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Lewis J.
Williams
,
Amy J.
Thompson
,
Philipp
Dijkstal
,
Martin
Appleby
,
Greta
Assmann
,
Florian S. N.
Dworkowski
,
Nicole
Hiller
,
Chia-Ying
Huang
,
Tom
Mason
,
Samuel
Perrett
,
Eduard
Prat
,
Didier
Voulot
,
Bill
Pedrini
,
John H.
Beale
,
Michael A.
Hough
,
Jonathan A. R.
Worrall
,
Robin L.
Owen
Open Access
Abstract: Serial femtosecond crystallography (SFX) exploits extremely brief X-ray free-electron laser pulses to obtain diffraction data before destruction of the crystal. However, during the pulse X-ray-induced site-specific radiation damage can occur, leading to electronic state and/or structural changes. Here, we present a systematic exploration of the effect of single-pulse duration and energy (and consequently different dose rates) on site-specific radiation damage under typical SFX room-temperature experimental conditions. For the first time in SFX we directly measured the photon pulse duration, varying from less than 10 fs to more than 50 fs, and used three pulse energies to probe in-pulse damage in two radiation-sensitive proteins: the iron-heme peroxidase DtpAa and the disulfide-rich thaumatin. While difference-map features arising from radiation damage are observed, they do not lead to significant change in refined atomic coordinates or key bond lengths. Our work thus provides experimental verification that average atomic coordinates are not significantly perturbed by radiation damage in typical SFX experiments.
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May 2025
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Metrology
Optics
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Open Access
Abstract: The Optics & Metrology group at Diamond Light Source has recently published a description of a bimorph deformable X-ray mirror operating in closed-loop using multi-beam interferometric feedback. This "adaptive" mirror can make fast and stabilised changes to the X-ray beam profile. Beam shaping at a rate of 1 Hz was achieved, a contrast to the now usual "set and forget" operation of "active" bimorph mirrors at synchrotrons. However, this breakthrough cannot be applied to synchrotron beamlines without a robust control system that allows the mirror to be rapidly and controllably deformed. Diamond has now responded to this need by taking an integrated approach, considering: the bimorph power supplies, the beamline control software, the beam imaging camera, the bimorph mirror optimisation software, and the bimorph mirror itself as part of a single system. In collaboration with CINEL, new HV-ADAPTOS high-voltage power supplies have been made available. The latest models contain new firmware that adds features not previously available, such as piezo-elastic creep compensation. Communication with the HV-ADAPTOS power supplies over Ethernet has been made more reliable by a new EPICS asynPortDriver interface developed at Diamond and rolled out to all appropriate Diamond beamlines. A new Bluesky/Ophyd plan for the measurement of the bimorph mirror's piezo response functions is under development and has undergone its preliminary tests. This plan is expected to be less affected by upgrades of the beamline control software than previous solutions. It relies on beam images produced by Gigabit Ethernet cameras and processed by the EPICS areaDetector driver. Finally, the need for strain-free clamping of the mirror has now been fully recognized and procedures for ensuring it have been put into practice. Although such a system is more complex than that required for a mechanically bent mirror, it gives bimorph mirrors an ability to operate rapidly and repeatably that other optics do not offer, and it lays a foundation for more advanced beam-shaping functions.
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May 2025
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Protein–inhibitor crystal structures aid medicinal chemists in efficiently improving the potency and selectivity of small-molecule inhibitors. It is estimated that a quarter of lead molecules in drug discovery projects are halogenated. Protein–inhibitor crystal structures have shed light on the role of halogen atoms in ligand binding. They form halogen bonds with protein atoms and improve shape complementarity of inhibitors with protein binding sites. However, specific radiation damage (SRD) can cause cleavage of carbon–halogen (C–X) bonds during X-ray diffraction data collection. This study shows significant C–X bond cleavage in protein–ligand structures of the therapeutic cancer targets B-cell lymphoma 6 (BCL6) and heat shock protein 72 (HSP72) complexed with halogenated ligands, which is dependent on the type of halogen and chemical structure of the ligand. The study found that metrics used to evaluate the fit of the ligand to the electron density deteriorated with increasing X-ray dose, and that SRD eliminated the anomalous signal from brominated ligands. A point of diminishing returns is identified, where collecting highly redundant data reduces the anomalous signal that may be used to identify binding sites of low-affinity ligands or for experimental phasing. Straightforward steps are proposed to mitigate the effects of C–X bond cleavage on structures of proteins bound to halogenated ligands and to improve the success of anomalous scattering experiments.
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Dec 2024
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B22-Multimode InfraRed imaging And Microspectroscopy
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[31420, 32898, 37199]
Open Access
Abstract: Hemoglycin, a space polymer of glycine and iron, has been identified in the carbonaceous chondritic meteorites Allende, Acfer 086, Kaba, Sutter's Mill and Orgueil. Its core form has a mass of 1494 Da and is basically an antiparallel pair of polyglycine strands linked at each end by an iron atom. The polymer forms two- and three- dimensional lattices with an inter-vertex distance of 4.9 nm. Here the extraction technique for meteorites is applied to a 2.1 Gya fossil stromatolite to reveal the presence of hemoglycin by mass spectrometry. Intact ooids from a recent (3000 Ya) stromatolite exhibited the same visible hemoglycin fluorescence in response to x-rays as an intact crystal from the Orgueil meteorite. X-ray analysis confirmed the existence in ooids of an internal three-dimensional lattice of 4.9 nm inter-vertex spacing, matching the spacing of lattices in meteoritic crystals. FTIR measurements of acid-treated ooid and a Sutter's Mill meteoritic crystal both show the presence, via the splitting of the Amide I band, of an extended anti-parallel beta sheet structure. It seems probable that the copious in-fall of carbonaceous meteoritic material, from Archaean times onward, has left traces of hemoglycin in sedimentary carbonates and potentially has influenced ooid formation.
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Oct 2024
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Abstract: In serial crystallography, large numbers of microcrystals are sequentially delivered to an X-ray beam and a diffraction pattern is obtained from each crystal. This serial approach was developed primarily for X-ray Free Electron Lasers (XFELs) where crystals are destroyed by the beam but is increasingly used in synchrotron experiments. The combination of XFEL and synchrotron-based serial crystallography enables time-resolved experiments over an extremely wide range of time domains - from femtoseconds to seconds - and allows intact or pristine structures free of the effects of radiation damage to be obtained. Several approaches have been developed for sample delivery with varying levels of sample efficiency and ease of use. In the fixed target approach, microcrystals are loaded onto a solid support which is then rastered through the X-ray beam. The key advantages of fixed targets are that every crystal loaded can be used for data collection, and that precise control of when crystals are moved into the beam allows for time-resolved experiments over a very wide range of time domains as well as multi-shot experiments characterising the effects of the X-ray beam on the sample. We describe the application of fixed targets for serial crystallography as implemented at beamline I24 at Diamond Light Source and at the SACLA XFEL. We discuss methodologies for time-resolved serial crystallography in fixed targets and describe best practices for obtaining high-quality structures covering sample preparation, data collection strategies and data analysis pipelines.
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Oct 2024
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I24-Microfocus Macromolecular Crystallography
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Abstract: Time-resolved X-ray crystallography experiments were first performed in the 1980s, yet they remained a niche technique for decades. With the recent advent of X-ray free electron laser (XFEL) sources and serial crystallographic techniques, time-resolved crystallography has received renewed interest and has become more accessible to a wider user base. Despite this, time-resolved structures represent < 1 % of models deposited in the world-wide Protein Data Bank, indicating that the tools and techniques currently available require further development before such experiments can become truly routine. In this chapter, we demonstrate how applying data multiplexing to time-resolved crystallography can enhance the achievable time resolution at moderately intense monochromatic X-ray sources, ranging from synchrotrons to bench-top sources. We discuss the principles of multiplexing, where this technique may be advantageous, potential pitfalls, and experimental design considerations.
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Oct 2024
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Copper nitrite reductases (CuNiRs) exhibit a strong pH dependence of their catalytic activity. Structural movies can be obtained by serially recording multiple structures (frames) from the same spot of a crystal using the MSOX serial crystallography approach. This method has been combined with on-line single crystal optical spectroscopy to capture the pH-dependent structural changes that accompany during turnover of CuNiRs from two Rhizobia species. The structural movies, initiated by the redox activation of a type-1 copper site (T1Cu) via X-ray generated photoelectrons, have been obtained for the substrate-free and substrate-bound states at low (high enzymatic activity) and high (low enzymatic activity) pH. At low pH, formation of the product nitric oxide (NO) is complete at the catalytic type-2 copper site (T2Cu) after a dose of 3 MGy (frame 5) with full bleaching of the T1Cu ligand-to-metal charge transfer (LMCT) 455 nm band (S(σ)Cys → T1Cu2+) which in itself indicates the electronic route of proton-coupled electron transfer (PCET) from T1Cu to T2Cu. In contrast at high pH, the changes in optical spectra are relatively small and the formation of NO is only observed in later frames (frame 15 in Br2DNiR, 10 MGy), consistent with the loss of PCET required for catalysis. This is accompanied by decarboxylation of the catalytic AspCAT residue, with CO2 trapped in the catalytic pocket.
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Jul 2024
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[27314]
Open Access
Abstract: Human gamma-D crystallin (HGD) is a major constituent of the eye lens. Aggregation of HGD contributes to cataract formation, the leading cause of blindness worldwide. It is unique in its longevity, maintaining its folded and soluble state for 50-60 years. One outstanding question is the structural basis of this longevity despite oxidative aging and environmental stressors including ultraviolet radiation (UV). Here we present crystallographic structures evidencing a UV-induced crystallin redox switch mechanism. The room-temperature serial synchrotron crystallographic (SSX) structure of freshly prepared crystallin mutant (R36S) shows no post-translational modifications. After aging for nine months in the absence of light, a thiol-adduct (dithiothreitol) modifying surface cysteines is observed by low-dose SSX. This is shown to be UV-labile in an acutely light-exposed structure. This suggests a mechanism by which a major source of crystallin damage, UV, may also act as a rescuing factor in a finely balanced redox system.
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Apr 2024
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I24-Microfocus Macromolecular Crystallography
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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.
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Mar 2024
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I24-Microfocus Macromolecular Crystallography
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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.
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Oct 2023
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