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Instruments / Technical Area	Publication Details	Published
	Guanosine hydrogel as a new injection matrix for protein serial X-ray crystallography Alessia Pepe , Ronald Rios-Santacruz , Sara Schianchi , Jovana Vitas , Elena Andreeva , Jacques-Philippe Colletier , Nicolas Coquelle , Elke De Zitter , Shibom Basu , Daniele De Sanctis , Julien Orlans , Ninon Zala , Kyprianos Hadjidemetriou , Kensuke Tono , Takehiko Tosha , Bruce Doak , Robert Shoeman , Martin Weik , Paolo Mariani , Giorgio Schirò Journal Of Applied Crystallography 59 DOI: 10.1107/S1600576725011276 Show Abstract ▼ Abstract: Serial X-ray crystallography is the method of choice for protein structural studies at X-ray free-electron lasers and is increasingly widely used at third- and fourth-generation synchrotron facilities. Sample delivery is a critical step in serial crystallography experiments, and the definition and control of delivery methods is especially important for time-resolved studies. Extruding protein crystals embedded in viscous matrices as a thin, stable jet is a delivery method that ensures low sample consumption. However, it requires specific characteristics, such as a constant speed, chemical compatibility and, for laser-triggered dynamic studies, optical transparency. Here, we present a new transparent carrier matrix for protein serial crystallography, based on a guanosine hydrogel. Crystal stability in the hydrogel, in terms of morphology, size and diffraction quality, along with optical properties, injection characteristics and X-ray background were tested at the ESRF synchrotron and compared with other commonly used matrices. Additionally, we present a high-quality serial femtosecond crystallography dataset collected at the Spring-8 Angstrom Compact Free-Electron Laser (Japan) using hen egg-white lysozyme microcrystals embedded in the hydrogel. The results establish that this new injection matrix is a suitable alternative to existing carriers. The main advantage is its stability, so that the composition of the aqueous phase inside the hydrogel can be easily modified and adapted to the crystallization conditions of the embedded crystals.	Feb 2026
I24-Microfocus Macromolecular Crystallography	Integrated structural dynamics uncover a new B12 photoreceptor activation mode 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 Nature 47 DOI: 10.1038/s41586-025-10074-2 Diamond Proposal Number(s): [24447, 31850] Show Abstract ▼ 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
I24-Microfocus Macromolecular Crystallography	Time-resolved serial synchrotron and serial femtosecond crystallography of heme proteins using photocaged nitric oxide 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 Iucrj 12 DOI: 10.1107/S2052252525006645 Diamond Proposal Number(s): [18565, 28583, 27313] Open Access Show Abstract ▼ 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	Double crossed? Structural and computational studies of an unusually crosslinked haem in Methylococcus capsulatus cytochrome P460 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 Chemical Science DOI: 10.1039/D5SC04213E Diamond Proposal Number(s): [27313] Open Access Show Abstract ▼ 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
I24-Microfocus Macromolecular Crystallography	A redox switch allows binding of Fe(II) and Fe(III) ions in the cyanobacterial iron-binding protein FutA from Prochlorococcus 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 Proceedings Of The National Academy Of Sciences 121 DOI: 10.1073/pnas.2308478121 Diamond Proposal Number(s): [15722, 14493, 23570] Open Access Show Abstract ▼ 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.	Mar 2024
I04-Macromolecular Crystallography VMXi-Versatile Macromolecular Crystallography in situ	Protein-to-structure pipeline for ambient-temperature in situ crystallography at VMXi Halina Mikolajek , Juan Sanchez-Weatherby , James Sandy , Richard J. Gildea , Ivan Campeotto , Harish Cheruvara , John D. Clarke , Toshana Foster , Sotaro Fujii , Ian T. Paulsen , Bhumika S. Shah , Michael A. Hough Iucrj 10 DOI: 10.1107/S2052252523003810 Open Access Show Abstract ▼ Abstract: The utility of X-ray crystal structures determined under ambient-temperature conditions is becoming increasingly recognized. Such experiments can allow protein dynamics to be characterized and are particularly well suited to challenging protein targets that may form fragile crystals that are difficult to cryo-cool. Room-temperature data collection also enables time-resolved experiments. In contrast to the high-throughput highly automated pipelines for determination of structures at cryogenic temperatures widely available at synchrotron beamlines, room-temperature methodology is less mature. Here, the current status of the fully automated ambient-temperature beamline VMXi at Diamond Light Source is described, and a highly efficient pipeline from protein sample to final multi-crystal data analysis and structure determination is shown. The capability of the pipeline is illustrated using a range of user case studies representing different challenges, and from high and lower symmetry space groups and varied crystal sizes. It is also demonstrated that very rapid structure determination from crystals in situ within crystallization plates is now routine with minimal user intervention.	Jul 2023
I23-Long wavelength MX	Serial femtosecond crystallography reveals that photoactivation in a fluorescent protein proceeds via the hula twist mechanism Alisia Fadini , Christopher D. M. Hutchison , Dmitry Morozov , Jeffrey Chang , Karim Maghlaoui , Samuel Perrett , Fangjia Luo , Jeslyn C. X. Kho , Matthew G. Romei , R. Marc L. Morgan , Christian Orr , Violeta Cordon-Preciado , Takaaki Fujiwara , Nipawan Nuemket , Takehiko Tosha , Rie Tanaka , Shigeki Owada , Kensuke Tono , So Iwata , Steven G. Boxer , Gerrit Groenhof , Eriko Nango , Jasper J. Van Thor Journal Of The American Chemical Society DOI: 10.1021/jacs.3c02313 Diamond Proposal Number(s): [23620] Open Access Show Abstract ▼ Abstract: Chromophore cis/trans photoisomerization is a fundamental process in chemistry and in the activation of many photosensitive proteins. A major task is understanding the effect of the protein environment on the efficiency and direction of this reaction compared to what is observed in the gas and solution phases. In this study, we set out to visualize the hula twist (HT) mechanism in a fluorescent protein, which is hypothesized to be the preferred mechanism in a spatially constrained binding pocket. We use a chlorine substituent to break the twofold symmetry of the embedded phenolic group of the chromophore and unambiguously identify the HT primary photoproduct. Through serial femtosecond crystallography, we then track the photoreaction from femtoseconds to the microsecond regime. We observe signals for the photoisomerization of the chromophore as early as 300 fs, obtaining the first experimental structural evidence of the HT mechanism in a protein on its femtosecond-to-picosecond timescale. We are then able to follow how chromophore isomerization and twisting lead to secondary structure rearrangements of the protein β-barrel across the time window of our measurements.	Jul 2023
I24-Microfocus Macromolecular Crystallography	Serial femtosecond crystallography reveals the role of water in the one- or two-electron redox chemistry of compound I in the catalytic cycle of the B-type dye-decolorizing peroxidase DtpB Marina Lucic , Matthew T. Wilson , Takehiko Tosha , Hiroshi Sugimoto , Anastasiia Shilova , Danny Axford , Robin L. Owen , Michael A. Hough , Jonathan A. R. Worrall Acs Catalysis 26, 13349 - 13359 DOI: 10.1021/acscatal.2c03754 Diamond Proposal Number(s): [25108, 28583] Open Access Show Abstract ▼ Abstract: Controlling the reactivity of high-valent Fe(IV)–O catalytic intermediates, Compounds I and II, generated in heme enzymes upon reaction with dioxygen or hydrogen peroxide, is important for function. It has been hypothesized that the presence (wet) or absence (dry) of distal heme pocket water molecules can influence whether Compound I undergoes sequential one-electron additions or a concerted two-electron reduction. To test this hypothesis, we investigate the role of water in the heme distal pocket of a dye-decolorizing peroxidase utilizing a combination of serial femtosecond crystallography and rapid kinetic studies. In a dry distal heme site, Compound I reduction proceeds through a mechanism in which Compound II concentration is low. This reaction shows a strong deuterium isotope effect, indicating that reduction is coupled to proton uptake. The resulting protonated Compound II (Fe(IV)–OH) rapidly reduces to the ferric state, giving the appearance of a two-electron transfer process. In a wet site, reduction of Compound I is faster, has no deuterium effect, and yields highly populated Compound II, which is subsequently reduced to the ferric form. This work provides a definitive experimental test of the hypothesis advanced in the literature that relates sequential or concerted electron transfer to Compound I in wet or dry distal heme sites.	Oct 2022
I24-Microfocus Macromolecular Crystallography	Complementarity of neutron, XFEL and synchrotron crystallography for defining the structures of metalloenzymes at room temperature Tadeo Moreno-Chicano , Leiah M. Carey , Danny Axford , John H. Beale , R. Bruce Doak , Helen M. E. Duyvesteyn , Ali Ebrahim , Robert W. Henning , Diana C. F. Monteiro , Dean A. Myles , Shigeki Owada , Darren A. Sherrell , Megan L. Straw , Vukica Šrajer , Hiroshi Sugimoto , Kensuke Tono , Takehiko Tosha , Ivo Tews , Martin Trebbin , Richard W. Strange , Kevin L. Weiss , Jonathan A. R. Worrall , Flora Meilleur , Robin L. Owen , Reza A. Ghiladi , Michael A. Hough Iucrj 9 DOI: 10.1107/S2052252522006418 Diamond Proposal Number(s): [14493] Open Access Show Abstract ▼ Abstract: Room-temperature macromolecular crystallography allows protein structures to be determined under close-to-physiological conditions, permits dynamic freedom in protein motions and enables time-resolved studies. In the case of metalloenzymes that are highly sensitive to radiation damage, such room-temperature experiments can present challenges, including increased rates of X-ray reduction of metal centres and site-specific radiation-damage artefacts, as well as in devising appropriate sample-delivery and data-collection methods. It can also be problematic to compare structures measured using different crystal sizes and light sources. In this study, structures of a multifunctional globin, dehaloperoxidase B (DHP-B), obtained using several methods of room-temperature crystallographic structure determination are described and compared. Here, data were measured from large single crystals and multiple microcrystals using neutrons, X-ray free-electron laser pulses, monochromatic synchrotron radiation and polychromatic (Laue) radiation light sources. These approaches span a range of 18 orders of magnitude in measurement time per diffraction pattern and four orders of magnitude in crystal volume. The first room-temperature neutron structures of DHP-B are also presented, allowing the explicit identification of the hydrogen positions. The neutron data proved to be complementary to the serial femtosecond crystallography data, with both methods providing structures free of the effects of X-ray radiation damage when compared with standard cryo-crystallography. Comparison of these room-temperature methods demonstrated the large differences in sample requirements, data-collection time and the potential for radiation damage between them. With regard to the structure and function of DHP-B, despite the results being partly limited by differences in the underlying structures, new information was gained on the protonation states of active-site residues which may guide future studies of DHP-B.	Sep 2022
I24-Microfocus Macromolecular Crystallography	Single crystal spectroscopy and multiple structures from one crystal (MSOX) define catalysis in copper nitrite reductases Samuel L. Rose , Seiki Baba , Hideo Okumura , Svetlana V. Antonyuk , Daisuke Sasaki , Tobias M. Hedison , Muralidharan Shanmugam , Derren J. Heyes , Nigel S. Scrutton , Takashi Kumasaka , Takehiko Tosha , Robert R. Eady , Masaki Yamamoto , S. Samar Hasnain Proceedings Of The National Academy Of Sciences 119 DOI: 10.1073/pnas.2205664119 Open Access Show Abstract ▼ Abstract: Many enzymes utilize redox-coupled centers for performing catalysis where these centers are used to control and regulate the transfer of electrons required for catalysis, whose untimely delivery can lead to a state incapable of binding the substrate, i.e., a dead-end enzyme. Copper nitrite reductases (CuNiRs), which catalyze the reduction of nitrite to nitric oxide (NO), have proven to be a good model system for studying these complex processes including proton-coupled electron transfer (ET) and their orchestration for substrate binding/utilization. Recently, a two-domain CuNiR from a Rhizobia species (Br2DNiR) has been discovered with a substantially lower enzymatic activity where the catalytic type-2 Cu (T2Cu) site is occupied by two water molecules requiring their displacement for the substrate nitrite to bind. Single crystal spectroscopy combined with MSOX (multiple structures from one crystal) for both the as-isolated and nitrite-soaked crystals clearly demonstrate that inter-Cu ET within the coupled T1Cu-T2Cu redox system is heavily gated. Laser-flash photolysis and optical spectroscopy showed rapid ET from photoexcited NADH to the T1Cu center but little or no inter-Cu ET in the absence of nitrite. Furthermore, incomplete reoxidation of the T1Cu site (∼20% electrons transferred) was observed in the presence of nitrite, consistent with a slow formation of NO species in the serial structures of the MSOX movie obtained from the nitrite-soaked crystal, which is likely to be responsible for the lower activity of this CuNiR. Our approach is of direct relevance for studying redox reactions in a wide range of biological systems including metalloproteins that make up at least 30% of all proteins.	Jul 2022

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