I24-Microfocus Macromolecular Crystallography
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Morgan
Thomas
,
Pierre G.
Matricon
,
Robert J.
Gillespie
,
Maja
Napiórkowska
,
Hannah
Neale
,
Jonathan S.
Mason
,
Jason
Brown
,
Kaan
Harwood
,
Charlotte
Fieldhouse
,
Nigel A.
Swain
,
Tian
Geng
,
Noel M.
O’boyle
,
Francesca
Deflorian
,
Andreas
Bender
,
Chris
De Graaf
Open Access
Abstract: Generative chemical language models (CLMs) have demonstrated success in learning language-based molecular representations for de novo drug design. Here, we integrate structure-based drug design (SBDD) principles with CLMs to go from protein structure to novel small-molecule ligands, without a priori knowledge of ligand chemistry. Using Augmented Hill-Climb, we successfully optimise multiple objectives within a practical timeframe, including protein-ligand complementarity. Resulting de novo molecules contain known or promising adenosine A2A receptor ligand chemistry that is not available in commercial vendor libraries, accessing commercially novel areas of chemical space. Experimental validation demonstrates a binding hit rate of 88%, with 50% having confirmed functional activity, including three nanomolar ligands and two novel chemotypes. The two strongest binders are co-crystallised with the A2A receptor, revealing their binding mechanisms that can be used to inform future iterations of structure-based de novo design, closing the AI SBDD loop.
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Jul 2025
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[28677, 36838]
Open Access
Abstract: Glycosphingolipids (GSLs) are crucial membrane components involved in essential cellular pathways. Complex GSLs, known as gangliosides, are synthesised by glycosyltransferase enzymes and imbalances in GSL metabolism cause severe neurological diseases. B4GALNT1 synthesises the precursors to the major brain gangliosides. Loss of B4GALNT1 function causes hereditary spastic paraplegia, while its overexpression is linked to cancers including childhood neuroblastoma. Here, we present crystal structures of the human homodimeric B4GALNT1 enzyme demonstrating dynamic remodelling of the substrate binding site during catalysis. We show that processing of lipid substrates by B4GALNT1 is severely compromised when surface loops flanking the active site are mutated from hydrophobic residues to polar. Molecular dynamics simulations support that these loops can insert into the lipid bilayer explaining how B4GALNT1 accesses and processes lipid substrates. By combining structure prediction and molecular simulations we propose that this mechanism of dynamic membrane insertion is exploited by other, structurally distinct GSL synthesising enzymes.
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Jul 2025
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[11265]
Open Access
Abstract: Botulinum neurotoxins are the causative agents of botulism, a lethal paralytic disease, but are also one of the most commonly used therapeutics for the treatment of numerous neuromuscular conditions. These toxins recognise motor nerve terminals with high specificity and affinity by using a dual binding mechanism involving gangliosides and protein receptors. The initial recognition of gangliosides is crucial for the toxins’ potency. In this study, we employed a synaptosome-binding screening strategy to identify BoNT/A mutants with enhanced ganglioside-binding which translated into improved potency. X-ray crystallography and receptor-binding assays were used to elucidate the molecular mechanisms underlying the increased affinity or altered ganglioside selectivity of these mutants. Our findings provide a basis for the development of BoNT/A variants with enhanced therapeutic potential.
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Jun 2025
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I24-Microfocus Macromolecular Crystallography
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Abstract: Time-resolved diffraction is becoming an established technique in large-scale facilities such as synchrotrons and XFELs. Alternative sources such as plasma X-ray source (PXS, Fig. 1A) [1] offer attractive pulse durations of units of picoseconds with lower operational cost than XFEL facilities. However, the downsides of PXS sources are the unstable flux and comparatively low brilliance. The latter may be overcome by a stroboscopic [2] or multiplexing [3] approaches, however, the majority of macromolecular samples are radiation sensitive and undergo irreversible reactions limiting the application of stroboscopic data collection.
Hadamard Time-Resolved Crystallography (HATRX) is a multiplexing technique, where diffraction is measured as an ensemble of individual time points (Fig. 1B). The individual time-resolved data are then reconstructed using the Hadamard transform. Multiplexing requires the ability to measure diffraction at distinct time-points utilising either detector-gating or a pulsed source. We present initial results demonstrating radiation damage during a HATRX experiment using the detector-gating technique at beamline I24 (Diamond Light Source). The necessary pulse-sequences may be introduced onto the driving laser of the PXS, which is advantageous over detector gating since the sample is only exposed to X-rays when data are being collected. We discuss the potentials and pitfalls of the PXS for HATRX studies.
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Jun 2025
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I24-Microfocus Macromolecular Crystallography
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Tomas
Akerud
,
Claudia
De Fusco
,
Peter
Brandt
,
Fredrik
Bergström
,
Patrik
Johansson
,
Margareta
Ek
,
Ulf
Börjesson
,
Anders
Johansson
,
Jakob
Danielsson
,
Martin
Bauer
,
Bertrand
Arnaud
,
Marie
Castaldo
,
Maria
Strömstedt
,
Birgitta
Rosengren
,
Frank
Jansen
,
Linda
Fredlund
Diamond Proposal Number(s):
[20016]
Open Access
Abstract: Nicotinamide N-methyl transferase (NNMT) is involved in the regulation of cellular nicotinamide adenine dinucleotide (NAD) and S-adenosyl-L-methionine (SAM) levels and has been implicated in a range of human diseases. Herein, we show that a class of NNMT inhibitors, analogs of the natural substrate nicotinamide (NAM), is turned over by the enzyme and that the methylated product is a potent inhibitor of the enzyme. The product inhibitor is, however, charged and has modest cellular potency. Utilizing this on-target biotransformation combines the cell permeability of the substrate with the high potency of the product resulting in highly efficient inhibition in vivo. First, we studied the structure-activity-relationship for both substrates and methylated products and solved structures using X-ray crystallography of representative inhibitors. Then we designed a new surface biosensor method to understand the structure-kinetic-relationship for the inhibitors. We were able to quantify the substrate binding kinetics to NNMT-SAM, catalysis rate, and rate of product release from NNMT-SAH in a single experiment. This is to our knowledge the first time an enzyme surface biosensor has been used to study and quantify catalysis in detail. Finally, by monitoring plasma concentrations of turnover inhibitor substrate, product, and the endogenous product, 1-Methyl nicotinamide (1-MNA), in the rat, we show that the turnover inhibitor mechanism of action is relevant in vivo.
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Jun 2025
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I24-Microfocus Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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Open Access
Abstract: Multi-crystal processing of X-ray diffraction data has become highly automated to keep pace with the current high-throughput capabilities afforded by beamlines. A significant challenge, however, is the automated clustering of such data based on subtle differences such as ligand binding or conformational shifts. Intensity-based hierarchical clustering has been shown to be a viable method of identifying such subtle structural differences, but the interpretation of the resulting dendrograms is difficult to automate. Using isomorphous crystals of bovine, porcine and human insulin, the existing clustering methods in the multi-crystal processing software xia2.multiplex were validated and their limits were tested. It was determined that weighting the pairwise correlation coefficient calculations with the intensity uncertainties was required for accurate calculation of the pairwise correlation coefficient matrix (correlation clustering) and dimension optimization was required when expressing this matrix as a set of coordinates representing data sets (cosine-angle clustering). Finally, the introduction of the OPTICS spatial density-based clustering algorithm into DIALS allowed the automatic output of species-pure clusters of bovine, porcine and human insulin data sets.
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Jun 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946, 28534]
Open Access
Abstract: The spillover of New World (NW) arenaviruses from rodent reservoirs into human populations poses a continued risk to human health. NW arenaviruses present a glycoprotein (GP) complex on the envelope surface of the virion, which orchestrates host cell entry and is a key target of the immune response arising from infection and immunization. Each protomer of the trimeric GP is composed of a stable signal peptide, a GP1 attachment glycoprotein, and a GP2 fusion glycoprotein. To glean insights into the architecture of this key therapeutic target, we determined the crystal structures of NW GP1−GP2 heterodimeric complexes from Junín virus and Machupo virus. Due to the metastability of the interaction between GP1 and GP2, structural elucidation required the introduction of a disulfide bond at the GP1−GP2 complex interface, but no other stabilizing modifications were required. While the overall assembly of NW GP1−GP2 is conserved with that presented by Old World (OW) arenaviruses, including Lassa virus and lymphocytic choriomeningitis virus, NW GP1−GP2 complexes are structurally distinct. Indeed, we note that when compared to the OW GP1−GP2 complex, the globular portion of NW GP1 undergoes limited structural alterations upon detachment from its cognate GP2. We further demonstrate that our engineered GP1−GP2 heterodimers are antigenically relevant and recognized by neutralizing antibodies. These data provide insights into the distinct assemblies presented by NW and OW arenaviruses, as well as provide molecular-level blueprints that may guide vaccine development.
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Jun 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Zak
Mciver
,
Alicia
Moraleda-Montoya
,
Zongjia
Chen
,
Ruwan
Epa
,
David
Starns
,
Matthew
Davy
,
Mikel
Garcia-Alija
,
Arnaud
Basle
,
Mario
Schubert
,
Didier
Ndeh
,
Beatriz
Trastoy
,
Spencer J.
Williams
,
Marcelo E.
Guerin
,
Alan
Cartmell
Diamond Proposal Number(s):
[18598, 30305, 21970]
Open Access
Abstract: Rhamnogalacturonan II is one of the most complex plant cell wall carbohydrates and is composed of 13 different sugars and 21 different glycosidic linkages. It is abundant in fruit and indulgence foods, such as chocolate and wine, making it common in the human diet. The human colonic commensal Bacteroides thetaiotaomicron expresses a consortium of 22 enzymes to metabolise rhamnogalacturonan II, some of which exclusively target sugars unique to rhamnogalacturonan II. Several of these enzyme families remain poorly described, and, consequently, our knowledge of rhamnogalacturonan II metabolism is limited. Chief among the poorly understood activities is glycoside hydrolase (GH) family 139, with targets α1,2-2O-methyl L-fucoside linkages, a sugar residue a sugar not found in any other plant cell wall complex glycans. Although the founding enzyme BT0984 was placed in the RG-II degradative pathway, no GH139 structure or catalytic blueprint had been available. We report the crystal structures of BT0984 and a second homologue, and reveal that the family operates with inverting stereochemistry. Using this data we undertook a mutagenic strategy, backed by molecular dynamics, to identify the important substrate binding and catalytic residues, mapping these residues throughout the GH139 family revealing the importance of the O2 methyl interaction of the substrate. We propose a catalytic mechanism that uses a non-canonical Asn as a catalytic base and shares similarity with L-fucosidases/L-galactosidases of family GH95.
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Jun 2025
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
VMXm-Versatile Macromolecular Crystallography microfocus
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Diamond Proposal Number(s):
[26803, 34438, 35338]
Abstract: Due to radiation damage, the majority of metalloproteins structures are incorrect. Radiation damage in X-ray crystallography manifests itself either globally or at specific radiation sensitive sites. Global damage can be monitored from data processing statistics, whereas specific damage is more clandestine and presents as structural changes within the electron density. Serial crystallography using an X-ray free-electron laser promises a pseudo zero dose structure, however, the paucity of beamlines means beamtime is highly competitive. Method development, therefore, is required to collect low-dose structures using synchrotron X-ray crystallography. One dose-reducing phenomenon is photoelectron escape, where the generated photoelectrons escape the crystal volume before depositing their energy.
This thesis conducted the first serial crystallography experiment at VMXm (Diamond Light Source, UK), where photoelectron escape is significant for the targeted microcrystal sizes. An oxidised iron intermediate in myoglobin, Compound II, was tested as FeIV-oxo “ferryl” intermediates, which are known to be particularly susceptible to radiation damage. Despite not being a formal heme peroxidase, myoglobin is an excellent model for testing dose-limiting techniques. An NADP+-specific glyceraldehyde 3’-phosphate dehydrogenase from the enteric pathogen Helicobacter pylori was also investigated. NADP+-specificity is unusual amongst GAPDHs and are therefore poised for therapeutic targets. The kinetics of GAPDHA were investigated, and amongst the first structures of an NADP+-specific GAPDH outside of photosynthetic organisms are reported. An underreported form of radiation damage was observed. Therefore, a transition to microcrystals for a prospective dose-series and time-resolved investigation was performed.
A Mix and Quench Microcrystal Reactor was developed to initiate a reaction within microcrystals with rapid mixing and to trap intermediates by quenching in liquid ethane. Current systems exist for time-resolved crystallography or time-resolved cryoEM; however, a system was developed to react and spray microcrystals onto a TEM grid for use on the specific goniometry at VMXm.
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May 2025
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B23-Circular Dichroism
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: A Capillary Zone Electrophoresis (CZE) fragment screening methodology was developed and applied to the human plasma protein Transthyretin (TTR), normally soluble, but could misfold and aggregate, causing amyloidosis. Termed Free Probe Peak Height Restoration (FPPHR), it monitors changes in the level of free ligand known to bind TTR (the Probe Ligand) in the presence of competing fragments. 129 fragments were screened, 12 of the 16 initial hits (12.4% hit rate) were co-crystallised with TTR, 11 were found at the binding site (92% confirmation rate). Subsequent analogue screens have identified a novel TTR-binding scaffold 4-(3H-pyrazol-4-yl)quinoline and its derived compounds were further studied by crystallography, circular dichroism (CD), isothermal titration calorimetry (ITC) and radiolabelled 125I-Thyroxine displacement assay in neat plasma. Two lead molecules had similar ITC Kd and 125I-Thyroxine displacement IC50 values to that of Tafamidis, adding another potential pipeline for transthyretin amyloidosis. The methodology is reproducible, procedurally simple, automatable, label-free without target immobilisation, non-fluorescence based and site-specific with low false positive rate, which could be applicable to fragment screening of many drug targets.
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May 2025
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