Structural Biology and Genomics Platform Didier Busso - April 26, 2007 Platform’s Technical Coordinator Structural Biology and Genomics Department IGBMC 1, rue Laurent Fries Illkirch cedex Structural Biology and Genomics Didier Busso Platform presentation

Structural Biology and Genomics Platform Didier Busso - April 26, 2007 General introduction

Structural Biology and Genomics Platform Didier Busso - April 26, 2007 The Bio-crystallography The protein of interest has to be isolated from a living organism (human, plant or animal). Usually, the protein is overproduce artificially: the DNA sequence encoding the protein is inserted into an expression vector which is transferred into an expression host that will produce the protein. Thus, the soluble protein is purified by chromatography and concentrated. The purified protein is transformed in crystal (which may contain up to 1 billion of billion of the protein). Crystals of TTNPB-SRC-1-hRARb LBD. Kammerer et al., Acta Cryst. (2004) D60:

Structural Biology and Genomics Platform Didier Busso - April 26, 2007 The Bio-crystallography At the Synchrotron, protein crystal is submitted to X-rays generating diffraction data sets. It is from this 3-D image that researchers will understand how the protein works. These researches have a primordial role in the development of new drugs. Diffraction data sets are analyzed by computers to reconstitute a three dimensional picture of the protein. Structure of RXR-LBD bound to DHA and co-activator peptide. Egea et al., Mol. Endocrinol. (2002) 16:

Structural Biology and Genomics Platform Didier Busso - April 26, 2007 Target selection and domains definition Cloning of Open Reading Frames Expression screening (culture conditions, fusion(s), additives, …) Biomass production Protein purification and characterization Protein crystallization Structure solving => Tidious for numerous ORFs The Bio-crystallography

Structural Biology and Genomics Platform Didier Busso - April 26, 2007 A Platform to ? Speed-up the discovery process in the new post-genomic context Tackle more difficult projects related to human health Miniaturize and parallelize experiments as much as possible to increase yield

Structural Biology and Genomics Platform Didier Busso - April 26, 2007 The Platform’s missions To be a center of technology and expertise To be a site of exchange and support To be an innovative center for the scientific community

Structural Biology and Genomics Platform Didier Busso - April 26, 2007 Cancéropole Génopole Contracts Programs linking european Structural Genomics centers of excellence. Structural Biology and Genomics Platform Its position Inventoried as operational RIO Platform since 2003 Academic users Industrial users

Structural Biology and Genomics Platform Didier Busso - April 26, 2007 Its structure within IGBMC facilities Structural Biology and Genomics Platform Proteomic Platform Bioinformatic Platform Imaging Platform Tight interaction with other Platforms and services Transcriptomic Platform Mouse Clinical Institute IGBMC common services

Structural Biology and Genomics Platform Didier Busso - April 26, 2007 Cartesian Robot Greiner CrystalQuick plate Leica macroscope wit x-y automated stage Examples Crystallization and Imaging Validation Electron Microscopy Its structure Cloning Gateway®: a powerful strategy Tecan Genesis Robot GScope: cloning tools Validation Databases Access tables / SQL server from cloning to crystals and Quality Controls Bar –coding + forms Analytical ultracentrifugation DLS NMR Gel filtration FPLC systems Purification and Characterization Validation Mass spectrometry Infors multi-fermentor 100 liters fermentor Culture flask (E. coli and Insect cells) Optimization and Production Validation SDS-PAGEDeep-well culture platesMicroplate sonicator Small-scale Expression Structure solving Bioinformatics

Structural Biology and Genomics Platform Didier Busso - April 26, 2007 General organization No Feasability Yes Structural Biology and Genomics Platform Know-how Protocols Maintenance Developments Database Support/service Platform advising Scientific Commitee Bench work Project Technical support

Structural Biology and Genomics Platform Didier Busso - April 26, 2007 General organization Project deposition

Structural Biology and Genomics Platform Didier Busso - April 26, 2007 General organization Users

Structural Biology and Genomics Platform Didier Busso - April 26, 2007 The Platform is a bridge between fundamental research and industry. Representative typical contracts with industry (R&D): - ASTRAZENECA 17/12/ /12/200X (renewable) Structure-function relations for the human nuclear receptor NURR1. - BAYER 21/12/ /12/200X Crystallization and X-ray structure analysis of the ligand binding domain (LBD) of the ecdysone receptor (EcR). - SCHERING 2004 – 200X (renewable) - CHUGAI 2004 – AVANTIS 2005 – 2006 Some services for industry: - ALIX 2004 – 200X (renewable) - SUMITOMO – Training in protein crystallography of nuclear receptors. - MOLECULAR ENGINE LABORATORIES Purification, crystallization and structure solving of the TCTP protein. General organization

Structural Biology and Genomics Platform Didier Busso - April 26, 2007 SBGP Website (restricted access) Vector information

Structural Biology and Genomics Platform Didier Busso - April 26, 2007 Protocols and user guides SBGP Website (restricted access)

Structural Biology and Genomics Platform Didier Busso - April 26, 2007 Vector information SBGP Website (restricted access)

Structural Biology and Genomics Platform Didier Busso - April 26, 2007 Bibliography SBGP Website (restricted access)

Structural Biology and Genomics Platform Didier Busso - April 26, 2007 Conclusions Integrated platforms from bioinformatic to structure solving Automated cloning and mini-expression screening Production facility for large scale culture Purification and characterization systems Automated crystallization and screen preparation Integrated data base from bioinformatic to structural determination Structure solving and modeling facilities

Structural Biology and Genomics Platform Didier Busso - April 26, 2007 Statistics Data from the Structural Biology and Genomics Department

Structural Biology and Genomics Platform Didier Busso - April 26, 2007 Technical Coordinator: Didier Busso Edouard Troesch Loubna Salim Production Module Matthieu Stierlé Crystallization Module Pierre Poussin- Courmontagne Structural Biology and Genomics Department IGBMC’s common services Dino Moras Jean-Claude Thierry Cloning-expression Module