Unit: Molecular Cryomicroscopy

Director: Gérard Pehau-Arnaudet

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 three 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 second is the structural study of the RC-LH1 super complex of Rubrivivax gelatinosus and the third of the 3D study of HBs subviral particles

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.

We are also engaged in different courses.

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…

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

Surface layers (S-layers) are components of the bacterial cell envelopes, consisting of protein molecules arranged in two-dimensional crystalline arrays. Bacillus anthracis synthesizes two abundant surface proteins of 94 kDa: Sap (Surface array protein) and EA1 (Extractable Antigen 1), which formed two independent crystals on the surface of Bacillus anthracis.

In our laboratory, we investigate the organization of the Sap protein in its S-layer by two dimensional electron crystallography. For this purpose we set up the techniques of two dimensional crystallization on lipid layer and adapt it to the studies of Sap recombinant protein, which have been deleted by its protein domain responsible for cell anchoring and have been replaced by a histidine-tag motif: using this approach, we reconstituted in vitro a two dimensional crystal with the same space group and crystalline arrangement as those formed by the wild-type protein on the bacterial surface in vivo. These crystals have been used to be study by cryo-electron crystallography. With image analysis, we obtained projection maps at 12 Å resolution, which was interpreted in terms of the overall molecular envelope of the protein and its spatial organisation in the S-layer.

In parallel, we carry out three dimensional crystallization experiments to allow X-ray crystallographic studies, in order to fit the atomic structure of the Sap protein to its molecular envelope resolved by electron crystallography.

Reconstituted crystal in vitro of Sap recombinant protein

on the left : filtered image of the two dimensional crystal of, obtained by cryo-electron microscopy ( about 220 nm x 220 nm). on the right : projection map of the Sap crystal at 12 Å resolution. The lattice is in red and contain two molecule reliable by a P2 symmetry. Each protein is composed by 4 domains, marked from 1 to 4. Unit cell parameters: a = 207,5 ± 1,4 Å, b = 87,8 ± 1,4 Å, γ = 96,5 ± 0,20°.

2-Understanding the transfer mechanism of the reduced quinone to its site of oxydation by structural analysis of low resolution electron microscopy data: J-L. Ranck (CR1, CNRS, UMR 168, Curie Institut, Paris), F. Reiss-Husson (DRCE, CNRS, CGM-UPR6067, Gif sur Yvette), G. Pehau-Arnaudet

Photosynthetic bacteria converts light energy (using light harvesting LH1and LH2) in a chemical form (reaction center RC). During this process, reduced quinones are produced at the RC level. Then, the quinone diffuse to the oxidative site located on the cytochrome bc1. From the RC to the cytbc1, the quinone has to diffuse in the midst of the plastic membrane through a LH1 ring surrounding the reaction center. A previous work on Rhodobacter sphaeroide, where the complex RC-LH1 forms, in vivo, large tubes with a two dimensional crystal organization has demonstrated that the LH1 ring is not complete, leaving the possibility for a free diffusion of the quinone. To generalyse this observation, we decided to work on another specie of bacteria, Rubrivivax gelatinosus, by isolating, purifying and reconstituting 2-D crystals, in spite to analyze by electron microscopy the structure of the LH1 ring. We also analyzed the complex RC-LH1 in solution. First results seem indicate, that the ring is incomplete. We are currently improving these structure by aligning and averaging a large number of pictures of supra molecular complexes.

3-3D structural study of the HBs sub-particles : I. Komla-Souka (PhD student, INTS, Paris), G. Pehau-Arnaudet, C. Sureau (CR1, CNRS, INTS, Paris)

In spite of extensive studies over the past 25 years, we are still lacking precise information on the tri-dimensional structure of the HBV envelope proteins present at the surface of the viral or subviral particles (also called HBs particles) Our understanding of the assembly and infectivity of HBV remains limited due, in part, to the absence of structural knowledge of the HBV surface. This represents an impediment to the development of new neutralizing molecules targeted to the outer elements of the particles. Our goal is to produce 3D structure of the HBs particles which are abundantly expressed during the course of a natural infection, and which serve also as a vaccine. The particles are made of only one type of glycoprotein, and they display the same surface proteins as that of the mature virion. We are planning to produce and purify HBs particles (INTS, Paris) for examination by electron microscopy using negatively stained or vitrified samples. Structural information should allow us to better understand the interactions between envelope proteins and neutralizing antibodies, and it should also facilitate the design of new antiviral strategies.

4- Facility

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.

Autumn 2002: the scientific committee, directed by F. Livolant approved our optimization settings and opened its access to the entire French scientific community. February 6th,, 2003, the new cryo-electron microscope was inaugurated: http://www.cnrs.fr/CMA/dyna/article.php3?id_article=244

After the agreement of the scientific committee, two scientists worked on the cryo-microscope during the past-year.

a- From LMCP, Nicolas Boisset PhD, (DR2, CNRS) (http://www.lmcp.jussieu.fr/%7Enboisset/ )

b- From Rennes, Patrick Bron, PhD, (MC, UMR 6026,Rennes University) (http://www.crm.univ-rennes1.fr/)

5- Courses

During the past year, we were engaged in two courses: an internal course of electron microscopy (organized by the Plate-forme de microscopie électronique), and in the Protein Biochemistry course, part of he postgraduate Advanced Master Degree (DEA) in the Structure, Function and Engineering of Proteins run by the Universities of Paris 6, Paris 7, and Paris 11.

Keywords: cryo-microscopy, electron microscopy, structural biology, Sap protein, RC-LH1 super complex, HBs

Activity Reports 2003 - Institut Pasteur

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