Extended Data Fig. 4: Validation of the orientation of the Nup159 complex.
From: In-cell architecture of the nuclear pore and snapshots of its turnover
a, Systematic fitting of the negative stain map of the Nup159 complex (yellow) into the S. cerevisiae NPC map (grey) using UCSF Chimera60 and Colores program from the Situs package61. As a negative control, also the mirror image of the negative stain map was fitted but did not lead to significant scores, further underlining the unambiguous nature of the fits. Each row shows the visualization of the top fits (left), the histogram of raw scores (middle), and a plot of the top five P values (right). In the P value plots, the statistically significant fits are coloured red (P < 0.05; P values were calculated using the two-sided test as implemented in fdrtool R-package, see Methods). All P values were adjusted for multiple comparisons using Benjamini-Hochberg procedure. The top fits are indicated in the histograms with an arrow and the score value. The number of sampled fits used to calculate P values after clustering of similar solutions was 585, 599, and 2243, for top, middle, and bottom rows respectively. b, Representative integrative Nup159 complex model from15 inside the in-cell S. cerevisiae NPC map (grey mesh) in the orientation determined in this work (left) versus the previously published orientation (right). The Nup159 complex model is shown in orange ribbons within yellow localization probability density from15 locally fitted with UCSF Chimera60. The previous orientation was reproduced by first fitting the entire model from15 to the in-cell cryo-EM map and then locally fitting the Nup159 complex to the density (which was needed to bring the Nup159 complex into the density and preserve the orientation). The dashed grey line indicates the flipping axis between the two fits. c, Superimposition of the crystal structure 3PBP27 onto the Nup82 β-propellers from the representative integrative Nup159 complex model from15 in the revised orientation predicts the position of Nup116, as confirmed by our knockout study (Fig. 2c). d, Visualization of two of the top resulting systematic fits of the 3PBP crystal structure into the cryo-ET map presented in this study confirms our nup116Δ structure (Fig. 2c). e, Crosslinks between the Nup159 complex and the Y-complex from15 support the new orientation (left) compared to the published orientation (right). Satisfied and violated crosslinks are depicted as blue and red bars respectively while the Nup159 complex and the Y-complex from15 are depicted within the relevant localization probability densities from15.
