These differences can be further reduced by applying orthogonal restraints, which reduces the RMSD between reference structures for umbrella windows to 0.5 nm for fast pulling and to 0.1 nm for slow pulling (Figure ). Slow pulling with orthogonal restraints leads to higher similarity between pathways, therefore avoiding one of the potential reasons for differences in PMF profiles. The importance of applying orthogonal restraints was recently discussed by Blazhynska and co-workers, who compared PMF profiles obtained from simulations with geometrical restraints (which according to Doudou and co-workers are similar to orthogonal restraints ) to those obtained from free simulations. They observed differences of several kcal/mol between individual repeats of simulations with no restraints, and none of those repeats converged to experimental values. The authors, however, did not report performing multiple repeats of restrained simulations. Nevertheless, the value they reported for a single restrained simulation was in good agreement with experimental values.