BioSimSpace was used to generate files for free energy calculations of the ligands in bound and unbound states. Free energy simulations were run with SOMD using both QUBE and AMBER/GAFF force field parameterization methods for comparison. Perturbation maps for each HIV-1 RT data set were constructed manually (Figure ). Each perturbation was carried out with two independent simulations, one in each direction. Each bound and unbound simulation was divided into 11 regularly spaced λ windows. The energy of the system was minimized for 1000 cycles by using the steepest descent method and each λ window was run for a total of 4 ns, using a time step of 2 fs. The first 5% of each simulation trajectory was discarded as equilibration. Table shows that the transformation of 2a to 2c (and the reverse) in group 2 varies by less than 0.3 kcal/mol with respect to changes in simulation time (in the range 2-4 ns). Free energy changes were calculated from the output using MBAR, and processed by the software FreeEnergyWorkflows 30 to produce the free energy estimates, and associated errors, reported in this manuscript. Further details may be found in the Supporting Information.