Atomic Structure of the Nuclear Pore Complex
EMSL Project ID
50998
Abstract
The nuclear pore complex (NPC) is the sole gateway for the transport of macromolecules between the nucleus and cytoplasm. NPCs are large (~120 MDa in humans) macromolecular assemblies embedded in the nuclear envelope, each containing ~1000 proteins termed nucleoporins. We adopt a divide-and-conquer strategy of reconstituting NPC subcomplexes, validating protein-protein interactions by biochemical and in vivo analyses in S. cerevisiae and human cell lines, and determining crystal structures or cryo-EM reconstructions of nucleoporins and nucleoporin complexes at side-chain resolution level. Over 15 years, our laboratory has determined >80 NPC-related crystal structures, including very challenging ~450 kDa heterohexameric and ~150 kDa heterotetrameric nucleoporin complexes. This effort allowed us to generate a composite structure of the human NPC symmetric core by quantitatively docking crystal structures into a ~23 Å cryo-ET reconstruction of the intact human NPC. Whereas the composite structure defines the spatial position of structured protein domains, the molecular details of the nucleoporin interactions giving rise to the overall NPC need to be determined by solving structures of NPC subcomplexes at side-chain resolution. In particular, most interactions that hold the inner ring of the NPC together involve flexible linear motifs of “linker” nucleoporins that bind multiple large structured nucleoporins. Owing to their linear nature, the structural elucidation of these motifs requires methods that can resolve single residue moieties. With our expertise in reconstituting and optimizing large NPC subcomplex samples, as well as preliminary data supporting their amenability to high resolution single particle cryo-EM reconstruction, we propose to harness the advantages of state-of-the-art equipment and cutting-edge cryo-EM expertise at PNCC in order to fulfil the goal of elucidating the architecture of the NPC symmetric core at side-chain resolution.
Project Details
Start Date
2019-08-01
End Date
2020-01-14
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members