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
VMXi-Versatile Macromolecular Crystallography in situ
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Open Access
Abstract: In vertebrates, a nuclear gene encoding mitochondrial cytochrome c (CYC) was duplicated and diverged into one with ubiquitous expression (somatic-type) and the other with testis-preferred expression (testis-type). In this study, synteny based phylogenetic analyses suggest that cyc ohnologs originated through whole genome duplication (WGD) events in the common ancestor of vertebrates and have been conserved for approximately 450–540 million years. Molecular evolutionary analyses revealed pervasive purifying selection and an approximate Phe/Tyr dichotomy at position 46 between somatic- and testis-type CYC proteins. This was the case in two closely related amphibians, Buergeria buergeri and its hot-spring-dwelling species, Buergeria japonica. The recombinant testis-type CYC proteins from B. buergeri (Bb-CYCT) and B. japonica (Bj-CYCT) exhibited significantly higher resistance to hydrogen peroxide (H2O2) than somatic-type counterpart proteins (Bb-CYCS and Bj-CYCS). Structural analysis showed that the Tyr-46 residues in the testis-type CYC proteins face the heme, suggesting a protective role against H2O2 through an oxidation of its hydroxyl group compared to the spatially corresponding Phe-46 residue in the somatic-type counterparts. When the Tyr-46 residue in Bj-CYCT was replaced with the Phe residue, the H2O2 resistance was significantly reduced, confirming the functional importance of Tyr-46. These results demonstrate that functional divergence between somatic- and testis-type Buergeria CYC proteins is at least partly attributable to the Phe-to-Tyr substitution, suggesting that ancient nuclear genome duplication contributed to tissue-specific divergence of mitochondrial function.
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Sep 2026
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VMXi-Versatile Macromolecular Crystallography in situ
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Elnaz
Aledavood
,
Sandra
Ramos-Inza
,
Jannis
Born
,
Megan
Lambert
,
Hannes
Berg
,
Jan Michael
Köhler
,
Anna
Niesteruk
,
Sridhar
Sreeramulu
,
Amy J.
Thompson
,
Halina
Mikolajek
,
James
Sandy
,
Juan
Sanchez-Weatherby
,
Siyi
Wang
,
Eleanor P.
Williams
,
Martin A.
Walsh
,
Michael A.
Hough
,
Harald
Schwalbe
,
Ana
Martínez
,
Carmen
Gil
Diamond Proposal Number(s):
[32304]
Open Access
Abstract: Fragment-based drug discovery (FBDD) is an effective approach for exploring chemical space using small, low-affinity fragments as starting points to facilitate development of lead compounds. Strategies to improve fragment potency include fragment merging and linking to generate higher-affinity inhibitors. Recently, artificial intelligence (AI) and machine learning (ML) have accelerated this process through structure-based optimization and generative compound design. Here, we present an AI-assisted FBDD workflow applied to the SARS-CoV-2 macrodomain (Mac1), a conserved viral protein involved in immune evasion and ADP-ribose metabolism. Using available structural data and previously identified fragments, we combined deep learning with molecular docking to design novel Mac1 binders. Selected compounds were synthesized and validated by NMR spectroscopy and X-ray crystallography, demonstrating improved binding relative to the original fragment hits with KD values in the range of 299-990 µM. This study demonstrates the advantages of integrating AI with FBDD to streamline molecular design, providing a data-driven framework for discovering new Mac1 inhibitors and guiding future antiviral drug development.
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Jul 2026
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I24-Microfocus Macromolecular Crystallography
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Marina
Lucic
,
Johan
Glerup
,
Pierre
Aller
,
Danny
Axford
,
Nicholas
Devenish
,
Jaehyun
Park
,
Anastasiia
Shilova
,
Arturo
Landeros De La Isla
,
Richard W.
Strange
,
Tiankun
Zhou
,
Robin L.
Owen
,
Jonathan A. R.
Worrall
,
Michael A.
Hough
Diamond Proposal Number(s):
[19458, 28583]
Open Access
Abstract: Metalloenzymes containing a heme cofactor catalyse a wide range of oxidative reactions critical to life. Understanding the structure and electronic states of the heme across the catalytic cycle is essential in understanding the oxidative chemistry performed on the substrate. This work demonstrates in crystallo manipulation of the heme-iron oxidation state in a B-type dye-decolourizing peroxidase from Streptomyces lividans (DtpB) using multiple, complementary, serial crystallography approaches. Fixed-target drop-on-chip serial femtosecond crystallography (SFX) together with dose-resolved serial synchrotron crystallography (SSX) allowed DtpB to be driven between multiple iron oxidation states. Drop-on-chip addition of hydrogen peroxide with fixed-target SFX is used to generate a ferryl [Fe(IV)=O] species, while the X-ray-driven approach modulates the iron oxidation state, with an apparent two-electron reduction leading to a return to a ferric state. The formation and dose response of the Fe(IV)—O state is highly variable between the chemically identical heme groups of the DtpB hexamer, highlighting the importance of understanding the effect of the crystalline lattice on observed changes in time- and dose-resolved crystallography.
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May 2026
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Lewis J.
Williams
,
Jos J. A. G.
Kamps
,
Adrian M. V.
Brânzanic
,
Maria
Lehene
,
Kristoffer J. M.
Lundgren
,
Ulf
Ryde
,
Kuntal
Chatterjee
,
Margaret D.
Doyle
,
Philipp S.
Simon
,
Hiroki
Makita
,
Amy J.
Thompson
,
Aaron S.
Brewster
,
Tiankun
Zhou
,
Marina
Lucic
,
Michael T.
Wilson
,
Pierre
Aller
,
Juan
Sanchez-Weatherby
,
Leland
Gee
,
Sebastian
Dehe
,
Sandra
Mous
,
Junko
Yano
,
Vittal K.
Yachandra
,
Michael A.
Hough
,
Allen M.
Orville
,
Jan F.
Kern
,
Radu L.
Silaghi-Dumitrescu
,
Jonathan A. R.
Worrall
Open Access
Abstract: The use of X-ray structures to determine and interpret the ferryl iron-oxygen bond order in molecular oxygen-activating heme enzymes has, in the past, been controversial. This has mainly stemmed from the susceptibility of ferryl species to X-ray-induced electronic state changes. In this work we establishe using time-resolved serial femtosecond X-ray crystallography (tr-SFX) on a dye-decolourising peroxidase that the ferryl intermediate species (Compounds I and II) captured following in situ mixing of microcrystals with H2O2 have single, rather than the double bond character expected. X-ray emission validated tr-SFX data with quantum refinement, time-dependent-DFT calculations and QM/MM geometry optimizations together support the concept that the single iron-oxygen bond character is not an indication of ferryl reduction or a protonated form (FeIV-OH) but is instead attributed to the existence of accessible excited states possessing ferric-oxyl (FeIII–O•–) character. Such states offer insight into the nature of ferryl heme.
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Feb 2026
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I24-Microfocus Macromolecular Crystallography
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Peter
Smyth
,
Sofia
Jaho
,
Lewis J.
Williams
,
Gabriel
Karras
,
Ann
Fitzpatrick
,
Amy J.
Thompson
,
Sinan
Battah
,
Danny
Axford
,
Sam
Horrell
,
Marina
Lucic
,
Kotone
Ishihara
,
Machika
Kataoka
,
Hiroaki
Matsuura
,
Kanji
Shimba
,
Kensuke
Tono
,
Takehiko
Tosha
,
Hiroshi
Sugimoto
,
Shigeki
Owada
,
Michael A.
Hough
,
Jonathan A. R.
Worrall
,
Robin L.
Owen
Diamond Proposal Number(s):
[18565, 28583, 27313]
Open Access
Abstract: Time-resolved X-ray crystallography is undergoing a renaissance due to the development of serial crystallography at synchrotron and XFEL beamlines. Crucial to such experiments are efficient and effective methods for uniformly initiating time-dependent processes within microcrystals, such as ligand binding, enzymatic reactions or signalling. A widely applicable approach is the use of photocaged substrates, where the photocage is soaked into the crystal in advance and then activated using a laser pulse to provide uniform initiation of the reaction throughout the crystal. This work characterizes photocage release of nitric oxide and binding of this ligand to two heme protein systems, cytochrome c′-β and dye-decolourizing peroxidase B using a fixed target sample delivery system. Laser parameters for photoactivation are systematically explored, and time-resolved structures over timescales ranging from 100 µs to 1.4 s using synchrotron and XFEL beamlines are described. The effective use of this photocage for time-resolved crystallography is demonstrated and appropriate illumination conditions for such experiments are determined.
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Sep 2025
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I24-Microfocus Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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Hans E.
Pfalzgraf
,
Aditya G.
Rao
,
Kakali
Sen
,
Hannah R.
Adams
,
Marcus
Edwards
,
You
Lu
,
Chin
Yong
,
Sofia
Jaho
,
Takehiko
Tosha
,
Hiroshi
Sugimoto
,
Sam
Horrell
,
James
Beilsten-Edmands
,
Robin L.
Owen
,
Colin R.
Andrew
,
Jonathan A. R.
Worrall
,
Ivo
Tews
,
Adrian J.
Mulholland
,
Michael A.
Hough
,
Thomas W.
Keal
Diamond Proposal Number(s):
[27313]
Open Access
Abstract: Cytochromes P460 oxidise hydroxylamine within the nitrogen cycle and contain as their active site an unusual catalytic c-type haem where the porphyrin is crosslinked to the protein via a lysine residue in addition to the canonical cross links from cysteine residues. Understanding how enzymes containing P460 haem oxidise hydroxylamine into either nitrous oxide or nitric oxide has implications for climate change. Interestingly the P460-containing hydroxylamine oxidoreductase utilises a tyrosine cross link to haem and performs similar chemistry. Previous crystal structures of cytochrome P460 from Nitrosomonas europaea (NeP460) clearly show the existence of a single crosslink between the NZ atom of lysine and the haem porphyrin, with mutagenesis studies indicating roles for the crosslink in positioning a proton transfer residue and/or influencing the distortion of the haem. Here we describe the evidence for a novel double crosslink between lysine and haem in the cytochrome P460 from Methylococcus capsulatus (Bath). In order to understand the complexities of this enzyme system we applied high resolution structural biology approaches at synchrotron and XFEL sources paired with crystal spectroscopies. Linked to this, we carried out QM/MM simulations that enabled the prediction of electronic absorption spectra providing a crucial validation to linking simulations and experimental structures. Our work demonstrates the feasibility of a double crosslink in McP460 and provides an opportunity to investigate how simulations can interact with experimental structures.
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Aug 2025
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VMXi-Versatile Macromolecular Crystallography in situ
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Abstract: Macromolecular X-ray crystallography is a powerful tool enabling modern structure- based drug design, where structures of protein-ligand complexes provide a basis for rational design decisions to improve the potency of ligands. However, like the protein itself, small molecules within protein crystals are subject to specific radiation damage (SRD) during the collection of X-ray diffraction data, but the effect of SRD on small molecule ligands has not yet been extensively described. This study is following on recently published work1 investigating SRD to halogenated ligands in protein-ligand structures of the therapeutic cancer target B-cell lymphoma 6 protein (BCL6), which found significant cleavage of carbon-halogen (C-X) bonds during X-ray diffraction data collection. The present work is making use of an improved set of BCL6 ligands to gain further understanding of the role of the halogen type and substitution position on the susceptibility of the C-X bond to SRD. Additionally, diffraction data will not only be collected at cryogenic temperature, but also at room temperature at the VMXi beamline at Diamond Light Source to investigate the impact of data collection temperature on SRD to small molecule ligands.
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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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Lewis J.
Williams
,
Amy J.
Thompson
,
Philipp
Dijkstal
,
Martin
Appleby
,
Greta
Assmann
,
Florian S. N.
Dworkowski
,
Nicole
Hiller
,
Chia-Ying
Huang
,
Tom
Mason
,
Samuel
Perrett
,
Eduard
Prat
,
Didier
Voulot
,
Bill
Pedrini
,
John H.
Beale
,
Michael A.
Hough
,
Jonathan A. R.
Worrall
,
Robin L.
Owen
Open Access
Abstract: Serial femtosecond crystallography (SFX) exploits extremely brief X-ray free-electron laser pulses to obtain diffraction data before destruction of the crystal. However, during the pulse X-ray-induced site-specific radiation damage can occur, leading to electronic state and/or structural changes. Here, we present a systematic exploration of the effect of single-pulse duration and energy (and consequently different dose rates) on site-specific radiation damage under typical SFX room-temperature experimental conditions. For the first time in SFX we directly measured the photon pulse duration, varying from less than 10 fs to more than 50 fs, and used three pulse energies to probe in-pulse damage in two radiation-sensitive proteins: the iron-heme peroxidase DtpAa and the disulfide-rich thaumatin. While difference-map features arising from radiation damage are observed, they do not lead to significant change in refined atomic coordinates or key bond lengths. Our work thus provides experimental verification that average atomic coordinates are not significantly perturbed by radiation damage in typical SFX experiments.
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May 2025
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