Institut de Biologie StructuraleGrenoble / France

Macromolecular complexes

Macromolecular Assemblies

Most of the experiments are in collaboration with other groups (mainly IBS, EMBL, iRTSV) with expertise in the biochemistry of the samples.
We are interested in understanding how macromolecular assemblies function by determining their 3D structures using cryo-EM. The samples we study are varied and include entire viruses (EBV, adenovirus, bacteriophages …), virus polymerase (Influenza virus, VSV), ESCRT complex, protease, peptidase, secretin, microtubules, ribosome complexes, and nanotubes. We perform classical (cryo-)EM and image analysis to obtain the highest possible resolution 3D structure. There is a great demand at the IBS for EM and we will continue to support other groups in their research. To this end, we will make the best use of our new instruments and image analysis techniques. We are especially interested in negative stranded RNA viruses, which we study in collaboration with the Ruigrok/Jamin group at IBS and the Cusack group at EMBL. We are in the process of solving the 3D structure of small particles (< 250 kDa). Because they are considered very small for EM analysis and thus require specific strategies to ensure that the correct structure is solved. We will use the new detector to improve their structures once the method is validated with catalase, a complex equivalent in size but with a known structure.
Our development activity in the experimental part will be focused on electron tomography for single particle (applied to C1q/C1 and phages) but also coupled with cell sectioning. This later part is currently under development on the Golgi proteins (H Lorta-Jacob) and the ESCRT system (W Weissenhorn), respectively. At mid term, we also aim to develop correlative light electron microscopy and will use the adenovirus model (see P Fender) to achieve this.

Examples of achievements:

ESCRT system

See Weissenhorn Group
3D reconstruction of a 485 Å average diameter tube. Two helically related asymmetric units are colored green and red.

Team Members : Christine Moriscot, Grégory Effantin et Guy Schoehn
Collaboration : Groupe EBEV IBS Winfried Weissenhorn

Negative staining EM image of CdvA filaments (scale bar: left 50 nm; right 20 nm)

Reference :
Moriscot C, Gribaldo S, Jault JM, Krupovic M, Arnaud J, Jamin M, Schoehn G, Forterre P, Weissenhorn W and Renesto P (2011). Crenarchaeal CdvA forms double-helical filaments containing DNA and interacts with ESCRT-III-like CdvB. PLoS One 6(7): e21921

Using sucrose density gradient ultracentrifugation and negative staining electron microscopy, we were able reinforce the concept that Cdv proteins were closely related to the eukaryotic ESCRT-III counterparts and suggested that the organization of the ESCRT-III machinery at the Crenarchaeal cell division septum was organized by CdvA, an ancient cytoskeleton protein that might help to coordinate genome segregation.

Effantin G, Dordor A, Sandrin V, Martinelli N, Sundquist WI, Schoehn G, Weissenhorn W. (2013). ESCRT-III CHMP2A and CHMP3 form variable helical polymers in vitro and act synergistically during HIV-1 budding. Cell Microbiol. 15(2):213-26.
We have solved the endosomal sorting complex required for transport-III (ESCRT-III) CHMP2A–CHMP3 structure at 22 Å resolution using cryo-EM after sorting the tubes according to their diameters and helical parameters . This complex is essential for budding of some enveloped viruses, for the formation of intraluminal vesicles at the endosome, and for the abscission step of cytokinesis. Using siRNA knockdown experiments and surface plasmon resonance affinity measurements, we have also shown that the CHMP2A–CHMP3 polymer observed in vitro contributes to HIV-1 budding by assembling on CHMP4B polymers.

Sporulation complex SpoIIIA

Team Members : Emmanuelle Neumann et Guy Schoehn
Collaboration : Pneumococcus group IBS (Cécile Morlot)

3D Reconstruction 3D of SpoIIIA complex with a background of negative stained image of the same complex

Bacterial sporulation is a good model of morphologic differenciation leading to the formation of a spore resistant to extreme environmental conditions.
One of the key steps of the spore development cycle is the assembly of an hetero-multimeric complex enclosing at least 9 distinct proteins (SpoIIIAA-AH et SpoIIQ) going through the mother cell and prespore membranes.
Several groups at IBS (PG, MEM and RMN) in collaboration with David Rudner in Harvard Medical School Boston, have produced complementary data in structural and cell biology which have shown that SpoIIIAG forms an ogilomeric ring essential for the development of the prespore.
The 3D reconstruction by cryo-EM of this ring revealed an architecture "cup and saucer" with a central hole of 6 nm.

Reference :
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.

Symmetrization of small proteins to solve their structure by electron microscopy

Team Members : Léandro Estrozi, Hélène Malet, Guy Schoehn
Collaboration : VIC group IBS (Carlo Petosa)

This project has shown that it was possible to symmetrize a protein using a scaffolding formed by an oligomeric protein. We solved the structure of the
Maltose Binding Protein (40 kDa) by electron microscopy with a resolution of 6-8 Å.

3D reconstruction of MBP and glutamine synthetase complex.
The right part represented in detail a part from GS

Reference :
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.

The C1 Complex

W.L. Ling, 2010

Team Members : Christine Moriscot, Wai Li Ling, Guy Schoehn
Collaboration : IRPAS group IBS (Nicole Thielens)

In collaboration with the "Immune Response to Pathogens and altered-self" group, we study the C1 complex of the innate immune system and its target recognition unit C1q with various ligands, including proteins such as enzymatically modified low density lipoprotein, calreticulin, prion protein, and also objects foreign to the body such as carbon nanotubes commonly employed in nanotechnology.

References :
Gaboriaud C, Ling WL, Thielens NM, Bally I, Rossi V (2014). Deciphering the fine details of c1 assembly and activation mechanisms: "mission impossible"? Front Immunol. 2014 Nov 6;5:565.
Bally I, Ancelet S, Moriscot C, Gonnet F, Mantovani A, Daniel R, Schoehn G, Arlaud GJ, Thielens NM (2013). Expression of recombinant human complement C1q allows identification of the C1r/C1s-binding sites. Proc Natl Acad Sci U S A. 2013 May 21;110(21):8650-5.