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23 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3 [Next/Last]

Instruments / Technical Area	Publication Details	Published
	Damage before destruction? X-ray-induced changes in single-pulse serial femtosecond crystallography 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 Iucrj 12 DOI: 10.1107/S2052252525002660 Open Access Show Abstract ▼ 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.	May 2025
VMXi-Versatile Macromolecular Crystallography in situ	Efficient in situ screening of and data collection from microcrystals in crystallization plates Amy J. Thompson , Juan Sanchez-Weatherby , Lewis J. Williams , Halina Mikolajek , James Sandy , Jonathan A. R. Worrall , Michael A. Hough Acta Crystallographica Section D Structural Biology 80 DOI: 10.1107/S2059798324001955 Open Access Show Abstract ▼ Abstract: A considerable bottleneck in serial crystallography at XFEL and synchrotron sources is the efficient production of large quantities of homogenous, well diffracting microcrystals. Efficient high-throughput screening of batch-grown microcrystals and the determination of ground-state structures from different conditions is thus of considerable value in the early stages of a project. Here, a highly sample-efficient methodology to measure serial crystallography data from microcrystals by raster scanning within standard in situ 96-well crystallization plates is described. Structures were determined from very small quantities of microcrystal suspension and the results were compared with those from other sample-delivery methods. The analysis of a two-dimensional batch crystallization screen using this method is also described as a useful guide for further optimization and the selection of appropriate conditions for scaling up microcrystallization.	Apr 2024
I24-Microfocus Macromolecular Crystallography	A redox switch allows binding of Fe(II) and Fe(III) ions in the cyanobacterial iron-binding protein FutA from Prochlorococcus 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 Proceedings Of The National Academy Of Sciences 121 DOI: 10.1073/pnas.2308478121 Diamond Proposal Number(s): [15722, 14493, 23570] Open Access Show Abstract ▼ 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
I04-Macromolecular Crystallography	New insights into controlling radical migration pathways in heme enzymes gained from the study of a dye-decolorising peroxidase Jonathan A. R. Worrall , Marina Lucic , Michael Wilson , Jacob Pullin , Michael A. Hough , Dimitri Svistunenko Chemical Science DOI: 10.1039/D3SC04453J Diamond Proposal Number(s): [25108] Open Access Show Abstract ▼ Abstract: In heme enzymes, such as members of the dye-decolorising peroxidase (DyP) family, the formation of the highly oxidising catalytic Fe(IV)-oxo intermediates following reaction with hydrogen peroxide can lead to free radical migration (hole hopping) from the heme to form cationic tyrosine and/or tryptophan radicals. These species are highly oxidising (~ 1 V vs NHE) and under certain circumstances can catalyse the oxidation of organic substrates. Factors that govern which specific tyrosine or tryptophan the free radical migrates to in heme enzymes are not well understood, although in the case of tyrosyl radical formation the nearby proximity of a proton acceptor is a recognised facilitating factor. By using an A-type member of the DyP family (DtpAa) as an exemplar, we combine protein engineering, X-ray crystallography, hole-hopping calculations, EPR spectroscopy and kinetic modelling to provide compelling new insights into the control of radical migration pathways following reaction of the heme with hydrogen peroxide. We demonstrate that the presence of a tryptophan/tyrosine dyad motif displaying a T-shaped orientation of aromatic rings on the proximal side of the heme dominates the radical migration landscape in wild-type DtpAa and continues to do so following the rational engineering into DtpAa of a previously identified radical migration pathway in an A-type homolog on the distal side of the heme. Only on disrupting the proximal dyad, through removal of an oxygen atom, does the radical migration pathway then switch to the engineered distal pathway to form the desired tyrosyl radical. Implications for protein design and biocatalysis are discussed.	Oct 2023
	Perspective: Structure determination of protein-ligand complexes at room temperature using X-ray diffraction approaches Michael A. Hough , Filippo Prischi , Jonathan A. R. Worrall Frontiers In Molecular Biosciences 10 DOI: 10.3389/fmolb.2023.1113762 Open Access Show Abstract ▼ Abstract: The interaction between macromolecular proteins and small molecule ligands is an essential component of cellular function. Such ligands may include enzyme substrates, molecules involved in cellular signalling or pharmaceutical drugs. Together with biophysical techniques used to assess the thermodynamic and kinetic properties of ligand binding to proteins, methodology to determine high-resolution structures that enable atomic level interactions between protein and ligand(s) to be directly visualised is required. Whilst such structural approaches are well established with high throughput X-ray crystallography routinely used in the pharmaceutical sector, they provide only a static view of the complex. Recent advances in X-ray structural biology methods offer several new possibilities that can examine protein-ligand complexes at ambient temperature rather than under cryogenic conditions, enable transient binding sites and interactions to be characterised using time-resolved approaches and combine spectroscopic measurements from the same crystal that the structures themselves are determined. This Perspective reviews several recent developments in these areas and discusses new possibilities for applications of these advanced methodologies to transform our understanding of protein-ligand interactions.	Jan 2023
	Serial femtosecond crystallography approaches to understanding catalysis in iron enzymes Jonathan A. R. Worrall , Michael A. Hough Current Opinion In Structural Biology 77 DOI: 10.1016/j.sbi.2022.102486 Open Access Show Abstract ▼ Abstract: Enzymes with iron-containing active sites play crucial roles in catalysing a myriad of oxidative reactions essential to aerobic life. Defining the three-dimensional structures of iron enzymes in resting, oxy-bound intermediate and substrate-bound states is particularly challenging, not least because of the extreme susceptibility of the Fe(III) and Fe(IV) redox states to radiation-induced chemistry caused by intense X-ray or electron beams. The availability of novel sources such as X-ray free electron lasers has enabled structures that are effectively free of the effects of radiation-induced chemistry and allows time-resolved structures to be determined. Important to both applications is the ability to obtain in crystallo spectroscopic data to identify the redox state of the iron in any particular structure or timepoint.	Dec 2022
I24-Microfocus Macromolecular Crystallography	Serial femtosecond crystallography reveals the role of water in the one- or two-electron redox chemistry of compound I in the catalytic cycle of the B-type dye-decolorizing peroxidase DtpB Marina Lucic , Matthew T. Wilson , Takehiko Tosha , Hiroshi Sugimoto , Anastasiia Shilova , Danny Axford , Robin L. Owen , Michael A. Hough , Jonathan A. R. Worrall Acs Catalysis 26, 13349 - 13359 DOI: 10.1021/acscatal.2c03754 Diamond Proposal Number(s): [25108, 28583] Open Access Show Abstract ▼ Abstract: Controlling the reactivity of high-valent Fe(IV)–O catalytic intermediates, Compounds I and II, generated in heme enzymes upon reaction with dioxygen or hydrogen peroxide, is important for function. It has been hypothesized that the presence (wet) or absence (dry) of distal heme pocket water molecules can influence whether Compound I undergoes sequential one-electron additions or a concerted two-electron reduction. To test this hypothesis, we investigate the role of water in the heme distal pocket of a dye-decolorizing peroxidase utilizing a combination of serial femtosecond crystallography and rapid kinetic studies. In a dry distal heme site, Compound I reduction proceeds through a mechanism in which Compound II concentration is low. This reaction shows a strong deuterium isotope effect, indicating that reduction is coupled to proton uptake. The resulting protonated Compound II (Fe(IV)–OH) rapidly reduces to the ferric state, giving the appearance of a two-electron transfer process. In a wet site, reduction of Compound I is faster, has no deuterium effect, and yields highly populated Compound II, which is subsequently reduced to the ferric form. This work provides a definitive experimental test of the hypothesis advanced in the literature that relates sequential or concerted electron transfer to Compound I in wet or dry distal heme sites.	Oct 2022
I24-Microfocus Macromolecular Crystallography	Complementarity of neutron, XFEL and synchrotron crystallography for defining the structures of metalloenzymes at room temperature Tadeo Moreno-Chicano , Leiah M. Carey , Danny Axford , John H. Beale , R. Bruce Doak , Helen M. E. Duyvesteyn , Ali Ebrahim , Robert W. Henning , Diana C. F. Monteiro , Dean A. Myles , Shigeki Owada , Darren A. Sherrell , Megan L. Straw , Vukica Šrajer , Hiroshi Sugimoto , Kensuke Tono , Takehiko Tosha , Ivo Tews , Martin Trebbin , Richard W. Strange , Kevin L. Weiss , Jonathan A. R. Worrall , Flora Meilleur , Robin L. Owen , Reza A. Ghiladi , Michael A. Hough Iucrj 9 DOI: 10.1107/S2052252522006418 Diamond Proposal Number(s): [14493] Open Access Show Abstract ▼ Abstract: Room-temperature macromolecular crystallography allows protein structures to be determined under close-to-physiological conditions, permits dynamic freedom in protein motions and enables time-resolved studies. In the case of metalloenzymes that are highly sensitive to radiation damage, such room-temperature experiments can present challenges, including increased rates of X-ray reduction of metal centres and site-specific radiation-damage artefacts, as well as in devising appropriate sample-delivery and data-collection methods. It can also be problematic to compare structures measured using different crystal sizes and light sources. In this study, structures of a multifunctional globin, dehaloperoxidase B (DHP-B), obtained using several methods of room-temperature crystallographic structure determination are described and compared. Here, data were measured from large single crystals and multiple microcrystals using neutrons, X-ray free-electron laser pulses, monochromatic synchrotron radiation and polychromatic (Laue) radiation light sources. These approaches span a range of 18 orders of magnitude in measurement time per diffraction pattern and four orders of magnitude in crystal volume. The first room-temperature neutron structures of DHP-B are also presented, allowing the explicit identification of the hydrogen positions. The neutron data proved to be complementary to the serial femtosecond crystallography data, with both methods providing structures free of the effects of X-ray radiation damage when compared with standard cryo-crystallography. Comparison of these room-temperature methods demonstrated the large differences in sample requirements, data-collection time and the potential for radiation damage between them. With regard to the structure and function of DHP-B, despite the results being partly limited by differences in the underlying structures, new information was gained on the protonation states of active-site residues which may guide future studies of DHP-B.	Sep 2022
I24-Microfocus Macromolecular Crystallography	Aspartate or arginine? Validated redox state X-ray structures elucidate mechanistic subtleties of FeIV = O formation in bacterial dye-decolorizing peroxidases Marina Lucic , Michael T. Wilson , Dimitri A. Svistunenko , Robin L. Owen , Michael A. Hough , Jonathan A. R. Worrall Jbic Journal Of Biological Inorganic Chemistry 65 DOI: 10.1007/s00775-021-01896-2 Diamond Proposal Number(s): [18565] Open Access Show Abstract ▼ Abstract: Structure determination of proteins and enzymes by X-ray crystallography remains the most widely used approach to complement functional and mechanistic studies. Capturing the structures of intact redox states in metalloenzymes is critical for assigning the chemistry carried out by the metal in the catalytic cycle. Unfortunately, X-rays interact with protein crystals to generate solvated photoelectrons that can reduce redox active metals and hence change the coordination geometry and the coupled protein structure. Approaches to mitigate such site-specific radiation damage continue to be developed, but nevertheless application of such approaches to metalloenzymes in combination with mechanistic studies are often overlooked. In this review, we summarize our recent structural and kinetic studies on a set of three heme peroxidases found in the bacterium Streptomyces lividans that each belong to the dye decolourizing peroxidase (DyP) superfamily. Kinetically, each of these DyPs has a distinct reactivity with hydrogen peroxide. Through a combination of low dose synchrotron X-ray crystallography and zero dose serial femtosecond X-ray crystallography using an X-ray free electron laser (XFEL), high-resolution structures with unambiguous redox state assignment of the ferric and ferryl (FeIV = O) heme species have been obtained. Experiments using stopped-flow kinetics, solvent-isotope exchange and site-directed mutagenesis with this set of redox state validated DyP structures have provided the first comprehensive kinetic and structural framework for how DyPs can modulate their distal heme pocket Asp/Arg dyad to use either the Asp or the Arg to facilitate proton transfer and rate enhancement of peroxide heterolysis.	Sep 2021
I24-Microfocus Macromolecular Crystallography Data acquisition	Fixed target serial data collection at Diamond Light Source Sam Horrell , Danny Axford , Nicholas E. Devenish , Ali Ebrahim , Michael A. Hough , Darren A. Sherrell , Selina L. S. Storm , Ivo Tews , Jonathan A. R. Worrall , Robin L. Owen Journal Of Visualized Experiments DOI: 10.3791/62200 Open Access Show Abstract ▼ Abstract: Serial data collection is a relatively new technique for synchrotron users. A user manual for fixed target data collection at I24, Diamond Light Source is presented with detailed step-by-step instructions, figures, and videos for smooth data collection.	Feb 2021

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