Here, P b and P u are the fraction of simulation time in which the ligand is bound and unbound to the binding site of interest respectively, v is the volume of the simulation box (L), c o is the standard-state concentration (1 mol L -1 ), N Av is Avogadro's number, k B is Boltzmann's constant and T is the temperature (K). We define the ligand to be in the bound state when the first two closest distances between the heavy atoms of the ligand and the side chain heavy atoms of the binding site residues is <5 A. All other frames are defined as unbound.