I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Serial synchrotron crystallography (SSX) enables structure determination from microcrystals under near-physiological, room-temperature conditions but is limited in part due to the inevitable onset of radiation damage. The ability to reduce the absorbed dose while retaining, or even improving, data quality is an attractive means of mitigating this limitation. Advances in detector technology have made the use of high-energy X-rays a routine approach in MX, improving diffraction efficiency and enhancing overall data quality. Here, we systematically evaluate low-dose SSX data collected at five different X-ray energies from 12.4 to 25 keV using a CdTe Eiger2 detector while maintaining a constant dose. Higher photon energies increased the mean diffracted intensity and signal-to-noise ratio per unit dose, and facilitated higher-resolution structure determination, even with limited crystal numbers. These findings highlight the advantages of high-energy X-rays and provide practical guidance for optimizing SSX experiments in probing protein dynamics.
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Mar 2026
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I24-Microfocus Macromolecular Crystallography
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Ronald
Rios-Santacruz
,
Harshwardhan
Poddar
,
Kevin
Pounot
,
Derren J.
Heyes
,
Nicolas
Coquelle
,
Megan J.
Mackintosh
,
Linus O.
Johannissen
,
Sara
Schianchi
,
Laura N.
Jeffreys
,
Elke
De Zitter
,
Rory
Munro
,
Martin
Appleby
,
Danny
Axford
,
Emma V.
Beale
,
Matthew J.
Cliff
,
María C.
Dávila-Miliani
,
Sylvain
Engilberge
,
Guillaume
Gotthard
,
Kyprianos
Hadjidemetriou
,
Samantha J. O.
Hardman
,
Sam
Horrell
,
Jochen S.
Hub
,
Kotone
Ishihara
,
Sofia
Jaho
,
Gabriel
Karras
,
Machika
Kataoka
,
Ryohei
Kawakami
,
Thomas
Mason
,
Hideo
Okumura
,
Shigeki
Owada
,
Robin L.
Owen
,
Antoine
Royant
,
Annica
Saaret
,
Michiyo
Sakuma
,
Muralidharan
Shanmugam
,
Hiroshi
Sugimoto
,
Kensuke
Tono
,
Ninon
Zala
,
John H.
Beale
,
Takehiko
Tosha
,
Jacques-Philippe
Colletier
,
Matteo
Levantino
,
Sam
Hay
,
Pawel M.
Kozlowski
,
David
Leys
,
Nigel S.
Scrutton
,
Martin
Weik
,
Giorgio
Schirò
Diamond Proposal Number(s):
[24447, 31850]
Abstract: Photoreceptor proteins regulate fundamental biological processes such as vision, photosynthesis and circadian rhythms1. A large photoreceptor subfamily uses vitamin B12 derivatives for light sensing2, contrasting with the well-established mode of action of these organometallic derivatives in thermally activated enzymatic reactions3. The exact molecular mechanism of B12 photoreception and how this differs from the thermal pathways remains unknown. Here we provide a detailed description of photoactivation in the prototypical B12 photoreceptor CarH4,5 from nanoseconds to seconds, combining time-resolved and temperature-resolved structural and spectroscopic methods with quantum chemical calculations. Building on the crystal structures of the initial tetrameric dark and final monomeric light-activated states5, our structural snapshots of key intermediates in the truncated B12-binding domain illustrate how photocleavage of a cobalt–carbon (Co–C) bond within the B12 chromophore adenosylcobalamin triggers a series of structural changes that propagate throughout CarH. Breakage of the photolabile Co–C5′ bond leads to the formation of a previously unknown adduct that links the C4′ position of the adenosyl moiety to the Co ion and can subsequently be cleaved thermally over longer timescales to allow release of the adenosyl group, ultimately causing tetramer dissociation4,5. This adduct, which differentiates CarH from thermally activated B12 enzymes, steers the photoactivation pathway and acts as the molecular bridge between photochemical and photobiological timescales. The biological relevance of our study is corroborated by kinetic data on full-length CarH in the presence of DNA. Our results offer a spatiotemporal understanding of CarH photoactivation and pave the way for designing B12-dependent photoreceptors for optogenetic applications.
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Feb 2026
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[37045]
Open Access
Abstract: Objective: To determine the high-resolution structure of human Copper-Zinc superoxide dismutase (hSOD1), an antioxidant enzyme whose mutations cause amyotrophic lateral sclerosis (ALS), under near-physiological conditions. Because SOD1 is intrinsically dynamic, capturing its structure at ambient temperature is key to understanding how temperature modulates its conformational flexibility, ensemble and functional states relevant to both catalysis and disease.
Materials and Methods: Recombinant hSOD1 was expressed in E. coli, purified by affinity and size-exclusion chromatography, and crystallized at ambient temperature. Serial synchrotron crystallography (SSX) data were collected at 293 K at the EMBL P14-2 Time-Resolved Experiments with Crystallography (T-REXX) beamline at PETRA III, and compared with a 100 K cryogenic at the Diamond Light Source beamline (I03). Both datasets were processed and refined using CCP4 suite and PHENIX packages. B-factor distributions, per-residue RMSD values, and conformational differences were analyzed to quantify temperature-dependent effects.
Results: The ambient-temperature SOD1SSX structure was determined at 2.3 Å resolution (PDB ID:9XJ0 this work) and closely matched its 2.37 Å cryogenic counterpart (SOD1CRYO, PDB ID:9XJI this work), both obtained from identical crystallization conditions in the hexagonal P6₃ space group. Cryocooling caused a 3.8% contraction in unit-cell volume, consistent with lattice densification and a 5.2% reduction in molecular surface volume. Despite the overall similarities, the ambient-temperature model revealed localized conformational differences in solvent-exposed loop residues, particularly Ser25-Asn26, Leu67-Glu77, Ile99, and the Asp109-His110-Cys111 triad, and a distinct side-chain orientation of Asn53 was observed at the dimerization interface. While the β-barrel core remained rigid, these regions correspond to redox- and metal-responsive sites implicated in aggregation/fiber formation and putative drug binding.
Conclusions: Temperature perturbs local dynamics in SOD1 structure without altering its native dimeric form. The ambient-temperature model reveals flexible, chemically accessible regions that act as druggable hotspots and coincide with ALS-linked mutation sites driving misfolding and aggregation. Considering temperature effects is crucial for structure-based drug design, ensuring candidate molecules engage physiologically relevant conformations. This structure lays the groundwork for future time-resolved crystallography of SOD1 folding and ligand interactions.
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Dec 2025
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I24-Microfocus Macromolecular Crystallography
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Abstract: Observing transient structural intermediates remains a central challenge in enzymology. Little is known about these despite their pivotal role in catalytic function. Time-resolved serial crystallography at synchrotron and X-ray free electron laser (XFEL) sources offers a promising avenue to capture these dynamic events. However, experimental success hinges on rigorous control of crystallisation, ligand delivery, and integration with beamline infrastructure. This thesis explores the use of droplet microfluidic and microcrystal strategies to address these bottlenecks and establish pipelines for future time-resolved studies.
A high-throughput crystallisation system was developed to generate uniform microcrystals within discrete aqueous droplets, leveraging a seeding strategy to overcome the low probability of nucleation at diminishing volumes. In parallel, a droplet micromixing device was engineered to initiate ligand-triggered reactions on millisecond timescales by exploiting convection within droplets as a means for rapidly mixing microcrystals with ligands. Flow parameters and mixing efficiency were characterised, followed by iterative design and fabrication of a X-ray transmissible device suitable for deployment at a synchrotron beamline.
Arabidopsis thaliana Pyridoxal 5'-phosphate synthase subunit 1.3 (AtPdx1.3) microcrystal slurries were validated for time-resolved studies using static serial femtosecond crystallography (SFX) at the SPring-8 angstrom compact free electron laser (SACLA). High-resolution radiation damage-free structures of apo and ligand-bound AtPdx1.3 were obtained at room temperature, representing the first XFEL structures of this enzyme. Diffraction from 20 μm crystals yielded resolution comparable to or better than previous larger crystals at cryogenic temperatures. Notably, only minimal structural differences were observed relative to cryotrapped structures, indicating strong conformational consistency. Soaking protocols enabled rapid ligand incorporation, capturing R5P, PLP and the crucial I320 intermediate within 15 minutes. These result establish robust workflows for intermediate state trapping and future dynamic studies. Taken together, the platforms developed in this thesis represent a significant step towards realising dynamic structural studies of enzymes at synchrotron and XFEL sources.
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Nov 2025
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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
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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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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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I19-Small Molecule Single Crystal Diffraction
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Diamond Proposal Number(s):
[33014, 35357]
Open Access
Abstract: The increasing availability of ultrabright Light Sources is facilitating the study of smaller crystals at faster timescales but with an increased risk of severe X-ray damage, leading to developments in multi-crystal methods such as serial crystallography (SX). SX studies on crystals with small unit cells are challenging as very few reflections are recorded in a single data image, making it difficult to determine the orientation matrix for each crystal and thus preventing the combination of the data from all crystals for structure solution. We herein present a Small-Rotative Fixed-Target Serial Synchrotron Crystallography (SR-FT-SSX) methodology, in which rotation of the serial target through a small diffraction angle at each crystal delivers high-quality data, facilitating ab initio unit cell determination and atomic-scale structure solution. The method is benchmarked using microcrystals of the small-molecule photoswitch sodium nitroprusside dihydrate, obtaining complete data to dmin = 0.6 Å by combining just 66 partial datasets selected against rigorous quality criteria.
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Nov 2024
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I24-Microfocus Macromolecular Crystallography
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Abstract: This chapter describes additions to the DIALS software package for processing serial still-shot crystallographic data, and the implementation of a pipeline, xia2.ssx, for processing and merging serial crystallography data using DIALS programs. To integrate partial still-shot diffraction data, a 3D gaussian profile model was developed that can describe anisotropic spot shapes. This model is optimised by maximum likelihood methods using the pixel-intensity distributions of strong diffraction spots, enabling simultaneous refinement of the profile model and Ewald-sphere offsets. We demonstrate the processing of an example SSX dataset where the improved partiality estimates lead to better model statistics compared with post-refined isotropic models. We also demonstrate some of the workflows available for merging SSX data, including processing time/dose resolved data series, where data can be separated at the point of merging after scaling and discuss the program outputs used to investigate the data throughout the pipeline.
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Nov 2024
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[260673]
Open Access
Abstract: Structure-based drug design is highly dependent on the availability of structures of the protein of interest in complex with lead compounds. Ideally, this information can be used to guide the chemical optimization of a compound into a pharmaceutical drug candidate. A limitation of the main structural method used today – conventional X-ray crystallography – is that it only provides structural information about the protein complex in its frozen state. Serial crystallography is a relatively new approach that offers the possibility to study protein structures at room temperature (RT). Here, we explore the use of serial crystallography to determine the structures of the pharmaceutical target, soluble epoxide hydrolase. We introduce a new method to screen for optimal microcrystallization conditions suitable for use in serial crystallography and present a number of RT ligand-bound structures of our target protein. From a comparison between the RT structural data and previously published cryo-temperature structures, we describe an example of a temperature-dependent difference in the ligand-binding mode and observe that flexible loops are better resolved at RT. Finally, we discuss the current limitations and potential future advances of serial crystallography for use within pharmaceutical drug discovery.
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Sep 2024
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