimensional geometric morphometrics and evolutionary modeling to test whether eye positions, orientations, and interocular angles correlate with hunting strategies in 52 species across the spider phylogeny. We demonstrate that eye configurations diversified from an ancestral medial cluster, as seen in modern trapdoor spiders, to a halo-like configuration in orb-weavers, and to the frontal clustering of eyes in several members of derived spider lineages. We show that visual hunters have the highest disparity and evolutionary rates in the configuration of their eyes but display a distinct morphological signature with multiple eye pairs concentrated at the front of the carapace. Moreover, we quantify the extent to which eye configuration is modular and show that the position and orientation of the eye pairs evolve semi-independently of each other. Our findings demonstrate that modularity in the spider visual system facilitates not only the specialization of individual eyes but also the whole architecture of the visual system, in line with different hunting strategies, body plans, and ecological niches.
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Jul 2026
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B22-Multimode InfraRed imaging And Microspectroscopy
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Open Access
Abstract: Microplastics (MPs) are persistent pollutants that are of growing concern to animals and humans. Morphological changes, oxidative stress, cyto- and genotoxicity are just a few effects reported in cells. Due to the large variety in MPs, there is an urgent need to find new analytical methods that can quickly identify affected cellular components and guide subsequent targeted analysis. Here, we propose Fourier-transform infrared microspectroscopy (µFTIR) with ZnS hemisphere solid immersion lenses as a screening tool for measuring the impact of MP exposure in single living cells. Macrophage-like (J774A.1) and lung epithelial (A549) cells were exposed to polystyrene (PS) beads of varying sizes (100 nm – 1 µm) and surface functionalizations (aminated, carboxylated, unfunctionalized) to demonstrate the approach. µFTIR spectra were collected from cells, and principal component analysis (PCA) was used to detect changes following exposure. In J774A.1 cells, 1 µm aminated and carboxylated MPs decreased cell viability and significant spectral changes were observed, across all PS types, including non-toxic ones. In A549 cells, no strong decrease in cell viability was detected, but PCA results showed significant spectral changes following exposure to 500 nm and 100 nm PS particles. Additionally, APS also altered specific cellular biomarkers, suggesting interaction between the MPs and cells due to the surface functionalization. Comparisons with cellular effects of MNPs described in literature (such as phagocytosis or endocytosis mechanisms) are in accordance with the spectral changes observed. These results demonstrate that single-live-cell µFTIR is a fast and cost-effective screening tool to detect biochemical changes in cells, find potential biomarkers, and contribute to understanding the cellular metabolism.
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Jul 2026
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Abstract: Plant lectins are carbohydrate-binding proteins with diverse biological functions and growing biomedical relevance due to their ability to recognize specific glycan structures. Here, we report the high-resolution crystallographic structure of a lectin from D. altissima seeds (DAL), resolved by X-ray diffraction at 2.21 Å (PDB ID: 7LJG). The structure reveals a homodimeric fold consistent with other lectins from the Dioclea genus, reinforcing its conserved molecular architecture. Other lectins reported within the Dioclea genus share the conserved β-sandwich fold typical of this group, while DAL exhibits unique amino acid substitutions at the carbohydrate-binding site that may influence its flexibility and interaction with other proteins. To explore DAL's potential antiviral activity against SARS-CoV-2, we performed molecular docking and molecular dynamics simulations targeting the main viral protease (Mpro). The results indicated that DAL forms a stable complex with Mpro, engaging key residues within the active site and exhibiting stronger predicted binding affinity than Nirmatrelvir, the currently approved COVID-19 therapeutic. Together, these findings highlight DAL as a promising scaffold for antiviral drug development, bridging structural insights and computational analysis to guide future experimental exploration.
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Jul 2026
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[21970]
Open Access
Abstract: Copper-containing nitrite reductases (CuNiRs) catalyse the reduction of nitrite to nitric oxide and are a key enzyme in the anaerobic ammonium oxidation and denitrification steps of the nitrogen cycle. The recent recognition of the widespread distribution of three-domain CuNiRs where cognate redox partners are fused to the core NiR enzyme offered the possibility of studying coordinated events (e.g. proton-coupled electron transfer) in a conformationally stable donor–acceptor complex. The C-terminal cytochrome c tethered domain of the CuNiR from Ralstonia pickettii (RpNiR) has been well studied. Reverse engineering of RpNiR undertaken to remove the cognate partner domain showed that the presence of the additional domain resulted in significant differences in the apparent Km for nitrite and the reduction potentials of the Cu centres when compared with the core enzyme. The oxidation state of the haem centre and the position of the tethering linker have also been shown to control access of substrate to the active site. A key feature of this control is a conserved tyrosine residue (Tyr323 in RpNiR) located in the tethering linker between the fused domain and the core enzyme. To gain insight into this control, we have undertaken targeted mutations of RpNiR to probe the so-called primary proton channel and perturb putative electron transfer routes from the haem to the `gatekeeper' Tyr323 and to the T1Cu centre. The resolution of our crystallographic data to better than 1.2 Å enabled us to apply unrestrained SHELXL refinement of the structures. Our data provide a significant advance in our understanding of catalysis and modulation of electron transfer in these tethered systems, with wider implications for these fundamental processes in other protein complexes.
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Jul 2026
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