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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
Open Access
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.
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Apr 2026
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Open Access
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.
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Mar 2026
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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
Open Access
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.
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Feb 2026
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[32727, 31353]
Open Access
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.
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Feb 2026
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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
Open Access
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.
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Nov 2025
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Open Access
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.
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May 2025
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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
Open Access
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.
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May 2025
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NONE-No attached Diamond beamline
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Open Access
Abstract: We have developed sample-efficient delivery and reaction initiation strategies that use room temperature microcrystal slurries and serial crystallography methods for time-resolved studies [1-3]. However, interpreting electron density maps from reaction cycle intermediates can be challenging when mixtures of species are present in the data. Therefore, to help reduce ambiguity we and our collaborators have also pioneered strategies to simultaneously collect time-resolved serial crystallography (tr- SSX/tr-SFX) diffraction data in the forward direction, and X-ray emission spectroscopy (tr-XES) data at ∼ 90°, using either XFEL (tr-SFX) or synchrotron (tr-SSX) sources. The resulting atomic and electronic structures are fully correlated and have been applied to a range of enzymes [1, 2, 4-8]. For instance, isopenicillin N synthase (IPNS) uses nonheme iron to catalyse the O2- dependent conversion of its tripeptide substrate delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) into isopenicillin N (IPN, the precursor of all penicillin/cephalosporin beta-lactam antibiotics). The unique four electron oxidation reaction leading to the beta-lactam bicyclic ring proceeds via two high-valent iron species, an Fe(III)-superoxo and a high-spin Fe(IV)=O oxyferryl species. These enable two sequential C-H bond cleavage steps that each exhibit large kinetic isotope effects (KIE). Our recent tr- SFX and tr-XES studies have characterised the Fe(III)-superoxo species and revealed unexpected, correlated motions throughout the whole protein caused by O2 binding [4].
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Mar 2025
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Abstract: Dynamic structural biology enables studying biological events at the atomic scale from 10’s of femtoseconds to a few seconds duration. With the advent of X-ray Free Electron Lasers (XFELs) and 4th generation synchrotrons, serial crystallography is becoming a major player for time-resolved experiments in structural biology. Despite significant progress, challenges such as obtaining sufficient amounts of protein to produce homogeneous microcrystal slurry, remain. Given this, it has been paramount to develop instrumentation that reduces the amount of microcrystal slurry required for experiments. Tape-drive systems use a conveyor belt made of X-ray transparent material as a motorized solid-support to steer deposited microcrystals into the beam. For efficient sample consumption on-demand ejectors can be synchronized with the X-ray pulses to expose crystals contained in droplets deposited on the tape. Reactions in the crystals can be triggered via various strategies, including pump-probe, substrate/ligand mixing, or gas incubation in the space between droplet ejection and X-ray illumination. Another challenge in time-resolved serial crystallography is interpreting the resulting electron density maps. This is especially difficult for metalloproteins where the active site metal is intimately involved in catalysis and often proceeds through multiple oxidation states during enzymatic catalysis. The unrestricted space around tape-drive systems can be used to accommodate complementary spectroscopic equipment. Here, we highlight tape-drive sample delivery systems for complementary and simultaneous X-ray diffraction (XRD) and X-ray emission spectroscopy (XES) measurements. We describe how the combination of both XRD and XES is a powerful tool for time-resolved experiments at XFELs and synchrotrons.
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Oct 2024
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I23-Long wavelength MX
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Yishun
Lu
,
Ramona
Duman
,
James
Beilsten-Edmands
,
Graeme
Winter
,
Mark
Basham
,
Gwyndaf
Evans
,
Jos J. A. G.
Kamps
,
Allen M.
Orville
,
Hok-Sau
Kwong
,
Konstantinos
Beis
,
Wesley
Armour
,
Armin
Wagner
Open Access
Abstract: rocessing of single-crystal X-ray diffraction data from area detectors can be separated into two steps. First, raw intensities are obtained by integration of the diffraction images, and then data correction and reduction are performed to determine structure-factor amplitudes and their uncertainties. The second step considers the diffraction geometry, sample illumination, decay, absorption and other effects. While absorption is only a minor effect in standard macromolecular crystallography (MX), it can become the largest source of uncertainty for experiments performed at long wavelengths. Current software packages for MX typically employ empirical models to correct for the effects of absorption, with the corrections determined through the procedure of minimizing the differences in intensities between symmetry-equivalent reflections; these models are well suited to capturing smoothly varying experimental effects. However, for very long wavelengths, empirical methods become an unreliable approach to model strong absorption effects with high fidelity. This problem is particularly acute when data multiplicity is low. This paper presents an analytical absorption correction strategy (implemented in new software AnACor) based on a volumetric model of the sample derived from X-ray tomography. Individual path lengths through the different sample materials for all reflections are determined by a ray-tracing method. Several approaches for absorption corrections (spherical harmonics correction, analytical absorption correction and a combination of the two) are compared for two samples, the membrane protein OmpK36 GD, measured at a wavelength of λ = 3.54 Å, and chlorite dismutase, measured at λ = 4.13 Å. Data set statistics, the peak heights in the anomalous difference Fourier maps and the success of experimental phasing are used to compare the results from the different absorption correction approaches. The strategies using the new analytical absorption correction are shown to be superior to the standard spherical harmonics corrections. While the improvements are modest in the 3.54 Å data, the analytical absorption correction outperforms spherical harmonics in the longer-wavelength data (λ = 4.13 Å), which is also reflected in the reduced amount of data being required for successful experimental phasing.
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Jun 2024
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