After finding that the SepTop approach worked well on this test system, we used this system as a test case to help us optimize our protocol and increase its efficiency. We found that running each λ window for 10 ns vs. 20 ns gave relative free energies which were statistically indistinguishable, so we decided to use a simulation time of 10 ns per λ window in future systems. Our initial protocol used 45 λ windows for calculations in the binding site, but we found that we could reduce the number of windows to 20 while retaining good overlap between neighboring states. We used the overlap matrix as implemented in alchemlyb to help find a reasonable alchemical pathway and spacing with sufficient overlap.