To answer this question, we compared experimentally determined thermodynamic stability data for alanine substitution mutations at nearly every position in the β1 domain of Streptococcal protein G (G β1) to in silico calculations in Lasergene Protein, as well as calculations from five other software tools. Our results show that these tools vary widely in the accuracy of hot spot detection, especially at low error thresholds.

Compared to PopMuSic, FoldX, and three Rosetta methods, Lasergene has the most correct predictions within all error tolerances at or below 1.5 kcal/mol.