Valine 111 reorients upon binding of 3-iodotoluene. Slow conformational changes in the protein prevent convergence in both methods on the timescale of our simulation. 3-Iodotoluene, being more bulky than toluene, induces a rearrangement of the valine 111 (Val111) side chain upon binding (Figure ). The timescale for this reorientation is relatively slow (a couple of nanoseconds) and consequently results are highly sensitive to the initial protein conformation (Figure ). The predicted free energy change is either too favorable or too unfavorable depending on which structure was used as a starting point. If this conformational change is not sampled properly in the free energy calculation, running simulations starting in the apo protein structure results in a predicted free energy which is too unfavorable. The contribution of protein rearrangement upon binding and potential steric clashes with the protein are not accounted for in this scenario. On the other hand, the free energy change is predicted as too favorable when simu-lations are started in the holo protein structure. The protein is deformed upon binding and the energetic cost of this protein strain energy is missing. 5.3.2. This side chain rearrangement is not sampled adequately in NEQ and EQ calculations. In the NEQ approach it is important to sample the correct orientation of Val111 in the end states at equilibrium and to run the transitions between the end states slowly enough to allow for a rearrangement at least in some transitions (as discussed in Sec. 5.2.2). Although the free energy estimate should be independent of the switching rate, provided that a sufficient number of work values was collected, we found that the efficiency of a protocol can dependent on the switching rate. Performing the transitions at a slower rate increases the probability of observing the reorientation of a side chain, because the transition has less dissipation and follows closer the free energy gradient. Here, dissipation was large, and reducing the switching rate can be more efficient than running faster transitions (see Figure ) which would require a longer total simulation time. Simulations were started in the bound protein conformation. The end state simulation of the decoupled state has therefore be run long enough so that the Val111 side chain can reorient adequately to obtain a correct equilibrium distribution of conformations. Here, the alternate rotamer (after reorientation) should be dominant in the unbound ensemble, so we discard all frames before reorientation to equilibration. This would not be necessary if the end state simulation was run long enough to capture the equilibrium distribution of conformations instead of being biased by the starting configuration of Val111.