The ligand transformation for the solvent leg of the RBFE calculation was also performed using NES simulation. We generated 100 starting structures from the last 4 ns of the 6 ns production runs of the two ligands in solvent (generated according to the procedure described in Section 2.6.3). From these starting structures of the two ligands, we ran NES switches of 500 ps simulation time (∆λ = 0.000004) transforming one ligand to the other. 2.6.5. Free energy calculation and uncertainty estimates. For both the ABFE and the RBFE thermodynamic cycles, the trajectories of the unrestrained stages needed to be post-processed to obtain the correct values of the various restraint potentials implemented in the simulation protocols (see Sections 2.3 for details). After post-processing the trajectories, we calculated the free energies of the equilibrium free energy simulations using the MBAR implementation in the Alchemlyb software package version 0.7. For the free energy calculations of the NES simulations in the ABFE and RBFE thermodynamic cycles, and in the solvent leg of the RBFE calculations, we used the BAR implementation in the PMX software package. We simulated three independent replicates of the ABFE and RBFE thermodynamic cycles for each of the systems studied here, as well as of the solvent leg of each of the ligand RBFE calculations. We calculated the uncertainties in our ABFE or RBFE estimates using the formula: