Notwithstanding this fact, no systematic evaluation of this effect has been undertaken. Here, we provide a critical assessment of this implementation. Although it is usually promoted as a major benefit of the NEQ approach over the EQ approach for ABFE calculations, we find the picture to be far more nuanced. Figure compares ∆G predictions using the NEQ approach in two cases: (a) Holo -when using holo protein structure to generate conformations for initiating transitions at both end-points, and (b) Apowhen using apo protein structure at the end-point with fully decoupled ligand and holo protein structure at the opposite end-point to generate conformations for initiating transitions from respective end-points.