For a given amino acid substitution in a protein, PolyPhen-2 extracts various sequence and structure-based features of the substitution site and feeds them to a probabilistic classifier.
Mapping of amino acid replacement to the known 3D structure reveals whether the replacement is likely to destroy the hydrophobic core of a protein, electrostatic interactions, interactions with ligands or other important features of a protein. If the spatial structure of a query protein is unknown, one can use the homologous proteins with known structure. Further analysis performed by PolyPhen-2 is based on the use of several structural parameters. Importantly, although all parameters are reported in the output, only some of them are used in the final decision rules.
For a mutation, PolyPhen-2 calculates theprobability that this mutation is damaging, and reports estimates of false positive rate (FPR, the chance that the mutation is classified as damaging when it is in fact non-damaging) and true positive rate (TPR, the chance that the mutation is classified as damaging when it is indeed damaging). A mutation is also appraised qualitatively, asbenign,possibly damaging,orprobably damaging, based on pairs of false positive rate (FPR) thresholds, optimized separately for each model