The protein's residues are mutated one by one to identify residue clusters that are well-ordered in the folded transition state. These residues' interactions can be checked by "double-mutant-cycle formula_3" "analysis", in which the single-site mutants' effects are compared to the double mutants'. Most mutations are conservative and replace the original residue with a smaller one ("cavity-creating mutations") like alanine, though tyrosine-to-phenylalanine, isoleucine-to-valine and threonine-to-serine mutants can be used too. Chymotrypsin inhibitor, SH3 domains, individual domains of proteins L and G, ubiquitin, and barnase have all been studied by formula_3 analysis.