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  Director : Gérard Pehau-Arnaudet (gpehau@pasteur.fr)



Since April 2002, the molecular cryo-microscopy platform is devoted to introduce a modern tool of structural biology in the Institut Pasteur, the cryo-electron microscopy. This new approach complete others approaches (X-rays, NMR), and consist in the observation of frozen hydrated specimens. Combined with image analysis, this technique can provide structural information at medium resolution (between 7 and 30 Å). We are currently working on two different topics: the first one supported by electron crystallography, is the analysis of one S layer of bacillus anthracis (with M Mock's team).

The other-one (with K.M.Kean's team) concern the genome of HCV. We want to test the validity of a model of circularisation of RNA to increase the efficiency of translation.

In December 2001 we started to use a new field emission gun (FEG) cryo-microscope, Jeol 2010F, and to receive external research teams who require this technique to perform structural studies at high and medium resolution.



The platform for molecular cryo-microscopy was created in April 2002. Its vocation is to bring the advantages of electron microscopy into the field of structural biology, thus complementing more classical approaches such as NMR or X ray analysis that are already available on the campus at the Institut Pasteur.

Developed during the early 1980s, cryo-microscopy is one of the most powerful techniques of structural biology. It is based on the high speed freezing of biological specimens in their fully hydrated state. The speed of freezing avoids formation of hexagonal or cubic ice, the samples being embedded in a film of vitreous ice. Analysis of the specimen under the microscope is performed at a temperature of -180°C using low doses of electrons (less than 10 electrons by Å2).

Structural data is obtained after image treatment by computational analysis. One of the major interests of cryo-microscopy is that it applies to many types of biological samples such as individual proteins (PM ≥ 600kDa), 2D crystals of proteins (PM ≥ 30kDa), viruses, DNA, RNAs…

We are currently working on two different topics.

1-Structure of Bacillus anthracis S-layer using electron crystallographyXavier Hagnerelle, Gérard Pehau Arnaudet, Agnès Fouet et Pedro Alzari

S-layers form the outermost layers of many Bacteria and Archaea, and consist of protein molecules arranged in two dimensional crystalline arrays (1). Bacillus anthracis synthesizes two abundant surface proteins of 94 kDa: Sap (Surface array protein) and EA1 (Extractable Antigen 1). Both proteins are arranged in two different crystal forms in a growth phase-dependant manner(2,3). Recent work showed that these two proteins might act as transcriptional repressors of eag (EA1 gene) (2).

In our laboratory, we investigate the structure of the Sap protein and its organization within the S-layer by two dimensional electron crystallography, in order to complement available genetic and biochemical data. For this purpose we set up a strategy of two dimensional crystallization on lipid layer (4) in order to reconstitute in vitro the same arrangement found at the bacterial surface. Using this approach, we obtained projection maps of two dimensional crystals at 15Å resolution and we are currently testing quality to record higher resolution data. In parallel with these efforts, the Sap protein has been over-expressed in recombinant form and three dimensional crystallization experiments are currently in progress to allow X-ray crystallographic studies.

(1) U. B. Sleytr, P. Messner, D. Pum, and M. Sara. Crystalline Bacterial Cell Surface Layers. Springer Verlag Berlin Heidelberg, 1988.

(2) T. Mignot, S. Mesnage, E. Couture-Tosi, M. Mock; A. Fouet.  Molecular Microbiology,  43(6), 1615-1627, 2002. 

(3)  E. Couture-Tosi, H. Delacroix, T. Mignot, S. Mesnage, M. Chami, A. Fouet  and G. Mosser, Journal of Bacteriology, 6448-6456, 2002

(4)  E. E. Uzgiris, R. D. Kornberg, Nature, 301(5896),125-129, 1983.

Projection map of Sap array at 15 Å resolution.

Unit cell parameters : a = 200 A, b = 86 Å, angle = 96° Insert : diffraction pattern, the first circle corresponds to 1/17Å

2-Circularisation mechanism of viral mRNA : a potential target for a wide-spectrum anti-viral strategy? Gérard Pehau-Arnaudet, Sylvie Paulous, Katherine M. Kean

In eukaryotic cells efficient translation initiation on cellular mRNAs requires a functional interaction between the 5' cap and the 3' poly(A) tail, mediated by interactions between the proteins that bind the ends of the mRNA (eIF4E and PABP for poly(A)-binding protein) respectively, both bound to the scaffold protein eIF4G.(1,2,3,4) These interactions allow the physical circularisation of cellular mRNAs.

This effect of the RNP mediated 3' to 5' interaction, and consequently the role of mRNA circularisation to enhance initiation of the translation, would be a property of a number eukaryote pathogens such as viruses. The M.C. Kean team (Institut Pasteur) has developed some experimental models to study the viral RNA translation.

The hepatitis C virus genome (3' uncapped and 5'non-polydenylated) was chosen for two major reasons. First, the importance of such virus in public health and second, the seemingly simplicity of the model. Using nuclease- treated rabbit reticulocytes, it was demonstrated a "communication" between the 5' IRES end and a site near the 3' end of the genome.

Our goal is to develop visualisation conditions of the RNP-RNA complexes using electron microscopy to validate or invalidate the circularisation model.

(1) Gallie DR., Genes DEv., (1991), 5, 2108-21016.

(2) Hentze M. W., Science, (1997), 275, 500-501.

(3) Imataka H., Gradi A., Sonenberg N., EMBO J., (1998), 17, 7480-7489.

(4) Wells SE., Hillner PE., Vale RD., Sachs AB., Molecular Cell., (1998), 2, 135-140.

In December 2001, a project co-financed by the Institut Pasteur, the Centre National pour la Recherche Scientifique and the Région Ile de France allowed acquisition of a 200kV Jeol electron microscope with a field emission gun that is entirely equipped for high-resolution cryo-microscopy. This project was born from the will of some groups of scientists from the GDR2368 who wanted to acquire an up-to-date microscope. Since January 2002, we have tested and optimized the performances of this microscope. In autumn 2002, the scientific committee, directed by F. Livolant approved our optimization settings and opened its access to the entire French scientific community.

Photo 1:Projection map of Sap array at 15 Å resolution.Unit cell parameters : a = 200 A, b = 86 Å, angle = 96° Insert : diffraction pattern, the first circle corresponds to 1/17Å

Photo 2: Jeol 2010F, cryo-electron microscope, 200kV



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        Gérard Pehau-Arnaudet, IE2, CNRS,gpehau@pasteur.fr

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