Interpreting phase diagrams
The predicted phase diagrams generated here are constructed by first calculating the overall mean-field homotypic intermolecular interaction parameter (epsilon), converting this into a Flory-Chi parameter, and solving the phase diagram using the analytical approach developed by Qian, Michaels, and Knowles (Qian et al. 2022). The most helpful application here is comparing two sequences that differ in terms of mutations to assess if/how mutations are expected to impact phase behavior. We note that these phase diagrams should be interpreted as a qualitative description of the phase behavior and not as a quantitative prediction of the phase boundaries. Moreover, there are several important considerations when considering the meaning of these phase diagrams:
Phase diagram temperatures here are reported in volume fraction vs. a reduced temperature. This reduced temperature is a normalized temperature, normalized by the critical temperature of sequence 1. Because of this, the absolute value of the reduced temperature is meaningless other than comparing sequence 1 to sequence 2. However, if the phase behavior of sequence 1 is known, this can be a way to assess if we should expect sequence 2 to behave similarly or differently.
WARNING
Because we are always comparing phase diagrams in reduced temperature, it is possible to find two sequences with VERY weak intermolecular interactions and see a robust-looking phase diagram. This is because the reduced temperature is normalized by the critical temperature of sequence 1 - EVEN if both sequence 1 and sequence 2 are very weakly interacting, sequence 1 will still have a critical temperature of 1.0. With this in mind, all comparisons should be considered relative to one another. PLEASE do not input your sequence into this page, see a phase diagram, and conclude that your protein will phase separate under physiological conditions.
To circumvent this, it can be informative to compare a sequence of interest with a known sequence that has been experimentally characterized. One such option here is the well-characterized N-terminal domain of the RNA binding protein FUS (shown below), although we caution that if sequences are dramatically different in terms of length, this may not be such a useful comparison.
MASNDYTQQA TQSYGAYPTQ PGQGYSQQSS QPYGQQSYSG YSQSTDTSGY
GQSSYSSYGQ SQNTGYGTQS TPQGYGSTGG YGSSQSSQSS YGQQSSYPGY
GQQPAPSSTS GSYGSSSQSS SYGQPQSGSY SQQPSYGGQQ QSYGQQQSYN
PPQGYGQQNQ YNS
References
If you use phase diagram predictions, please cite the FINCHES paper along with the theoretical derivation of the Flory-Huggins model we use.
Qian, D., Michaels, T. C. T. & Knowles, T. P. J. Analytical Solution to the Flory-Huggins Model. J. Phys. Chem. Lett. 13, 7853–7860 (2022).