Crystallogenesis and X-Ray Diffraction - CNRS URA 2185  

  HEADDr. HAOUZ Ahmed /
  MEMBERSFRAYSSE Jocelyne / MIRAS Isabelle / NAVAZA Rafael/ Dr. SHEPARD William / WEBER Patrick

  Annual Report

The goal of the platform 6 (PF6) is to provide research teams working on macromolecular crystallography at Pasteur with the equipment and technology required for automated crystallogenesis, computing and X ray diffraction measurements, and develop high-throughput methodologies for gene cloning, protein production and crystallization by automating the different steps of the process for structural genomics studies. Funding from the Genopole®Ile de France, the French Ministry of Research, the European Commission and the Pasteur Institute has allowed us to make significant investments in equipment for the project on the structural genomics of mycobacteria (2001-2007). New or upgraded equipment includes a complete X-ray diffraction facility (MicroMax007 rotating anode generator, MarResearch 345dtb Image Plate detector, cryogenic systems), two automatic workstations for protein crystallization (robot TECAN Genesis150, Cartesian Technologies nanodispenser), a microscope with motorized plate (Nikon Eclipse E600) for crystal visualization, and central computing facilities for crystallography and LIMS databases. Our latest acquisition, the Matrix Maker automatic workstation (Emerald BioSystems), is now open to research teams working on macromolecular crystallography for crystal optimization.

Each year, more than 250 different proteins sample have been submitted to automatic crystallization assays, with a success rate (diffracting crystals or exploitable hints) of roughly one of every three proteins assayed. Most of these proteins (about 40%) originated from the Structural Genomics of Mycobacteria project directed by Prof. Pedro ALZARI (IP), As of december 2007, 598 mycobacterial genes have been cloned into bacterial expression vectors using Gateway or related technologies, 236 gene products were expressed as soluble proteins and 85 of these purified to homogeneity and subjected to crystallization trials. We have determined so far 20 crystal structures, mostly using SAD or MAD techniques on SeMet-labeled proteins, and diffraction data has been collected for 5 other proteins for which structure determination is in progress.

The PF6 also participates in several other research projects (PTR, GPH,....) involving structural studies of single proteins or protein complexes. These projects arise from the crystallography labs or from direct collaborations of the PF6 with biology labs at the Pasteur Institute and outside .

Keywords: Protein crystallography, Structural genomics, automated crystallogenesis, X-ray diffraction, tuberculosis, leprosy


Crystal of CysK protein from Bacillus subtilis, PTR 256, obtained at the PF6


Bellinzoni M, Haouz A, Grana M, Munier-Lehmann H, Shepard W, Alzari PM.(2006). The crystal structure of Mycobacterium tuberculosis adenylate kinase in complex with two molecules of ADP and Mg2+ supports an associative mechanism for phosphoryl transfer. Protein Science15:1489-93, PMID: 16672241.

Geerlof A, Brown J, Coutard B, Egloff MP, Enguita FJ, Fogg MJ, Gilbert RJ, Groves MR, Haouz A, Nettleship JE, Nordlund P, Owens RJ, Ruff M, Sainsbury S, Svergun DI, Wilmanns M.(2006). The impact of protein characterization in structural proteomics.Acta Crystallogr D Biol Crystallogr. 62 :1125-36. PMID: 17001090.

Delarue M; Duclert-Savatier N, Miclet E, Haouz A,Giganti D; Ouazzani J, Lopez P, Nilges, and M; Stoven V . (2007). Three dimensional structure and implications for the catalytic mechanism of 6- phosphogluconolactonase from Trypanosoma brucei.Journal of Molecular Biology 366 : 868-881. PMID: 17196981

Shepard W, Haouz A, Grana M, Buschiazzo A, Betton J-M, Cole ST, Alzari PM. The crystal structure of Rv0813c from Mycobacterium tuberculosisreveals a new family of FABP-like proteins in bacteria. Journal of Bacteriology, 189: 1899-1904. PMID: 17172346

Topalis,D., Kumamoto, K., Amaya-Velasco, MF., Dugué, L., Haouz, A.,Alexandre, J., Gallois-Montbrun, S., Alzari P.M., Pochet, S., Agrofoglio L. and Deville-Bonne, D. (2007).Nucleotide binding to human UMP-CMP kinase using fluorescent derivatives, a screening approach based on affinity for UMP-CMP binding site.FEBS Journal.274: 3704-3714. PMID: 17608725

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Activity Reports 2007 - Institut Pasteur
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