The thermodynamic cycle for a TI RBFE simulation is depicted below in Fig. . The difference in 𝛥𝐺 𝑏𝑖𝑛𝑑 ° between ligands A and B, ∆∆G bind 𝐴→𝐵 , is computed by performing an alchemical mutation of the ligands from one to the other in both the bound and unbound states: