Extended Data Fig. 6: Characterization of positive charges and their positions in IDR for EB1 phase separation.

(a) Schematic diagram of three classes of EB1 mutants: 1) positive charges in IDR were mutated (KR1Q-KR6Q); 2) the positions of charges in IDR were rearranged in cluster as 1-10 K/R; 3) a scramble sequence of IDR (scr9). The positive charges and mutations in IDR were annotated. All the sequences of EB1 mutants were listed in Supplementary Table 2. (b) Ultra-coarse-grained computational modeling to elucidate the phase separation behaviors of the three classes of EB1 mutants as in (a). (c) The random phase approximation (RPA) theory was applied to estimate the IDR electrostatic effects on EB1 phase separation. (d) The in vitro phase separation assay of EB1 mutants (10 μM) in the presence of 150 mM KCl. Scale bar, 5 µm. (e) The phase diagram of EB1 mutants generated by in vitro phase separation assay. The saturation concentration was measured by turbidity assay at indicated salt and protein concentrations. The red circles indicate phase separation (above saturation concentration), and the blue circles indicate no phase separation (below saturation concentration). Data are representative from three independent replicates.