Some of the simulations included a bubbleligand which overlaps with the normal ligand but interacts only with water molecules. The bubble-ligand interacts with the water (not including the three buried water molecules) through Lennard-Jones interactions while not interacting with any other molecules in the system, and is designed (Sec. 5.5.3) to alleviate water sampling problems in the binding site at intermediate lambda values. The Lennard-Jones parameters σ and of this bubble-ligand were set to 0.33 nm and 0.41 kJ/mol for all atoms of the bubble-ligand (see topology file SI). We also introduced position restraints to prevent the side chains of a nearby protein loop from filling the vacuum of the empty binding site created by the bubble-ligand. The restraints were applied to the side chain backbone atoms using a force constant of 1000 kJ/(mol * nm 2 ). The three buried water molecules which do not interact with the bubble-ligand were restrained with position restraints as well. The restraints were turned on and off using 15 λ windows with varying force constants. 4.3. We obtained uncertainty estimates by running independent repeats. In order to obtain reliable error estimates, we performed independent replicates, both for the EQ and the NEQ approach. Given that MD simulations are sensitive to the initial conditions, two trajectories from the same system can diverge quickly. The divergence of an ensemble of independent simulations from various starting conditions provides insight into sensitivity to initial conditions. Although in the NEQ approach the fast-switching transitions are already independent of one another (at least to the extent that the equilibrium snapshots from which they are started are assumed uncorrelated), we also ran repeats of the end state simulations. Extracting snapshots from independent shorter MD simulations rather than one larger one can ensure better sampling of phase space. Bootstrapping of NEQ work values was used for the individual replicates but we found that it underestimated the uncertainty.