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Instruments / Technical Area	Publication Details	Published
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
	Structures of the intermediates of Kok’s photosynthetic water oxidation clock Jan Kern , Ruchira Chatterjee , Iris D. Young , Franklin D. Fuller , Louise Lassalle , Mohamed Ibrahim , Sheraz Gul , Thomas Fransson , Aaron S. Brewster , Roberto Alonso-Mori , Rana Hussein , Miao Zhang , Lacey Douthit , Casper De Lichtenberg , Mun Hon Cheah , Dmitry Shevela , Julia Wersig , Ina Seuffert , Dimosthenis Sokaras , Ernest Pastor , Clemens Weninger , Thomas Kroll , Raymond G. Sierra , Pierre Aller , Agata Butryn , Allen M. Orville , Mengning Liang , Alexander Batyuk , Jason E. Koglin , Sergio Carbajo , Sébastien Boutet , Nigel W. Moriarty , James M. Holton , Holger Dobbek , Paul D. Adams , Uwe Bergmann , Nicholas K. Sauter , Athina Zouni , Johannes Messinger , Junko Yano , Vittal K. Yachandra Nature 11 DOI: 10.1038/s41586-018-0681-2 Show Abstract ▼ Abstract: Inspired by the period-four oscillation in flash-induced oxygen evolution of photosystem II discovered by Joliot in 1969, Kok performed additional experiments and proposed a five-state kinetic model for photosynthetic oxygen evolution, known as Kok’s S-state clock or cycle1,2. The model comprises four (meta)stable intermediates (S0, S1, S2 and S3) and one transient S4 state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (Mn4CaO5) cluster in the oxygen-evolving complex3,4,5,6,7. This reaction is coupled to the two-step reduction and protonation of the mobile plastoquinone QB at the acceptor side of PSII. Here, using serial femtosecond X-ray crystallography and simultaneous X-ray emission spectroscopy with multi-flash visible laser excitation at room temperature, we visualize all (meta)stable states of Kok’s cycle as high-resolution structures (2.04–2.08 Å). In addition, we report structures of two transient states at 150 and 400 µs, revealing notable structural changes including the binding of one additional ‘water’, Ox, during the S2→S3 state transition. Our results suggest that one water ligand to calcium (W3) is directly involved in substrate delivery. The binding of the additional oxygen Ox in the S3 state between Ca and Mn1 supports O–O bond formation mechanisms involving O5 as one substrate, where Ox is either the other substrate oxygen or is perfectly positioned to refill the O5 position during O2 release. Thus, our results exclude peroxo-bond formation in the S3 state, and the nucleophilic attack of W3 onto W2 is unlikely.	Nov 2018
	Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers Franklin D Fuller , Sheraz Gul , Ruchira Chatterjee , E. Sethe Burgie , Iris D. Young , Hugo Lebrette , Vivek Srinivas , Aaron Brewster , Tara Michels-Clark , Jonathan A Clinger , Babak Andi , Mohamed Ibrahim , Ernest Pastor , Casper De Lichtenberg , Rana Hussein , Christopher J Pollock , Miao Zhang , Claudiu A Stan , Thomas Kroll , Thomas Fransson , Clemens Weninger , Markus Kubin , Pierre Aller , Louise Lassalle , Philipp Braeuer , Mitchell D. Miller , Muhamed Amin , Sergey Koroidov , Christian G. Roessler , Marc Allaire , Raymond G Sierra , Peter T. Docker , James M. Glownia , Silke Nelson , Jason E Koglin , Diling Zhu , Matthieu Chollet , Sanghoon Song , Henrik Lemke , Mengning Liang , Dimosthenis Sokaras , Roberto Alonso-Mori , Athina Zouni , Johannes Messinger , Uwe Bergmann , Amie K. Boal , J. Martin Bollinger , Carsten Krebs , Martin Högbom , George N. Phillips , Richard D. Vierstra , Nicholas K Sauter , Allen M. Orville , Jan Kern , Vittal K Yachandra , Junko Yano Nature Methods 5 DOI: 10.1038/nmeth.4195 Show Abstract ▼ Abstract: X-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy, both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing insights into the interplay between the protein structure and dynamics and the chemistry at an active site. The implementation of such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly affects the data quality. We present here a robust way of delivering controlled sample amounts on demand using acoustic droplet ejection coupled with a conveyor belt drive that is optimized for crystallography and spectroscopy measurements of photochemical and chemical reactions over a wide range of time scales. Studies with photosystem II, the phytochrome photoreceptor, and ribonucleotide reductase R2 illustrate the power and versatility of this method.	Feb 2017
	Structure of photosystem II and substrate binding at room temperature Iris D. Young , Mohamed Ibrahim , Ruchira Chatterjee , Sheraz Gul , Franklin D. Fuller , Sergey Koroidov , Aaron S. Brewster , Rosalie Tran , Roberto Alonso-Mori , Thomas Kroll , Tara Michels-Clark , Hartawan Laksmono , Raymond G. Sierra , Claudiu A. Stan , Rana Hussein , Miao Zhang , Lacey Douthit , Markus Kubin , Casper De Lichtenberg , Long Vo Pham , Håkan Nilsson , Mun Hon Cheah , Dmitriy Shevela , Claudio Saracini , Mackenzie A. Bean , Ina Seuffert , Dimosthenis Sokaras , Tsu-Chien Weng , Ernest Pastor , Clemens Weninger , Thomas Fransson , Louise Lassalle , Philipp Bräuer , Pierre Aller , Peter T. Docker , Babak Andi , Allen M. Orville , James M. Glownia , Silke Nelson , Marcin Sikorski , Diling Zhu , Mark S. Hunter , Thomas J. Lane , Andy Aquila , Jason E. Koglin , Joseph Robinson , Mengning Liang , Sébastien Boutet , Artem Y. Lyubimov , Monarin Uervirojnangkoorn , Nigel W. Moriarty , Dorothee Liebschner , Pavel V. Afonine , David G. Waterman , Gwyndaf Evans , Philippe Wernet , Holger Dobbek , William I. Weis , Axel T. Brunger , Petrus H. Zwart , Paul D. Adams , Athina Zouni , Johannes Messinger , Uwe Bergmann , Nicholas K. Sauter , Jan Kern , Vittal K. Yachandra , Junko Yano Nature DOI: 10.1038/nature20161 Show Abstract ▼ Abstract: Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the Mn4CaO5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4)1, in which S1 is the dark-stable state and S3 is the last semi-stable state before O–O bond formation and O2 evolution2, 3. A detailed understanding of the O–O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site4, 5, 6. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S1), two-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL7 provided a damage-free view of the S1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions8, 9, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn4CaO5 cluster in the S2 and S3 states10. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site10, 11, 12, 13. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O–O bond formation mechanisms.	Nov 2016

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