Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[21005]
Open Access
Abstract: The dynamic nature of protein and macromolecular complexes means that the capture of multiple sequential states along a reaction pathway can provide much greater insight into function than that obtained from a single static structure. We present a set of modular, easy-to-implement tools and workflows for optical excitation, on-grid characterization and tightly coupled rapid vitrification, establishing a proof-of-principle framework for time-resolved cryoEM and cryo-electron tomography (cryoET). We apply this framework to E. coli chemotaxis, in which serine-sensitive chemoreceptors initiate signalling upon ligand binding and undergo critical conformational changes within the chemosensory arrays. Using DMNB-caged serine [O-(4,5-dimethoxy-2-nitrobenzyl)-L-serine] as a model trigger, we quantified its photophysical properties and uncaging efficiency using UV–Vis spectroscopy and two-dimensional gas chromatography mass spectrometry (GC×GC-MS). Coupling a femtosecond-pulsed laser to a Vitrobot enabled reproducible reaction-to-vitrification delays of ∼150 ms, yielding intact E. coli minicells with well-preserved chemotaxis arrays suitable for in situ structural analysis by cryoET. This integrated approach provides a robust and generalisable framework for millisecond time-resolved cryoET, laying the groundwork for capturing transient conformational states in their native cellular context.
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Jul 2026
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I24-Microfocus Macromolecular Crystallography
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Jos J. A. G.
Kamps
,
Philip
Hinchliffe
,
Johan
Glerup
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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
Diamond Proposal Number(s):
[25260]
Open Access
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.
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Jul 2026
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I24-Microfocus Macromolecular Crystallography
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Marina
Lucic
,
Johan
Glerup
,
Pierre
Aller
,
Danny
Axford
,
Nicholas
Devenish
,
Jaehyun
Park
,
Anastasiia
Shilova
,
Arturo
Landeros De La Isla
,
Richard W.
Strange
,
Tiankun
Zhou
,
Robin L.
Owen
,
Jonathan A. R.
Worrall
,
Michael A.
Hough
Diamond Proposal Number(s):
[19458, 28583]
Open Access
Abstract: Metalloenzymes containing a heme cofactor catalyse a wide range of oxidative reactions critical to life. Understanding the structure and electronic states of the heme across the catalytic cycle is essential in understanding the oxidative chemistry performed on the substrate. This work demonstrates in crystallo manipulation of the heme-iron oxidation state in a B-type dye-decolourizing peroxidase from Streptomyces lividans (DtpB) using multiple, complementary, serial crystallography approaches. Fixed-target drop-on-chip serial femtosecond crystallography (SFX) together with dose-resolved serial synchrotron crystallography (SSX) allowed DtpB to be driven between multiple iron oxidation states. Drop-on-chip addition of hydrogen peroxide with fixed-target SFX is used to generate a ferryl [Fe(IV)=O] species, while the X-ray-driven approach modulates the iron oxidation state, with an apparent two-electron reduction leading to a return to a ferric state. The formation and dose response of the Fe(IV)—O state is highly variable between the chemically identical heme groups of the DtpB hexamer, highlighting the importance of understanding the effect of the crystalline lattice on observed changes in time- and dose-resolved crystallography.
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May 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
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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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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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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
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.
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Nov 2023
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