Figure (2).

A thermodynamic cycle for the binding of two ligands a and b to a protein P. In the experiment the ligands are transferred from the solvent to the active site where one measures the difference ΔΔF = ΔF_{aw →Pa} - ΔF_{bw →Pb}. In simulations the nonphysical transformation a→b is carried out in the protein and in solution and the corresponding free energies ΔF_Pa→Pb and ΔF_{aw →bw} are calculated. Because the free energy of the entire cycle is zero, the desired ΔΔF is obtained in terms of the nonphysical free energy differences ΔΔF = ΔF_Pa→Pb - ΔF_{aw →bw}.