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Instruments / Technical Area	Publication Details	Published
	Serial-femtosecond crystallography reveals how a phytochrome variant couples chromophore and protein structural changes Luisa Sauthof , Michal Szczepek , Andrea Schmidt , Asmit Bhowmick , Medhanjali Dasgupta , Megan J. Mackintosh , Sheraz Gul , Franklin D. Fuller , Ruchira Chatterjee , Iris D. Young , Norbert Michael , Nicolas Andreas Heyder , Brian Bauer , Anja Koch , Isabel Bogacz , In-Sik Kim , Philipp S. Simon , Agata Butryn , Pierre Aller , Volha U. Chukhutsina , James M. Baxter , Christopher D. M. Hutchison , Dorothee Liebschner , Billy Poon , Nicholas K. Sauter , Mitchell D. Miller , George N. Phillips , Roberto Alonso-Mori , Mark S. Hunter , Alexander Batyuk , Shigeki Owada , Kensuke Tono , Rie Tanaka , Jasper J. Van Thor , Norbert Krauß , Tilman Lamparter , Aaron S. Brewster , Igor Schapiro , Allen M. Orville , Vittal K. Yachandra , Junko Yano , Peter Hildebrandt , Jan F. Kern , Patrick Scheerer Science Advances 11 DOI: 10.1126/sciadv.adp2665 Open Access Show Abstract ▼ Abstract: The photoreaction and commensurate structural changes of a chromophore within biological photoreceptors elicit conformational transitions of the protein promoting the switch between deactivated and activated states. We investigated how this coupling is achieved in a bacterial phytochrome variant, Agp2-PAiRFP2. Contrary to classical protein crystallography, which only allows probing (cryo-trapped) stable states, we have used time-resolved serial femtosecond x-ray crystallography (tr-SFX) and pump-probe techniques with various illumination and delay times with respect to photoexcitation of the parent Pfr state. Thus, structural data for seven time frames were sorted into groups of molecular events along the reaction coordinate. They range from chromophore isomerization to the formation of Meta-F, the intermediate that precedes the functional relevant secondary structure transition of the tongue. Structural data for the early events were used to calculate the photoisomerization pathway to complement the experimental data. Late events allow identifying the molecular switch that is linked to the intramolecular proton transfer as a prerequisite for the following structural transitions.	May 2025
I19-Small Molecule Single Crystal Diffraction	Ligand-mediated quantum yield enhancement in 1-D silver organothiolate metal–organic chalcogenolates Mariya Aleksich , Yeongsu Cho , Daniel W. Paley , Maggie C. Willson , Hawi N. Nyiera , Patience A. Kotei , Vanessa Oklejas , David W. Mittan-Moreau , Elyse A. Schriber , Kara Christensen , Ichiro Inoue , Shigeki Owada , Kensuke Tono , Michihiro Sugahara , Satomi Inaba-Inoue , Mohammad Vakili , Christopher J. Milne , Fabio Dallantonia , Dmitry Khakhulin , Fernando Ardana-Lamas , Frederico Lima , Joana Valerio , Huijong Han , Tamires Gallo , Hazem Yousef , Oleksii Turkot , Ivette J. Bermudez Macias , Thomas Kluyver , Philipp Schmidt , Luca Gelisio , Adam R. Round , Yifeng Jiang , Doriana Vinci , Yohei Uemura , Marco Kloos , Adrian P. Mancuso , Mark Warren , Nicholas K. Sauter , Jing Zhao , Tess Smidt , Heather J. Kulik , Sahar Sharifzadeh , Aaron S. Brewster , J. Nathan Hohman Advanced Functional Materials 17 DOI: 10.1002/adfm.202414914 Diamond Proposal Number(s): [35300] Show Abstract ▼ Abstract: X-ray free electron laser (XFEL) microcrystallography and synchrotron single-crystal crystallography are used to evaluate the role of organic substituent position on the optoelectronic properties of metal–organic chalcogenolates (MOChas). MOChas are crystalline 1D and 2D semiconducting hybrid materials that have varying optoelectronic properties depending on composition, topology, and structure. While MOChas have attracted much interest, small crystal sizes impede routine crystal structure determination. A series of constitutional isomers where the aryl thiol is functionalized by either methoxy or methyl ester are solved by small molecule serial femtosecond X-ray crystallography (smSFX) and single crystal rotational crystallography. While all the methoxy examples have a low quantum yield (0-1%), the methyl ester in the ortho position yields a high quantum yield of 22%. The proximity of the oxygen atoms to the silver inorganic core correlates to a considerable enhancement of quantum yield. Four crystal structures are solved at a resolution range of 0.8–1.0 Å revealing a collapse of the 2D topology for functional groups in the 2- and 3- positions, resulting in needle-like crystals. Further analysis using density functional theory (DFT) and many-body perturbation theory (MBPT) enables the exploration of complex excitonic phenomena within easily prepared material systems.	Dec 2024
	In situ structural observation of a substrate- and peroxide-bound high-spin ferric-hydroperoxo intermediate in the P450 enzyme CYP12 Romie C. Nguyen , Ian Davis , Medhanjali Dasgupta , Yifan Wang , Philipp S. Simon , Agata Butryn , Hiroki Makita , Isabel Bogacz , Kednerlin Dornevil , Pierre Aller , Asmit Bhowmick , Ruchira Chatterjee , In-Sik Kim , Tiankun Zhou , Derek Mendez , Daniel W. Paley , Franklin Fuller , Roberto Alonso Mori , Alexander Batyuk , Nicholas K. Sauter , Aaron S. Brewster , Allen M. Orville , Vittal K. Yachandra , Junko Yano , Jan F. Kern , Aimin Liu Journal Of The American Chemical Society 10 DOI: 10.1021/jacs.3c04991 Show Abstract ▼ Abstract: The P450 enzyme CYP121 from Mycobacterium tuberculosis catalyzes a carbon–carbon (C–C) bond coupling cyclization of the dityrosine substrate containing a diketopiperazine ring, cyclo(l-tyrosine-l-tyrosine) (cYY). An unusual high-spin (S = 5/2) ferric intermediate maximizes its population in less than 5 ms in the rapid freeze-quenching study of CYP121 during the shunt reaction with peracetic acid or hydrogen peroxide in acetic acid solution. We show that this intermediate can also be observed in the crystalline state by EPR spectroscopy. By developing an on-demand-rapid-mixing method for time-resolved serial femtosecond crystallography with X-ray free-electron laser (tr-SFX-XFEL) technology covering the millisecond time domain and without freezing, we structurally monitored the reaction in situ at room temperature. After a 200 ms peracetic acid reaction with the cocrystallized enzyme–substrate microcrystal slurry, a ferric-hydroperoxo intermediate is observed, and its structure is determined at 1.85 Å resolution. The structure shows a hydroperoxyl ligand between the heme and the native substrate, cYY. The oxygen atoms of the hydroperoxo are 2.5 and 3.2 Å from the iron ion. The end-on binding ligand adopts a near-side-on geometry and is weakly associated with the iron ion, causing the unusual high-spin state. This compound 0 intermediate, spectroscopically and structurally observed during the catalytic shunt pathway, reveals a unique binding mode that deviates from the end-on compound 0 intermediates in other heme enzymes. The hydroperoxyl ligand is only 2.9 Å from the bound cYY, suggesting an active oxidant role of the intermediate for direct substrate oxidation in the nonhydroxylation C–C bond coupling chemistry.	Nov 2023
	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
	XFEL microcrystallography of self-assembling silver n-alkanethiolates Mariya Aleksich , Daniel W. Paley , Elyse A. Schriber , Will Linthicum , Vanessa Oklejas , David W. Mittan-Moreau , Ryan P. Kelly , Patience A. Kotei , Anita Ghodsi , Raymond G. Sierra , Andrew Aquila , Frédéric Poitevin , Johannes P. Blaschke , Mohammad Vakili , Christopher J. Milne , Fabio Dall’antonia , Dmitry Khakhulin , Fernando Ardana-Lamas , Frederico Lima , Joana Valerio , Huijong Han , Tamires Gallo , Hazem Yousef , Oleksii Turkot , Ivette J. Bermudez Macias , Thomas Kluyver , Philipp Schmidt , Luca Gelisio , Adam R. Round , Yifeng Jiang , Doriana Vinci , Yohei Uemura , Marco Kloos , Mark Hunter , Adrian P. Mancuso , Bryan D. Huey , Lucas R. Parent , Nicholas K. Sauter , Aaron S. Brewster , J. Nathan Hohman Journal Of The American Chemical Society DOI: 10.1021/jacs.3c02183 Show Abstract ▼ Abstract: New synthetic hybrid materials and their increasing complexity have placed growing demands on crystal growth for single-crystal X-ray diffraction analysis. Unfortunately, not all chemical systems are conducive to the isolation of single crystals for traditional characterization. Here, small-molecule serial femtosecond crystallography (smSFX) at atomic resolution (0.833 Å) is employed to characterize microcrystalline silver n-alkanethiolates with various alkyl chain lengths at X-ray free electron laser facilities, resolving long-standing controversies regarding the atomic connectivity and odd–even effects of layer stacking. smSFX provides high-quality crystal structures directly from the powder of the true unknowns, a capability that is particularly useful for systems having notoriously small or defective crystals. We present crystal structures of silver n-butanethiolate (C4), silver n-hexanethiolate (C6), and silver n-nonanethiolate (C9). We show that an odd–even effect originates from the orientation of the terminal methyl group and its role in packing efficiency. We also propose a secondary odd–even effect involving multiple mosaic blocks in the crystals containing even-numbered chains, identified by selected-area electron diffraction measurements. We conclude with a discussion of the merits of the synthetic preparation for the preparation of microdiffraction specimens and compare the long-range order in these crystals to that of self-assembled monolayers.	Jul 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
	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

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