I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[39189]
Open Access
Abstract: The enzymes Cannabichromenic Acid Synthase (CBCAS), Cannabidiolic Acid Synthase (CBDAS) and Tetrahydrocannabinolic Acid Synthase (THCAS) are together the major cannabinoid synthase enzymes responsible for the biosynthesis of their respective metabolites from a common precursor Cannabigerolic Acid (CBGA). As the catalysts responsible for generating biological molecules of significant pharmaceutical value, there has been considerable interest in the enzymes with respect to heterologous production, mechanism, and incorporation into synthetic biology pathways for the facile industrial production of these molecules. The enzymes share high degrees of homology, and therefore their distinct specificities are governed by very subtle differences in sequence and therefore structure, although, until now, only a structure for THCAS has been reported. In this report, we present structures of CBCAS, CBDAS and a structure of THCAS at a higher resolution than the known structure, each in complex with their flavin coenzyme FAD. The structures reveal active site differences that may be responsible for the complementary activities observed, in terms of both first-shell amino acid substitutions, but also in more remote residues that influence active site topology through referred effects, or that have effects on substrate access. The structures provide a useful and informative platform for the rational engineering of improved or altered chemoselectivity in these enzymes.
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Dec 2026
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[34182]
Open Access
Abstract: Chikungunya virus (CHIKV) outbreaks impose significant burdens on healthcare systems and raise an urgent need for effective antiviral therapies. So far there are no specific drugs against CHIKV infection. CHIKV-encoded macrodomain is critical for virulence and counteracts the host immune response, representing a promising antiviral drug target. Here, we describe small molecule inhibitors targeting the CHIKV macrodomain. Compound 1 (MDOLL-0273) was identified through a high-throughput screening using a fluorescence resonance energy transfer based assay, exhibiting an IC50 of 8.9 μM, and its inhibitory activity was validated through multiple orthogonal assays. The compound features a thiobarbiturate-indole scaffold and shows high selectivity over a panel of human and viral ADP-ribose binding and hydrolyzing proteins. X-ray crystallography revealed that the inhibitor occupies an adenine binding site of the macrodomain and extends into a novel cryptic pocket. Guided by structure-activity relationship studies, compound 11 (MDOLL-0591) of the developed series with similar IC50 of 10 μM but with increased lipophilicity was discovered to have antiviral activity against CHIKV in cell culture, demonstrating that macrodomain could be targeted in virus infections.
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Nov 2026
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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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Open Access
Abstract: Motivated by the need for a convenient way to demonstrate the growth of crystals in glass plates by the lipid cubic phase method at crystallization workshops and courses, we developed a lightweight, portable, compact and inexpensive digital microscope device. The device is built around hand-held Wi-Fi and hardwired digital microscopes that are available commercially at low cost and that can use cell phones and personal computers for imaging without the need for unwieldy eyepieces. The device provides illumination from above and below the crystallization plate over a range of brightness levels. It can be used with regular white light and with polarized light to detect birefringent crystals. While not the focus of this study, the new device has been shown to also work with batch and vapour-diffusion plates. Remote monitoring of crystallization plates in a cold cabinet or walk-in refrigerator at 4 °C is possible with the Wi-Fi microscope. Crystals grown in lipid cubic phase and vapour-diffusion crystallization plates were harvested with the aid of the new device and used for diffraction data collection, leading to structure determination at high resolution. Most parts of the device were 3D printed in polylactic acid plastic. The corresponding STL files, included as part of this publication, can be used to make replicates of the current microscope and to produce bespoke devices suited to the specific needs of the user. We envision using the new device for outreach activities at primary, secondary and third-level schools, and at science fairs in support of the citizen scientist. Relatedly, in this study examples are included of recorded images of plant and animal tissue sections. One reveals birefringent materials in a tomato leaf. Another shows optically active crystals of calcium oxalate in onion tunic tissue.
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Oct 2026
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[36570]
Open Access
Abstract: Leishmaniasis is a neglected disease that affects around two million people every year. Current treatments are often highly toxic or prone to resistance, underscoring the urgent need for new therapeutic strategies. PROTACs may offer a promising alternative, as they can potentially mitigate both toxicity and resistance. However, very little is known about the ubiquitin–proteasome system (UPS) in Leishmania. Notably, only two E3 ligases containing a CULT domain have been identified so far, and none carrying a von Hippel–Lindau (VHL) domain─the classical E3 ligase used by clinically advanced PROTACs. In this work, we take an important step toward understanding the UPS in Leishmania, and we propose that in this organism the UbC4 E2 enzyme, rather than an E3, may be directly exploited to develop a PROTAC able to engage a protein of interest. Here, we report the biochemical and structural characterization of the Leishmania major ubiquitin-conjugating enzyme 4 (UbC4). Through a fragment screening campaign, we identified 10 fragments binding to distinct cavities on UbC4. Among these, five interact with the same noncatalytic pocket that is poorly conserved in humans, while one fragment binds near the catalytic cysteine. Using DeepFrag predictions and molecular docking, we explored fragment elongation strategies to enhance affinity for their respective binding sites, with the goal of guiding the development of E2-recruiting PROTACs or UPS inhibitors for the treatment of leishmaniasis.
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Sep 2026
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I03-Macromolecular Crystallography
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Open Access
Abstract: Carbapenem-resistant Enterobacterales (CRE), including Escherichia coli and Klebsiella pneumoniae are increasingly common pathogens. Resistance in CRE is often associated with production of K. pneumoniae (KPC) carbapenemases. We report a structurally modified meropenem derivative, JDB-1-200 (i.e., 8-epi-meropenem, in which the stereochemistry of the C8 hydroxyl is inverted), displaying enhanced activity relative to meropenem against KPC-producing CRE. JDB-1-200 alone displayed superior activity (MICs of 0.5 to 1 mg/L, compared with >8 mg/L for meropenem) against several clinically relevant Enterobacterales, including KPC-producing K. pneumoniae and Citrobacter freundii. Crystallographic analysis of JDB-1-200 complexes with the E166Q deacylation-deficient KPC-2 mutant, compared with an analogous meropenem (8R-hydroxyl) complex, indicates that the improvement arises from the JDB 8S-hydroxyl group acting as a hydrogen-bond donor to the hydrolytic water, slowing acyl-enzyme hydrolysis, with similar interactions observed in a CTX-M-15:JDB-1-200 complex. Kinetic inhibition data for KPC-2 and CTX-M-15 indicate that JDB-1-200 could be used alone or alongside a β-lactamase inhibitor for effective CRE treatment.
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Sep 2026
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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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Krios II-Titan Krios II at Diamond
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Zak
Mciver
,
Didi
He
,
Jennifer
Ross
,
Marta
Cozzaglio
,
Cecilia
Piergentili
,
Aritha
Dornau
,
Natasha
Sumpner
,
Finn
Brady
,
Kathleen
Bialik
,
Thomas
Mccorvie
,
Claudia
Sissi
,
Arnaud
Basle
,
David J.
Clarke
,
Jon
Marles-Wright
Diamond Proposal Number(s):
[31827]
Open Access
Abstract: Encapsulins are self-assembling protein nanocompartments found in bacteria and archaea that encapsulate cargo enzymes to protect the cell from their toxic reaction products or intermediates. Developments in cryo-electron microscopy (cryo-EM) data processing strategies have enabled encapsulins and their cargo proteins to be investigated together in greater detail. In this study, we present the single particle cryo-EM structure of the Rhodospirillum rubrum encapsulin in both the presence and absence of its partner encapsulated ferritin (EncFtn). Single particle icosahedral reconstructions of empty and loaded encapsulins revealed a higher degree of conformational flexibility at the five-fold pore in the cargo loaded encapsulin. We applied a new non-point group averaging workflow to analyze the encapsulated ferritins within the encapsulin nanocompartment, to produce the first fully refined in situ atomic model of the EncFtn at 2.8 Å resolution. Masked 2D classification and particle subtraction demonstrate that cargo loading is heterogeneous in this recombinant complex, with the encapsulin able to house up to five of the decameric EncFtn complexes. Our data provides new insights into the dynamics and cargo arrangement in encapsulins and demonstrates an adaptable workflow for high resolution reconstruction of encapsulin cargoes.
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Sep 2026
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I23-Long wavelength MX
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Open Access
Abstract: Preventing collisions during automated sample exchange is critical for synchrotron beamlines, particularly for complex cryogenic in-vacuum endstations where recovery from hardware damage may take days. GoniOwl, a compact convolutional neural network (CNN) model, classifies sample-pin presence on the goniometer from a live camera feed on the long-wavelength macromolecular crystallography beamline I23 at Diamond Light Source. Trained on over 8700 manually verified images spanning two years of routine operation and augmented for robustness to illumination changes, camera shifts and occlusions, the model achieves >99% accuracy with millisecond-level inference. A confidence-gating mechanism routes uncertain predictions to a fail-safe path requiring operator confirmation, ensuring suitability for machine-protection control. Integrated via Experimental Physics and Industrial Control System (EPICS) process variables, GoniOwl runs in real time within the automated sample-change sequence. In shadow-mode deployment, the CNN matched or exceeded both the legacy histogram method and operator confirmations, which each achieved 96% accuracy. A closed-loop disagreement-audit workflow automatically collects divergent cases for targeted retraining and verification. The approach is readily transferable to other beamline environments where camera-based vision systems can provide an additional software machine protection layer.
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Sep 2026
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NONE-No attached Diamond beamline
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Daniel
Muñoz-Reyes
,
Kate K.
Fieseler
,
Max
Winokan
,
Mathew
Golding
,
Eda
Capkin
,
Matteo
Ferla
,
Sara
Pérez-Suárez
,
Charles W. E.
Tomlinson
,
Peter G.
Marples
,
Celia
Miró-Rodríguez
,
Lorena
Aguado
,
Alicia
Mansilla
,
Daren
Fearon
,
Warren A.
Thompson
,
Frank
Von Delft
,
Maria Jose
Sanchez Barrena
Diamond Proposal Number(s):
[31306]
Open Access
Abstract: Efficient drug discovery workflows ideally generate data that directly address key translational milestones, including confirmation of target engagement and binding pose, structure-activity relationships (SAR), and biological relevance, within rapid and resource-efficient experimental cycles. Here, we describe a data-driven, automation-assisted, customizable framework for fragment-to-hit progression that directly delivers structurally validated hit series primed for rapid SAR exploration, by exploiting the high-throughput crystallography available at synchrotrons. This recently evolved direct-to-biology approach combines X-ray crystallographic fragment screening with algorithmically-guided fragment merging and reagent prioritization; low-cost robotic array synthesis and reaction production assessment by LC-MS; and finally orthogonal biophysical evaluation of crude reaction mixtures for binding assessment and 3D binding pose using respectively grating-coupled interferometry and crystallography. We demonstrated the effectiveness of the strategy on a challenging target class, by collectively progressing a large set of fragment hits through a single DMTA cycle comprising over 250 synthetically diverse compounds, enabling rapid, resource- and cost-effective exploration of the off-catalogue chemical space. This led to the discovery of protein-protein interaction modulators of Neuronal Calcium Sensor 1 (NCS-1), a key regulator in the central nervous system with therapeutic relevance, which contains a large interaction pocket capable of accommodating multiple protein partners. We advanced fragments into scaffold series that selectively engage biologically validated subpockets and, importantly, revealed allosteric and cryptic binding sites, critical for achieving specificity in target modulation and subsequent hit-to-lead generation. The approach is general, engineerable and scalable, and provides proof-of-principle for how to expand the scope of fragment-based hit discovery.
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Sep 2026
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