Phylogenetic tree created using the structure-based multiple sequence alignments in each fold. The thermophilic proteins are shown in boxes. The thermophilic-mesophilic protein pairs sharing a close evolutionary relationship that have been selected for further analysis are marked with black stars for each of the three folds: (A) (α/β)8, (B) β-jelly roll, and (C) (α/α)6. (D) A fragment of the pairwise alignment between the thermophilic (pdb id: 2e4t) and mesophilic (pdb id: 3ik2) (α/β)8 pair is shown to demonstrate the sequence differences for the statistically significant amino acid arginine in the thermophilic protein. Four arginine positions (shown in boxes) in the thermophilic protein are substituted by different amino acids in the mesophilic protein.