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Instruments / Technical Area	Publication Details	Published
	Structure of a ribonucleotide reductase R2 protein radical Hugo Lebrette , Vivek Srinivas , Juliane John , Oskar Aurelius , Rohit Kumar , Daniel Lundin , Aaron S. Brewster , Asmit Bhowmick , Abhishek Sirohiwal , In-Sik Kim , Sheraz Gul , Cindy Pham , Kyle D. Sutherlin , Philipp Simon , Agata Butryn , Pierre Aller , Allen M. Orville , Franklin D. Fuller , Roberto Alonso-Mori , Alexander Batyuk , Nicholas K. Sauter , Vittal K. Yachandra , Junko Yano , Ville R. I. Kaila , Britt-Marie Sjöberg , Jan Kern , Katarina Roos , Martin Högbom Science 382, 109 - 113 DOI: 10.1126/science.adh8160 Show Abstract ▼ Abstract: Aerobic ribonucleotide reductases (RNRs) initiate synthesis of DNA building blocks by generating a free radical within the R2 subunit; the radical is subsequently shuttled to the catalytic R1 subunit through proton-coupled electron transfer (PCET). We present a high-resolution room temperature structure of the class Ie R2 protein radical captured by x-ray free electron laser serial femtosecond crystallography. The structure reveals conformational reorganization to shield the radical and connect it to the translocation path, with structural changes propagating to the surface where the protein interacts with the catalytic R1 subunit. Restructuring of the hydrogen bond network, including a notably short O–O interaction of 2.41 angstroms, likely tunes and gates the radical during PCET. These structural results help explain radical handling and mobilization in RNR and have general implications for radical transfer in proteins.	Oct 2023
	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
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
	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
I24-Microfocus Macromolecular Crystallography	Photoreversible interconversion of a phytochrome photosensory module in the crystalline state E. Sethe Burgie , Jonathan A. Clinger , Mitchell D. Miller , Aaron S. Brewster , Pierre Aller , Agata Butryn , Franklin D. Fuller , Sheraz Gul , Iris D. Young , Cindy C. Pham , In-Sik Kim , Asmit Bhowmick , Lee J. O’riordan , Kyle D. Sutherlin , Joshua V. Heinemann , Alexander Batyuk , Roberto Alonso-Mori , Mark S. Hunter , Jason E. Koglin , Junko Yano , Vittal K. Yachandra , Nicholas K. Sauter , Aina E. Cohen , Jan Kern , Allen M. Orville , George N. Phillips , Richard D. Vierstra Proceedings Of The National Academy Of Sciences 37 DOI: 10.1073/pnas.1912041116 Diamond Proposal Number(s): [19458] Open Access Show Abstract ▼ Abstract: A major barrier to defining the structural intermediates that arise during the reversible photointerconversion of phytochromes between their biologically inactive and active states has been the lack of crystals that faithfully undergo this transition within the crystal lattice. Here, we describe a crystalline form of the cyclic GMP phosphodiesterases/adenylyl cyclase/FhlA (GAF) domain from the cyanobacteriochrome PixJ in Thermosynechococcus elongatus assembled with phycocyanobilin that permits reversible photoconversion between the blue light-absorbing Pb and green light-absorbing Pg states, as well as thermal reversion of Pg back to Pb. The X-ray crystallographic structure of Pb matches previous models, including autocatalytic conversion of phycocyanobilin to phycoviolobilin upon binding and its tandem thioether linkage to the GAF domain. Cryocrystallography at 150 K, which compared diffraction data from a single crystal as Pb or after irradiation with blue light, detected photoconversion product(s) based on Fobs − Fobs difference maps that were consistent with rotation of the bonds connecting pyrrole rings C and D. Further spectroscopic analyses showed that phycoviolobilin is susceptible to X-ray radiation damage, especially as Pg, during single-crystal X-ray diffraction analyses, which could complicate fine mapping of the various intermediate states. Fortunately, we found that PixJ crystals are amenable to serial femtosecond crystallography (SFX) analyses using X-ray free-electron lasers (XFELs). As proof of principle, we solved by room temperature SFX the GAF domain structure of Pb to 1.55-Å resolution, which was strongly congruent with synchrotron-based models. Analysis of these crystals by SFX should now enable structural characterization of the early events that drive phytochrome photoconversion.	Dec 2019
I11-High Resolution Powder Diffraction	A co-templating route to the synthesis of Cu SAPO STA-7, giving an active catalyst for the selective catalytic reduction of NO A. Lorena Picone , Stewart J. Warrender , Alexandra M. Z. Slawin , Daniel M. Dawson , Sharon E. Ashbrook , Paul A. Wright , Stephen P. Thompson , Lucia Gaberova , Philip L. Llewellyn , Beatrice Moulin , Alexandre Vimont , Marco Daturi , Min Bum Park , Sam Kyung Sung , In-Sik Nam , Suk Bong Hong Microporous And Mesoporous Materials 146, 36 - 47 DOI: 10.1016/j.micromeso.2011.04.039 Show Abstract ▼ Abstract: Copper cyclam (cyclam = 1,4,8,11-tetraazacyclotetradecane) and tetraethylammonium (TEA+) act as co-templates for the hydrothermal crystallisation of the silicoaluminophosphate SAPO STA-7, as determined by UV–visible, ESR and solid-state MAS NMR spectroscopies, powder and single crystal X-ray diffraction and chemical analysis. Calcination of the as-prepared solid in flowing oxygen removes all organics to leave Cu(II),H-SAPO STA-7 (hereafter Cu-SAPO STA-7) in which the presence of bridging hydroxyl groups is confirmed by IR and the presence of multiple environments for Cu2+ is shown by IR using NO as a probe molecule. Rietveld analysis of synchrotron X-ray powder diffraction data collected over the temperature range 293–673 K locates Cu2+ cations distributed over four sites: above six membered rings (6MRs) and in the three different 8MR windows of the STA-7 structure. Cu-SAPO STA-7 is a very good catalyst for the selective catalytic reduction of NO with NH3, in the presence or absence of water vapour, so that this approach represents an efficient and effective route to copper-containing SAPO catalysts that obviates the need for an aqueous Cu2+ ion exchange step during preparation.	Dec 2011

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