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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
I04-1-Macromolecular Crystallography (fixed wavelength)	Fragment binding to the Nsp3 macrodomain of SARS-CoV-2 identified through crystallographic screening and computational docking Marion Schuller , Galen J. Correy , Stefan Gahbauer , Daren Fearon , Taiasean Wu , Roberto Efraín Díaz , Iris D. Young , Luan Carvalho Martins , Dominique H. Smith , Ursula Schulze-Gahmen , Tristan W. Owens , Ishan Deshpande , Gregory E. Merz , Aye C. Thwin , Justin T. Biel , Jessica K. Peters , Michelle Moritz , Nadia Herrera , Huong T. Kratochvil , Anthony Aimon , James Bennett , Jose Brandao Neto , Aina E. Cohen , Alexandre Dias , Alice Douangamath , Louise Dunnett , Oleg Fedorov , Matteo P. Ferla , Martin R. Fuchs , Tyler J. Gorrie-Stone , James M. Holton , Michael G. Johnson , Tobias Krojer , George Meigs , Alisa J. Powell , Johannes Gregor Matthias Rack , Victor Rangel , Silvia Russi , Rachael E. Skyner , Clyde A. Smith , Alexei S. Soares , Jennifer L. Wierman , Kang Zhu , Peter O’brien , Natalia Jura , Alan Ashworth , John J. Irwin , Michael C. Thompson , Jason E. Gestwicki , Frank Von Delft , Brian K. Shoichet , James S. Fraser , Ivan Ahel Science Advances 7 DOI: 10.1126/sciadv.abf8711 Diamond Proposal Number(s): [27001] Open Access Show Abstract ▼ Abstract: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate–ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.	Apr 2021
	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
	Improving signal strength in serial crystallography with DIALS geometry refinement Aaron S. Brewster , David G. Waterman , James Parkhurst , Richard J. Gildea , Iris D. Young , Lee J. O'Riordan , Junko Yano , Graeme Winter , Gwyndaf Evans , Nicholas K. Sauter Acta Crystallographica Section D Structural Biology 74, 877 - 894 DOI: 10.1107/S2059798318009191 Open Access Show Abstract ▼ Abstract: The DIALS diffraction-modeling software package has been applied to serial crystallography data. Diffraction modeling is an exercise in determining the experimental parameters, such as incident beam wavelength, crystal unit cell and orientation, and detector geometry, that are most consistent with the observed positions of Bragg spots. These parameters can be refined by nonlinear least-squares fitting. In previous work, it has been challenging to refine both the positions of the sensors (metrology) on multipanel imaging detectors such as the CSPAD and the orientations of all of the crystals studied. Since the optimal models for metrology and crystal orientation are interdependent, alternate cycles of panel refinement and crystal refinement have been required. To simplify the process, a sparse linear algebra technique for solving the normal equations was implemented, allowing the detector panels to be refined simultaneously against the diffraction from thousands of crystals with excellent computational performance. Separately, it is shown how to refine the metrology of a second CSPAD detector, positioned at a distance of 2.5 m from the crystal, used for recording low-angle reflections. With the ability to jointly refine the detector position against the ensemble of all crystals used for structure determination, it is shown that ensemble refinement greatly reduces the apparent nonisomorphism that is often observed in the unit-cell distributions from still-shot serial crystallography. In addition, it is shown that batching the images by timestamp and re-refining the detector position can realistically model small, time-dependent variations in detector position relative to the sample, and thereby improve the integrated structure-factor intensity signal and heavy-atom anomalous peak heights.	Sep 2018
I19-Small Molecule Single Crystal Diffraction	DIALS : implementation and evaluation of a new integration package Graeme Winter , David G. Waterman , James M. Parkhurst , Aaron S. Brewster , Richard J. Gildea , Markus Gerstel , Luis Fuentes-Montero , Melanie Vollmar , Tara Michels-Clark , Iris D. Young , Nicholas K. Sauter , Gwyndaf Evans Acta Crystallographica Section D Structural Biology 74, 85 - 97 DOI: 10.1107/S2059798317017235 Open Access Show Abstract ▼ Abstract: The DIALS project is a collaboration between Diamond Light Source, Lawrence Berkeley National Laboratory and CCP4 to develop a new software suite for the analysis of crystallographic X-ray diffraction data, initially encompassing spot finding, indexing, refinement and integration. The design, core algorithms and structure of the software are introduced, alongside results from the analysis of data from biological and chemical crystallography experiments.	Feb 2018
	High-speed fixed-target serial virus crystallography Philip Roedig , Helen M. Ginn , Tim Pakendorf , Geoff Sutton , Karl Harlos , Thomas S. Walter , Jan Meyer , Pontus Fischer , Ramona Duman , Ismo Vartiainen , Bernd Reime , Martin Warmer , Aaron S. Brewster , Iris D. Young , Tara Michels-Clark , Nicholas K. Sauter , Abhay Kotecha , James Kelly , David J. Rowlands , Marcin Sikorsky , Silke Nelson , Daniel S. Damiani , Roberto Alonso-Mori , Jingshan Ren , Elizabeth E. Fry , Christian David , David I. Stuart , Armin Wagner , Alke Meents Nature Methods 8 DOI: 10.1038/nmeth.4335 Show Abstract ▼ Abstract: We report a method for serial X-ray crystallography at X-ray free-electron lasers (XFELs), which allows for full use of the current 120-Hz repetition rate of the Linear Coherent Light Source (LCLS). Using a micropatterned silicon chip in combination with the high-speed Roadrunner goniometer for sample delivery, we were able to determine the crystal structures of the picornavirus bovine enterovirus 2 (BEV2) and the cytoplasmic polyhedrosis virus type 18 polyhedrin, with total data collection times of less than 14 and 10 min, respectively. Our method requires only micrograms of sample and should therefore broaden the applicability of serial femtosecond crystallography to challenging projects for which only limited sample amounts are available. By synchronizing the sample exchange to the XFEL repetition rate, our method allows for most efficient use of the limited beam time available at XFELs and should enable a substantial increase in sample throughput at these facilities.	Jun 2017
	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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