Conformational restraints were applied on the host. These are optional in APR calculations to facilitate sampling during the pulling phase . For CB8, eight distance "jack" restraints were used on the carbonyls to enlarge the cavity. Jack restraints with a distance of 13.5 Å were used in previous calculations of CB7 and we applied a slightly larger distance for CB8. From our initial calculations, we found that a distance of 14 Å was enough to achieve good overlap between neighboring windows. For TEMOA, four distance jack restraints were used on the upper phenyl groups of the cavity. The same four distance jack restraints were used for TEETOA on the upper phenyl groups, with an additional two diagonal restraints on the ethyl groups to keep the groups as far apart as possible. The free energy contribution of applying conformational restraints on the host molecule was calculated in the same manner as the guest restraints and was simultaneously scaled in the attach phase. However, the free energy cost of releasing the conformational restraints in the unbound state was calculated explicitly by scaling the force constants to zero in 15 windows. The parameters used for the conformational restraints were: (a) jack distance = 14.0 Å; (b) force