Starting NEQ transitions from structures where Val111 had not reoriented yet led falsely to good overlap of the work distributions but a wrong estimate of the free energy difference (Figure ). Only a few transitions starting from the "wrong" structure (the overpopulated minor rotamer) had a huge impact on the result since the tails of the distribution have a large statistical weight for the ∆G estimate. When as few as 17 transitions starting from the wrong Val111 rotamer were included (Figure , discard 60), the free energy did not converge to the same result as when starting all transitions in the correct dominant rotamer (Figure , discard 80). We attribute this to the fact that the alternate rotamer ought to be present to a vanishingly small degree at equilibrium.