There is an intermediate regime around λ = 0.1 where the ligand slowly exchanges between both conformations, substantially increasing the uncertainty of the free energy differences between runs. This produces a spike in the allocated simulation time. The main effect of the differing allocation of sampling time is to accelerate equilibration (Figure ). At longer sampling times, the adaptive protocol produces estimates which are significantly closer to the final 30 ns estimate than the non-adaptive protocol. At very short timescales this trend is reversed, which suggests that fast relaxations are better sampled by the equal-time protocol, while the adaptive protocol more effectively samples the slow exchange between different ring orientations.