|
|
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.
|
Nov 2025
|
|
|
|
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.
|
May 2025
|
|
|
|
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.
|
May 2025
|
|
NONE-No attached Diamond beamline
|
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].
|
Mar 2025
|
|
|
|
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.
|
Oct 2024
|
|
I23-Long wavelength MX
|
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.
|
Jun 2024
|
|
I24-Microfocus Macromolecular Crystallography
|
Rachel
Bolton
,
Moritz M.
Machelett
,
Jack
Stubbs
,
Danny
Axford
,
Nicolas
Caramello
,
Lucrezia
Catapano
,
Martin
Maly
,
Matthew J.
Rodrigues
,
Charlotte
Cordery
,
Graham J.
Tizzard
,
Fraser
Macmillan
,
Sylvain
Engilberge
,
David
Von Stetten
,
Takehiko
Tosha
,
Hiroshi
Sugimoto
,
Jonathan A. R.
Worrall
,
Jeremy S.
Webb
,
Mike
Zubkov
,
Simon
Coles
,
Eric
Mathieu
,
Roberto A.
Steiner
,
Garib
Murshudov
,
Tobias E.
Schrader
,
Allen M.
Orville
,
Antoine
Royant
,
Gwyndaf
Evans
,
Michael A.
Hough
,
Robin L.
Owen
,
Ivo
Tews
Diamond Proposal Number(s):
[15722, 14493, 23570]
Open Access
Abstract: The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes generally encode two different types of FutA iron-binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here, we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray-induced photoreduction of the iron center with observation of the Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump–probe approach capture the transition between Fe(III) and Fe(II) states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins.
|
Mar 2024
|
|
|
|
Jack
Stubbs
,
Theo
Hornsey
,
Niall
Hanrahan
,
Luis Blay
Esteban
,
Rachel
Bolton
,
Martin
Maly
,
Shibom
Basu
,
Julien
Orlans
,
Daniele
De Sanctis
,
Jung-Uk
Shim
,
Patrick D.
Shaw Stewart
,
Allen M.
Orville
,
Ivo
Tews
,
Jonathan
West
Open Access
Abstract: Serial crystallography requires large numbers of microcrystals and robust strategies to rapidly apply substrates to initiate reactions in time-resolved studies. Here, we report the use of droplet miniaturization for the controlled production of uniform crystals, providing an avenue for controlled substrate addition and synchronous reaction initiation. The approach was evaluated using two enzymatic systems, yielding 3 µm crystals of lysozyme and 2 µm crystals of Pdx1, an Arabidopsis enzyme involved in vitamin B6 biosynthesis. A seeding strategy was used to overcome the improbability of Pdx1 nucleation occurring with diminishing droplet volumes. Convection within droplets was exploited for rapid crystal mixing with ligands. Mixing times of <2 ms were achieved. Droplet microfluidics for crystal size engineering and rapid micromixing can be utilized to advance time-resolved serial crystallography.
|
Mar 2024
|
|
|
|
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.
|
Nov 2023
|
|
I24-Microfocus Macromolecular Crystallography
|
James
Birch
,
Tristan O. C.
Kwan
,
Peter J.
Judge
,
Danny
Axford
,
Pierre
Aller
,
Agata
Butryn
,
Rosana
Reis
,
Juan F.
Bada Juarez
,
Javier
Vinals
,
Robin L.
Owen
,
Eriko
Nango
,
Rie
Tanaka
,
Kensuke
Tono
,
Yasumasa
Joti
,
Tomoyuki
Tanaka
,
Shigeki
Owada
,
Michihiro
Sugahara
,
So
Iwata
,
Allen M.
Orville
,
Anthony
Watts
,
Isabel
Moraes
Diamond Proposal Number(s):
[19152]
Open Access
Abstract: Serial crystallography has emerged as an important tool for structural studies of integral membrane proteins. The ability to collect data from micrometre-sized weakly diffracting crystals at room temperature with minimal radiation damage has opened many new opportunities in time-resolved studies and drug discovery. However, the production of integral membrane protein microcrystals in lipidic cubic phase at the desired crystal density and quantity is challenging. This paper introduces VIALS (versatile approach to high-density microcrystals in lipidic cubic phase for serial crystallography), a simple, fast and efficient method for preparing hundreds of microlitres of high-density microcrystals suitable for serial X-ray diffraction experiments at both synchrotron and free-electron laser sources. The method is also of great benefit for rational structure-based drug design as it facilitates in situ crystal soaking and rapid determination of many co-crystal structures. Using the VIALS approach, room-temperature structures are reported of (i) the archaerhodopsin-3 protein in its dark-adapted state and 110 ns photocycle intermediate, determined to 2.2 and 1.7 Å, respectively, and (ii) the human A2A adenosine receptor in complex with two different ligands determined to a resolution of 3.5 Å.
|
Oct 2023
|
|
