where ∆𝐺 bind A/B is the absolute binding free energy of ligands A/B, ∆𝐺 A→B prot is the change in free energy of mutating ligand A into ligand B in complex with the protein, and ∆𝐺 A→B wat is the change in free energy of mutating ligand A into ligand B in solution (Fig. ). Thus, TI RBFE calculations consist of two alchemical transformations: from one ligand to the other while in complex with the protein, and from one ligand to the other while in solution. While in principle any mutation could be accomplished, only minor mutations are commonly performed due to rapid accumulation of error in large transformations. Therefore, RBFE simulations are typically restricted to sets of congeneric ligands that share a common substructure.