Electrostatic interactions of the disappearing atoms were linearly decoupled from the simulations between λ values of 0 and 0.55 and completely turned off beyond that, while those of the appearing atoms were linearly coupled to the simulations from λ value 0.45 to 1 and completely extinguished otherwise. While the coordinates were recorded every 10 ps, energy derivatives with respect to λ were recorded every 2 ps. When the enhanced sampling REST2 protocol was employed, all alchemical atoms constituted the "hot" region in the ligand simulations, whereas all alchemical atoms along with all protein residues falling within 3 Å of the alchemical atoms constituted the "hot" region in ligand-protein simulations. The maximum "effective" temperature used was 600 K.