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

Instruments / Technical Area	Publication Details	Published
	Successful sample preparation for serial crystallography experiments John H. Beale , Rachel Bolton , Stephen A. Marshall , Emma V. Beale , Stephen B. Carr , Ali Ebrahim , Tadeo Moreno-chicano , Michael A. Hough , Jonathan A. R. Worrall , Ivo Tews , Robin L. Owen Journal Of Applied Crystallography 52, 1385 - 1396 DOI: 10.1107/S1600576719013517 Open Access Show Abstract ▼ Abstract: Serial crystallography, at both synchrotron and X-ray free-electron laser light sources, is becoming increasingly popular. However, the tools in the majority of crystallization laboratories are focused on producing large single crystals by vapour diffusion that fit the cryo-cooled paradigm of modern synchrotron crystallography. This paper presents several case studies and some ideas and strategies on how to perform the conversion from a single crystal grown by vapour diffusion to the many thousands of micro-crystals required for modern serial crystallography grown by batch crystallization. These case studies aim to show (i) how vapour diffusion conditions can be converted into batch by optimizing the length of time crystals take to appear; (ii) how an understanding of the crystallization phase diagram can act as a guide when designing batch crystallization protocols; and (iii) an accessible methodology when attempting to scale batch conditions to larger volumes. These methods are needed to minimize the sample preparation gap between standard rotation crystallography and dedicated serial laboratories, ultimately making serial crystallography more accessible to all crystallographers.	Dec 2019
	High-throughput structures of protein–ligand complexes at room temperature using serial femtosecond crystallography Tadeo Moreno Chicano , Ali Ebrahim , Danny Axford , Martin V. Appleby , John H. Beale , Amanda K. Chaplin , Helen M. E. Duyvesteyn , Reza A. Ghiladi , Shigeki Owada , Darren A. Sherrell , Richard Strange , Hiroshi Sugimoto , Kensuke Tono , Jonathan A. R. Worrall , Robin L. Owen , Michael A. Hough Iucrj 6 DOI: 10.1107/S2052252519011655 Open Access Show Abstract ▼ Abstract: High-throughput X-ray crystal structures of protein–ligand complexes are critical to pharmaceutical drug development. However, cryocooling of crystals and X-ray radiation damage may distort the observed ligand binding. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) can produce radiation-damage-free room-temperature structures. Ligand-binding studies using SFX have received only modest attention, partly owing to limited beamtime availability and the large quantity of sample that is required per structure determination. Here, a high-throughput approach to determine room-temperature damage-free structures with excellent sample and time efficiency is demonstrated, allowing complexes to be characterized rapidly and without prohibitive sample requirements. This yields high-quality difference density maps allowing unambiguous ligand placement. Crucially, it is demonstrated that ligands similar in size or smaller than those used in fragment-based drug design may be clearly identified in data sets obtained from <1000 diffraction images. This efficiency in both sample and XFEL beamtime opens the door to true high-throughput screening of protein–ligand complexes using SFX.	Nov 2019
I24-Microfocus Macromolecular Crystallography	Dose-resolved serial synchrotron and XFEL structures of radiation-sensitive metalloproteins Ali Ebrahim , Tadeo Moreno-chicano , Martin V. Appleby , Amanda K. Chaplin , John Beale , Darren A. Sherrell , Helen M. E. Duyvesteyn , Shigeki Owada , Kensuke Tono , Hiroshi Sugimoto , Richard W. Strange , Jonathan Worrall , Danny Axford , Robin L. Owen , Michael A. Hough Iucrj 6 DOI: 10.1107/S2052252519003956 Diamond Proposal Number(s): [14493] Open Access Show Abstract ▼ Abstract: An approach is demonstrated to obtain, in a sample- and time-efficient manner, multiple dose-resolved crystal structures from room-temperature protein microcrystals using identical fixed-target supports at both synchrotrons and X-ray free-electron lasers (XFELs). This approach allows direct comparison of dose-resolved serial synchrotron and damage-free XFEL serial femtosecond crystallography structures of radiation-sensitive proteins. Specifically, serial synchrotron structures of a heme peroxidase enzyme reveal that X-ray induced changes occur at far lower doses than those at which diffraction quality is compromised (the Garman limit), consistent with previous studies on the reduction of heme proteins by low X-ray doses. In these structures, a functionally relevant bond length is shown to vary rapidly as a function of absorbed dose, with all room-temperature synchrotron structures exhibiting linear deformation of the active site compared with the XFEL structure. It is demonstrated that extrapolation of dose-dependent synchrotron structures to zero dose can closely approximate the damage-free XFEL structure. This approach is widely applicable to any protein where the crystal structure is altered by the synchrotron X-ray beam and provides a solution to the urgent requirement to determine intact structures of such proteins in a high-throughput and accessible manner.	Jul 2019
I04-Macromolecular Crystallography	A histidine residue and a tetranuclear cuprous‐thiolate cluster dominate the copper loading landscape of a copper storage protein from Streptomyces lividans Megan L. Straw , Michael A. Hough , Michael T. Wilson , Jonathan A. R. Worrall Chemistry – A European Journal DOI: 10.1002/chem.201901411 Diamond Proposal Number(s): [13467] Show Abstract ▼ Abstract: The chemical basis for protecting organisms against the toxic effect imposed by excess cuprous ions is to constrain this through high‐affinity binding sites that employ cuprous‐thiolate coordination chemistries. In bacteria, a family of cysteine rich four helix‐bundle proteins utilise thiolate chemistry to bind up to 80 cuprous ions. These proteins have been termed copper storage proteins (Csp). The present study investigates cuprous ion loading to the Csp from Streptomyces lividans (SlCsp) using a combination of X‐ray crystallography, site‐directed mutagenesis and stopped‐flow reaction kinetics with either aquatic cuprous ions or a chelating donor. We illustrate that at low cuprous ion concentrations, copper is loaded exclusively into an outer core region of SlCsp via one end of the four helix‐bundle, facilitated by a set of three histidine residues. X‐ray crystallography reveals the existence of polynuclear cuprous‐thiolate clusters culminating in the assembly of a tetranuclear [Cu4(μ2‐S‐Cys)4(Νδ1‐His)] cluster in the outer core. As more cuprous ions are loaded, the cysteine lined inner core of SlCsp fills with cuprous ions but in a fluxional and dynamic manner with no evidence for the assembly of further intermediate polynuclear cuprous‐thiolate clusters as observed in the outer core. Using site‐directed mutagenesis a key role for His107 in the efficient loading of cuprous ions from a donor is established. A model of copper loading to SlCsp is proposed and discussed.	May 2019
I02-Macromolecular Crystallography	A cytosolic copper storage protein provides a second level of copper tolerance in Streptomyces lividans Megan L. Straw , Amanda K. Chaplin , Michael A. Hough , Jordi Paps , Vassiliy N. Bavro , Michael T. Wilson , Erik Vijgenboom , Jonathan A. R. Worrall Metallomics 4 DOI: 10.1039/C7MT00299H Diamond Proposal Number(s): [13467] Show Abstract ▼ Abstract: Streptomyces lividans has a distinct dependence on the bioavailability of copper for its morphological development. A cytosolic copper resistance system is operative in S. lividans that serves to preclude deleterious copper levels. This system comprises of several CopZ-like copper chaperones and P1-type ATPases, predominantly under the transcriptional control of a metalloregulator from the copper sensitive operon repressor (CsoR) family. In the present study, we discover a new layer of cytosolic copper resistance in S. lividans that involves a protein belonging to the newly discovered family of copper storage proteins, which we have named Ccsp (cytosolic copper storage protein). From an evolutionary perspective, we find Ccsp homologues to be widespread in Bacteria and extend through into Archaea and Eukaryota. Under copper stress Ccsp is upregulated and consists of a homotetramer assembly capable of binding up to 80 cuprous ions (20 per protomer). X-ray crystallography reveals 18 cysteines, 3 histidines and 1 aspartate are involved in cuprous ion coordination. Loading of cuprous ions to Ccsp is a cooperative process with a Hill coefficient of 1.9 and a CopZ-like copper chaperone can transfer copper to Ccsp. A Δccsp mutant strain indicates that Ccsp is not required under initial copper stress in S. lividans, but as the CsoR/CopZ/ATPase efflux system becomes saturated, Ccsp facilitates a second level of copper tolerance.	Dec 2017
I03-Macromolecular Crystallography	Active site maturation and activity of the copper-radical oxidase GlxA is governed by a tryptophan residue Amanda K. Chaplin , Dimitri A. Svistunenko , Michael Hough , Michael T. Wilson , Erik Vijgenboom , Jonathan A. R. Worrall Biochemical Journal DOI: 10.1042/BCJ20160968 Diamond Proposal Number(s): [7461] Show Abstract ▼ Abstract: GlxA from Streptomyces lividans is a mononuclear copper-radical oxidase and a member of the auxiliary activity family 5 (AA5). Its domain organisation and low sequence homology make it a distinct member of the AA5 family in which the fungal galactose 6-oxidase (Gox) is the best-characterized. GlxA is a key cuproenzyme in the copper-dependent morphological development of S. lividans with a function that is linked to the processing of an extracytoplasmic glycan. The catalytic site in GlxA and Gox contain two distinct one-electron acceptors comprising the copper ion and a 3'-(S-cysteinyl) tyrosine. The latter is formed post-translationally through a covalent bond between a cysteine and a copper coordinating tyrosine ligand and houses a radical. In GlxA and Gox a second coordination sphere tryptophan residue (Trp288 in GlxA) is present, but the orientation of the indole ring differs between the two enzymes creating a marked difference in the π-π stacking interaction of the benzyl ring with the 3'-(S-cysteinyl) tyrosine. Differences in the spectroscopic and enzymatic activity have been reported between GlxA and Gox with the indole orientation suggested as a reason. Here we report a series of in vivo and in vitro studies using the W288F and W288A variants of GlxA to assess the role of Trp288 on the morphology, maturation, spectroscopic and enzymatic properties. Our findings point towards a salient role for Trp288 in the kinetics of copper loading and maturation of GlxA, with its presence essential for stabilising the metalloradical site required for coupling catalytic activity and morphological development.	Jan 2017
I03-Macromolecular Crystallography	Heterogeneity in the Histidine-brace Copper Coordination Sphere in Auxiliary Activity Family 10 (AA10) Lytic Polysaccharide Monooxygenases Amanda Chaplin , Michael T. Wilson , Mike Hough , Dimitri A. Svistunenko , Glyn R. Hemsworth , Paul Walton , Erik Vijgenboom , Jonathan Worrall Journal Of Biological Chemistry 291, 12838 - 12850 DOI: 10.1074/jbc.M116.722447 Diamond Proposal Number(s): [7461] Show Abstract ▼ Abstract: Copper-dependent lytic polysaccharide monooxygenases (LPMOs) are enzymes that oxidatively deconstruct polysaccharides. The active site copper in LPMOs is coordinated by a histidine-brace. This utilizes the amino group and side chain of the N-terminal His residue with the side chain of a second His residue to create a T-shaped arrangement of nitrogen ligands. We report a structural, kinetic, and thermodynamic appraisal of copper binding to the histidine-brace in an auxiliary activity family 10 (AA10) LPMO from Streptomyces lividans (SliLPMO10E). Unexpectedly, we discovered the existence of two apo-SliLPMO10E species in solution that can each bind copper at a single site with distinct kinetic and thermodynamic (exothermic and endothermic) properties. The experimental EPR spectrum of copper-bound SliLPMO10E requires the simulation of two different line shapes, implying two different copper-bound species, indicative of three and two nitrogen ligands coordinating the copper. Amino group coordination was probed through the creation of an N-terminal extension variant (SliLPMO10E-Ext). The kinetics and thermodynamics of copper binding to SliLPMO10E-Ext are in accord with copper binding to one of the apo-forms in the wild-type protein, suggesting that amino group coordination is absent in the two-nitrogen coordinate form of SliLPMO10E. Copper binding to SliLPMO10B was also investigated, and again it revealed the presence of two apo-forms with kinetics and stoichiometry of copper binding identical to that of SliLPMO10E. Our findings highlight that heterogeneity exists in the active site copper coordination sphere of LPMOs that may have implications for the mechanism of loading copper in the cell.	Jun 2016
I03-Macromolecular Crystallography	Structural insights into conformational switching in the copper metalloregulator CsoR from T. V. Porto , M. A. Hough , J. A. R. Worrall Acta Crystallographica Section D Biological Crystallography 71, 1872 - 1878 DOI: 10.1107/S1399004715013012 PMID: 26327377 Diamond Proposal Number(s): [9475] Show Abstract ▼ Abstract: Copper-sensitive operon repressors (CsoRs) act to sense cuprous ions and bind them with a high affinity under copper stress in many bacteria. The binding of copper(I) leads to a conformational change in their homotetramer structure, causing disassembly of the operator DNA-CsoR complex and evoking a transcriptional response. Atomic-level structural insight into the conformational switching mechanism between the apo and metal-bound states is lacking. Here, a new X-ray crystal structure of the CsoR from Streptomyces lividans is reported and compared with a previously reported S. lividans CsoR X-ray structure crystallized under different conditions. Based on evidence from this new X-ray structure, it is revealed that the conformational switching between states centres on a concertina effect at the C-terminal end of each α2 helix in the homotetramer. This drives the Cys104 side chain, a copper(I)-ligating residue, into a position enabling copper(I) coordination and as a result disrupts the α2-helix geometry, leading to a compacting and twisting of the homotetramer structure. Strikingly, the conformational switching induces a redistribution of electrostatic surface potential on the tetrameric DNA-binding face, which in the copper(I)-bound state would no longer favour interaction with the mode of operator DNA binding.	Sep 2015
I03-Macromolecular Crystallography	GlxA is a new structural member of the radical copper oxidase family and is required for glycan deposition at hyphal tips and morphogenesis of Streptomyces lividans Amanda Chaplin , Marloes Petrus , Giulia Mangiameli , Mike Hough , Dimitri Svistunenko , Peter Nicholls , Dennis Claessen , Eric Vijgenboom , Jonathan Worrall Biochemical Journal DOI: 10.1042/BJ20150190 PMID: 26205496 Diamond Proposal Number(s): [7461] Show Abstract ▼ Abstract: Streptomyces lividans displays a distinct dependence on copper to fully initiate morphological development. Evidence has accumulated to implicate the participation of an extracytoplasmic cuproenzyme in morphogenesis. Here we show that GlxA fulfils all criteria to be that cuproenzyme. GlxA is membrane associated and has an active site consisting of a mononuclear copper and a cross-linked Tyr-Cys co-factor. The domain organisation of the tertiary structure defines GlxA as a new structural member of the mono-copper oxidase family, with copper coordination geometry similar to, but spectroscopically distinct from fungal galactose oxidase. Electron paramagnetic resonance spectroscopy reveals that the oxidation of cupric GlxA generates a protein radical residing on the Tyr-Cys cross-link. A variety of canonical galactose oxidase substrates (including D-galactose) were tested but none were readily turned over by GlxA. A glxA null-mutant leads to loss of glycan accumulation at hyphal tips and consequently a drastically changed morphology both on solid substrates and in liquid-grown environments, a scenario similarly observed in the absence of the neighbouring glycan synthase CslA. In addition the glxA mutant has lost the stimulation of development by copper, supporting a model whereby the enzymatic action of GlxA on the glycan is required for development and morphology. From a biotechnology perspective the open mycelium morphology observed with the glxA mutant in submerged culture has implications for use as an enzyme production host.	Jun 2015
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structural and mechanistic insights into an extracytoplasmic copper trafficking pathway in Streptomyces lividans Mike Hough , Katie Blundell , Jonathan Worrall , Erik Vijgenboom Biochemical Journal DOI: 10.1042/BJ20140017 PMID: 24548299 Diamond Proposal Number(s): [7641] Show Abstract ▼ Abstract: In Streptomyces lividans an extracytoplasmic copper-binding Sco protein plays a role in two unlinked processes: (i) initiating a morphological development switch and (ii) facilitating the co-factoring of the CuA domain of CcO (cytochrome c oxidase). How Sco obtains copper once secreted to the extracytoplasmic environment is unknown. In the present paper we report on a protein possessing an HX6MX21HXM motif that binds a single cuprous ion with subfemtomolar affinity. High-resolution X-ray structures of this extracytoplasmic copper chaperone-like protein (ECuC) in the apo- and Cu(I)-bound states reveal that the latter possesses a surface-accessible cuprous-ion-binding site located in a dish-shaped region of ?-sheet structure. A cuprous ion is transferred under a favourable thermodynamic gradient from ECuC to Sco with no back transfer occurring. The ionization properties of the cysteine residues in the Cys86xxxCys90 copper-binding motif of Sco, together with their positional locations identified from an X-ray structure of Sco, suggests a role for Cys86 in initiating an inter-complex ligand-exchange reaction with Cu(I)–ECuC. Generation of the genetic knockouts, ?sco, ?ecuc and ?sco/ecuc, and subsequent in vivo assays lend support to the existence of a branched extracytoplasmic copper-trafficking pathway in S. lividans. One branch requires both Sco and to a certain extent ECuC to cofactor the CuA domain, whereas the other uses only Sco to deliver copper to a cuproenzyme to initiate morphological development.	Feb 2014

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