Daresbury Synchrotron Research Articles

In the beginning was the U1A protein: a personal reflection.

In the beginning was the U1A protein: a personal reflection.

Crystallization and preliminary X-ray analysis of a bifunctional catalase-phenol oxidase from Scytalidium thermophilum.

Catalase-phenol oxidase from Scytalidium thermophilum is a bifunctional enzyme: its major activity is the catalase-mediated decomposition of hydrogen peroxide, but it also catalyzes phenol oxidation. To understand the structural basis of this dual functionality, the enzyme, which has been shown to be a tetramer in solution, has been purified by anion-exchange and gel-filtration chromatography and has been crystallized using the hanging-drop vapour-diffusion technique. Streak-seeding was used to obtain larger crystals suitable for X-ray analysis. Diffraction data were collected to 2.8 A resolution at the Daresbury Synchrotron Radiation Source. The crystals belonged to space group P2(1) and contained one tetramer per asymmetric unit.

Time-resolved diffraction studies into the pressure acid leaching of nickel laterite ores: a comparison of laboratory and synchrotron X-ray experiments

This paper compares time-resolved diffraction experiments that have been performed using laboratory and synchrotron X-ray sources. The experiments investigated the mechanism and kinetics of pressure acid leaching of nickel laterite ores. The sample environment was a purpose-built capillary reaction vessel, and extensive method development was conducted in the laboratory using Mo Kα radiation prior to repeating and extending the experiments at the Daresbury Synchrotron Radiation Source, beamline MPW6.2. In general, the synchrotron results confirmed the findings from previously reported laboratory work and also confirmed the presence of a minor phase that had been ambiguous in the laboratory experiments,i.e.the formation of hematite in the pressure acid leaching of saprolite. The synchrotron measurements also extended the experimental programme to include poorly diffracting laterite components that could not be examined in the laboratory,e.g.nontronite. The results from these components supported the reaction mechanisms determined fromex-situanalyses conducted in larger scale autoclaves.

Analysis of crystalline phases in airborne particulate matter by two-dimensional X-ray diffraction (XRD2)

In this work, the potentiality of two-dimensional X-ray diffraction (XRD(2)) to characterise aerosol particles collected on commercial glass filters is presented. Indeed, even if routine analysis usually requires only mass determination, and rarely chemical composition, phase determination is fundamental to recognize the primary or secondary origin of the particulate matter and thus to determine the main sources of the pollution and to model contamination events. The experiments were performed at Daresbury Synchrotron (UK) Laboratory on 14.1 Beamline. The analysis of filters collected in Tuscany (Italy) is discussed with particular attention to the presence of arsenic sulfide. The first results of these experiments are very promising, showing the presence of unexpected compounds in the particulate matter of the investigated area.

Design and practices at four European synchrotron radiation facilities

The European Directive 96/29/Euratom of 13/05/1996, based on the ICRP 60 Recommendations, defines basic safety standards for the protection of the health of workers and the general public against the dangers arising from ionising radiation. Member states of the European Union have had to integrate these basic standards in their national legislation. Synchrotron radiation facilities are affected by this new radiation protection legislation. In the UK, the Daresbury Synchrotron Radiation Source (SRS) operates under the Ionising Radiations Regulations 1999, and the third generation Diamond Light Source (DLS) project is largely benefiting from their experience. In France, the ESRF case is presently being used to define detailed prescriptions, that will also apply to future sources in this country, for example the third generation light source Synchrotron Soleil, presently under construction. This paper summarises general policies and practices of these four SR facilities: SRS, European Synchrotron Radiation Facility (ESRF), DLS and Synchrotron Soleil.

A Comparative Study of Uracil-DNA Glycosylases from Human and Herpes Simplex Virus Type 1

Uracil-DNA glycosylase (UNG) is the key enzyme responsible for initiation of base excision repair. We have used both kinetic and binding assays for comparative analysis of UNG enzymes from humans and herpes simplex virus type 1 (HSV-1). Steady-state fluorescence assays showed that hUNG has a much higher specificity constant (k(cat)/K(m)) compared with the viral enzyme due to a lower K(m). The binding of UNG to DNA was also studied using a catalytically inactive mutant of UNG and non-cleavable substrate analogs (2'-deoxypseudouridine and 2'-alpha-fluoro-2'-deoxyuridine). Equilibrium DNA binding revealed that both human and HSV-1 UNG enzymes bind to abasic DNA and both substrate analogs more weakly than to uracil-containing DNA. Structure determination of HSV-1 D88N/H210N UNG in complex with uracil revealed detailed information on substrate binding. Together, these results suggest that a significant proportion of the binding energy is provided by specific interactions with the target uracil. The kinetic parameters for human UNG indicate that it is likely to have activity against both U.A and U.G mismatches in vivo. Weak binding to abasic DNA also suggests that UNG activity is unlikely to be coupled to the subsequent common steps of base excision repair.

Crystal structure of dihydrodipicolinate synthase (BA3935) from Bacillus anthracis at 1.94 Å resolution

Introduction. As part of a structural genomics program, we are determining structures of proteins from the causative agent of anthrax, Bacillus anthracis, a Grampositive spore-forming bacterium. Among our initial candidates for crystallographic analysis are the products of essential genes based on knock-out studies in Bacillus subtilis. The BA3935 gene of B. anthracis (www.tigr.org), annotated as DapA2, encodes a putative protein consisting of 292 amino acid residues with a subunit molecular weight of 31,233 Da. The predicted protein has 60% identity with dihydrodipicolinate synthase (DHDPS or DapA) from B. subtilis and 40% amino acid sequence identity to its orthologue in Escherichia coli. dapA is one of only 271 out of the total of 4118 genes in B. subtilis that are indispensable for growth of the organism on standard laboratory media. 1 DHDPS catalyses the condensation of aspartate semialdehyde and pyruvate and is the first committed step on the pathway to diaminopimelate and L-lysine in prokaryotes, some fungi, and higher plants (Scheme 1). The product released from the E. coli enzyme has been shown to be 4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid (HTDPA) rather than L-2,3-dihydrodipicolinic acid (DHDPA); as the name of the enzyme suggests, 2 DHDPA can be formed spontaneously from HTDPA by elimination of water. The steps of aspartate semialdehyde synthesis from aspartate are shared with the biosynthetic pathways leading methionine and threonine. The diaminopimelate/ lysine pathway is thought to be of particular importance in Gram-positive bacteria because diaminopimelate makes up a higher proportion of the dry cell weight than it does in Gram-negative bacteria, a consequence of the thicker cell wall in the former. In Bacilli, the product of the DHDPS reaction is further reduced by dipicolinate synthase to dipicolinate, which makes up 10% of the dry weight of spores. Crystal structures have been determined of DHDPS from E. coli, 3 Nicotiana sylvestris, 4 and most recently from Thermotoga maritima. 5 In this article, we report the crystal structure of DHDPS from B. anthracis (Ba DHDPS), the first structure of a dihydrodipicolinate synthase from a Gram-positive bacterium. The structure of Ba DHDPS was determined by molecular replacement using the coordinate set for the E. coli orthologue (PDB code 1DHP) as the search model. 3 Two structures have been determined to 1.9 and 2.2 A resolution in different orthorhombic crystal forms. Data collection, refinement, and model-building statistics are summarized in Table I. The structures in the two crystal forms are very similar and unless otherwise stated, the commentary here will refer to the structure refined to higher resolution

Crystal structure of PurE (BA0288) from Bacillus anthracis at 1.8 Å resolution

Crystal structure of PurE (BA0288) from <i>Bacillus anthracis</i> at 1.8 Å resolution

Probing the Mechanism of Ligand Recognition in Family 29 Carbohydrate-binding Modules

The recycling of photosynthetically fixed carbon, by the action of microbial plant cell wall hydrolases, is integral to one of the major geochemical cycles and is of considerable industrial importance. Non-catalytic carbohydrate-binding modules (CBMs) play a key role in this degradative process by targeting hydrolytic enzymes to their cognate substrate within the complex milieu of polysaccharides that comprise the plant cell wall. Family 29 CBMs have, thus far, only been found in an extracellular multienzyme plant cell wall-degrading complex from the anaerobic fungus Piromyces equi, where they exist as a CBM29-1:CBM29-2 tandem. Here we present both the structure of the CBM29-1 partner, at 1.5 A resolution, and examine the importance of hydrophobic stacking interactions as well as direct and solvent-mediated hydrogen bonds in the binding of CBM29-2 to different polysaccharides. CBM29 domains display unusual binding properties, exhibiting specificity for both beta-manno- and beta-gluco-configured ligands such as mannan, cellulose, and glucomannan. Mutagenesis reveals that "stacking" of tryptophan residues in the n and n+2 subsites plays a critical role in ligand binding, whereas the loss of tyrosine-mediated stacking in the n+4 subsite reduces, but does not abrogate, polysaccharide recognition. Direct hydrogen bonds to ligand, such as those provided by Arg-112 and Glu-78, play a pivotal role in the interaction with both mannan and cellulose, whereas removal of water-mediated interactions has comparatively little effect on carbohydrate binding. The interactions of CBM29-2 with the O2 of glucose or mannose contribute little to binding affinity, explaining why this CBM displays dual gluco/manno specificity.

Characterisation of a new VUV beamline at the Daresbury SRS using a dispersed fluorescence apparatus incorporating CCD detection

The design and performance of a new normal incidence monochromator at the Daresbury Synchrotron Radiation Source, optimised for experiments requiring high flux of vacuum-UV radiation, are described. The re-developed beamline 3.1, based on the Wadsworth design of monochromator, is the source of tunable vacuum-UV photons in the range 4–31 eV, providing over two orders of magnitude more flux than the vacuum-UV, Seya monochromator in its previous manifestation. The undispersed and dispersed fluorescence spectra resulting from photoexcitation of N 2, CO 2, CF 4 and C 6F 6 are presented. Emitting species observed were N 2 + B 2 Σ u + – X 2 Σ g + , CO 2 + A ˜ 2 Π u – X ˜ 2 Π g and B ˜ 2 Σ u + – X ˜ 2 Π g , CF 4 + C ˜ 2 T 2 – X ˜ 2 T 1 and C ˜ 2 T 2 – A ˜ 2 T 2 , CF 3 ∗ 2 A 2 ′ – 2 A 2 , and C 6 F 6 + B ˜ 2 A 2 u – X ˜ 2 E 1 g . A CCD multi-channel detector has significantly reduced the time period needed to record dispersed fluorescence spectra with a comparable signal-to-noise ratio.

Measurements of angular distribution for photoionization of mercury into the 5d9 2D5/2 ionic state over the energy range from 15 eV to 17 eV

Differential cross sections for photoionization into the 5d96s2 2D5/2 ionic state of mercury have been obtained over the photon energy range from 15 eV to 17 eV. By measuring the photoelectron emission at the angles of 0o and 90o with respect to the major axis of the polarization ellipse the variation of the asymmetry β-parameter for the 2D5/2 ionic state of mercury has been determined as a continuous function of photon energy over the above range. This energy range contains three series of autoionizing states converging to the 2D3/2 ionization limit. The measurements have been performed using the 5 m McPherson normal incidence monochromator at the Daresbury Synchrotron Radiation Source with a photon energy spread of typically 3 meV.

Diffraction-enhanced imaging at the UK synchrotron radiation source

The Diffraction-Enhanced Imaging (DEI) system, which shares access to Beamline 7.6 on the Daresbury Synchrotron Radiation Source (SRS), is now in its third year of existence. The system was developed under a European Commission grant PHase Analyser SYstem (PHASY), won during the Fourth Framework. Typical applications continue to be the imaging of small biological specimens, using a beam of 12–17 keV after monochromation and up to 40 mm in width and 1–2 mm in height, although it is planned to investigate other materials as opportunity permits and time becomes available for more routine scientific use. Recent improvements have been made to the optical alignment procedure for setting up the station before imaging: a small laser device can now be set up to send a beam down the X-ray path through the four crystals, and a small photodiode, which has much better signal-to-noise characteristics than the ion chamber normally used for alignment, has been trailed successfully. A 3-D tomographic reconstruction capability has recently been developed and tested for DEI projection image sets, and will be applied to future imaging work on the SRS, in conjunction with volume visualization software. The next generation of DEI system, planned to operate at up to 60 keV on an SRS wiggler station, is in its design stage; it will feature much improved mechanics and mountings, especially for angular control, and a simplified alignment procedure to facilitate the necessary sharing of the SRS station.

Crystal structure of mahnertite

Mahnertite, (Na 0.88 Ca 0.12 )Cu 2.75 [AsO 4 ] 2 Cl 0.62 ·3.63 H 2 O, M = 565.17, is tetragonal, with space group I 4/ mmm, a = 10.037(1), c = 23.739(1) A, V = 2391.5(4) A 3 , Z = 8, ρ o = 3.32, ρ c = 3.14 g/cm 3 , μ = 9.78 mm -1 . The crystal structure of mahnertite was solved and refined against data collected at the Daresbury Synchrotron Radiation Source, using a 20 × 20 × 2 μm 3 single crystal, Λ = 0.6898 A, F (000) = 2150, R = 0.049 for 639 reflections with I > 2σ( I ). The structure consists of sheets parallel to (001), which comprise two symmetrically independent distorted Cu 5-vertex square pyramids Cuϕ 5 (ϕ =O, Cl or H 2 O) as well as AsO 4 tetrahedra. Four Cuϕ 5 pyramids located around the 4-fold axis share edges to form clusters linked by AsO 4 tetrahedra. The sheets are linked by the common apical vertex of the Cu(2)ϕ 5 polyhedra. As a result, a heterogeneous framework is formed. (Na,Ca) atoms, additional Cu(3) cations and H 2 O molecules are located in the interlayers. The mineral is structurally related to zdenekite, NaPbCu 5 (AsO 4 ) 4 Cl·5H 2 O.

Mechanical Deformation of Polyurethanes

The mechanical properties of thermoplastic polyurethanes (TPU) depend upon their composition and the complex two‐phase morphologies, which originate from microphase separation of chains segments. In the present work, poly(ether‐urethanes) were prepared with hard segment contents from 36% to 71% by weight, by systematically varying the length of the soft‐segment macrodiol. Samples were prepared by hot pressing or solvent casting, and the resulting hard‐ and soft‐segment morphologies were characterized by using small‐angle x‐ray scattering (SAXS) and transmission electron microscopy (TEM). Environmental scanning electron microscopy (ESEM) was used to study the fracture surfaces of TPU samples. The deformation behavior of the morphology was studied in real time, by using 2‐dimensional SAXS (2D‐SAXS) at the Daresbury synchrotron radiation source. Two distinct mechanisms were identified, with the dominant mechanism in a given material dependent on the copolymer composition and the extent of microphase separation, which developed during processing.

The Mobile Luminescence End-Station, MoLES: a new public facility at Daresbury Synchrotron.

A new mobile end-station is described for use on multiple beamlines at the Daresbury synchrotron radiation source (overall excitation range 5 eV to 70 keV) that allows for the detection and dispersion of photoluminescence from solid-state samples in the emission range 190-1000 nm (1.2-6.5 eV). The system is fully self-contained and includes sample-cooling facilities for the temperature range 8-330 K using a closed-cycle refrigerator, thus eliminating the need for liquid cryogens. The system also includes solid-state laser sources for use with a variety of pump-probe-type experiments, and an Ar(+) surface-cleaning facility. In order to demonstrate the various capabilities of the system, the results of a variety of experiments are summarized, carried out over the excitation range 5-5000 eV on beamlines 3.2, MPW6.1 and 4.2. These include the optical detection of XAS of L-edge structure in natural minerals and archaeological ceramics, band-gap determinations of wide-band-gap silicates, and pump-probe studies of quartz.

Expression, purification and crystallization of the Plasmodium falciparum enoyl reductase.

New hope has been gained in the control of the malaria parasite Plasmodium falciparum (pf) with the discovery that the parasite contains a prokaryotic type II fatty-acid synthase (FAS). Since enzymes of this type are absent in humans, they are potential targets for the development of new drugs. The enoyl reductase enzyme (ENR) belonging to this pathway is of particular interest because it has been shown to be inhibited by submicromolar concentrations of the antimicrobial agent triclosan. Here, the development of an efficient overexpression system for pfENR as a fusion protein with maltose-binding protein, its simple one-step purification and cleavage from its fusion protein and crystallization under new conditions with bound NAD(+) cofactor and triclosan are reported. The crystals belong to the space group P2(1), with approximate unit-cell parameters a = 88.2, b = 82.4, c = 94.8 A, beta = 90.77 degrees, and contain a tetramer in the asymmetric unit. Cryocooled crystals (100 K) diffracted to beyond 2.2 A resolution at the Daresbury Synchrotron Radiation Source.

Software for automatic calibration of synchrotron powder diffractometers.

An automatic procedure to calibrate angular-dispersive monochromatic diffraction instruments has been developed at the Daresbury Synchrotron Radiation Source. The procedure uses a macro Language to control the powder diffraction instruments to locate Bragg reflections and perform peak-centre refinement from a standard reference material. The information obtained is used to refine the wavelength of the radiation used and the angular offset of the detector arm. The concept and implementation of the new software are described with applications to demonstrate its viability. The results of a reliability and accuracy study are also presented.

Crystal structure of indium monosulfide, InS, at 7.9 GPa

InS, monoclinic, Pl2i/nl (No. 14), a = 3.8630(8) Â, b = 10.7668(9) k,c = 3.8554(7) k, s = 90.880(7)°, V = 160.3 Â, Ζ = 4, wR(P) = 0.120, R(I) = 0.062, T= 295 Κ, Ρ = 7.9 GPa. Source of material The starting material were dark red pieces of InS ingots. Powdering was performed by grinding of the crystalline material at ambient conditions. tionship of tetragonal HgCl and orthorhombic as well as monoclinic InS. Detailed discussions of the crystal chemical similarities of InS, HgCl, GeS, and Til have been given earlier [1-3]. The present results [space group No. 14, standard setting P2\!c a = 386.25(1) pm,?>= 1076.6( 1 ) pm, c=5.4991(4),/? = 135.488(8)°; In (0.018(3), 0.1189(5), -0.037(2)), S (0.02(1), 0.359(1), 0.000(6))] are in disagreement with earlier work which supposes hp-InS to crystallize in the tetragonal space group lAlmmm [4]. Table 1. Data collection and handling. Powder: dark red Wavelength: synchrotron radiation (0.4654 Â) μ: 12.5 cm Diffractometer: Daresbury Station 9.1, φ scan, transmission 2dmax, stepwidth: 26°, 0.02° N(points) measured: 1001 N(hkl)measarai·· 108 N(param)ref,ned'· 9 Programs: CSD [5], ATOMS [6] Table 2. Atomic coordinates and displacement parameters (in Â). Atom Site X y ζ t/iso In 4c S 4e 0.055(2) 0.019(5) 0.1191(4) -0.038(2) 0.359(1) -0.001(6) 0.0143(3) 0.0139(5) Experimental details High pressures were generated using gasketed diamond anvil cells with a 4:1 methanol/ethanol mixture as a pressure transmitting medium. For pressure calibration we used the ruby luminescence method. Discussion The figure shows the crystal structure of a high-pressure modification of InS in a projection along the a axis. Both atom types, indium and sulphur, realise a distorted octahedron as a first coordination sphere. The crystal structure is a distorted variety of the tetragonal calomel type. It can be described as slightly distorted NaCl-type double-layers which are interconnected by homonuclear In bonds. In comparison to the orthorhombic low-pressure modification, the inclination angle between the In-dumbbells and the long axis is pronouncedly reduced in the monoclinic high-pressure phase. The selected non-standard setting P2\!n points out the metrical rela* e-mail: schwarz@cpfs.mpg.de Acknowledgments. Major parts of the work were performed at the MPI fur Festkorperforschung (Stuttgart). The author gratefully acknowledges support with fruitful discussions and beamtime at the Daresbury Synchrotron Radiation Department by K. Syassen, R. Nelmes and M. I. McMahon and is indebted to N. M. Gasanly for InS single crystals.

Near K-edge linear attenuation coefficients for amorphous and crystalline GaAs

We present tabulated near K-edge linear attenuation coefficients for stoichiometric crystalline and amorphous gallium arsenide. The coefficients were derived from total electron yield photocurrent measurements at the Daresbury Synchrotron Radiation Source and show considerable near-edge structure when compared to curves generated from standard atomic data tables. However, very little difference is found between the crystalline and amorphous samples, suggesting that the bulk of the structure arises from the local coordination environment.

On the experimental determination of the Fano factor in Si at soft X-ray wavelengths

We have investigated the experimental determination of the Fano factor in silicon using a low-noise CCD detector. We have tested the hypothesis of Fraser et al. (Nucl. Instr. and Meth. A 350 (1994) 365) that the distribution of secondary electrons generated by low-energy X-ray interactions is not normally distributed, leading to an asymmetry in the electron number distribution with energy. This in turn, leads to systematically low values of the Fano factor when derived using a traditional analysis of the energy resolution function. Based on measurements taken at the Daresbury Synchrotron Radiation Source (SRS) we find that monoenergetic energy-loss distributions are indeed non-Gaussian to a similar degree predicted by Fraser et al. (1994). The Fano factors determined from a probability analysis of the pulse height data are typically (0.155±0.002) which are significantly different from the value of (0.143±0.001) derived from a Gaussian decomposition of the energy resolution function. The results are in excellent agreement with the theoretical values of ∼0.158 derived from the photoionization theory of Fraser et al. (1994). Lastly, we show that for practical detection systems, failure to correct for a finite energy threshold can also lead to an underestimate in the derived value of F—by as much as ∼5% in the present case.