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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
	Gold Standard for macromolecular crystallography diffraction data Herbert J. Bernstein , Andreas Forster , Asmit Bhowmick , Aaron S. Brewster , Sandor Brockhauser , Luca Gelisio , David R. Hall , Filip Leonarski , Valerio Mariani , Gianluca Santoni , Clemens Vonrhein , Graeme Winter Iucrj 7 DOI: 10.1107/S2052252520008672 Open Access Show Abstract ▼ Abstract: Macromolecular crystallography (MX) is the dominant means of determining the three-dimensional structures of biological macromolecules. Over the last few decades, most MX data have been collected at synchrotron beamlines using a large number of different detectors produced by various manufacturers and taking advantage of various protocols and goniometries. These data came in their own formats: sometimes proprietary, sometimes open. The associated metadata rarely reached the degree of completeness required for data management according to Findability, Accessibility, Interoperability and Reusability (FAIR) principles. Efforts to reuse old data by other investigators or even by the original investigators some time later were often frustrated. In the culmination of an effort dating back more than two decades, a large portion of the research community concerned with high data-rate macromolecular crystallography (HDRMX) has now agreed to an updated specification of data and metadata for diffraction images produced at synchrotron light sources and X-ray free-electron lasers (XFELs). This `Gold Standard' will facilitate the processing of data sets independent of the facility at which they were collected and enable data archiving according to FAIR principles, with a particular focus on interoperability and reusability. This agreed standard builds on the NeXus/HDF5 NXmx application definition and the International Union of Crystallography (IUCr) imgCIF/CBF dictionary, and it is compatible with major data-processing programs and pipelines. Just as with the IUCr CBF/imgCIF standard from which it arose and to which it is tied, the NeXus/HDF5 NXmx Gold Standard application definition is intended to be applicable to all detectors used for crystallography, and all hardware and software developers in the field are encouraged to adopt and contribute to the standard.	Sep 2020
	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
	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
	Towards in cellulo virus crystallography Helen M. E. Duyvesteyn , Helen M. Ginn , Maija K. Pietila , Armin Wagner , Johan Hattne , Jonathan M. Grimes , Elina Hirvonen , Gwyndaf Evans , Marie-laure Parsy , Nicholas K. Sauter , Aaron S. Brewster , Juha Huiskonen , David I. Stuart , Geoff Sutton , Dennis H. Bamford Scientific Reports 8 DOI: 10.1038/s41598-018-21693-3 Open Access Show Abstract ▼ Abstract: Viruses are a significant threat to both human health and the economy, and there is an urgent need for novel anti-viral drugs and vaccines. High-resolution viral structures inform our understanding of the virosphere, and inspire novel therapies. Here we present a method of obtaining such structural information that avoids potentially disruptive handling, by collecting diffraction data from intact infected cells. We identify a suitable combination of cell type and virus to accumulate particles in the cells, establish a suitable time point where most cells contain virus condensates and use electron microscopy to demonstrate that these are ordered crystalline arrays of empty capsids. We then use an X-ray free electron laser to provide extremely bright illumination of sub-micron intracellular condensates of bacteriophage phiX174 inside living Escherichia coli at room temperature. We have been able to collect low resolution diffraction data. Despite the limited resolution and completeness of these initial data, due to a far from optimal experimental setup, we have used novel methodology to determine a putative space group, unit cell dimensions, particle packing and likely maturation state of the particles.	Feb 2018

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