Developments in optics and performance at BL13-XALOC, the macromolecular crystallography beamline at the Alba Synchrotron

Juanhuix, Jordi; Gil-Ortiz, F.; Cuní, Guifre; Colldelram, Carles; Nicolás, Josep; Lidón, Julio; Boter, E.; Ruget, Claude; Ferrer, S.; Benach, J.

doi:10.1107/s160057751400825x

Cited by 173 publications

(149 citation statements)

References 28 publications

(20 reference statements)

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“…Prior to data collection, the crystals were transferred to the cryosolution (50 mM Tris-HCl (pH 7.7), 0.2 M NaF, 19% PEG 3350, 20% glycerol) and flash-cooled in liquid nitrogen. Data from a native crystal were collected at 100 K on the Xaloc beam line of the ALBA synchrotron (Barcelona, Spain) (53). Data from the EMTS-derivatized crystal were collected at 100 K using a rotating anode generator.…”

Section: Methodsmentioning

confidence: 99%

The Structure of the Plakin Domain of Plectin Reveals an Extended Rod-like Shape

Ortega

Manso

Buey

et al. 2016

Journal of Biological Chemistry

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Plakins are large multi-domain proteins that interconnect cytoskeletal structures. Plectin is a prototypical plakin that tethers intermediate filaments to membrane-associated complexes. Most plakins contain a plakin domain formed by up to nine spectrin repeats (SR1-SR9) and an SH3 domain. The plakin domains of plectin and other plakins harbor binding sites for junctional proteins. We have combined x-ray crystallography with small angle x-ray scattering (SAXS) to elucidate the structure of the plakin domain of plectin, extending our previous analysis of the SR1 to SR5 region. Two crystal structures of the SR5-SR6 region allowed us to characterize its uniquely wide inter-repeat conformational variability. We also report the crystal structures of the SR7-SR8 region, refined to 1.8 Å, and the SR7-SR9 at lower resolution. The SR7-SR9 region, which is conserved in all other plakin domains, forms a rigid segment stabilized by uniquely extensive inter-repeat contacts mediated by unusually long helices in SR8 and SR9. Using SAXS we show that in solution the SR3-SR6 and SR7-SR9 regions are rod-like segments and that SR3-SR9 of plectin has an extended shape with a small central kink. Other plakins, such as bullous pemphigoid antigen 1 and microtubule and actin cross-linking factor 1, are likely to have similar extended plakin domains. In contrast, desmoplakin has a two-segment structure with a central flexible hinge. The continuous versus segmented structures of the plakin domains of plectin and desmoplakin give insight into how different plakins might respond to tension and transmit mechanical signals.Plakins are a family of very large proteins that interconnect and organize the intermediate filaments (IF), 4 microtubules, and microfilaments of the cytoskeleton and tether them to membrane-associated structures (1, 2). So far, seven plakins have been described in mammals; these are plectin, bullous pemphigoid antigen 1 (BPAG1), desmoplakin, microtubule and actin cross-linking factor 1 (MACF1, also known as ACF7), envoplakin, periplakin, and epiplakin. Invertebrates have a more reduced plakin repertoire; for example Caenorhabditis elegans and Drosophila melanogaster each have a single plakin gene encoding VAB-10 and Shot (also known as Short Stop or kakapo), respectively. Most of the plakin genes produce multiple isoforms that increase the structural and functional versatility of these proteins.Plectin is expressed in a large variety of cell types in which it acts as a highly polyvalent cytolinker that contributes to cell adhesion and the organization of the cytoskeleton. Plectin cross-links IFs to microtubules and actin filaments and mediates the attachment of IFs to cell-cell and cell-matrix junctional complexes such as hemidesmosomes, desmosomes, Z-lines, and focal contacts. Plectin also connects IFs to organelles such as the nucleus and mitochondria (3). Defects in the PLEC gene cause various forms of the blistering disease epidermolysis bullosa simplex (EBS), which may occur only with skin fragility, as found in EBS Og...

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Section: Methodsmentioning

confidence: 99%

The Structure of the Plakin Domain of Plectin Reveals an Extended Rod-like Shape

Ortega

Manso

Buey

et al. 2016

Journal of Biological Chemistry

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“…Red crystals were obtained after 3-4 weeks and were flash-frozen in liquid nitrogen using 10% w/v PEG200 as a cryoprotectant. X-ray diffraction data were collected on the ID29 at the European Synchrotron Research Facility (Grenoble, France) [25] and on the BL13-XALOC beamline at ALBA synchrotron (Cerdanyola del Vallès, Spain) [26]. Data were processed using XDS [27] and scaled using AIMLESS [28].…”

Section: Methodsmentioning

confidence: 99%

Subtle structural changes in the Asp251Gly/Gln307His P450 BM3 mutant responsible for new activity toward diclofenac, tolbutamide and ibuprofen

Nardo

Dell'Angelo

Catucci

et al. 2016

Archives of Biochemistry and Biophysics

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This paper reports the structure of the double mutant Asp251Gly/Gln307His (here called A2), generated by random mutagenesis, is able to produce 4'-hydroxydiclofenac, 2-hydroxyibuprofen and 4-hydroxytolbutamide from diclofenac, ibuprofen and tolbutamide, respectively. Here, we report the crystal structure of the heme domain of the mutant in the substrate-free form and in complex with the substrate N-palmitoylglycine, together with its impact on thermal stability, reduction potential and electron transfer.The substrate-free structure adopts a conformation more similar to the closed one found in the substrate-bound wild type enzyme, but with a higher degree of disorder in the region of the G-helix and F-G loop, part of the substrate access channel. This is due to the mutation Asp251Gly that breaks the salt bridge between Aps251 on Ihelix and Lys224 on G-helix, allowing the G-helix to move away from I-helix and conferring a higher degree of flexibility to this element. This subtle structural change is accompanied by long-range structural rearrangements of the active site with the rotation of Phe87 and a reorganization of catalytically important water molecules.Differential scanning calorimetry shows a population destabilized by 2.2°C compared to the wild type protein, probably reflecting the increased flexibility of part of the protein compared to WT. Moreover, a shift of the heme reduction potential by 50 mV toward positive values, a 2-folds higher first electron transfer rate in the absence of oxygen and a 4-folds higher NADPH consumption rate in the presence of oxygen as the only electron acceptors, when compared to wild type protein.The data demonstrate that a single mutation far away from the active site is able to trigger an increase in protein flexibility in a key region of the substrate access channel, shifting the conformational equilibrium toward the closed form of the protein that is ready to accept electrons and enter the P450 catalytic cycle as soon as a substrate is accepted.

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“…20 μm thick microtomed cross sections of the samples were measured with a focused beam of 50x6μm 2 (FWHM), 1s acquisition time and 12.6 keV energy in a virtually noise free Pilatus 6M (Dectris) detector with a large (424x435 mm 2 , 6 Mpixels) active area 11 . The diffraction patterns from all the layers were obtained by scanning the sample over ca.…”

Section: Ray Diffractionmentioning

confidence: 99%

Low molecular weight organic acid salts, markers of old fungi activity in wall paintings

et al. 2016

Self Cite

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Micro Infrared Spectroscopy (µSR-FTIR) and X-Ray diffraction (µSR-XRD) with synchrotron light, Gas Chromatography/Mass Spectrometry (CG/MS), Optical Microscopy (OM) and Scanning Electron Microscopy (SEM/EDS) were used to identify and obtain the distribution of complex mixtures of calcium salts of low molecular weight organic acids (LMWOA) in micro-layered micro-samples. Filamentous fungi produce LMWOA that can react with metal cations producing stable salts. These substances were found in the dark spots covering the surfaces of Saint Michael's Chapel wall paintings of the Royal Monastery of Pedralbes in Barcelona linking them to old fungi activity. The presence of glycerol likewise related to the fungi activity is also identified in the layers.

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Developments in optics and performance at BL13-XALOC, the macromolecular crystallography beamline at the Alba Synchrotron

Abstract: The design, performance and first results of the new macromolecular crystallography beamline BL13-XALOC at the ALBA Synchrotron are described, including new developments in optics and control.

Cited by 173 publications

References 28 publications

The Structure of the Plakin Domain of Plectin Reveals an Extended Rod-like Shape

The Structure of the Plakin Domain of Plectin Reveals an Extended Rod-like Shape

Subtle structural changes in the Asp251Gly/Gln307His P450 BM3 mutant responsible for new activity toward diclofenac, tolbutamide and ibuprofen

Low molecular weight organic acid salts, markers of old fungi activity in wall paintings

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