Fig. 1: Engineering of a perfectly repeated GLFG domain with authentic phase behavior and NPC-typical selectivity.

a Sequences of the Tetrahymena thermophila MacNup98A (“Mac98A”) FG domain and simplified variants. Mac98A contains an intervening 44-residue GLEBS domain (binding site for the mRNA export mediator Gle2p) that was kept unchanged in these variants. For space economy, only the N-terminal ≈400 residues up to the GLEBS domain are shown. The C-terminal sequences of the FG domains (≈270 residues not shown) follow the same design principle (see Supplementary Note 1 for complete sequences and Supplementary Table 1 for amino acid compositions). b Indicated FG domains were dissolved at a concentration of 1 mM in 4 M guanidinium hydrochloride, and phase separation was initiated by a rapid 50-fold dilution with assay buffer (50 mM Tris/HCl pH 7.5 50 mM, 150 mM NaCl, 5 mM DTT), followed by another fourfold dilution in buffer +6 µM mCherry and 1 µM Alexa Fluor 488-labeled rat NTF2. Samples were analyzed by confocal laser-scanning microscopy (CLSM). All FG domain variants phase-separated to µm-sized, spherical FG particles that excluded mCherry (red) very well and accumulated the NTR NTF2 (green) to very high partition coefficients. Note that phase separation occurred here already at 5 µM FG domain concentration, which is at least 100 times lower than the most conservative estimate for the local Nup98 FG domain concentration at NPCs (48 copies in a cylinder of 70 nm diameter and 40 nm height).