Another approach that we introduced in this study allows to circumvent the need of an exhaustive apo state sampling by probing multiple initial apo states (when they are available) with the absolute ∆G calculation protocol (Fig ). This method does not require any prior knowledge of the experimentally measured binding affinities and it allows estimating relative free energies for the apo states by relating them one to another via a common holo reference state. The ∆G value for apo state structures calculated this way represents only one component of the overall free energy of the conformers, as only a contribution that is experienced by the ligand binding is considered. Nevertheless, this method allows identifying the most likely apo state for the use in the absolute binding free energy calculations.