I24-Microfocus Macromolecular Crystallography
|
Jos J. A. G.
Kamps
,
Philip
Hinchliffe
,
Johan
Glerup
,
Emily I.
Freeman
,
Pauline A.
Lang
,
Catherine
Tooke
,
Michael
Beer
,
Laura
Parkinson
,
Do-Heon
Gu
,
Sehan
Park
,
Nicholas
Devenish
,
Tiankun
Zhou
,
Anastasya
Shilova
,
Samanpreet
Kaur
,
Patrick
Rabe
,
Christopher J.
Schofield
,
James
Spencer
,
Jaehyun
Park
,
Robin L.
Owen
,
Allen M.
Orville
,
Pierre
Aller
Diamond Proposal Number(s):
[25260]
Open Access
Abstract: We describe the design and implementation of a drop-on-fixed-target method for time-resolved serial crystallography at both synchrotron and XFEL facilities. A piezoelectric droplet dispensing pipette is employed for addition of picolitre volume aqueous droplets (∼40–90 pl; ∼40–55 µm diameter sphere), containing (co-)substrate(s) or ligand(s), onto enzyme microcrystals previously loaded into the trapezoidal wells of an etched crystalline silicon fixed-target chip containing 25 600 wells in a high-density, square grid with 125 µm centre-to-centre well spacing. These features demand exquisite accuracy and thereby constrain motion controls to enable robust time-resolved crystallographic studies. The system was tested with three enzyme systems, comprising lysozyme and two β-lactamases, CTX-M-15 and AmpCEC. Mitigation strategies for cross-well contamination, including the implementation of interleaved controls, are described; the overall performance of the system at synchrotron and X-ray free-electron laser facilities was evaluated. This drop-on-fixed-target method is a reliable framework for time-resolved crystallography and will improve the consistency of measurements across facilities.
|
Jul 2026
|
|
Krios I-Titan Krios I at Diamond
|
Diamond Proposal Number(s):
[21005]
Open Access
Abstract: The dynamic nature of protein and macromolecular complexes means that the capture of multiple sequential states along a reaction pathway can provide much greater insight into function than that obtained from a single static structure. We present a set of modular, easy-to-implement tools and workflows for optical excitation, on-grid characterization and tightly coupled rapid vitrification, establishing a proof-of-principle framework for time-resolved cryoEM and cryo-electron tomography (cryoET). We apply this framework to E. coli chemotaxis, in which serine-sensitive chemoreceptors initiate signalling upon ligand binding and undergo critical conformational changes within the chemosensory arrays. Using DMNB-caged serine [O-(4,5-dimethoxy-2-nitrobenzyl)-L-serine] as a model trigger, we quantified its photophysical properties and uncaging efficiency using UV–Vis spectroscopy and two-dimensional gas chromatography mass spectrometry (GC×GC-MS). Coupling a femtosecond-pulsed laser to a Vitrobot enabled reproducible reaction-to-vitrification delays of ∼150 ms, yielding intact E. coli minicells with well-preserved chemotaxis arrays suitable for in situ structural analysis by cryoET. This integrated approach provides a robust and generalisable framework for millisecond time-resolved cryoET, laying the groundwork for capturing transient conformational states in their native cellular context.
|
Jul 2026
|
|
I24-Microfocus Macromolecular Crystallography
|
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.
|
May 2026
|
|
I24-Microfocus Macromolecular Crystallography
|
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.
|
Mar 2026
|
|
I24-Microfocus Macromolecular Crystallography
Detectors
|
John
Matheson
,
Danny
Axford
,
Anna
Bergamaschi
,
Maria
Carulla
,
Nicholas
Devenish
,
Noemi
Frisina
,
Viktoria
Hinger
,
Vadym
Kedych
,
Christopher
Lane
,
Aldo
Mozzanica
,
Eva
Gimenez-Navarro
,
James
O'Hea
,
Dominic
Oram
,
Robin L.
Owen
,
David
Perl
,
Adam
Prescott
,
Bernd
Schmitt
,
Shane
Scully
,
Adam
Taylor
,
Gary
Yendell
,
Graeme
Winter
Open Access
Abstract: A Jungfrau-1M detector has undergone testing at Diamond Light Source. The Jungfrau series of detectors from PSI use integration and adaptive gain, to offer very high frame rate and dynamic range, suitable for high-flux and time-resolved measurements. They are becoming more widely used, to take advantage of increasing light source brightness. We report on our experiences in testing the performance of a Jungfrau-1M without illumination, with a laboratory X-ray tube and on a microfocus beamline. The Jungfrau-1M was found to be able to resolve single photons in the laboratory and on the beamline. It was confirmed that range switching from high to intermediate gain is associated with a discontinuity in the detector response. Two methods of dark frame subtraction were compared for their effect on minimizing this discontinuity. The Jungfrau-1M was found to be very effective for recording macromolecular crystallography diffraction patterns, with no apparent detriment from the discontinuity. The Diamond machine will be upgraded in 2028–9 and will operate at significantly higher flux than at present, necessitating increased use of integrating detectors, such as Jungfrau, in the future.
|
Mar 2026
|
|
I24-Microfocus Macromolecular Crystallography
|
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.
|
Feb 2026
|
|
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
VMXm-Versatile Macromolecular Crystallography microfocus
|
Open Access
Abstract: Structure determination by X-ray diffraction is limited by crystal size and can be compromised by radiation damage when using very intense X-ray radiation. X-ray structure determination from partial diffraction data sets combined from multiple crystals is a potential solution, but its exploitation in chemistry and materials science is largely unrealized. Here we report the use of synchrotron radiation for multi-crystal X-ray diffraction (MCXRD) adapted for structure determination of metal-organic framework (MOF) materials with crystal dimensions too small for conventional single-crystal diffraction studies. We further show that radiation-induced chemical changes and degradation of diffraction quality can be alleviated. Our approach encompasses both rotation- and stationary-MCXRD measurements for 10 to 1000s of crystals with software-optimized combination of the multiple data sets. We report the crystal structures of six MOFs: MOF-919(Sc/Cu), MET-2, MIL-88B(Cr)-1,4-NDC, PCN-260(Sc), UiO-66, and UiO-66-MoO4 with unit cell dimensions ranging from 18−114 Å and crystal sizes from 0.5−480 µm3. This approach can address the challenges of structure determination in a regime of particle size and sample radiation sensitivity that lies between existing single-crystal X-ray diffraction and the emerging field of electron diffraction. MCXRD can provide accurate atomic-resolution structure determination for some of the most challenging cases in chemistry and materials science.
|
Jan 2026
|
|
I24-Microfocus Macromolecular Crystallography
|
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.
|
Sep 2025
|
|
I24-Microfocus Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
|
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.
|
Aug 2025
|
|
Metrology
Optics
|
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.
|
May 2025
|
|
