EMBL Grenoble

Grenoble, France

EMBL Grenoble

Grenoble, France
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Delageniere S.,European Synchrotron Radiation Facility | Brenchereau P.,European Synchrotron Radiation Facility | Launer L.,European Synchrotron Radiation Facility | Ashton A.W.,Diamond Light Source | And 14 more authors.
Bioinformatics | Year: 2011

Motivation: Individual research groups now analyze thousands of samples per year at synchrotron macromolecular crystallography (MX) resources. The efficient management of experimental data is thus essential if the best possible experiments are to be performed and the best possible data used in downstream processes in structure determination pipelines. Information System for Protein crystallography Beamlines (ISPyB), a Laboratory Information Management System (LIMS) with an underlying data model allowing for the integration of analyses down-stream of the data collection experiment was developed to facilitate such data management. Results: ISPyB is now a multisite, generic LIMS for synchrotron-based MX experiments. Its initial functionality has been enhanced to include improved sample tracking and reporting of experimental protocols, the direct ranking of the diffraction characteristics of individual samples and the archiving of raw data and results from ancillary experiments and post-experiment data processing protocols. This latter feature paves the way for ISPyB to play a central role in future macromolecular structure solution pipelines and validates the application of the approach used in ISPyB to other experimental techniques, such as biological solution Small Angle X-ray Scattering and spectroscopy, which have similar sample tracking and data handling requirements. © The Author 2011. Published by Oxford University Press. 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.


PubMed | CNRS Institute of Pharmacology and Structural Biology, Jingjie PTM Biolab Hangzhou Co., Rockefeller University, University of Chicago and 3 more.
Type: Journal Article | Journal: Molecular cell | Year: 2016

Recently discovered histone lysine acylation marks increase the functional diversity of nucleosomes well beyond acetylation. Here, we focus on histone butyrylation in the context of sperm cell differentiation. Specifically, we investigate the butyrylation of histone H4 lysine 5 and 8 at gene promoters where acetylation guides the binding of Brdt, a bromodomain-containing protein, thereby mediating stage-specific gene expression programs and post-meiotic chromatin reorganization. Genome-wide mapping data show that highly active Brdt-bound gene promoters systematically harbor competing histone acetylation and butyrylation marks at H4 K5 and H4K8. Despite acting as a direct stimulator of transcription, histone butyrylation competes with acetylation, especially at H4 K5, to prevent Brdt binding. Additionally, H4 K5K8 butyrylation also marks retarded histone removal during late spermatogenesis. Hence, alternating H4 acetylation and butyrylation, while sustaining direct gene activation and dynamic bromodomain binding, could impact the final male epigenome features.


Reynoird N.,French Institute of Health and Medical Research | Schwartz B.E.,Brigham and Women's Hospital | Delvecchio M.,EMBL Grenoble | Sadoul K.,French Institute of Health and Medical Research | And 9 more authors.
EMBO Journal | Year: 2010

In a subset of poorly differentiated and highly aggressive carcinoma, a chromosomal translocation, t(15;19)(q13;p13), results in an in-frame fusion of the double bromodomain protein, BRD4, with a testis-specific protein of unknown function, NUT (nuclear protein in testis). In this study, we show that, after binding to acetylated chromatin through BRD4 bromodomains, the NUT moiety of the fusion protein strongly interacts with and recruits p300, stimulates its catalytic activity, initiating cycles of BRD4-NUT/p300 recruitment and creating transcriptionally inactive hyperacetylated chromatin domains. Using a patient-derived cell line, we show that p300 sequestration into the BRD4-NUT foci is the principal oncogenic mechanism leading to p53 inactivation. Knockdown of BRD4-NUT released p300 and restored p53-dependent regulatory mechanisms leading to cell differentiation and apoptosis. This study demonstrates how the off-context activity of a testis-specific factor could markedly alter vital cellular functions and significantly contribute to malignant cell transformation. © 2010 European Molecular Biology Organization.


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.


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.


Larabi A.,EMBL Grenoble | Devos J.M.,EMBL Grenoble | Ng S.-L.,Harvard University | Nanao M.H.,EMBL Grenoble | And 3 more authors.
Cell Reports | Year: 2013

Tank-binding kinase I (TBK1) plays a key role in the innate immune system by integrating signals from pattern-recognition receptors. Here, we report the X-ray crystal structures of inhibitor-bound inactive and active TBK1 determined to 2.6 å and 4.0 å resolution, respectively. The structures reveal a compact dimer made up of trimodular subunits containing an N-terminal kinase domain (KD), a ubiquitin-like domain (ULD), and an α-helical scaffold dimerization domain (SDD). Activation rearranges the KD into an active conformation while maintaining the overall dimer conformation. Low-resolution SAXS studies reveal that the missing C-terminal domain (CTD) extends away from the main body of the kinase dimer. Mutants that interfere with TBK1 dimerization show significantly reduced trans-autophosphorylation but retain the ability to bind adaptor proteins through the CTD. Our results provide detailed insights into the architecture of TBK1 and the molecular mechanism of activation.


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.


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.


PubMed | EMBL Grenoble, Radboud Institute of Molecular Life science, University of Bristol and University of Cambridge
Type: Journal Article | Journal: Journal of molecular biology | Year: 2016

The nucleosome remodeling deacetylase (NuRD) complex is a highly conserved regulator of chromatin structure and transcription. Structural studies have shed light on this and other chromatin modifying machines, but much less is known about how they assemble and whether stable and functional sub-modules exist that retain enzymatic activity. Purification of the endogenous Drosophila NuRD complex shows that it consists of a stable core of subunits, while others, in particular the chromatin remodeler CHD4, associate transiently. To dissect the assembly and activity of NuRD, we systematically produced all possible combinations of different components using the MultiBac system, and determined their activity and biophysical properties. We carried out single-molecule imaging of CHD4 in live mouse embryonic stem cells, in the presence and absence of one of core components (MBD3), to show how the core deacetylase and chromatin-remodeling sub-modules associate in vivo. Our experiments suggest a pathway for the assembly of NuRD via preformed and active sub-modules. These retain enzymatic activity and are present in both the nucleus and the cytosol, an outcome with important implications for understanding NuRD function.

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