Thermodynamic cycles of calculating protein–ligand binding free energy in FEP methods. (A) Calculating the relative binding free energy of two ligands (⁠|$L$| and |$L^{\prime}$|⁠) binding to a protein (⁠|$P$|⁠) in water. (B) Calculating the absolute binding free energy between a ligand |$L$| and a protein |$P$| in water.