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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
I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	PGRP-LB: An inside view into the mechanism of the amidase reaction Julien Orlans , Carole Vincent-Monegat , Isabelle Rahioui , Catherine Sivignon , Agata Butryn , Laurent Soulère , Anna Zaidman-Remy , Allen M. Orville , Abdelaziz Heddi , Pierre Aller , Pedro Da Silva International Journal Of Molecular Sciences 22 DOI: 10.3390/ijms22094957 Diamond Proposal Number(s): [19737, 19458, 23571] Open Access Show Abstract ▼ Abstract: Peptidoglycan recognition proteins (PGRPs) are ubiquitous among animals and play pivotal functions in insect immunity. Non-catalytic PGRPs are involved in the activation of immune pathways by binding to the peptidoglycan (PGN), whereas amidase PGRPs are capable of cleaving the PGN into non-immunogenic compounds. Drosophila PGRP-LB belongs to the amidase PGRPs and downregulates the immune deficiency (IMD) pathway by cleaving meso-2,6-diaminopimelic (meso-DAP or DAP)-type PGN. While the recognition process is well analyzed for the non-catalytic PGRPs, little is known about the enzymatic mechanism for the amidase PGRPs, despite their essential function in immune homeostasis. Here, we analyzed the specific activity of different isoforms of Drosophila PGRP-LB towards various PGN substrates to understand their specificity and role in Drosophila immunity. We show that these isoforms have similar activity towards the different compounds. To analyze the mechanism of the amidase activity, we performed site directed mutagenesis and solved the X-ray structures of wild-type Drosophila PGRP-LB and its mutants, with one of these structures presenting a protein complexed with the tracheal cytotoxin (TCT), a muropeptide derived from the PGN. Only the Y78F mutation abolished the PGN cleavage while other mutations reduced the activity solely. Together, our findings suggest the dynamic role of the residue Y78 in the amidase mechanism by nucleophilic attack through a water molecule to the carbonyl group of the amide function destabilized by Zn2+.	May 2021
	Dynamic structural biology experiments at XFEL or synchrotron sources Pierre Aller , Allen M. Orville DOI: 10.1007/978-1-0716-1406-8_11 Show Abstract ▼ Abstract: Macromolecular crystallography (MX) leverages the methods of physics and the language of chemistry to reveal fundamental insights into biology. Often beautifully artistic images present MX results to support profound functional hypotheses that are vital to entire life science research community. Over the past several decades, synchrotrons around the world have been the workhorses for X-ray diffraction data collection at many highly automated beamlines. The newest tools include X-ray-free electron lasers (XFELs) located at facilities in the USA, Japan, Korea, Switzerland, and Germany that deliver about nine orders of magnitude higher brightness in discrete femtosecond long pulses. At each of these facilities, new serial femtosecond crystallography (SFX) strategies exploit slurries of micron-size crystals by rapidly delivering individual crystals into the XFEL X-ray interaction region, from which one diffraction pattern is collected per crystal before it is destroyed by the intense X-ray pulse. Relatively simple adaptions to SFX methods produce time-resolved data collection strategies wherein reactions are triggered by visible light illumination or by chemical diffusion/mixing. Thus, XFELs provide new opportunities for high temporal and spatial resolution studies of systems engaged in function at physiological temperature. In this chapter, we summarize various issues related to microcrystal slurry preparation, sample delivery into the X-ray interaction region, and some emerging strategies for time-resolved SFX data collection.	May 2021
I24-Microfocus Macromolecular Crystallography	Anaerobic fixed-target serial crystallography Patrick Rabe , John Beale , Agata Butryn , Pierre Aller , Anna Dirr , Pauline A. Lang , Danny N. Axford , Stephen Carr , Thomas M. Leissing , Michael A. Mcdonough , Bradley Davy , Ali Ebrahim , Julien Orlans , Selina L. S. Storm , Allen M. Orville , Christopher J. Schofield , Robin L. Owen Iucrj 7 DOI: 10.1107/S2052252520010374 Diamond Proposal Number(s): [19458] Open Access Show Abstract ▼ Abstract: Cryogenic X-ray diffraction is a powerful tool for crystallographic studies on enzymes including oxygenases and oxidases. Amongst the benefits that cryo-conditions (usually employing a nitrogen cryo-stream at 100 K) enable, is data collection of dioxygen-sensitive samples. Although not strictly anaerobic, at low temperatures the vitreous ice conditions severely restrict O2 diffusion into and/or through the protein crystal. Cryo-conditions limit chemical reactivity, including reactions that require significant conformational changes. By contrast, data collection at room temperature imposes fewer restrictions on diffusion and reactivity; room-temperature serial methods are thus becoming common at synchrotrons and XFELs. However, maintaining an anaerobic environment for dioxygen-dependent enzymes has not been explored for serial room-temperature data collection at synchrotron light sources. This work describes a methodology that employs an adaptation of the `sheet-on-sheet' sample mount, which is suitable for the low-dose room-temperature data collection of anaerobic samples at synchrotron light sources. The method is characterized by easy sample preparation in an anaerobic glovebox, gentle handling of crystals, low sample consumption and preservation of a localized anaerobic environment over the timescale of the experiment (<5 min). The utility of the method is highlighted by studies with three X-ray-radiation-sensitive Fe(II)-containing model enzymes: the 2-oxoglutarate-dependent L-arginine hydroxylase VioC and the DNA repair enzyme AlkB, as well as the oxidase isopenicillin N synthase (IPNS), which is involved in the biosynthesis of all penicillin and cephalosporin antibiotics.	Sep 2020
I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	High-throughput in situ experimental phasing Joshua M. Lawrence , Julien Orlans , Gwyndaf Evans , Allen M. Orville , James Foadi , Pierre Aller Acta Crystallographica Section D Structural Biology 76, 790 - 801 DOI: 10.1107/S2059798320009109 Diamond Proposal Number(s): [14449, 16818] Open Access Show Abstract ▼ Abstract: In this article, a new approach to experimental phasing for macromolecular crystallography (MX) at synchrotrons is introduced and described for the first time. It makes use of automated robotics applied to a multi-crystal framework in which human intervention is reduced to a minimum. Hundreds of samples are automatically soaked in heavy-atom solutions, using a Labcyte Inc. Echo 550 Liquid Handler, in a highly controlled and optimized fashion in order to generate derivatized and isomorphous crystals. Partial data sets obtained on MX beamlines using an in situ setup for data collection are processed with the aim of producing good-quality anomalous signal leading to successful experimental phasing.	Aug 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
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
I24-Microfocus Macromolecular Crystallography	Reducing sample consumption for serial crystallography using acoustic drop ejection Bradley Davy , Danny Axford , John H. Beale , Agata Butryn , Peter Docker , Ali Ebrahim , Gabriel Leen , Allen M. Orville , Robin L. Owen , Pierre Aller Journal Of Synchrotron Radiation 26 DOI: 10.1107/S1600577519009329 Diamond Proposal Number(s): [14493, 19458] Open Access Show Abstract ▼ Abstract: Efficient sample delivery is an essential aspect of serial crystallography at both synchrotrons and X-ray free-electron lasers. Rastering fixed target chips through the X-ray beam is an efficient method for serial delivery from the perspectives of both sample consumption and beam time usage. Here, an approach for loading fixed targets using acoustic drop ejection is presented that does not compromise crystal quality, can reduce sample consumption by more than an order of magnitude and allows serial diffraction to be collected from a larger proportion of the crystals in the slurry.	Sep 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

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