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Instruments / Technical Area	Publication Details	Published
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
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
	XFEL crystal structures of peroxidase compound II Hanna Kwon , Jaswir Basran , Chinar Pathak , Mahdi Hussain , Samuel L. Freeman , Alistair J. Fielding , Anna J. Bailey , Natalia Stefanou , Hazel A. Sparkes , Takehiko Tosha , Keitaro Yamashita , Kunio Hirata , Hironori Murakami , Go Ueno , Hideo Ago , Kensuke Tono , Masaki Yamamoto , Hitomi Sawai , Yoshitsugu Shiro , Hiroshi Sugimoto , Emma Raven , Peter C. E. Moody Angewandte Chemie International Edition DOI: 10.1002/anie.202103010 Open Access Show Abstract ▼ Abstract: Oxygen activation in all heme enzymes requires the formation of high oxidation states of iron, usually referred to as ferryl heme. There are two known intermediates: Compound I and Compound II. The nature of the ferryl heme – and whether it is an Fe IV =O or Fe IV ‐OH species – is important for controlling reactivity across groups of heme enzymes. The most recent evidence for Compound I indicates that the ferryl heme is an unprotonated Fe IV =O species. For Compound II, the nature of the ferryl heme is not unambiguously established. Here, we report 1.06 Å and 1.50 Å crystal structures for Compound II intermediates in cytochrome c peroxidase (C c P) and ascorbate peroxidase (APX), collected using the X‐ray free electron laser at SACLA. The structures reveal differences between the two peroxidases. The iron‐oxygen bond length in C c P (1.76 Å) is notably shorter than in APX (1.87 Å). The results indicate that the ferryl species is finely tuned across Compound I and Compound II species in closely related peroxidase enzymes. We propose that this fine‐tuning is linked to the functional need for proton delivery to the heme.	Apr 2021
	Serial femtosecond zero dose crystallography captures a water‐free distal heme site in a dye‐decolourising peroxidase to reveal a catalytic role for an arginine in FeIV=O formation Marina Lucic , Dimitri Svistunenko , Michael Wilson , Amanda Chaplin , Bradley Davy , Ali Ebrahim , Danny Axford , Takehiko Tosha , Hiroshi Sugimoto , Shigeki Owada , Florian Dworkowski , Ivo Tews , Robin Owen , Michael Hough , Jonathan A. R. Worrall Angewandte Chemie International Edition DOI: 10.1002/anie.202008622 Open Access Show Abstract ▼ Abstract: Obtaining structures of intact redox states of metal centres derived from zero dose X‐ray crystallography can advance our mechanistic understanding of metalloenzymes. In dye‐decolourising heme peroxidases (DyPs), controversy exists regarding the mechanistic role of the distal heme residues, aspartate and arginine, in the heterolysis of peroxide to form the catalytic intermediate compound I (Fe IV =O and a porphyrin cation radical). Using serial femtosecond X‐ray (SFX) crystallography, we have determined the pristine structures of the Fe III and Fe IV =O redox states of a B‐type DyP. These structures reveal a water‐free distal heme site, which together with the presence of an asparagine, infer the use of the distal arginine as a catalytic base. A combination of mutagenesis and kinetic studies corroborate such a role. Our SFX approach thus provides unique insight into how the distal heme site of DyPs can be tuned to select aspartate or arginine for the rate enhancement of peroxide heterolysis.	Aug 2020
NONE-No attached Diamond beamline	High-throughput structures of protein–ligand complexes at room temperature using serial femtosecond crystallography Tadeo Moreno Chicano , Ali Ebrahim , Danny Axford , Martin V. Appleby , John H. Beale , Amanda K. Chaplin , Helen M. E. Duyvesteyn , Reza A. Ghiladi , Shigeki Owada , Darren A. Sherrell , Richard Strange , Hiroshi Sugimoto , Kensuke Tono , Jonathan A. R. Worrall , Robin L. Owen , Michael A. Hough Iucrj 6 DOI: 10.1107/S2052252519011655 Open Access Show Abstract ▼ Abstract: High-throughput X-ray crystal structures of protein–ligand complexes are critical to pharmaceutical drug development. However, cryocooling of crystals and X-ray radiation damage may distort the observed ligand binding. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) can produce radiation-damage-free room-temperature structures. Ligand-binding studies using SFX have received only modest attention, partly owing to limited beamtime availability and the large quantity of sample that is required per structure determination. Here, a high-throughput approach to determine room-temperature damage-free structures with excellent sample and time efficiency is demonstrated, allowing complexes to be characterized rapidly and without prohibitive sample requirements. This yields high-quality difference density maps allowing unambiguous ligand placement. Crucially, it is demonstrated that ligands similar in size or smaller than those used in fragment-based drug design may be clearly identified in data sets obtained from <1000 diffraction images. This efficiency in both sample and XFEL beamtime opens the door to true high-throughput screening of protein–ligand complexes using SFX.	Nov 2019
I24-Microfocus Macromolecular Crystallography	Dose-resolved serial synchrotron and XFEL structures of radiation-sensitive metalloproteins Ali Ebrahim , Tadeo Moreno-Chicano , Martin V. Appleby , Amanda K. Chaplin , John Beale , Darren A. Sherrell , Helen M. E. Duyvesteyn , Shigeki Owada , Kensuke Tono , Hiroshi Sugimoto , Richard W. Strange , Jonathan Worrall , Danny Axford , Robin L. Owen , Michael A. Hough Iucrj 6 DOI: 10.1107/S2052252519003956 Diamond Proposal Number(s): [14493] Open Access Show Abstract ▼ Abstract: An approach is demonstrated to obtain, in a sample- and time-efficient manner, multiple dose-resolved crystal structures from room-temperature protein microcrystals using identical fixed-target supports at both synchrotrons and X-ray free-electron lasers (XFELs). This approach allows direct comparison of dose-resolved serial synchrotron and damage-free XFEL serial femtosecond crystallography structures of radiation-sensitive proteins. Specifically, serial synchrotron structures of a heme peroxidase enzyme reveal that X-ray induced changes occur at far lower doses than those at which diffraction quality is compromised (the Garman limit), consistent with previous studies on the reduction of heme proteins by low X-ray doses. In these structures, a functionally relevant bond length is shown to vary rapidly as a function of absorbed dose, with all room-temperature synchrotron structures exhibiting linear deformation of the active site compared with the XFEL structure. It is demonstrated that extrapolation of dose-dependent synchrotron structures to zero dose can closely approximate the damage-free XFEL structure. This approach is widely applicable to any protein where the crystal structure is altered by the synchrotron X-ray beam and provides a solution to the urgent requirement to determine intact structures of such proteins in a high-throughput and accessible manner.	Jul 2019

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