Fig. 4: Peripheral binding and translocation.

a, Volume-corrected radial density map for all MINFLUX localizations of Imp α–JF549 (2,678 tracks, n = 48,603 localizations). b, Confocal image of a permeabilized U2OS cell nuclear envelope decorated with Imp β1–mEosEM (excitation = 478 nm). The punctate pattern indicates localization to NPCs. Typical result from n = 41 cells. c, Volume-corrected radial density map of Imp β1–mEosEM showing peripheral binding within the pore. Reference NPC scaffolds were identified as described earlier⁷, and mEosEM was localized by astigmatism imaging (excitation = 561 nm) after photoactivation (excitation = 408 nm; n = 5,500 localizations, 48 cells, 412 NPCs; see Extended Data Fig. 9c,d and Supplementary Video 5). d, Volume-corrected radial density map for translocation-arrested import complexes (Imp β1/Imp α/NLS–BFP–mEosEM). Binding within the pore was largely at the periphery, yielding a largely vacant centre (n = 4,361 localizations, 39 cells, 391 NPCs), similar to Imp β1 alone (c). The white curved lines in a,c,d approximate the locations of the nuclear envelope. The image in a is scaled from 0 (black) to maximum density (yellow); c,d are scaled to 70% and 60% of maximum, respectively, due to the hotspots at approximately 100 nm and approximately 170 nm above the pore centres (see Extended Data Fig. 9e,f for full scale). Average localization densities from a,c,d were calculated for |z | ≤ 15 nm. e, Localization densities for bound Imp β1, translocation-arrested import complexes and actively transiting Imp α. f, Annular zone model. Three distinct binding behaviours for Imp β1 were observed: (I) an empty centre (no binding); (II) a transport active zone (low-affinity binding of import complexes); and (III) a high-affinity binding zone for Impβ1 and import complexes. INM, inner nuclear membrane; ONM, outer nuclear membrane. The NPC structure was adapted from ref. ⁴⁸, Cell Press.

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