Another possible approach to overcome this issue is to increase/enhance the conformational sampling that may yield a good mix of both apo and holo conformations at all intermediate states. In this study, we have shown the effect of extended simulation duration on the systematic errors in ABFEs (Table ). It is clear from our research that such measures do not guarantee a reduction in ∆G prot conf and any such effect varies substantially across systems. This is expected as sometimes the energy barriers between apo and holo states are too high to be overcome with standard MD simulations. Further, a standard simulation length or ensemble size cannot be recommended as each system will have its own requirements, if the errors can be reduced at all. Enhanced sampling techniques may be helpful in some cases but the same issues persist with their application too. Comparing predictions from the NEQ approach obtained using holo structures at both end-points (denoted as Holo) against those using apo structures at one end-point and holo structures at the other end-point (denoted as Apo). The top and bottom panels display data before and after adjustment with ∆G prot conf , respectively. A subset of 137 complexes spanning across 8 protein systems have been compared. The solid red line denotes the perfect correlation, whereas the blue, orange and red shaded regions represent ±1, ± 1-2 and ± 2-3 kcal/mol ranges.