Running NEQ with this bubble approach resulted in overlapping work distributions (already with 1 ns/transition, see SI Figure ) which supports our hypothesis that water sampling was causing the overlap problems. However, in both the EQ and NEQ approaches the standard deviation across independent replicates was high (0.3-0.5 kcal/mol, SI Figure ). This was due to slow side chain rearrangement of Asn36 (Section 5.5.2). Rotamer populations of this side chain in the unbound state differed among independent replicates which showed that sampling time was not sufficient to sample the correct Boltzmann weight of the rotamers (see SI). Both NEQ work values and dH/dλ values in the EQ approach correlated with the orientation of this side chain (SI).