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43 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4, 5 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I24-Microfocus Macromolecular Crystallography VMXi-Versatile Macromolecular Crystallography in situ	Droplet-on-demand tape drive and X-ray emission spectroscopy prototypes for time-resolved serial crystallography on VMXi at Diamond Light Source Pierre Aller , Juan Sanchez-Weatherby , Abbey Telfer , Robert Bosman , Nicholas Devenish , Philip Hinchliffe , Sam Horrell , Jophy Ip , Richard Littlewood , Andrew Male , Eva Gimenez-Navarro , Urszula Neuman , Jos J. A. G. Kamps , David Omar , Laura Parkinson , Muthraj Pandi , Nico Rubies , James Sandy , Anastasya Shilova , James Spencer , Jonathan Spiers , John P. Sutter , Amy J. Thompson , Catherine L. Tooke , Ben Williams , Tiankun Zhou , Michael A. Hough , Allen M. Orville Iucrj 13 DOI: 10.1107/S2052252526005658 Open Access Show Abstract ▼ Abstract: Time-resolved X-ray crystallography is experiencing a resurgence, in part because of serial methods that readily allow scientists to create stop-motion movies of the macromolecular functions of photoactivation, enzyme-catalysed reactions and ligand-induced conformational changes triggering further downstream signalling events. While some reactions can be initiated with light, either naturally or using photocaged compounds, a more generally applicable approach is to mix microcrystals with reagents at varying time points prior to exposure to the X-ray beam. A powerful approach has been to combine droplet-on-demand `tape drive' sample delivery with X-ray emission spectroscopy (XES) that correlates atomic structure with the electronic states of metal ions within the sample. To the best of our knowledge, such a combined methodology has not been deployed previously on a synchrotron beamline but has been restricted to X-ray free-electron lasers. Here we describe two independent prototype experiments along the development pathway to a combined droplet-on-demand diffraction and XES system on the microfocus synchrotron beamline VMXi at Diamond Light Source. We demonstrate the collection of a high-quality serial diffraction data set from microcrystals within droplets having a volume of hundreds of picolitres deposited on a moving tape. In separate experiments on VMXi, we collected XES data from microcrystals of a copper enzyme delivered using a high-viscosity extruder. Together, these results demonstrate the feasibility of combined droplet-on-demand serial crystallography and XES experiments using a third-generation synchrotron beamline.	Sep 2026
I24-Microfocus Macromolecular Crystallography	Drop-on-fixed-target reaction initiation approach for serial and time-resolved crystallography Jos J. A. G. Kamps , Philip Hinchliffe , Johan Glerup , Emily I. Freeman , Pauline A. Lang , Catherine Tooke , Michael Beer , Laura Parkinson , Do-Heon Gu , Sehan Park , Nicholas Devenish , Tiankun Zhou , Anastasya Shilova , Samanpreet Kaur , Patrick Rabe , Christopher J. Schofield , James Spencer , Jaehyun Park , Robin L. Owen , Allen M. Orville , Pierre Aller Iucrj 13 DOI: 10.1107/S2052252526003489 Diamond Proposal Number(s): [25260] Open Access Show Abstract ▼ Abstract: We describe the design and implementation of a drop-on-fixed-target method for time-resolved serial crystallography at both synchrotron and XFEL facilities. A piezoelectric droplet dispensing pipette is employed for addition of picolitre volume aqueous droplets (∼40–90 pl; ∼40–55 µm diameter sphere), containing (co-)substrate(s) or ligand(s), onto enzyme microcrystals previously loaded into the trapezoidal wells of an etched crystalline silicon fixed-target chip containing 25 600 wells in a high-density, square grid with 125 µm centre-to-centre well spacing. These features demand exquisite accuracy and thereby constrain motion controls to enable robust time-resolved crystallographic studies. The system was tested with three enzyme systems, comprising lysozyme and two β-lactamases, CTX-M-15 and AmpCEC. Mitigation strategies for cross-well contamination, including the implementation of interleaved controls, are described; the overall performance of the system at synchrotron and X-ray free-electron laser facilities was evaluated. This drop-on-fixed-target method is a reliable framework for time-resolved crystallography and will improve the consistency of measurements across facilities.	Jul 2026
	Compact tape-driven sample delivery system for serial femtosecond crystallography Jungmin Kang , Yoshiaki Shimazu , Fangjia Luo , Ayumi Yamashita , Tomoyuki Tanaka , Yuichi Inubushi , Kensuke Tono , Nipawan Nuemket , Allen M. Orville , So Iwata , Eriko Nango , Makina Yabashi Journal Of Applied Crystallography 59 DOI: 10.1107/S1600576726000063 Open Access Show Abstract ▼ Abstract: We have developed a compact tape drive (CoT) with on-demand sample delivery for time-resolved serial femtosecond crystallography (SFX) experiments, which can deliver sample droplets and/or initiate reactions with a drop-on-drop strategy. Two disposable piezoelectric injectors are positioned in tandem along the tape to produce a queue of nanolitre-scale droplets. X-ray free-electron laser pulses arrive perpendicular to and pass through the broad face of the tape. The pulse is synchronized and aligned to the droplets, thereby enabling highly efficient SFX data collection. The tape transport speed and the delivery distance can be varied to control the mixing time from approximately 130 ms to tens of seconds. We conducted time-resolved SFX experiments utilizing a basic enzymatic reaction model of hen egg white lysozyme (HEWL) and N-acetyl-D-glucosamine (GlcNAc) to demonstrate the drop-on-drop capabilities of the CoT, and the full binding process of GlcNAc to HEWL was observed at 1.3–9.7 s.	Apr 2026
	Automated microbatch-under-oil phase diagrams to rationalize serial crystallography sample preparation Jack Stubbs , Courtney J. Tremlett , Abigail Waitman , Nicholas J. Harmer , Allen M. Orville , Ivo Tews , Stefan Kolek , Patrick D. Shaw Stewart Iucrj 13 DOI: 10.1107/S2052252526000448 Open Access Show Abstract ▼ Abstract: Serial crystallography relies on the reproducible production of high-density suspensions of microcrystals, yet sample optimization remains a resource-intensive bottleneck. While phase diagrams provide a theoretical framework for controlling crystal size and number, experimental mapping is traditionally hindered by relatively high sample consumption. We present an automated microbatch-under-oil crystallization approach that rapidly maps phase boundaries using only 15–60 µl (∼0.15–3.8 mg) of protein. While this workflow is ideally suited for refining existing hits, it serves as a standalone platform for characterizing the crystallization landscape of new protein targets. The power of this approach lies in the integration of three distinct strategies that exploit the stable chemical environment of microbatch-under-oil. Firstly, we utilize an ingenious diagonal sampling strategy that traverses the phase boundary parallel to the solubility curve by systematically varying protein-to-precipitant ratios, identifying primary nucleation zones with far greater efficiency than traditional orthogonal grids. Secondly, we employ a linked variation of multiple precipitants to reveal morphology-specific regions, such as the rod versus plate transitions crucial for time-resolved experiments. Finally, we incorporate automated seed-stock titration to precisely define the metastable zone, enabling the predictive rescue of nucleation-limited systems. The synergy of these three strategies enables the systematic decoupling of nucleation from growth, providing a rational route to optimize microcrystal density, size and lattice order. Crucially, by eliminating the evaporation-related variables inherent in vapor diffusion, this method ensures that the chemical coordinates identified during screening remain constant during scale-up to larger volumes. This workflow transforms empirical serial crystallography sample preparation into a rational, reproducible and highly efficient process applicable to both the optimization of known conditions and the de novo development of microcrystal suspensions, tailored to the rigorous demands of modern serial diffraction experiments.	Mar 2026
I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structural basis of the promiscuity of the unusual Fe(II) and 2-oxoglutarate dependent human aspartate/asparagine-β-hydroxylase Mariska De Munnik , Amelia Brasnett , Tiankun Zhou , William Myers , Yicheng Wang , Kuntal Chatterjee , Anthony Tumber , Stephen A. Marshall , Philipp S. Simon , Pierre Aller , Anastasiia Shilova , Danny Axford , Hiroki Makita , Daniel W. Paley , Vandana Tiwari , Alexander T. Stead , Sebastian Dehe , Humberto Sanchez , Daniel J. Rosenberg , Roberto Alonso-Mori , Asmit Bhowmick , Junko Yano , Vittal K. Yachandra , Jaehyun Park , Sehan Park , Allen M. Orville , Lennart Brewitz , Jan F. Kern , Christopher J. Schofield , Patrick Rabe Nature Communications 30 DOI: 10.1038/s41467-026-69425-w Diamond Proposal Number(s): [32727, 31353] Open Access Show Abstract ▼ Abstract: Protein-hydroxylation catalysed by Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases is an important regulatory mechanism in human biology. Such oxygenases typically coordinate their Fe(II) cofactor via a conserved triad of an aspartate- or glutamate- and two histidine-residues. By contrast, aspartate/asparagine β-hydroxylase (AspH), which catalyses asparagine/aspartate-residue oxidation in epidermal growth factor-like domains (EGFDs), has only two histidine-residues (H679, H725), with a water occupying the site normally occupied by an aspartate- or glutamate-residue. We describe mechanistic studies with catalytically active AspH crystals. Turnover studies with single crystals under cryogenic conditions give (3 R)-hydroxylated EGFDs with the product alcohol coordinating Fe(II) trans to H725. Time-resolved serial crystallography of microcrystals using an acoustic droplet ejection system, coupled to X-ray emission analyses, demonstrate turnover within 1.5 s, giving a product complex in which Fe(II) is regenerated. Solution and crystallographic studies with the O2 surrogate nitric oxide imply O2 binds to Fe(II) trans to H725. The additional Fe-chelating water is maintained throughout AspH catalysis and is not directly involved in substrate hydroxylation, because O2 is the sole oxygen source in alcohol products, as shown by 18O labelling studies. The results reveal how AspH accommodates both aspartate- and asparagine-substrates and will assist in efforts targeting AspH for cancer treatment.	Feb 2026
	Monooxygenase activity of indoleamine 2,3-dioxygenase Ali B. Lubis , Anna J. Bailey , Marko Hanževački , Christopher Williams , Mehul Jesani , Lola González-Sánchez , Christopher J. Arthur , Hannah C. Wilson , Andrea E. Gallio , Peter C. E. Moody , Matthew P. Crump , Adrian J. Mulholland , Allen M. Orville , Jonathan Clayden , Emma L. Raven Journal Of The American Chemical Society 148, 6178 - 6187 DOI: 10.1021/jacs.5c17552 Open Access Show Abstract ▼ Abstract: Indoleamine 2,3-dioxygenase (IDO) is a heme-dependent enzyme that catalyzes the first, rate-limiting step of the kynurenine pathway─the oxidation of l-tryptophan to N-formylkynurenine (NFK). IDO-catalyzed depletion of tryptophan levels and accumulation of kynurenine pathway metabolites is an important control mechanism of the immune responses in cells. IDO has been considered as a dioxygenase because two atoms of oxygen are inserted into the substrate. Here, we use LC-MS and NMR to examine the reactivity of human IDO (hIDO) with l-tryptophan (l-Trp) and several other tryptophan analogues. Alongside dioxygenase activity, we identify a concurrent pathway of heme-dependent monooxygenase activity in the reaction of hIDO with l-Trp, leading to the formation of a cyclic 3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid (HPIC) species. Reaction profiles for the reaction of hIDO with other tryptophan analogues are likewise examined. Formation of HPIC from l-Trp is reproduced in HeLa cells induced to overexpress hIDO, indicating that this dual dioxygenase/monooxygenase reactivity also occurs biologically. Notably, the reaction of hIDO with β-[3-benzo(b)thienyl]-l-alanine (S-l-Trp)─a known inhibitor ─yielded only the cyclic HPIC analogue, suggesting that IDO activity can be selectively directed toward the monooxygenase pathway. Molecular dynamics simulations underscore the critical role of substrate plasticity within the active site of hIDO, while DFT calculations provide a mechanistic rationalization for the observed product distributions. Together, the data demonstrate dual dioxygenase/monooxygenase functionality for human IDO. As the overall gatekeeper for control of tryptophan levels in cells, the findings provide mechanistic information on relevance to therapeutic strategies focused on IDO inhibition.	Feb 2026
	Can ferric-oxyl excited states explain elongated iron-oxygen bonds in heme peroxidase catalytic intermediates? Lewis J. Williams , Jos J. A. G. Kamps , Adrian M. V. Brânzanic , Maria Lehene , Kristoffer J. M. Lundgren , Ulf Ryde , Kuntal Chatterjee , Margaret D. Doyle , Philipp S. Simon , Hiroki Makita , Amy J. Thompson , Aaron S. Brewster , Tiankun Zhou , Marina Lucic , Michael T. Wilson , Pierre Aller , Juan Sanchez-Weatherby , Leland Gee , Sebastian Dehe , Sandra Mous , Junko Yano , Vittal K. Yachandra , Michael A. Hough , Allen M. Orville , Jan F. Kern , Radu L. Silaghi-Dumitrescu , Jonathan A. R. Worrall Nature Communications 114 DOI: 10.1038/s41467-026-69192-8 Open Access Show Abstract ▼ Abstract: The use of X-ray structures to determine and interpret the ferryl iron-oxygen bond order in molecular oxygen-activating heme enzymes has, in the past, been controversial. This has mainly stemmed from the susceptibility of ferryl species to X-ray-induced electronic state changes. In this work we establishe using time-resolved serial femtosecond X-ray crystallography (tr-SFX) on a dye-decolourising peroxidase that the ferryl intermediate species (Compounds I and II) captured following in situ mixing of microcrystals with H2O2 have single, rather than the double bond character expected. X-ray emission validated tr-SFX data with quantum refinement, time-dependent-DFT calculations and QM/MM geometry optimizations together support the concept that the single iron-oxygen bond character is not an indication of ferryl reduction or a protonated form (FeIV-OH) but is instead attributed to the existence of accessible excited states possessing ferric-oxyl (FeIII–O•–) character. Such states offer insight into the nature of ferryl heme.	Feb 2026
	The single-particle, clusters and biomolecules and serial femtosecond crystallography instrument of the European X-ray Free-Electron Laser: Interaction region downstream at atmospheric pressure (IRD) Adam Round , Pierre Aller , Richard Bean , Johan Bielecki , Agata Butryn , Nicholas E. Devenish , Raphael De Wijn , Thomas Dietze , Katerina Doerner , Fabio Dall'Antonia , Gabriele Giovanetti , Huijong Han , Vincent Hennicke , Chan Kim , Yoonhee Kim , Marco Kloos , Jayanath C. P. Koliyadu , Gabriel Leen , Romain Letrun , Luis Lopez Morillo , Allen M. Orville , Tim Pakendorf , Marco Ramilli , Nadja Reimers , Patrick Reinke , Juan Sanchez-Weatherby , Tokushi Sato , Robin Schubert , Joachim Schulz , Cedric Signe Takem , Marcin Sikorski , Prasad Thute , Fabian Trost , Oleksii Turkot , Patrik Vagovic , Mohammad Vakili , Raul Villanueva Guerrero , Henry N. Chapman , Alke Meents , Serguei Molodtsov , Sakura Pascarelli , Thomas Tschentschera , Adrian Mancuso , Pontus Fischer , Sebastian Guenther Journal Of Synchrotron Radiation 33 DOI: 10.1107/S1600577525008999 Open Access Show Abstract ▼ Abstract: The Single-Particle, Clusters and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) scientific instrument at the European X-Ray Free-Electron Laser (EuXFEL) became operational with user experiments in September 2017. The unique properties and capabilities of the EuXFEL, enabling megahertz data collection rates, provide more rapid data collection with improved statistics compared with other XFEL facilities. This improves the feasibility of obtaining multiple data points in time-resolved experiments and hence enables the observation of reactions in greater detail (molecular movies). In collaboration with the SFX User Consortium (SFX UC), the SPB/SFX instrument was designed to further increase user access and research outcomes. Focusing the pulses downstream of the first interaction region [described previously (Mancuso et al., 2019)], a second experiment plane is enabled, which allows for greater optimization and more efficient usage of available beam time. Additionally, the SFX UC provided further instrumentation to provide improved capabilities on SPB/SFX. The aim for additional and extended functionality for the second interaction region was to enable sample-efficient data collection at atmospheric pressure in an environment where the sample temperature and humidity can be controlled. This paper describes the extended capabilities of the downstream interaction region of the SPB/SFX instrument and its major components, in particular its X-ray focusing optics, vacuum to atmospheric pressure out-coupling, available sample delivery methods and 2D detector, and the supporting optical laser systems for pump–probe experiments.	Nov 2025
	Small but mighty: the power of microcrystals in structural biology Courtney J. Tremlett , Jack Stubbs , William S. Stuart , Patrick D. Shaw Stewart , Jonathan West , Allen M. Orville , Ivo Tews , Nicholas J. Harmer Iucrj 12 DOI: 10.1107/S2052252525001484 Open Access Show Abstract ▼ Abstract: Advancements in macromolecular crystallography, driven by improved sources and cryocooling techniques, have enabled the use of increasingly smaller crystals for structure determination, with microfocus beamlines now widely accessible. Initially developed for challenging samples, these techniques have culminated in advanced beamlines such as VMXm. Here, an in vacuo sample environment improves the signal-to-noise ratio in X-ray diffraction experiments, and thus enables the use of submicrometre crystals. The advancement of techniques such as microcrystal electron diffraction (MicroED) for atomic-level insights into charged states and hydrogen positions, along with room-temperature crystallography to observe physiological states via serial crystallography, has driven a resurgence in the use of microcrystals. Reproducibly preparing small crystals, especially from samples that typically yield larger crystals, requires considerable effort, as no one singular approach guarantees optimal crystals for every technique. This review discusses methods for generating such small crystals, including mechanical crushing and batch crystallization with seeding, and evaluates their compatibility with microcrystal data-collection modalities. Additionally, we examine sample-delivery methods, which are crucial for selecting appropriate crystallization strategies. Establishing reliable protocols for sample preparation and delivery opens new avenues for macromolecular crystallography, particularly in the rapidly progressing field of time-resolved crystallography.	May 2025
	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

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