Fig. 4: Most (18 out of 24) tested proteins form high confidence condensates in cellulo.

a Representative images of the U2OS cells expressing the tested proteins, N-terminally tagged with an iRFP fluorescent tag. Formation of mesoscale cellular condensates are highlighted in the inset. All images are scaled to the scale bar 10 μm (shown on upper left image). We found 21 out of the 24 tested proteins (87.5%) formed mesoscale foci without any stressors, while 3 proteins (C1ORF52, SPAG7 and CWC27, encircled in red) localized to the nucleoplasm without forming any detectable foci (foci were defined by exhibiting a fluorescent intensity ratio >1). Notice the presence of rim-like structures in case of H1T and H2A1H. Using size, shape and the fraction of cells forming mesoscale foci as a deciding characteristic, ~75% i.e., 18 proteins (encircled in blue) form high confidence condensates, and 3 proteins (encircled in orange) form low confidence condensates (foci with longest diameter <350 nm. Fig. S9). The experiments were repeated at least twice to ascertain the reproducibility of the results. More representative images are shown in Fig. S10 and the raw images are provided as Dataset S4. b Wide range of secondary structural motifs covered in the test proteins; AlphaFold2 structural models of the proteins are colored according to secondary structures. Notice the wide range of structural motifs, alpha-helical (red), beta stranded (yellow) to largely disordered (green) proteins (AlphaFold2 structures are provided as Dataset S6). c Disorder content (computed as mean IUPred score or reverse pLDDT score (1 - pLDDT)) of the tested protein does not correlate with the ability to form condensates. d Comparison of the predictions provided by sequence-based predictors (PICNIC, PdPS-8fea, PdPS-10fea, PSAP, and DeePhase) of protein condensates. PICNIC exhibits the lowest misclassification rate for the tested 24 proteins. Source data are provided as a Source Data file.