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Grenoble, France

Blanchet C.E.,EMBL Hamburg | Zozulya A.V.,EMBL Hamburg | Kikhney A.G.,EMBL Hamburg | Kikhney A.G.,German Electron Synchrotron | And 9 more authors.
Journal of Applied Crystallography | Year: 2012

A setup is presented for automated high-throughput measurements of small-angle X-ray scattering (SAXS) from macromolecular solutions on the bending-magnet beamline X33 of EMBL at the storage ring DORIS-III (DESY, Hamburg). A new multi-cell compartment allows for rapid switching between in-vacuum and in-air operation, for digital camera assisted control of cell filling and for colour sample illumination. The beamline is equipped with a Pilatus 1 M-W pixel detector for SAXS and a Pilatus 300 k-W for wide-angle scattering (WAXS), and results from the use of the Pilatus detectors for scattering studies are reported. The setup provides a broad resolution range from 100 to 0.36 nm without the necessity of changing the sample-to-detector distance. A new optimized robotic sample changer is installed, permitting rapid and reliable automated sample loading and cell cleaning with a required sample volume of 40 l. All the devices are fully integrated into the beamline control software system, ensuring fully automated and user-friendly operation (attended, unattended and remote) with a throughput of up to 15 measurements per hour. © 2012 International Union of Crystallography Printed in Singapore-all rights reserved.

Laffly E.,University of Maryland Baltimore County | Garzoni F.,EMBL Grenoble | Fontecilla-Camps J.C.,CNRS Institute of Pharmacology and Structural Biology | Cavazza C.,CNRS Institute of Pharmacology and Structural Biology
International Journal of Hydrogen Energy | Year: 2010

The need of an efficient and well-characterized heterologous expression system of [FeFe]-hydrogenase for the production of O 2-resistant mutants prompted us to explore the use of Escherichia coli as a possible expression system. O 2-resistant hydrogenase mutants could be instrumental when coupling oxygenic photosynthesis with hydrogen bio-production. In general, expression of Desulfovibrio vulgaris Hildenborough active enzyme in E. coli was very modest indicating that the co-expression of the HydE, HydF and HydG maturases with hydrogenase structural genes in this bacterium is not optimal. A 28-fold increase in activity was obtained when these proteins were co-expressed with the Iron-Sulfur Cluster operon, indicating that one of the problems with over-expression is the correct insertion of FeS clusters. However, the measured activity is still about 4000-fold lower than the one measured in the native hydrogenase indicating that additional, so far unidentified factors may be necessary for optimal heterologous expression of [FeFe]-hydrogenase. © 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.

Bowler M.W.,EMBL Grenoble | Bowler M.W.,French National Center for Scientific Research
FEBS Letters | Year: 2013

Domain motions are essential to many catalytic mechanisms in enzymes but they are often difficult to study. X-ray crystal structures can provide molecular details of snapshots of catalysis but many states important in the cycle remain inaccessible using this technique. Phosphoglycerate kinase (PGK) undergoes large domain movements in order to catalyse the production of ATP. PGK is the enzyme responsible for the first ATP generating step of glycolysis and has been implicated in oncogenesis and the in vivo activation of l-nucleoside pro-drugs effective against retroviruses. Its mechanism requires considerable hinge bending to bring the substrates into proximity in order for phosphoryl transfer to occur. The enzyme has been the subject of intense study for decades but new crystal structures, methods in solution scattering and modelling techniques are throwing light on the dynamics of catalysis of this archetypal kinase. Here, I argue that Brownian forces acting on the protein are the dominant factor in the catalytic cycle and that the enzyme has evolved measures to harness this force for efficient catalysis. © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Estrozi L.F.,EMBL Grenoble | Navaza J.,CNRS Institute of Pharmacology and Structural Biology
Journal of Structural Biology | Year: 2010

A protocol to attain high-resolution single-particle reconstructions is presented. The protocol is the concatenation of two procedures: one to obtain an ab initio low-resolution reconstruction, the other to determine a fixed point of the consecutive applications of fast projection matching and 3D reconstruction. It is a reciprocal space formulation where the Fourier coefficients of the 3D scattering density are expressed in terms of symmetry adapted functions and the 2D particle images are represented by their Fourier-Bessel transforms. The new protocol shows advantages in terms of speed and accuracy when compared to other methods currently in use. We illustrate its performance as applied to high-resolution cryo-electron micrographs of rotavirus. © 2010 Elsevier Inc.

Han L.,Karolinska Institutet | Monne M.,Karolinska Institutet | Okumura H.,Karolinska Institutet | Okumura H.,Meijo University | And 5 more authors.
Cell | Year: 2010

ZP3, a major component of the zona pellucida (ZP) matrix coating mammalian eggs, is essential for fertilization by acting as sperm receptor. By retaining a propeptide that contains a polymerization-blocking external hydrophobic patch (EHP), we determined the crystal structure of an avian homolog of ZP3 at 2.0 Å resolution. The structure unveils the fold of a complete ZP domain module in a homodimeric arrangement required for secretion and reveals how EHP prevents premature incorporation of ZP3 into the ZP. This suggests mechanisms underlying polymerization and how local structural differences, reflected by alternative disulfide patterns, control the specificity of ZP subunit interaction. Close relative positioning of a conserved O-glycan important for sperm binding and the hypervariable, positively selected C-terminal region of ZP3 suggests a concerted role in the regulation of species-restricted gamete recognition. Alternative conformations of the area around the O-glycan indicate how sperm binding could trigger downstream events via intramolecular signaling. © 2010 Elsevier Inc.

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