The majority of this uncertainty can be attributed to the entry of water to the binding site. Binding site water was defined as being simultaneously within 8 Å of the N atom in Pro1A, and CG2 in Val106A, which are on opposite sides of the binding site. At the start of the vanishing stage there were no waters in the binding site, which increased to an average of approximately 4.5 after decoupling, in good agreement with the crystal structure of free MIF (PDB ID 1gd0). A sudden jump in water occupancy of the binding site occurred around λ = 0.4, the region of divergence of the PMFs for B1. Here, it was found that water may only enter the binding site by "forcing" the partially vanished ligand to the side of the binding site (Figure ).