EMBL Grenoble Outstation

Grenoble, France

EMBL Grenoble Outstation

Grenoble, France
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Nurizzo D.,European Synchrotron Radiation Facility | Bowler M.W.,EMBL Grenoble Outstation | Guichard N.,European Synchrotron Radiation Facility | McSweeney S.,European Synchrotron Radiation Facility | And 5 more authors.
AIP Conference Proceedings | Year: 2016

The European Synchrotron Radiation Facility has a long standing history in the automation of experiments in Macromolecular Crystallography. MASSIF-1 (Massively Automated Sample Screening and evaluation Integrated Facility), a beamline constructed as part of the ESRF Upgrade Phase I program, has been open to the external user community since July 2014 and offers a unique completely automated data collection service to both academic and industrial structural biologists. © 2016 Author(s).


Mourao A.,Helmholtz Center Munich | Mourao A.,TU Munich | Mourao A.,European Molecular Biology Laboratory EMBL | Varrot A.,EMBL Grenoble Outstation | And 6 more authors.
RNA | Year: 2010

Small nuclear and small nucleolar RNAs (snRNAs and snoRNAs) are critical components of snRNPs and snoRNPs and play an essential role in the maturation of, respectively, mRNAs and rRNAs within the nucleus of eukaryotic cells. Complex and specific pathways exist for the assembly of snRNPs and snoRNPs, involving, for instance, nucleocytoplasmic transport of snRNAs and intranuclear transport between compartments of snoRNAs. The phosphorylated adaptor for nuclear export (PHAX) is required for nuclear export of snRNAs in metazoans and also involved in the intranuclear transport of snoRNAs to Cajal bodies. PHAX contains a conserved single-stranded nucleic acid binding domain (RNA-GG-bind domain) with no sequence homology with any other known RNA-binding module. Here, we report NMR and X-ray crystallography studies that elucidate the structural basis for RNA recognition by the PHAX RNA-binding domain (PHAX-RBD). The crystal structure of the RNA-GG-bind domain from the parasite Cryptosporidium parvum (Cp RBD) forms well-folded dimers in solution in the absence of any ligand. The human PHAX-RBD is monomeric and only adopts a tertiary fold upon RNA binding. The PHAX-RBD represents a novel helical fold and binds single-stranded RNA with micromolar affinity without sequence specificity. RNA recognition by human PHAXRBD is consistent with mutational analysis that affects RNA binding and PHAX-mediated nuclear export. Our data suggest that the PHAX-RBD mediates auxiliary RNA contacts with the snRNA and snoRNA substrates that are required for transport and/or substrate release. Copyright © 2010 RNA Society.


Guedich S.,University of Strasbourg | Puffer-Enders B.,University of Strasbourg | Baltzinger M.,University of Strasbourg | Hoffmann G.,EMBL Grenoble Outstation | And 7 more authors.
RNA Biology | Year: 2016

Riboswitches are non-coding elements upstream or downstream of mRNAs that, upon binding of a specific ligand, regulate transcription and/or translation initiation in bacteria, or alternative splicing in plants and fungi. We have studied thiamine pyrophosphate (TPP) riboswitches regulating translation of thiM operon and transcription and translation of thiC operon in E. coli, and that of THIC in the plant A. thaliana. For all, we ascertained an induced-fit mechanism involving initial binding of the TPP followed by a conformational change leading to a higher-affinity complex. The experimental values obtained for all kinetic and thermodynamic parameters of TPP binding imply that the regulation by A. thaliana riboswitch is governed by mass-action law, whereas it is of kinetic nature for the two bacterial riboswitches. Kinetic regulation requires that the RNA polymerase pauses after synthesis of each riboswitch aptamer to leave time for TPP binding, but only when its concentration is sufficient. A quantitative model of regulation highlighted how the pausing time has to be linked to the kinetic rates of initial TPP binding to obtain an ON/OFF switch in the correct concentration range of TPP. We verified the existence of these pauses and the model prediction on their duration. Our analysis also led to quantitative estimates of the respective efficiency of kinetic and thermodynamic regulations, which shows that kinetically regulated riboswitches react more sharply to concentration variation of their ligand than thermodynamically regulated riboswitches. This rationalizes the interest of kinetic regulation and confirms empirical observations that were obtained by numerical simulations. © 2016 The Author(s). Published with license by Taylor & Francis Group, LLC


De Sanctis D.,European Synchrotron Radiation Facility | Beteva A.,European Synchrotron Radiation Facility | Caserotto H.,European Synchrotron Radiation Facility | Dobias F.,European Synchrotron Radiation Facility | And 15 more authors.
Journal of Synchrotron Radiation | Year: 2012

ID29 is an ESRF undulator beamline with a routinely accessible energy range of between 20.0 keV and 6.0 keV (λ = 0.62 Å to 2.07 Å) dedicated to the use of anomalous dispersion techniques in macromolecular crystallography. Since the beamline was first commissioned in 2001, ID29 has, in order to provide an improved service to both its academic and proprietary users, been the subject of almost continuous upgrade and refurbishment. It is now also the home to the ESRF Cryobench facility, ID29S. Here, the current status of the beamline is described and plans for its future are briefly outlined. © 2012 International Union of Crystallography.


Hutin S.,CNRS Institute of Pharmacology and Structural Biology | Brennich M.,European Synchrotron Radiation Facility | Maillot B.,European Synchrotron Radiation Facility | Maillot B.,Institute Of Genetique Et Of Biologie Moleculaire Et Cellulaire | And 3 more authors.
Acta Crystallographica Section D: Structural Biology | Year: 2016

Biological small-angle X-ray scattering (BioSAXS) is a powerful technique to determine the solution structure, particle size, shape and surface-to-volume ratio of macromolecules. However, a drawback is that the sample needs to be monodisperse. To ensure this, size-exclusion chromatography (SEC) has been implemented on many BioSAXS beamlines. Here, the integration of ionexchange chromatography (IEC) using both continuous linear and step gradients on a beamline is described. Background subtraction for continuous gradients by shifting a reference measurement and two different approaches for step gradients, which are based on interpolating between two background measurements, are discussed. The results presented here serve as a proof of principle for online IEC and subsequent data treatment.


Thierry E.,University Grenoble Alpes | Thierry E.,French National Center for Scientific Research | Brennich M.,European Synchrotron Radiation Facility | Round A.,EMBL Grenoble Outstation | And 5 more authors.
Virus Genes | Year: 2015

The helicase–primase complex is part of the lytic DNA replication machinery of herpesviruses, but up to now, almost nothing is known about its structure. For Epstein–Barr virus it consists in the helicase BBLF4, the primase BSLF1 and the accessory protein BBLF2/3. The accessory protein shows only weak sequence homology within the herpesvirus family but may be related to an inactive B-family polymerase. BSLF1 belongs to the archaeo-eukaryotic primase family, whereas the helicase BBLF4 has been related either to Dda helicases of caudovirales or to Pif1 helicases. We produced the helicase–primase complex in insect cells using a baculovirus coding for all three proteins simultaneously. The soluble monomeric helicase–primase complex containing the three proteins with 1:1:1 stoichiometry showed ATPase activity, which is strongly stimulated in the presence of ssDNA oligomers. Furthermore, we expressed BBLF2/3 as soluble monomeric protein and performed small-angle X-ray scattering experiments which yielded an envelope whose shape is compatible with B-family polymerases. © 2015, Springer Science+Business Media New York.


Theveneau P.,European Synchrotron Radiation Facility | Baker R.,European Synchrotron Radiation Facility | Barrett R.,European Synchrotron Radiation Facility | Beteva A.,European Synchrotron Radiation Facility | And 26 more authors.
Journal of Physics: Conference Series | Year: 2013

Automation and advances in technology are the key elements in addressing the steadily increasing complexity of Macromolecular Crystallography (MX) experiments. Much of this complexity is due to the inter-and intra-crystal heterogeneity in diffraction quality often observed for crystals of multi-component macromolecular assemblies or membrane proteins. Such heterogeneity makes high-throughput sample evaluation an important and necessary tool for increasing the chances of a successful structure determination. The introduction at the ESRF of automatic sample changers in 2005 dramatically increased the number of samples that were tested for diffraction quality. This "first generation" of automation, coupled with advances in software aimed at optimising data collection strategies in MX, resulted in a three-fold increase in the number of crystal structures elucidated per year using data collected at the ESRF. In addition, sample evaluation can be further complemented using small angle scattering experiments on the newly constructed bioSAXS facility on BM29 and the micro-spectroscopy facility (ID29S). The construction of a second generation of automated facilities on the MASSIF (Massively Automated Sample Screening Integrated Facility) beam lines will build on these advances and should provide a paradigm shift in how MX experiments are carried out which will benefit the entire Structural Biology community. © Published under licence by IOP Publishing Ltd.


PubMed | University Grenoble Alpes, EMBL Grenoble Outstation, Joseph Fourier University and European Synchrotron Radiation Facility
Type: Journal Article | Journal: Virus genes | Year: 2015

The helicase-primase complex is part of the lytic DNA replication machinery of herpesviruses, but up to now, almost nothing is known about its structure. For Epstein-Barr virus it consists in the helicase BBLF4, the primase BSLF1 and the accessory protein BBLF2/3. The accessory protein shows only weak sequence homology within the herpesvirus family but may be related to an inactive B-family polymerase. BSLF1 belongs to the archaeo-eukaryotic primase family, whereas the helicase BBLF4 has been related either to Dda helicases of caudovirales or to Pif1 helicases. We produced the helicase-primase complex in insect cells using a baculovirus coding for all three proteins simultaneously. The soluble monomeric helicase-primase complex containing the three proteins with 1:1:1 stoichiometry showed ATPase activity, which is strongly stimulated in the presence of ssDNA oligomers. Furthermore, we expressed BBLF2/3 as soluble monomeric protein and performed small-angle X-ray scattering experiments which yielded an envelope whose shape is compatible with B-family polymerases.


Reuter M.,EMBL Grenoble Outstation | Pillai R.S.,EMBL Grenoble Outstation
Methods in Molecular Biology | Year: 2014

Small RNAs associate with members of the Argonaute family to function in gene regulation, transposon control, and creation of silent chromatin domains. In this partnership, small RNAs act as guides for the bound Argonaute and other associated proteins. Complementary base pairing of small RNAs to target nucleic acid molecules allow specificity for the small RNA-mediated functions. One key activity of some Argonaute protein family members is their small RNA-guided endonuclease activity called Slicer action. Here we describe a protocol that can be used to probe slicer activity in endogenous Piwi complexes isolated from mouse testes. © 2014 Springer Science+Business Media, LLC.


PubMed | EMBL Grenoble Outstation
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2013

Small RNAs associate with members of the Argonaute family to function in gene regulation, transposon control, and creation of silent chromatin domains. In this partnership, small RNAs act as guides for the bound Argonaute and other associated proteins. Complementary base pairing of small RNAs to target nucleic acid molecules allow specificity for the small RNA-mediated functions. One key activity of some Argonaute protein family members is their small RNA-guided endonuclease activity called Slicer action. Here we describe a protocol that can be used to probe slicer activity in endogenous Piwi complexes isolated from mouse testes.

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