Presentation of the LMES

Electron Microscopy in Structural Biology

Negative Staining

This technique, fast and easy to perform, allows direct visualization of proteins (from 50-100 kDa). The embedding of these proteins with heavy atoms yields highly contrasted images. Although this procedure only gives structural information from the surface of the object, it is required for checking any biological sample before a cryo-electron microscopy study or any crystallization step.

Cryo-Electron Microscopy and 3D Analysis

This technique does not require any chemical fixation or stain and thus better preserves the structure of the object. More complicated to run than the negative stain procedure, it consists in freezing thin hydrated samples very fast in liquid ethane, in order to fix them in amorphous ice. This method both allows to preserve the native structure of the specimen and to access the internal structure of macromolecular complexes. Since no stain is added, the contrast obtained on the images is very low and 3D analysis is then required. During 3D analysis low contrasted images are averaged in order to increase the signal to noise ratio. Images are 2D projections of a same object in different orientations. These projections are mathematically combined to obtain a 3D structure of the object of interest.

Aims of the laboratory

Our laboratory is interested in determining the structure of biological molecular complexes in order to reveal the molecular interactions and their biological implications. We employ cryo-electron microscopy and 3D image reconstruction to obtain medium resolution maps which we combine with the X-ray crystallography atomic structures of the component molecules. The development, validation and application of novel computational methods play a key role in our research. We provide efficient and user-friendly tools (and support) for structure determination and interpretation for both the expert and novice researcher.

Keywords

negative staining, cryo-electron microscopy, 3D image reconstruction, virus, microtubules, motor proteins, MAPs, MR with 3DEM probes, X-ray/EM fit

Major Publications

• Neumann E, Garcia-Saez I, Debonis S, Wade RH, Kozielski F, Conway JF (2006) J Mol Biol 362(2), 203-11. Human Kinetochore-associated Kinesin CENP-E Visualized at 17 Resolution Bound to Microtubules.(abstract)

• Trapani S, Navaza, J (2006) Acta Cryst A62, 262-269. Calculation of spherical harmonics and Wigner d functions by FFT. Applications to fast rotational matching in molecular replacement and implementation into AMoRe. ( abstract )

• Navaza J (2003) J Struct Biol 144, 13-23. On the three-dimensional reconstruction of icosahedral particles. ( abstract )

• Hewat EA, Neumann E & Blaas D (2002) Mol Cell 10, 317-326. The concerted conformational changes during human rhinovirus 2 uncoating. ( abstract )

• Hewat EA, Neumann E, Conway JF, Moser R, Ronacher B, Marlovits TC, Blaas D (2000) EMBO J 19(23), 6317-25. The cellular receptor to human rhinovirus 2 binds around the 5-fold axis and not in the canyon: a structural view. (abstract)

(The list of all the publications of the laboratory is available here)