Fig. 3. Ph SAM, but not its polymerization activity, is essential for condensate formation in vitro.

a Schematic diagram of Mini-Ph truncations. b Structure of the Ph SAM–SAM interface indicating the position of the ML and EH mutations that impair SAM polymerization. The EH mutation (Leu 1565 Arg) has a stronger effect on polymerization than the single ML mutation (Leu 1547 Arg). The figure was prepared from the structure of the ML mutant, PDB 1D 1KW4. c Summary of filter-binding experiments to measure DNA binding. Points show the mean ± SEM (independent experiments: Mini-Ph n = 10; Mini-Ph-ML n = 3; Mini-Ph EH n = 5; Mini-PhΔSAM n = 4). d Kd_app with 95% CI for each protein, calculated from the data shown in C using Eq. (2). e Both the SAM and the FCS/HD1 region are required for formation of phase-separated condensates with chromatin. f Both the SAM and the FCS/HD1 region are required for formation of phase-separated condensates with DNA. g Representative images of condensates formed by Mini-Ph or the polymerization mutants (ML and EH) in the presence of chromatin (1-h incubation). h The concentration of nucleosomes in condensates formed by wild-type (WT) and polymerization mutants (ML and EH) is similar. i, j EH forms smaller condensates with chromatin than WT Mini-Ph, as determined by measuring the average size of the condensates (I, not significant), or the % area covered by condensates (j). p Values for h–j are for one-way ANOVA comparing each sample to the WT control, with Dunnett’s correction for multiple comparisons. Bars show the mean ± SEM of three experiments; nine images were analyzed for each experiment. See also Supplementary Fig. 7.