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Institut de Biologie StructuraleGrenoble / France

Contact person(s) related to this article / NEUMANN Emmanuelle / SCHOEHN Guy

Presentation of the Methods and Electron Microscopy group

Head of group: Guy SCHOEHN

The group is also in charge of the EM platform.


The group is divided into 4 teams:

Team Guy Schoehn : Macromolecular assemblie’s structure
Members: Maria Bacia, Daphna Fenel, Grégory Effantin, Leandro Estrozi, Benoit Gallet, Wai-Li Ling, Emmanuelle Neumann, Hélène Malet, Christine Moriscot, Stéphanie Hutin, Guy Schoehn

Team Irina Gutsche : Bacterial stress response and viral nucleocapsid
Members: Irina Gutsche, Isai Kandiah, Diego Carriel, Jan Félix, Angélique Fraudeau, Matthew Jessop, Clarissa Liesche, Karine Huard, Megghane Baulard

Team Jean-Luc Pellequer : AFM
Members : Jean-Luc Pellequer, Jean-Marie Teulon

Team Pascal Fender : Adenovirology
Members : Pascal Fender, Emilie Stermann, Charles Vragniau

Aims of the group

The group is involved in the structure determination of biological macromolecular complexes and in the understanding of structure-function relationships. Two teams use electron microscopy as main tool. The third team mainly uses AFM studies and the last one is specialized in adenovirology.

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. We then combine these volumes 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.

Equipments

Transmission electron microscopes:
- FEI Tecnai12 120kV LaB6 with Orius GATAN camera (negative staining)
- FEI Tecnai F20 200kV FEG with 4kx4k Ceta FEI camera (cryo and tomography), Windows 7
- FEI Tecnai Polara 300kV FEG with K2 Summit GATAN electron counting direct detection camera, GMS 3.2 (automatic data collection) and 2kx2k GATAN GIF (cryo and tomography)
Microscopy associated instruments:
- Carbon evaporator
- Glow discharger
- FEI Vitrobot (automated vitrification machine)
- Film scanner

Instruments for cellular sample preparation:
- Automated high-pressure freezer Leica HPM100
- Automated freeze substitution machine Leica AFS2 + FSP
- (Cryo-)ultra-microtome Leica UC7/FC7
- Automated immuno-labeling machine Leica IGL

Microscopes à Force Atomique :
- Multimode 8, Nanoscope V (Bruker)
- Dimension 3100, Nanoscope V (Bruker)
- Instruments associés aux microscopes :
- Microscope fluo, Nikon Eclipse TE2000
- Lampe UV/Ozone

Keywords

Negative Staining, Cryo-Electron Microscopy, 3D image reconstruction, Cryo-Tomography, Virus, Microtubules, Macromolecular Complexes, Methodology, Direct detection ef electrons, AFM, Adenovirology.

New Major References

- Rodrigues CD, Henry X, Neumann E, Kurauskas V, Bellard L, Fichou Y, Schanda P, Schoehn G, Rudner DZ, Morlot C. (2016). A ring-shaped conduit connects the mother cell and forespore during sporulation in Bacillus subtilis. Proc Natl Acad Sci U S A. 113(41):11585-11590.

- Kandiah E, Carriel D, Perard J, Malet H, Bacia M, Liu K, Chan SW, Houry WA, Ollagnier de Choudens S, Elsen S, Gutsche I. (2016). Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA. Sci Rep. 6:24601.

- Effantin G, Estrozi LF, Aschman N, Renesto P, Stanke N, Lindemann D, Schoehn G, Weissenhorn W. (2016). Cryo-electron Microscopy Structure of the Native Prototype Foamy Virus Glycoprotein and Virus Architecture. PLoS Pathog. 12(7):e1005721.

- Costa L, Andriatis A, Brennich M, Teulon JM, Chen SW, Pellequer JL, Round A. (2016). Combined small angle X-ray solution scattering with atomic force microscopy for characterizing radiation damage on biological macromolecules. BMC Struct Biol. 16(1):18.

- Coscia F, Estrozi LF, Hans F, Malet H, Noirclerc-Savoye M, Schoehn G, Petosa C. (2016). Fusion to a homo-oligomeric scaffold allows cryo-EM analysis of a small protein. Sci Rep. 6:30909.

- Chen SW, Teulon JM, Godon C, Pellequer JL. (2016). Atomic force microscope, molecular imaging, and analysis. J Mol Recognit. 29(1):51-5.

- Richter M, Yumul R, Wang H, Saydaminova K, Ho M, May D, Baldessari A, Gough M, Drescher C, Urban N, Roffler S, Zubieta C, Carter D, Fender P, Lieber A.(2015) Preclinical safety and efficacy studies with an affinity-enhanced epithelial junction opener and PEGylated liposomal doxorubicin. Mol Ther Methods Clin Dev. 11 ;2:15005.

- Gutsche I, Desfosses A, Effantin G, Ling WL, Haupt M, Ruigrok R, Sachse C, Schoehn G. Near-atomic cryo-EM structure of the helical measles virus nucleocapsid. Science (2015) 348 (6235):704-7

- Sumarheni S, Hong SS, Josserand V, Coll JL, Boulanger P, Schoehn G, Fender P. (2014) Human Full-Length Coagulation Factor X and a GLA Domain-Derived 40-mer Polypeptide Bind to Different Regions of the Adenovirus Serotype 5 Hexon Capsomer. Hum Gene Ther. 25(4):339-49.

- Malet H, Liu K, El Bakkouri M, Chan SW, Effantin G, Bacia M, Houry WA, Gutsche I. (2014) Assembly principles of a unique cage formed by hexameric and decameric E. coli proteins. Elife. 3:e03653.

- Gebhardt R, Teulon JM, Pellequer JL, Burghammer M, Colletier JP, Riekel C. (2014) Virus particle assembly into crystalline domains enabled by the coffee ring effect. Soft Matter. 10(30):5458-62.

- Lu ZZ, Wang H, Zhang Y, Cao H, Li Z, Fender P and Lieber A. (2013) Penton-Dodecahedral Particles Trigger Opening of Intercellular Junctions and Facilitate Viral Spread during Adenovirus Serotype 3 Infection of Epithelial Cells. PLoS Pathog. 9(10):e1003718

The list of all the publications of the group is available here.