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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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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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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B22-Multimode InfraRed imaging And Microspectroscopy
I19-Small Molecule Single Crystal Diffraction
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Open Access
Abstract: The latest developments in acoustic technology and their integration of standing waves offers the potential to eject and trap tiny amounts of both liquid and solids with precision and stability in free space. This is appealing for experimenters at Light Sources who are traditionally relying on the use of containers or on the manual attachment of the samples to solid supports for analysis with X rays or IR. Here we present a brief overview of applications of acoustics and levitation for sample manipulation and delivery currently under investigation at Diamond Light source.
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May 2025
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Metrology
Optics
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Open Access
Abstract: The Optics & Metrology group at Diamond Light Source has recently published a description of a bimorph deformable X-ray mirror operating in closed-loop using multi-beam interferometric feedback. This "adaptive" mirror can make fast and stabilised changes to the X-ray beam profile. Beam shaping at a rate of 1 Hz was achieved, a contrast to the now usual "set and forget" operation of "active" bimorph mirrors at synchrotrons. However, this breakthrough cannot be applied to synchrotron beamlines without a robust control system that allows the mirror to be rapidly and controllably deformed. Diamond has now responded to this need by taking an integrated approach, considering: the bimorph power supplies, the beamline control software, the beam imaging camera, the bimorph mirror optimisation software, and the bimorph mirror itself as part of a single system. In collaboration with CINEL, new HV-ADAPTOS high-voltage power supplies have been made available. The latest models contain new firmware that adds features not previously available, such as piezo-elastic creep compensation. Communication with the HV-ADAPTOS power supplies over Ethernet has been made more reliable by a new EPICS asynPortDriver interface developed at Diamond and rolled out to all appropriate Diamond beamlines. A new Bluesky/Ophyd plan for the measurement of the bimorph mirror's piezo response functions is under development and has undergone its preliminary tests. This plan is expected to be less affected by upgrades of the beamline control software than previous solutions. It relies on beam images produced by Gigabit Ethernet cameras and processed by the EPICS areaDetector driver. Finally, the need for strain-free clamping of the mirror has now been fully recognized and procedures for ensuring it have been put into practice. Although such a system is more complex than that required for a mechanically bent mirror, it gives bimorph mirrors an ability to operate rapidly and repeatably that other optics do not offer, and it lays a foundation for more advanced beam-shaping functions.
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May 2025
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Abstract: In serial crystallography, large numbers of microcrystals are sequentially delivered to an X-ray beam and a diffraction pattern is obtained from each crystal. This serial approach was developed primarily for X-ray Free Electron Lasers (XFELs) where crystals are destroyed by the beam but is increasingly used in synchrotron experiments. The combination of XFEL and synchrotron-based serial crystallography enables time-resolved experiments over an extremely wide range of time domains - from femtoseconds to seconds - and allows intact or pristine structures free of the effects of radiation damage to be obtained. Several approaches have been developed for sample delivery with varying levels of sample efficiency and ease of use. In the fixed target approach, microcrystals are loaded onto a solid support which is then rastered through the X-ray beam. The key advantages of fixed targets are that every crystal loaded can be used for data collection, and that precise control of when crystals are moved into the beam allows for time-resolved experiments over a very wide range of time domains as well as multi-shot experiments characterising the effects of the X-ray beam on the sample. We describe the application of fixed targets for serial crystallography as implemented at beamline I24 at Diamond Light Source and at the SACLA XFEL. We discuss methodologies for time-resolved serial crystallography in fixed targets and describe best practices for obtaining high-quality structures covering sample preparation, data collection strategies and data analysis pipelines.
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Oct 2024
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[27314]
Open Access
Abstract: Human gamma-D crystallin (HGD) is a major constituent of the eye lens. Aggregation of HGD contributes to cataract formation, the leading cause of blindness worldwide. It is unique in its longevity, maintaining its folded and soluble state for 50-60 years. One outstanding question is the structural basis of this longevity despite oxidative aging and environmental stressors including ultraviolet radiation (UV). Here we present crystallographic structures evidencing a UV-induced crystallin redox switch mechanism. The room-temperature serial synchrotron crystallographic (SSX) structure of freshly prepared crystallin mutant (R36S) shows no post-translational modifications. After aging for nine months in the absence of light, a thiol-adduct (dithiothreitol) modifying surface cysteines is observed by low-dose SSX. This is shown to be UV-labile in an acutely light-exposed structure. This suggests a mechanism by which a major source of crystallin damage, UV, may also act as a rescuing factor in a finely balanced redox system.
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Apr 2024
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I24-Microfocus Macromolecular Crystallography
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Rachel
Bolton
,
Moritz M.
Machelett
,
Jack
Stubbs
,
Danny
Axford
,
Nicolas
Caramello
,
Lucrezia
Catapano
,
Martin
Maly
,
Matthew J.
Rodrigues
,
Charlotte
Cordery
,
Graham J.
Tizzard
,
Fraser
Macmillan
,
Sylvain
Engilberge
,
David
Von Stetten
,
Takehiko
Tosha
,
Hiroshi
Sugimoto
,
Jonathan A. R.
Worrall
,
Jeremy S.
Webb
,
Mike
Zubkov
,
Simon
Coles
,
Eric
Mathieu
,
Roberto A.
Steiner
,
Garib
Murshudov
,
Tobias E.
Schrader
,
Allen M.
Orville
,
Antoine
Royant
,
Gwyndaf
Evans
,
Michael A.
Hough
,
Robin L.
Owen
,
Ivo
Tews
Diamond Proposal Number(s):
[15722, 14493, 23570]
Open Access
Abstract: The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes generally encode two different types of FutA iron-binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here, we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray-induced photoreduction of the iron center with observation of the Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump–probe approach capture the transition between Fe(III) and Fe(II) states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins.
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Mar 2024
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I24-Microfocus Macromolecular Crystallography
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James
Birch
,
Tristan O. C.
Kwan
,
Peter J.
Judge
,
Danny
Axford
,
Pierre
Aller
,
Agata
Butryn
,
Rosana
Reis
,
Juan F.
Bada Juarez
,
Javier
Vinals
,
Robin L.
Owen
,
Eriko
Nango
,
Rie
Tanaka
,
Kensuke
Tono
,
Yasumasa
Joti
,
Tomoyuki
Tanaka
,
Shigeki
Owada
,
Michihiro
Sugahara
,
So
Iwata
,
Allen M.
Orville
,
Anthony
Watts
,
Isabel
Moraes
Diamond Proposal Number(s):
[19152]
Open Access
Abstract: Serial crystallography has emerged as an important tool for structural studies of integral membrane proteins. The ability to collect data from micrometre-sized weakly diffracting crystals at room temperature with minimal radiation damage has opened many new opportunities in time-resolved studies and drug discovery. However, the production of integral membrane protein microcrystals in lipidic cubic phase at the desired crystal density and quantity is challenging. This paper introduces VIALS (versatile approach to high-density microcrystals in lipidic cubic phase for serial crystallography), a simple, fast and efficient method for preparing hundreds of microlitres of high-density microcrystals suitable for serial X-ray diffraction experiments at both synchrotron and free-electron laser sources. The method is also of great benefit for rational structure-based drug design as it facilitates in situ crystal soaking and rapid determination of many co-crystal structures. Using the VIALS approach, room-temperature structures are reported of (i) the archaerhodopsin-3 protein in its dark-adapted state and 110 ns photocycle intermediate, determined to 2.2 and 1.7 Å, respectively, and (ii) the human A2A adenosine receptor in complex with two different ligands determined to a resolution of 3.5 Å.
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Oct 2023
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