In Fig we compare the relative binding free energies constructed from the absolute ∆G calculations to the values from Gapsys et al. obtained by an explicit relative ∆∆G calculation protocol. The absolute ∆G protocol indeed yields relative free energies comparable to those calculated via direct alchemical transformation of the ligands (AUE of 4.0±0.4 kJ/mol) (Fig 6 ), indicating that the relative binding free energies are captured properly even when considering additional challenges of the absolute ∆G estimation. Furthermore, the accuracy of relative free energies obtained from the absolute ∆G protocol is also in good agreement with experimental results (Fig This observation is encouraging for the prospective drug design studies. Absolute ∆G calculations can be reliably used for the cases where the main assumptions for estimating relative free energy differences do not hold, e.g. where binding pose changes occur or investigated ligand structures differ substantially. It is, however, important to take into account the computational time required by these methods: absolute ∆G estimates in this work required 10 times longer sampling in comparison to the ∆∆G calculations in . The difference in computational cost between these approaches suggests a natural delineation in their application. When exploring large chemical libraries by means of free energy calculations, it would be most efficient to evaluate structurally similar compounds by computing ∆∆G values, while absolute ∆G calculations could be performed less frequently for the cases that are not tractable by the relative free energy estimation.