Research Activities and Services in Structural Biology. EMBL Grenoble

Transcription

1 Research Activities and Services in Structural Biology EMBL Grenoble

2 Introduction to EMBL The European Molecular Biology Laboratory (EMBL) has five sites: the main laboratory in Heidelberg, Germany, and outstations in Hinxton, UK (the European Bioinformatics Institute), Grenoble, France, Hamburg, Germany and Monterotondo, Italy. EMBL science covers a spectrum of themes in basic life sciences research, from the study of sequences and structures of individual biological macromolecules, to their functions in cellular structures and networks, to their influence on the development and lives of organisms and the molecular basis of disease. The institute, founded in 1974, pursues a number of goals on behalf of the life science community in its member states: conducting basic research in molecular biology; providing state-of-the-art services and infrastructures to scientists; providing high-level training to staff, students and visitors; developing new instrumentation for biological research, and technology transfer. EMBL Grenoble The EMBL outstation in Grenoble, France, hosts 70 staff and shares a campus with the European Synchrotron Radiation Facility (ESRF) and the Institut Laue Langevin (ILL), a leading high flux neutron source. The outstation collaborates very closely with these international institutes in building and operating X-ray and neutron beamlines for macromolecular crystallography and developing associated instrumentation and techniques. In particular, EMBL Grenoble provides access for EMBL member states and associate scientific communities to ESRF beamlines for macromolecular crystallography (MX), each of which is equipped with an EMBL-designed high-precision diffractometer and frozen crystal sample changer for rapid crystal screening. The outstation also undertakes research in structural molecular biology, notably using crystallographic techniques to obtain atomic structures of proteins and nucleic acids. Research covers fundamental processes such as transcription, protein synthesis and non-coding RNAs as well as areas with application to human health, such as the structure of viruses and their replication, and the search for new antibiotics. Additional activities include the implementation of high-throughput technologies for automated expression and crystallisation of proteins, and development of diffraction instrumentation. Advanced training in structural molecular biology is provided for international PhD students and postdoctoral fellows, complemented by courses and workshops. The outstation actively participates in both local and international scientific research networks. EMBL Grenoble is a founder member of the Partnership for Structural Biology (PSB) together with the ESRF, ILL and the Institut de Biologie Structurale (IBS), a leading French national laboratory. 2

3 Research services and facilities Protein crystallography and small-angle scattering synchrotron beamlines The outstation collaborates with the ESRF to provide user access to MX synchrotron beamlines ID14 (two fixed energy, one MAD and one small-angle scattering endstation), ID23 (one MAD and one microfocus end station) and ID29 (MAD). Under an agreement with the UK Research Councils (MRC, BBSRC, EPSRC), EMBL Grenoble provides access to the BM14 MAD beamline to the EMBL member states and associate scientific communities. From 2009 India will join the Consortium running this beamline. Diffraction Instrumentation The Instrumentation group collaborates with the ESRF and ILL to develop state-of-the-art diffraction instrumentation, including the microdiffractometer (MD2), minidiffractometer (MD2M), automatic sample changer (SC3), the Mini-kappa goniometer head and a new crystal dehumidifier, all of which equip the beamlines as well as being available commercially. Expression and crystallisation Through the new EU-funded FP7 project PCUBE ( in 2009, we will provide access to our EMBL Grenoble protein production and crystallisation facilities, in particular: Expression screening of difficult proteins for soluble domains (ESPRIT): Random construct libraries of a target gene are synthesised by enzymatic DNA degradation and up to 30,000 constructs tested robotically for expression of soluble, purifiable protein; thus, we obtain well-behaved protein samples. Eukaryotic expression facility for insect cell expression (EEF): The newly installed EEF uses advanced multiexpression technologies developed by the Berger group for producing large proteins and multiprotein complexes. It is supported by EMBL and the EC projects 3D Repertoire and INSTRUCT (FP6,FP7). Crystallisation: The high-throughput crystallisation (HTX) laboratory provides fully automated crystallisation screening using nanovolume technology, which allows for extensive screening even with limited amounts of sample. A Crystallisation Data Management System provides real-time web access to results and experimental parameters. More than 350 scientists have used the HTX Lab. Training The outstation regularly organises and hosts courses and workshops covering various aspects of structural biology such as methods in protein crystallography and structural characterisation of macromolecular complexes. The outstation offers a stimulating environment for PhD students and postdoctoral fellows as part of the International EMBL PhD programme. Grenoble s Université Joseph Fourier is one of EMBL s partner universities, so students may register there to be awarded their PhD jointly with EMBL. Young researchers looking for postdoctoral opportunities should apply directly to the outstation or visit the website for details about the special EMBL inter-disciplinary postdoctoral fellowships (EIPOD). 3

4 Research groups and teams Head of outstation STEPHEN CUSACK s group uses X-ray crystallography to study the structural biology of protein-rna complexes involved in epigenetics (e.g. the dosage compensation complex), RNA metabolism (e.g. nonsense-mediated decay), protein synthesis (e.g. aminoacyl-trna synthetases) and viral infections. A major project involves studying the molecular basis for the mechanism of influenza virus RNA polymerase, how the polymerase mutates to adapt from avian to human hosts and how the innate immune system responds to viral RNA. EU projects: SPINE2-Complexes (FP6), FLUPOL (FP6), INSTRUCT (FP7) RAMESH PILLAI s group explores how micrornas act as guides for their associated proteins to bring them to their target mrnas. They inhibit a step very early in translation and lead to the accumulation of these targets in cytoplasmic structures called processing bodies (P-bodies). MiRNAs can also lead to target mrna degradation and deadenylation, which could be a consequence of accumulation in P-bodies. The group aims to understand the mechanism and biology of regulation of gene expression by noncoding RNAs. IMRE BERGER s group develops expression technologies to entirely automate the process of production for eukaryotic gene regulatory multiprotein complexes. They subject the complex specimens produced to electron microscopic analyses and use the homogeneous complexes identified in this way for X-ray crystallography. They also address a bottleneck in complex crystallography: the challenge of defining crystallisable core assemblies of multiprotein complexes in a reasonable time frame. EU projects: 3D Repertoire (FP6), INSTRUCT (FP7), PCUBE (FP7) DANIEL PANNE s group explores the switching mechanisms that underlie transcriptional regulation in eukaryotes, with a special interest on cooperative assembly of generic transcription factors into specific superstructures (sometimes also called enhanceosomes ). Using the interferon-β enhancer as a model system and X-ray crystallography, they focus on the structural analysis of these complex transcriptional assemblies, with an emphasis on systems that are important in the immune system. 4

5 FLORENT CIPRIANI s Diffraction Instrumentation Team collaborates closely with the ESRF and EMBL Hamburg in developing beamline instrumentation for structural biology. The team plays an important role in the standardisation and automation of the MX beamlines and makes their technology available to the scientific community through EMBLEM, EMBL s technology transfer company. A current major project is to develop an automated sample environment for the future biology-dedicated ESRF and EMBL@Petra3 SAXS beamlines. EU projects: SPINE2-Complexes (FP6), BIOXHIT (FP6) DARREN HART focuses on method development for difficult-to-express proteins. While structural genomics is effective at harvesting low-hanging fruit, many proteins remain refractory. The team has developed a high-throughput process, ESPRIT, in which all truncated variants of a target protein are synthesised as a random library and then screened as colony arrays to identify rare soluble variants. Thus proteins that resisted analysis due to poor recombinant expression may yield well-expressed domains for study. Targets include influenza proteins, human kinases and histone deacetylases. EU projects: SPINE2-Complexes, FLUPOL, SOUTH and 3D Repertoire (all FP6), PCUBE (FP7) JOSÉ MÁRQUEZ s high-throughput crystallisation (HTX) laboratory automates the process of extensive screening using extremely low volumes of sample through the introduction of liquid handling, crystallisation and crystalimaging robots. The platform, among the largest of its kind in Europe, is open to European researchers and now has more than 250 registered users, processing about a thousand samples per year. The lab also collaborates with the instrumentation group in the development of new crystallisation technologies. EU projects: BIOXHIT (FP6), PCUBE (FP7) ANDREW McCARTHY s synchrotron radiation team designs, constructs and operates macromolecular crystallography (MX) beamlines and improves the data collection techniques for macromolecular crystallography at third generation synchrotrons. The group develops hardware, software and novel methodologies for sample handling and data collection, and members are involved in collaborations on proteins involved in neuronal development and caffeine biosynthesis as well as DNA modifying and processing enzymes. EU projects: SPINE2-Complexes (FP6), BIOXHIT (FP6) CHRISTIANE SCHAFFITZEL s team uses cryo-em and single particle analysis to explore how the folding of a nascent polypeptide chain into its native structure is achieved. Synthesis and folding of proteins require concerted interactions of the translating ribosome with translation factors, processing enzymes, molecular chaperones and factors involved in the export of proteins. The functional and structural characterisation of such complexes provide important mechanistic insight into the molecular mechanism of protein sorting. 5

6 Partnership for Structural Biology Structural biology is increasingly dependent on integrated and highthroughput technologies to facilitate the determination of protein and protein-complex structures and to allow complementary functional studies. To exploit the differing expertise and to pursue joint projects, the Partnership for Structural Biology (PSB) was formally established in November 2002 by the following institutes: EMBL Grenoble, the European Synchrotron Radiation Facility (ESRF), the Institut Laue Langevin (ILL) and the Institut de Biologie Structurale (IBS). The PSB project received financial support under the EU FP6 Construction of New Infrastructures Initiative. The new Carl-Ivar Brändèn Building, on the ESRF/ILL site adjacent to the EMBL Grenoble building, was built jointly by the PSB partners to house many of the common platforms of the PSB and to facilitate inter-institute collaboration. There are currently twelve technical platforms at the PSB ranging from protein expression to biophysical methods based on large facilities. Protein production: standard protein expression (Robiomol); expression screening of difficult proteins for soluble domains (ESPRIT); eukaryotic expression facility for insect cell expression (EEF); isotopic labeling of proteins for neutron or NMR studies Protein characterisation: molecular interaction studies by surface plasmon resonance; sample quality control (mass spectroscopy, N-terminal sequencing, NMR, EM); in cristallo spectroscopy studies (Cryobench) Crystallisation: robotic nanovolume crystallisation (screening and optimisation) Structure Determination: synchrotron X-ray crystallography; Neutron Diffraction; small angle X-ray and neutron scattering (SAXS/SANS); negative-stain and cryo-electron microscopy; high-field NMR The major research focus areas of PSB scientists are host-pathogen interaction, membrane protein, extreme environments and fundamental cellular processes such as transcription and translation. Although basic research related to human health is a key impetus, developing new methodologies and techniques for the future of integrated structural biology is also a major concern. The PSB actively encourages academic visitors to make use of, through training courses or collaboration, the new infrastructures, particularly the X-ray and neutron beamlines, the protein production, characterisation and crystallisation facilities, as well as the deuterium labeling platform. 6

7 Projects and partnerships EMBL Grenoble collaborates in partnership with both national and international academic and research institutes. With campus partners ESRF, ILL and IBS, EMBL Grenoble promotes access to synchrotron radiation crystallography and small anglescattering beamlines and provides sample preparation and characterization platforms to offer fully-integrated structural biology facilities to 2,500 users per year (page 6). Partnership for Structural Biology (PSB) ESRF ILL IBS EMBL CNRS UJF Unit of Virus Host Cell Interactions (UVHCI) UVHCI The Unit of Virus Host Cell Interactions (UVHCI) was created in January 2007 through a Unité Mixte de Recherche (UMR 5233) to formalise the collaboration between the Université Joseph Fourier (UJF), EMBL Grenoble and the National Center for Scientific Research (CNRS) in the field of virus research. This has now been modified into an Unite Mixte Internationale (UMI 3265), the second to be created in France out of 14 worldwide. Its objective is to pursue high quality research in structural and molecular biology, focused, but not exclusively, on virus-host cell interactions. Research themes include virus structure, assembly and maturation, host and virus gene-expression mechanisms, cell biology of infected cells, innate immunity and anti-pathogen drug design. Viruses studied include influenza virus, rabies virus, Epstein-Barr virus and HIV. In addition, methods and technical developments in high throughput expression and crystallisation, synchrotron X-ray and neutron diffraction instrumentation and electron microscopy are pursued. EU projects EMBL Grenoble participates in the large-scale EU Integrated projects SPINE (Structural Proteomics in Europe) and its FP6 successor (SPINE2- Complexes), FLUPOL, BIOXHIT and 3D Repertoire. The ILL-EMBL Deuteration laboratory and user access to ESRF beamlines are financed respectively by the EU Neutron and Muon Integrated Infrastructure Initiative (NMI3) and the Integrating Activity on Synchrotron and Free Electron Laser Science (IASFS). A new EU-funded project in 2009, PCUBE, will provide user access to the EMBL Grenoble protein production facilities (ESPRIT and EEF). EMBL, including the Grenoble outstation, is one of the core centres in the preparatory phase of INSTRUCT (Integrated Structural Biology Infrastructure for Europe), one of the European Strategy Forum on Research Infrastructures (ESFRI) biomedical projects. 7

8 EMBL s member states: Austria Belgium Croatia Denmark Finland France Germany Greece Iceland Ireland Israel Italy Luxembourg Netherlands Norway Portugal Spain Sweden Switzerland United Kingdom Associated member state: Australia EMBL Grenoble 6, rue Jules Horowitz BP Grenoble cedex 9 France Phone: +33 (0) Fax: +33 (0) Founded: 1976 Number of staff: 83