Panel (A) shows a representative protein stability curve expressed as its Gibbs free energy of folding (ΔG_f) across temperature. The stability curve exhibits two melting points where it crosses the x-axis, and a peak of stability where the curve has its most negative y value. Horizontal and vertical arrows represent the changes to protein stability predicted by HoTMuSiC and PoPMuSiC respectively (ΔT_m and ΔΔG_f). Panel (B) shows the ΔT_m predicted by HoTMuSiC to enzymes from psychrophiles, mesophiles, and thermophiles as a result of single amino acid mutations. Panel (C) shows the ΔΔG_f predicted by PoPMuSiC to enzymes from psychrophiles, mesophiles, and thermophiles. All data points are plotted with the mean ± the SEM. * represent the statistical significance results from Dunnett’s T3 multiple comparisons tests for panel (B) and Tukey’s multiple comparisons tests for panel (C) (* = P<0.05, ** = P<0.01, *** = P<0.001, ns = not significant).