Figure 9.

Schematic representation of a thermodynamic cycle to calculate the relative binding free energy upon a ligand modification. The binding free energy of the ligand or protein in state A (ΔG1) and that in state B (ΔG2) are the physical binding processes that need to be determined, whereas (ΔG3) and (ΔG4) (the vertical legs) indicate the unphysical transformation of ligand A (yellow) to ligand B (green). However, (ΔG3) and (ΔG4) values are more accessible to the free energy perturbation methods. According to the conservation of energy, we can derive (ΔΔG = ΔG2 − ΔG1 = ΔG4 − ΔG3).