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Instruments / Technical Area	Publication Details	Published
	Redox-controlled reorganization and flavin strain within the ribonucleotide reductase R2b–NrdI complex monitored by serial femtosecond crystallography Juliane John , Oskar Aurelius , Vivek Srinivas , Patricia Saura , In-Sik Kim , Asmit Bhowmick , Philipp S. Simon , Medhanjali Dasgupta , Cindy Pham , Sheraz Gul , Kyle D. Sutherlin , Pierre Aller , Agata Butryn , Allen M. Orville , Mun Hon Cheah , Shigeki Owada , Kensuke Tono , Franklin D Fuller , Alexander Batyuk , Aaron S. Brewster , Nicholas K. Sauter , Vittal K Yachandra , Junko Yano , Ville R. I. Kaila , Jan Kern , Hugo Lebrette , Martin Högbom Elife 11 DOI: 10.7554/eLife.79226 Open Access Show Abstract ▼ Abstract: Redox reactions are central to biochemistry and are both controlled by and induce protein structural changes. Here, we describe structural rearrangements and crosstalk within the Bacillus cereus ribonucleotide reductase R2b–NrdI complex, a di-metal carboxylate-flavoprotein system, as part of the mechanism generating the essential catalytic free radical of the enzyme. Femtosecond crystallography at an X-ray free electron laser was utilized to obtain structures at room temperature in defined redox states without suffering photoreduction. Together with density functional theory calculations, we show that the flavin is under steric strain in the R2b–NrdI protein complex, likely tuning its redox properties to promote superoxide generation. Moreover, a binding site in close vicinity to the expected flavin O2 interaction site is observed to be controlled by the redox state of the flavin and linked to the channel proposed to funnel the produced superoxide species from NrdI to the di-manganese site in protein R2b. These specific features are coupled to further structural changes around the R2b–NrdI interaction surface. The mechanistic implications for the control of reactive oxygen species and radical generation in protein R2b are discussed.	Sep 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
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	X-ray free-electron laser studies reveal correlated motion during isopenicillin N synthase catalysis Patrick Rabe , Jos J. A. G. Kamps , Kyle D. Sutherlin , James D. S. Linyard , Pierre Aller , Cindy C. Pham , Mikako Makita , Ian Clifton , Michael A. Mcdonough , Thomas M. Leissing , Denis Shutin , Pauline A. Lang , Agata Butryn , Jurgen Brem , Sheraz Gul , Franklin D. Fuller , In-Sik Kim , Mun Hon Cheah , Thomas Fransson , Asmit Bhowmick , Iris D. Young , Lee O'Riordan , Aaron S. Brewster , Ilaria Pettinati , Margaret Doyle , Yasumasa Joti , Shigeki Owada , Kensuke Tono , Alexander Batyuk , Mark S. Hunter , Roberto Alonso-Mori , Uwe Bergmann , Robin L. Owen , Nicholas K. Sauter , Timothy D. W. Claridge , Carol V. Robinson , Vittal K. Yachandra , Junko Yano , Jan F. Kern , Allen M. Orville , Christopher J. Schofield Science Advances 7 DOI: 10.1126/sciadv.abh0250 Diamond Proposal Number(s): [23459, 19458] Open Access Show Abstract ▼ Abstract: Isopenicillin N synthase (IPNS) catalyzes the unique reaction of L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) with dioxygen giving isopenicillin N (IPN), the precursor of all natural penicillins and cephalosporins. X-ray free-electron laser studies including time-resolved crystallography and emission spectroscopy reveal how reaction of IPNS:Fe(II):ACV with dioxygen to yield an Fe(III) superoxide causes differences in active site volume and unexpected conformational changes that propagate to structurally remote regions. Combined with solution studies, the results reveal the importance of protein dynamics in regulating intermediate conformations during conversion of ACV to IPN. The results have implications for catalysis by multiple IPNS-related oxygenases, including those involved in the human hypoxic response, and highlight the power of serial femtosecond crystallography to provide insight into long-range enzyme dynamics during reactions presently impossible for nonprotein catalysts.	Aug 2021
I24-Microfocus Macromolecular Crystallography	An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography Agata Butryn , Philipp S. Simon , Pierre Aller , Philip Hinchliffe , Ramzi N. Massad , Gabriel Leen , Catherine L. Tooke , Isabel Bogacz , In-Sik Kim , Asmit Bhowmick , Aaron S. Brewster , Nicholas E. Devenish , Jurgen Brem , Jos J. A. G. Kamps , Pauline A. Lang , Patrick Rabe , Danny Axford , John H. Beale , Bradley Davy , Ali Ebrahim , Julien Orlans , Selina L. S. Storm , Tiankun Zhou , Shigeki Owada , Rie Tanaka , Kensuke Tono , Gwyndaf Evans , Robin L. Owen , Frances A. Houle , Nicholas K. Sauter , Christopher J. Schofield , James Spencer , Vittal K. Yachandra , Junko Yano , Jan F. Kern , Allen M. Orville Nature Communications 12 DOI: 10.1038/s41467-021-24757-7 Diamond Proposal Number(s): [19458, 25260] Open Access Show Abstract ▼ Abstract: Serial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of enzyme reactions with small molecule substrates.	Jul 2021
	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
	High resolution XFEL structure of the soluble methane monooxygenase hydroxylase complex with its regulatory component at ambient temperature in two oxidation states Vivek Srinivas , Rahul Banerjee , Hugo Lebrette , Jason C. Jones , Oskar Aurelius , In-Sik Kim , Cindy C. Pham , Sheraz Gul , Kyle Sutherlin , Asmit Bhowmick , Juliane John , Esra Bozkurt , Thomas Fransson , Pierre Aller , Agata Butryn , Isabel Bogacz , Philipp Stefan Simon , Stephen Keable , Alexander Britz , Kensuke Tono , Kyung-Sook Kim , Sang-Youn Park , Sang-Jae Lee , Jaehyun Park , Roberto Alonso-Mori , Franklin Fuller , Alexander Batyuk , Aaron S. Brewster , Uwe Bergmann , Nicholas Sauter , Allen M. Orville , Vittal K. Yachandra , Junko Yano , John D. Lipscomb , Jan F. Kern , Martin Högbom Journal Of The American Chemical Society DOI: 10.1021/jacs.0c05613 Show Abstract ▼ Abstract: Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme that catalyzes the conversion of methane to methanol at ambient temperature using a nonheme, oxygen-bridged dinuclear iron cluster in the active site. Structural changes in the hydroxylase component (sMMOH) containing the diiron cluster caused by complex formation with a regulatory component (MMOB) and by iron reduction are important for the regulation of O2 activation and substrate hydroxylation. Structural studies of metalloenzymes using traditional synchrotron-based X-ray crystallography are often complicated by partial X-ray-induced photoreduction of the metal center, thereby obviating determination of the structure of pure oxidation states. Here microcrystals of the sMMOH:MMOB complex from Methylosinus trichosporium OB3b were serially exposed to X-ray free electron laser (XFEL) pulses, where the ≦35 fs duration of exposure of an individual crystal yields diffraction data before photoreduction-induced structural changes can manifest. Merging diffraction patterns obtained from thousands of crystals generates radiation damage free, 1.95 Å resolution crystal structures for the fully oxidized and fully reduced states of the sMMOH:MMOB complex for the first time. The results provide new insight into the manner by which the diiron cluster and the active site environment are reorganized by the regulatory protein component in order to enhance the steps of oxygen activation and methane oxidation. This study also emphasizes the value of XFEL and serial femtosecond crystallography (SFX) methods for investigating the structures of metalloenzymes with radiation sensitive metal active sites.	Jul 2020
	Untangling the sequence of events during the S 2 → S 3 transition in photosystem II and implications for the water oxidation mechanism Mohamed Ibrahim , Thomas Fransson , Ruchira Chatterjee , Mun Hon Cheah , Rana Hussein , Louise Lassalle , Kyle D. Sutherlin , Iris D. Young , Franklin D. Fuller , Sheraz Gul , In-Sik Kim , Philipp S. Simon , Casper De Lichtenberg , Petko Chernev , Isabel Bogacz , Cindy C. Pham , Allen M. Orville , Nicholas Saichek , Trent Northen , Alexander Batyuk , Sergio Carbajo , Roberto Alonso-Mori , Kensuke Tono , Shigeki Owada , Asmit Bhowmick , Robert Bolotovsky , Derek Mendez , Nigel W. Moriarty , James M. Holton , Holger Dobbek , Aaron S. Brewster , Paul D. Adams , Nicholas K. Sauter , Uwe Bergmann , Athina Zouni , Johannes Messinger , Jan Kern , Vittal K. Yachandra , Junko Yano Proceedings Of The National Academy Of Sciences 766 DOI: 10.1073/pnas.2000529117 Open Access Show Abstract ▼ Abstract: In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S1, S2, S3, and S0, showing that a water molecule is inserted during the S2 → S3 transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O2 formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2 → S3 transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QA and QB, are observed. At the donor site, tyrosine YZ and His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a “water wheel”-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of ∼350 µs) during the S2 → S3 transition mirrors the appearance of OX electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.	May 2020
	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
	Crystallography on a chip – without the chip: sheet-on-sheet sandwich R. Bruce Doak , Gabriela Nass Kovacs , Alexander Gorel , Lutz Foucar , Thomas R. M. Barends , Marie Luise Grünbein , Mario Hilpert , Marco Kloos , Christopher M. Roome , Robert L. Shoeman , Miriam Stricker , Kensuke Tono , Daehyun You , Kiyoshi Ueda , Darren A. Sherrell , Robin Owen , Ilme Schlichting Acta Crystallographica Section D Structural Biology 74, 1000 - 1007 DOI: 10.1107/S2059798318011634 Open Access Show Abstract ▼ Abstract: Crystallography chips are fixed-target supports consisting of a film (for example Kapton) or wafer (for example silicon) that is processed using semiconductor-microfabrication techniques to yield an array of wells or through-holes in which single microcrystals can be lodged for raster-scan probing. Although relatively expensive to fabricate, chips offer an efficient means of high-throughput sample presentation for serial diffraction data collection at synchrotron or X-ray free-electron laser (XFEL) sources. Truly efficient loading of a chip (one microcrystal per well and no wastage during loading) is nonetheless challenging. The wells or holes must match the microcrystal size of interest, requiring that a large stock of chips be maintained. Raster scanning requires special mechanical drives to step the chip rapidly and with micrometre precision from well to well. Here, a `chip-less' adaptation is described that essentially eliminates the challenges of loading and precision scanning, albeit with increased, yet still relatively frugal, sample usage. The device consists simply of two sheets of Mylar with the crystal solution sandwiched between them. This sheet-on-sheet (SOS) sandwich structure has been employed for serial femtosecond crystallography data collection with micrometre-sized crystals at an XFEL. The approach is also well suited to time-resolved pump–probe experiments, in particular for long time delays. The SOS sandwich enables measurements under XFEL beam conditions that would damage conventional chips, as documented here. The SOS sheets hermetically seal the sample, avoiding desiccation of the sample provided that the X-ray beam does not puncture the sheets. This is the case with a synchrotron beam but not with an XFEL beam. In the latter case, desiccation, setting radially outwards from each punched hole, sets lower limits on the speed and line spacing of the raster scan. It is shown that these constraints are easily accommodated.	Oct 2018

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