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Content archived on 2024-06-18

Computing the structure and dynamics of protein assemblies in living cells by coupling sub-diffraction fluorescence microscopy with single-particle reconstruction: application to viral capsids

Objective

Electron microscopy (EM) is an invaluable tool for investigating the nanometer-scale organization of molecular assemblies such as viruses, but is restricted to dead cells, does not readily label targeted proteins, and is prone to fixation artefacts. Recently developed methods to break the diffraction limit in optical microscopy have the potential to resolve protein arrangements in living cells. However, their resolution is currently restricted to ~20-30 nm, still an order of magnitude removed from EM, and dynamic super-resolution imaging remains challenging. Here, we aim to reconstruct the protein arrangements of molecular structures at resolutions better than 20 nm by harnessing the power of statistics, i.e. by aggregating images from hundreds or thousands of copies of nearly-identical structures and when possible by exploiting their symmetry. To do this, we will adapt computational methods of single particle reconstruction from electron microscopy to super-resolution optical microscopy. After validation on synthetic data, we will test and apply these methods to nuclear pores and adenovirus capsids. These examples have been chosen because of their geometric features that work well with our approaches. Particularly, we are interested in obtaining novel insight into the dynamic structural changes occurring at the nuclear pore complex during active transport. Furthermore, we aim to decipher the sequence of events during viral capsid formation. This work has the potential to further push the resolution of optical microscopy towards that of electron microscopy for the analysis of ordered molecular assemblies. If successful, our project will open the door to structural investigations in living cells, including the assembly process of viral particles, or the plasticity of the nuclear pores and its role in nucleo-cytoplasmic transport.

Call for proposal

FP7-PEOPLE-2009-IEF
See other projects for this call

Coordinator

INSTITUT PASTEUR
EU contribution
€ 165 645,60
Address
RUE DU DOCTEUR ROUX 25-28
75724 Paris
France

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Region
Ile-de-France Ile-de-France Paris
Activity type
Research Organisations
Administrative Contact
Nadia Khelef (Dr.)
Links
Total cost
No data