Finally, we observe that there are systems such as ROS1 for which the standard NEQ protocol does not work. Unlike in the case of EQ, the ROS1 system exhibits substantially larger error bars in the case of NEQ which are related to zero overlap and large distances. For instance, the range of uncertainties for ROS1 system using the NEQ approach is 0.29-1.33 kcal/mol against 0.29-0.59 kcal/mol for the EQ approach. Furthermore, the distance values are non-zero even on incresing the ensemble size to 100, number of transitions to 10000 and transition length to 2 ns (see Figure ). Due to all these reasons, it is clear that the EQ method is preferable over the NEQ method for ABFE predictions.