I24-Microfocus Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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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
Open Access
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.
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Sep 2026
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I24-Microfocus Macromolecular Crystallography
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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
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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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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Mohammad
Vakili
,
Huijong
Han
,
Christina
Schmidt
,
Agnieszka
Wrona
,
Marco
Kloos
,
Iñaki
De Diego
,
Katerina
Dörner
,
Tian
Geng
,
Chan
Kim
,
Faisal H. M.
Koua
,
Diogo V. M.
Melo
,
Mathieu
Rappas
,
Adam
Round
,
Ekaterina
Round
,
Marcin
Sikorski
,
Joana
Valerio
,
Tiankun
Zhou
,
Kristina
Lorenzen
,
Joachim
Schulz
Open Access
Abstract: Time-resolved crystallography enables the visualization of protein molecular motion during a reaction. Although light is often used to initiate reactions in time-resolved crystallography, only a small number of proteins can be activated by light. However, many biological reactions can be triggered by the interaction between proteins and ligands. The sample delivery method presented here uses a mix-and-extrude approach based on 3D-printed microchannels in conjunction with a micronozzle. The diffusive mixing enables the study of the dynamics of samples in viscous media. The device design allows mixing of the ligands and protein crystals in 2 to 20 s. The device characterization using a model system (fluorescence quenching of iq-mEmerald proteins by copper ions) demonstrated that ligand and protein crystals, each within lipidic cubic phase, can be mixed efficiently. The potential of this approach for time-resolved membrane protein crystallography to support the development of new drugs is discussed.
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Aug 2023
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I24-Microfocus Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[19458, 25260]
Open Access
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.
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Jul 2021
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B21-High Throughput SAXS
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[12788]
Abstract: The cardiac ankyrin repeat protein (CARP) is up-regulated in the myocardium during cardiovascular disease and in response to mechanical or toxic stress. Stress-induced CARP interacts with the N2A spring region of the titin filament to modulate muscle compliance. We characterize the interaction between CARP and titin-N2A and show that the binding site in titin spans the dual domain UN2A-Ig81. We find that the unique sequence UN2A is not structurally disordered, but that it has a stable, elongated α-helical fold that possibly acts as a constant force spring. Our findings portray CARP/titin-N2A as a structured node and help to rationalize the molecular basis of CARP mechanosensing in the sarcomeric I-band.
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Sep 2016
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