In order to further assess the sensitivity of the sMD/MSM workflow to the effect of fragments, a model for untethered 3, 3u (with the K197C PTP1B mutant) was built. The covalent linkage was replaced by a flat-bottomed non-directional distance restraint to K197C (see Methods). The sMD/MSM produced a broad active state probability distribution with a median only slighly shifted down with respect to the reference system (Figure ). Comparison of the computed MSM ensembles for 3 and 3u shows that 3 mainly adopts a "upright" binding pose owing to the covalent tether (Figure ) that resembles the crystallographic pose observed for this fragment. This pose enables the fragment phenol moeity to engage in hydrogen bonding interactions with K150, a suggested allosteric residue . By contrast untethered fragment 3u is more mobile and adopts predominantly a "sideways" pose (Figure ). This causes the phenol group to interact with E200, which has not been flagged as a residue of interest to the allosteric network . The "upright" pose can still be detected albeit less frequently. These observations suggest that stabilisation of the "upright" pose could be a plausible design strategy to elaborate fragment 3 into a non-covalently bound allosteric inhibitor of wild-type PTP1B.