I24-Microfocus Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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Pierre
Aller
,
Juan
Sanchez-Weatherby
,
Abbey
Telfer
,
Robert
Bosman
,
Nicholas
Devenish
,
Philip
Hinchliffe
,
Sam
Horrell
,
Jophy
Ip
,
Richard
Littlewood
,
Andrew
Male
,
Eva
Gimenez-Navarro
,
Urszula
Neuman
,
Jos J. A. G.
Kamps
,
David
Omar
,
Laura
Parkinson
,
Muthraj
Pandi
,
Nico
Rubies
,
James
Sandy
,
Anastasya
Shilova
,
James
Spencer
,
Jonathan
Spiers
,
John P.
Sutter
,
Amy J.
Thompson
,
Catherine L.
Tooke
,
Ben
Williams
,
Tiankun
Zhou
,
Michael A.
Hough
,
Allen M.
Orville
Open Access
Abstract: Time-resolved X-ray crystallography is experiencing a resurgence, in part because of serial methods that readily allow scientists to create stop-motion movies of the macromolecular functions of photoactivation, enzyme-catalysed reactions and ligand-induced conformational changes triggering further downstream signalling events. While some reactions can be initiated with light, either naturally or using photocaged compounds, a more generally applicable approach is to mix microcrystals with reagents at varying time points prior to exposure to the X-ray beam. A powerful approach has been to combine droplet-on-demand `tape drive' sample delivery with X-ray emission spectroscopy (XES) that correlates atomic structure with the electronic states of metal ions within the sample. To the best of our knowledge, such a combined methodology has not been deployed previously on a synchrotron beamline but has been restricted to X-ray free-electron lasers. Here we describe two independent prototype experiments along the development pathway to a combined droplet-on-demand diffraction and XES system on the microfocus synchrotron beamline VMXi at Diamond Light Source. We demonstrate the collection of a high-quality serial diffraction data set from microcrystals within droplets having a volume of hundreds of picolitres deposited on a moving tape. In separate experiments on VMXi, we collected XES data from microcrystals of a copper enzyme delivered using a high-viscosity extruder. Together, these results demonstrate the feasibility of combined droplet-on-demand serial crystallography and XES experiments using a third-generation synchrotron beamline.
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Sep 2026
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Abstract: The nanofabrication of functional protein-based surfaces is challenging due to the chemical complexity of proteins and their unpredictable behavior at the solid–liquid interface. Many proteins of interest–such as antibodies or large enzymatic complexes–lack strong and dynamic protein–protein and protein–surface interactions necessary to drive self-assembly of stable arrays with high surface coverage. Additionally, adsorption-induced conformational changes at the solid–liquid interface could lead to a loss of activity and increase the risk of undesirable interfacial processes. Here we introduce SAKe, a Kelch-like designer protein, as a versatile platform to address these challenges. Ancestral sequence reconstruction led to high thermal stability, and the high symmetry allowed modularity of the protein’s core. Rational engineering of the bottom side allowed SAKe to form large (up to 5 μm in length), well-defined and pH-dependent two-dimensional assemblies while maintaining structural integrity, which is key for further development of functional materials. SAKe self-assembly was investigated through in-liquid atomic force microscopy on muscovite mica. High resolution imaging confirmed the integrity of the SAKe protein upon adsorption on the solid–liquid interface. These results showcase the SAKe protein as a platform for the further engineering of functional protein-based two-dimensional materials.
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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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Talos-Talos Arctica at Diamond
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Stuart R.
Castillo
,
Natalie M.
Robinson
,
Maksymilian
Dziura
,
Omotayo
Gbadamosi
,
Dominik
Dziura
,
Isabelle J.
Dib
,
Eilis C.
Bragginton
,
Emma
Buzzard
,
Karen M.
Davies
,
Najet
Mahmoudi
,
Piotr A.
Zolnierczuk
,
Elizabeth G.
Kelley
,
Drew
Marquardt
Diamond Proposal Number(s):
[40460]
Open Access
Abstract: Herein, we show compelling evidence of structural remodeling in mitochondrial membranes of cancerous colon cells when compared to their noncancerous counterparts. The physical characteristics of cancerous and noncancerous mitochondria were systematically investigated using small-angle neutron scattering (SANS), cryogenic electron tomography (cryo-ET), neutron spin–echo (NSE) spectroscopy, and high-throughput lipidomics. Our results exhibit evidence of both mechanical softening of the mitochondrial membrane, as seen by NSE, and ultrastructure rearrangements of the inner mitochondrial membranes, as seen by SANS and cryo-ET, in cancerous cells. These differences in properties seem to be correlated with changes in the lipidome, suggesting the changes are due to the limited number of lipid classes immediately available during the rapid proliferation of cancerous cells. Finally, we highlight a mechanism by which pancratistatin (PST), a natural anticancer therapeutic candidate, may selectively induce apoptosis by altering the mechanical properties and ultrastructure of cancerous mitochondria while sparing noncancerous mitochondria. Discovery of these structural anomalies will lead to improvements in diagnostics, therapeutic development, and the advancement of targeted approaches for traditionally complex cancers which evade our current treatments.
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Jul 2026
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[30763]
Open Access
Abstract: Disorders of the blood coagulation cascade continue to pose a major clinical challenge, necessitating the development of new therapeutic agents capable of modulating this process. Several oligonucleotide aptamers targeting coagulation factors have been developed, and some are undergoing preclinical or clinical evaluation. Among them, anti-thrombin anticoagulant aptamers are promising dual-targeting agents in that, in addition to inhibiting enzyme activity, they may limit thrombin generation by binding to its precursor, prothrombin. In the present study, combined calorimetric and spectroscopic analyses reveal that these aptamers recognize proexosite I of prothrombin and exosite I of thrombin through broadly similar thermodynamic binding mechanisms. Integration of structural SAXS studies and limited proteolysis shows that aptamer binding to proexosite I alters prothrombin structure, shifting the equilibrium from its more abundant closed form to the open conformation. Taken together, these results support the classification of these aptamers as dual-targeting agents capable of recognizing both thrombin and prothrombin and provide guidance for their continued development as anticoagulant therapeutics.
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Jul 2026
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Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[42268, 35335]
Abstract: Central to Alzheimer’s disease pathology are prefibrillar oligomer assemblies of amyloid-β (Aβ) peptide. A widely discussed hypothesis proposes that amyloid-β oligomers insert into neuronal lipid membranes, disrupting their integrity and causing a loss of cellular homeostasis in Alzheimer’s disease. This membrane disruption is believed to be a major source of Aβ-induced neurotoxicity. Cryo electron tomography (cryo-ET) has facilitated 3D nanoscale imaging of Aβ-membrane interactions under near-native conditions. Analyses of small extracellular vesicles (sEVs) reveals that Aβ oligomers including annular and curvilinear extended oligomers (CLEOs) exhibit extensive binding to cell-derived lipid membranes, including insertion into and carpeting of the lipid bilayer. Notably, these oligomeric assemblies were also internalized and concentrated within the cell-derived exosomes and other small sEVs. Enrichment of Aβ oligomers within the vesicles typically ranged between 5 to 20 times the external Aβ levels depending on the vesicle size and curvature. In contrast, monomeric and fibrillar forms of Aβ displayed minimal membrane interaction. Once internalized CLEOs appear to be trapped in an oligomeric form and do not readily go on to form fibrils. Studies with vesicles of brain lipid extract indicate the Aβ internalization does not require the presence of a membrane protein. Our in vitro studies underscore the membrane-disruptive capacity of oligomeric Aβ species and suggest a role of sEVs in concentrating toxic Aβ oligomers and transporting oligomers across the brain interstitium.
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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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B21-High Throughput SAXS
I03-Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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Maya
Noureddine
,
Halina
Mikolajek
,
Nathan
Cowieson
,
Nikos
Pinotsis
,
Paul
Robinson
,
Alexandre
Slater
,
Charles
Redwood
,
Siobhan
Loughna
,
Chris
Denning
,
Fiyaz
Mohammed
,
Katja
Gehmlich
Diamond Proposal Number(s):
[34438]
Open Access
Abstract: Hypertrophic cardiomyopathy (HCM) is a genetic disease associated with sudden cardiac death. Variants in alpha-actinin-2 (ACTN2), a Z-disc protein that anchors actin thin filaments have been implicated in HCM, yet their structural consequences remain poorly defined. Here, we characterise seventeen HCM-associated ACTN2 variants spanning multiple domains using an integrated and tiered workflow combining high-throughput assays, structural modelling and biophysical approaches. All variants display reduced solubility, with actin-binding domain (ABD) substitutions showing pronounced thermal instability by differential scanning fluorimetry. Modelling of nine variants predicts diverse pathogenic mechanisms including compromised actin-binding, impaired ABD regulatory conformations, disrupted dimerisation interfaces, and perturbed domain architecture. Crystal structures of two rod-domain variants reveal intact dimerisation despite modelling predictions. Actin-binding assays for ABD variants confirm altered actin engagement suggesting that binding dynamics may drive pathogenicity. Limited proteolysis indicates reduced structural stability across variants, while size-exclusion chromatography coupled with multi-angle light scattering or small-angle X-ray scattering (SEC-MALS/SAXS) shows a strong propensity for aggregation. Batch-mode SAXS further demonstrates early aggregation onset in selected ABD variants at elevated temperatures. Collectively, these findings establish that HCM-linked ACTN2 variants compromise protein integrity through multiple mechanisms, highlight the ABD as a hotspot of vulnerability and provide a potential framework for interpreting cardiomyopathy-associated variants.
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Jul 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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B21-High Throughput SAXS
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Maya
Noureddine
,
Bethany A. I.
Jones
,
Oseloka C. M.
Oliobi
,
Rylan
Beckingham
,
Elisabeth
Ehler
,
Halina
Mikolajek
,
Nathan
Cowieson
,
Paul
Robinson
,
Charles
Redwood
,
Alexandre
Slater
,
Siobhan
Loughna
,
Chris
Denning
,
Rachel
Myles
,
Caroline
Coats
,
Fiyaz
Mohammed
,
Katja
Gehmlich
Diamond Proposal Number(s):
[34438]
Open Access
Abstract: Filamin C (FLNC) is a key Z-disc protein that anchors actin filaments to membrane-bound complexes and is essential for sarcomeric stability and mechanotransduction. Genetic variants in FLNC are implicated in cardiomyopathy, a hereditary disease affecting heart muscle function. However, the structural and functional consequences of FLNC missense variants remain poorly defined, particularly those classified as variants of uncertain significance (VUS). Here, we used an integrated approach to investigate the FLNC missense variant M82K, identified in two unrelated patients presenting with dilated cardiomyopathy and associated clinical symptoms. Using structural modelling of the FLNC actin-binding domain (ABD), we predicted that the M82K variant may destabilise the ABD. Biophysical analyses of the E. coli-expressed FLNC-ABD-M82K variant displayed a ~ 10 °C decrease in melting temperature, accelerated thermolysin-mediated proteolysis, decreased solubility, and reduced actin-binding affinity. Further analysis using size exclusion chromatography coupled with and without small-angle X-ray scattering (SEC and SEC-SAXS), native PAGE, and mass photometry, all demonstrated a pronounced shift of mutant protein toward high-molecular-weight aggregates, with thermal SAXS confirming aggregation across elevated temperatures. Cellular analysis of GFP-tagged full-length FLNC-M82K showed reduced protein stability using cycloheximide. In neonatal rat cardiomyocytes there was no clear evidence of aggregation relative to wild-type in short-term transient transfections. Together, these findings support a pathogenic classification of FLNC-M82K and provide mechanistic insight into how FLNC destabilisation could lead to cardiomyopathy. More broadly, this multi-pronged strategy provides a framework for interpreting VUSs in cardiomyopathy-associated genes.
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Jun 2026
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