However, it is important to note that NEQ free energy calculations do not require switching transitions to sample all relevant conformational states; what is required is that end state sampling populate the correct equilibrium distribution of states, and switching transitions must be sufficiently numerous to provide an adequate average over possible work values. Thus, the presence of work values that correlate with a particular slow conformational degree of freedom does not necessarily indicate sampling problems; rather, it means that practitioners need to check that the end states in fact sample the correct equilibrium distribution of states. If they do not, results will be inaccurate or at least imprecise. 5.2.4. In the equilibrium FEC, the Ile78 orientation correlates with dH/dλ values. In the EQ approach, sampling problems can in some cases be detected by examining sudden changes in the dH/dλ values and identifying related structural degrees of freedom. We plotted the running average of the dH/dλ values over the simulation time for every state, picked a λ window where there was an obvious jump in the dH/dλ value and looked at frames from the trajectory near that jump. Once any potential sampling problem -such as a motion which might be responsible for the sudden jump in dH/dλ -was identified we correlated the motion with the dH/dλ curves of all λ windows to assess correlations between that structural degree of freedom and dH/dλ values.