SummaryNuclear pore complexes (NPCs) are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Elucidating their 110 MDa structure imposes a formidable challenge and requires in situ structural biology approaches. Fifteen out of about thirty nucleoporins (Nups) are structured and form the Y- and inner ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ∼60 nm in diameter 1. The scaffold is decorated with transport channel Nups that often contain phenylalanine (FG)-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y-complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here, we combined cryo electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modeling to generate the most comprehensive architectural model of the NPC to date. Our data suggest previously unknown protein interfaces across Y-complexes and to inner ring complex members. We demonstrate that the higher eukaryotic transport channel Nup358 (RanBP2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport channel Nups. We conclude that, similarly to coated vesicles, multiple copies of the same structural building block - although compositionally identical - engage in different local sets of interactions and conformations.

The nuclear pore complex (NPC) is a fundamental component of all eukaryotic cells that facilitates nucleocytoplasmic exchange of macromolecules. It is assembled from multiple copies of about 30 nucleoporins. Due to its size and complex composition, determining the structure of the NPC is an enormous challenge, and the overall architecture of the NPC scaffold remains elusive. In this study, we have used an integrated approach based on electron tomography, single-particle electron microscopy, and crosslinking mass spectrometry to determine the structure of a major scaffold motif of the human NPC, the Nup107 subcomplex, in both isolation and integrated into the NPC. We show that 32 copies of the Nup107 subcomplex assemble into two reticulated rings, one each at the cytoplasmic and nuclear face of the NPC. This arrangement may explain how changes of the diameter are realized that would accommodate transport of huge cargoes.

Nuclear pore complexes (NPCs) are 110-megadalton assemblies that mediate nucleocytoplasmic transport. NPCs are built from multiple copies of ~30 different nucleoporins, and understanding how these nucleoporins assemble into the NPC scaffold imposes a formidable challenge. Recently, it has been shown how the Y complex, a prominent NPC module, forms the outer rings of the nuclear pore. However, the organization of the inner ring has remained unknown until now. We used molecular modeling combined with cross-linking mass spectrometry and cryo-electron tomography to obtain a composite structure of the inner ring. This architectural map explains the vast majority of the electron density of the scaffold. We conclude that despite obvious differences in morphology and composition, the higher-order structure of the inner and outer rings is unexpectedly similar.

Cryo-EM tomography of wild-type and mutant cilia and flagella from Tetrahymena and Chlamydomonas reveals new information on the substructure of radial spokes.