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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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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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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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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I03-Macromolecular Crystallography
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Dylan
Kramer
,
Clarissa
Santos Rocha
,
Christopher A.
Gaulke
,
Marie
Nearing
,
Sumathi
Sankaran-Walters
,
Ikaika
Loque
,
Anugraha
Kidigannappa
,
Eric
Pham
,
Shuang
Hu
,
Patrawin
Wanakumjorn
,
Ramona
Abbattista
,
Abhaya
Dandekar
,
Roland
Faller
,
Satya
Dandekar
Open Access
Abstract: HIV infection disrupts gut epithelial barrier integrity and mucosal immunity, driving chronic inflammation and disease progression which are not fully resolved despite anti-retroviral therapy. Here we identify the microbiota-derived octadecanoid-hydroxy-fatty-acid metabolite 10-hydroxystearic acid (10-HSA), produced by Lactiplantibacillus plantarum, as a key mediator of gut epithelial barrier repair in human intestinal epithelial cells in vitro, ex vivo and in the non-human primate model of HIV/AIDS. X-ray crystallography and transcriptomics combined with functional analyses revealed that 10-HSA directly binds PPARα, inducing lipid metabolism, mitochondrial regeneration and subsequent epigenetic histone crotonylation, thereby promoting gut epithelial renewal. Co-administration of 10-HSA with anti-retroviral therapy in SIV-infected macaques accelerated viral suppression, resolved systemic inflammation, repaired gut epithelial integrity and recovered the gut microbiota. These findings identify a microbiota-derived lipid metabolite–PPARα–histone crotonylation axis that activates gut epithelial regeneration. This study defines a host–microbiome metabolic pathway that restores epithelial–immune homeostasis and enhances the efficacy of anti-retroviral therapy.
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Aug 2026
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[26794]
Open Access
Abstract: Crystallography has facilitated the development of inhibitors in modern drug discovery. Complex structures are traditionally obtained by co-crystallization or soaking methods, but they are not always successful; co-crystallization may lead to selective crystallization of the apoprotein, and compounds cannot necessarily be soaked into preformed crystals. Successful endeavours in this field include human tankyrases TNKS1 and TNKS2, which are promising drug targets controlling a wide range of signalling events. In the case of TNKS2, apo crystals have a closed conformation in the donor loop, delineating the NAD+-binding active site, and compound soaking requires a large conformational change, which often damages the crystals and deteriorates the diffraction quality. This work describes a crystal form of TNKS2 that can be used efficiently in replacement soaking (also known as cross-soaking) when TNKS2 is co-crystallized with a low-affinity inhibitor. Replacement soaking is demonstrated with recently described nanomolar inhibitors as well as a nonhydrolysable analogue of NAD+, which all occupy the substrate-binding site. The obtained complex structures offer new insight into the binding modes of the inhibitors and the substrate NAD+, and comparison to previously published complex structures of closely related PARP proteins with NAD+ analogues reveals key characteristics of the NAD+-binding site of TNKS2. In cases where traditional methods fail to yield complex structures, the replacement soaking method provides an alternative method, which was demonstrated here to be necessary for elucidating the complex structures.
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Aug 2026
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[34035]
Open Access
Abstract: The discovery of tubulysins sparked considerable interest due to their high cytotoxic activity against multidrug resistant tumors. Total synthesis of these complex natural products—peptidic metabolites from myxobacteria Archangium gephyra and Angiococcus disciformis—demonstrated the power of organic chemistry and paved the way for the development of simpler, more stable, and selective derivatives. Despite these significant achievements, tubulysins are not used as stand-alone drugs due to their toxic side effects. In this study, we outline the design, synthesis, and evaluation of photoswitchable tubulysin analogues, which could be starting points for development of photopharmacological therapies aimed at addressing the toxicity challenges associated with tubulysins. The cytotoxic activity of one of the key analogues was shown to be light-controllable. For the first time, we provide a comparative analysis of the crystal structures of both photoisomers of a diarylethene-containing compound complexed with target proteins. This comparison enables a mechanistic explanation for the experimentally observed differences in the target binding efficiency and respective activity between the two photoisomers of the photoswitchable tubulysin analogue.
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Aug 2026
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19880]
Abstract: AAA proteases are hexameric ATP-dependent metallopeptidases that perform crucial proteolytic activities within prokaryotic and eukaryotic membranes. Structurally, protomers are comprised of catalytically active C-terminal domains that are anchored to the membrane by an N-terminal autonomous folding unit. In this study, we determined the fold, stability, and oligomeric state of the N-terminal intermembrane domains of human spastic paraplegia type 7 (SPG7)/ paraplegin protein and its bacterial orthologue FtsH using circular dichroism (CD), small-angle X-ray scattering (SAXS), small-angle neutron scattering (SANS) and X-ray crystallography. Solution-state analysis revealed that the N-terminal domain of paraplegin is a monomer in solution whereas FtsH predominantly forms a dimer. Unexpectedly, the N-terminal domain of paraplegin presents as a domain-swapped homodimer in our crystal structure that involves the first helix and first two beta-strands from one monomer and beta-strand 3, helix 2 and beta-strand 4 from another symmetry-related molecule. However, together they form an assembly which is similar to protomers observed for the N-terminal regions of FtsH and AFG3L2. Drawing from our structural data, we postulate that domain-swapping interactions of the N-terminal regions contribute to stability of the AAA protease hexamer containing paraplegin, demonstrating the extensive flexibility of the N-terminal portion of this protein and its role in achieving the appropriate molecular architecture required for function.
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Aug 2026
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