The 3D distribution of interaction sites may indicate some native properties of the FG-Nups barrier. Moreover, we found that these interaction sites are spatially clustered into distinct groups in the periphery around a central axial channel with a diameter of approximately 10–20 nm in the NPC. Recently, we have shown that three-dimensional (3D) density maps of transient interactions between the FG-Nups barrier and a cargo-free or a cargo-bound transport receptor in native NPCs can be obtained by an advanced single-molecule fluorescence microscopy approach. Understanding the structure and function of the FG-Nups barrier under real-time trafficking conditions is still a formidable challenge due to the dynamic nature of a channeled membranous environment. ‘Natively unfolded’ nucleoporins (Nups) with domains rich in phenylalanine-glycine (FG) repeats form the selective permeability barrier and provide binding sites for mobile transport receptors in the NPC. The nuclear pore complex (NPC) acts as a selective gate that mediates the bidirectional transport of macromolecules between the cytoplasm and the nucleus of eukaryotic cells.