Extended Data Figure 5. Connection of the inner and outer Y-complexes.
(a) Four clamp-shaped densities (segmented red) emanate from the Nup96/107 region of both Y-complexes in the CR. Only the inner clamps connect both Y-complexes, whereas the outer ones protrude into a more complex substructure at the outer periphery of the CR (Fig. 2b). (b) Same as in Fig. 2c, d but for the NR. (c) Volcano plot visualizing shotgun proteomics data obtained of HeLa cells in the Nup358 knockdown (treatment) as compared to the control condition. (d) Nuclear transport assays of NLS-MBP-GFP in non-treated cells and the Nup358 knockdown condition. Cargos with a classical nuclear localization signal are imported in the absence of Nup358 as previously observed 27 although a lower efficacy cannot be excluded. (e) Classification of subtomograms of the knockdown condition reveals that approximately 5% of all asymmetric units contain an outer Y-complex in the CR, which is in excellent agreement with the knockdown efficiency of ∼95%. Classification was done on the level of asymmetric units. Transferred to the level of NPCs, it suggests that out of 920 NPCs observed under gene silencing conditions, 663 had no outer Y-complex in the CR, 183 had one, 49 had two, 14 had three, 4 had four, 6 had five, 0 had six, 1 had seven and 0 had eight. (e) The observed adjacency of outer Y-complexes in the CR under knockdown conditions was much higher than expected. The 5% of asymmetric units that contained outer Y-complexes in the CR were analyzed on the NPC level to determine whether their neighboring asymmetric units also contained outer Y-complexes in the CR. The observed frequency of adjacency is shown in dark blue. The respective total number of observed outer Y-complexes in the CR and the number of the ones that had adjacent partners is indicated as (n/m). The observed frequency is considerably elevated over the theoretical frequency (shown in bright blue) that would be expected if Y-complexes would bind to random subunits. This observation implies cooperativity for Y-complex assembly/maintenance within the CR that might arise through the head to tail contact of adjacent Y-complexes. NPCs with 0, 1 and 8 outer Y-complexes per CR are not shown because they cannot contain any adjacent Y-complexes or were not observed, respectively.