Unit: Macromolecular crystallisation and X-ray diffraction facility (Platform)
Director: ALZARI, Pedro M.
The goal of the platform is to provide the research teams working on macromolecular crystallography at Pasteur with the equipment and technology required for automatic crystallogenesis, computing and X ray diffraction, as well as to develop high-throughput methodologies for gene cloning, protein production and crystallization, by automating the different steps of the process. During the last eighteen months, over 100 proteins have been submitted to automatic crystallization assays, with a success rate (diffracting crystals or exploitable hints) of roughly one every three proteins. Most of the proteins originated from the project on the structural genomics of mycobacteria (about 60%) described below, while the rest came from the different labs of protein crystallography or direct collaborations of the PF6 with non-crystallographers.
Structural Genomics of Mycobacteria
Supported by grants from the National Genopole Network, the European Union (SPINE and X-TB contracts) and the Institut Pasteur, we have undertaken a structural genomics project on mycobacterial proteins, in collaboration with several laboratories within the Institute. The list of targets includes a number of proteins that are known to be important for mycobacterial survival and/or virulence, as well as several others of unknown biological function but whose relevance as potential therapeutic targets is highlighted by the comparative analysis of mycobacterial genomes. As an initial outcome of this project, two technical platforms have been setup for protein production (http://www.pasteur.fr/recherche/genopole/PF5) and high-throughput cloning and protein crystallization (http://www.pasteur.fr/recherche/genopole/PF6). At present, more than 400 mycobacterial genes have been subcloned into bacterial expression vectors, several purified proteins have been subjected to crystallization trials and to date 15 crystal structures have been determined at high resolution. Some of these correspond to mycobacterial genes of previously unknown function, for which the structural information has provided useful hints to elucidate their putative biochemical function.
Other ongoing projects
Through collaborations with other laboratories, the PF6 is also involved in a number of crystallographic studies of single proteins and protein complexes. Ongoing projects include several proteins involved in Trypanosoma cruzi metacyclogenesis (Amsud-Pasteur project), secreted Chlamydia proteins of unknown function that could be involved in pathogenicity (coll. A. Subtil, IP), human protein kinases in complex with specific inhibitors (coll. R. Biondi, Univ. Saarland, Germany), protective anti-cholera antibodies specific for the LPS antigen (coll. J.M. Fournier, IP), human UMP-CMP kinase and complexes with inhibitor (coll. D. Deville-Bonne, Institut Jacques Monod, Paris), P. falciparum serine-proteases (coll. J.C. Barale, PTR, IP), and Yak1 kinase from pathogenic Candida (coll. G. Janbon, PTR, IP).
Figure: Three proteins of unknown function from M. tuberculosis.
Keywords: structural genomics, gene cloning, protein purification, crystallogenesis, X-ray diffraction, LIMS