(a) The instantaneous difference of protocol work accumulation over 1000 switching steps. (b) The instantaneous difference of protocol work accumulation over 10,000 switching steps. (c) The amount of asymmetry for the protocol work accumulation in ((b)), showing the difference between the process of turning the ligand off and that for turning it back on. From 200 iterations of NCMC and MolDarting simulation, we took the average values of the protocol work at each step for 1000 and 10,000 switching steps. From these average values, we calculated the instantaneous difference between the work values, shown by the blue line. The standard deviation of these differences are shown in red. We can see that there is a large accumulation of protocol work when the ligand’s interactions are being turned back on (after the halfway point of the NCMC steps). This difference is further illustrated in (c), which looks at the absolute difference between forward and reverse work values for equivalent points in the protocol shown in (b). Essentially, this shows how asymmetric the protocol is, and helps us identify that the largest asymmetry and greatest problems originate from the steric component of the protocol.