To avoid protein tumbling and unrealistic backbone deformation which may be associated with the large dissipative force exerted on the system, positional harmonic restraints with K=0.01 kcal/mol/Å 2 are added to the protein C ɑ atoms in order to ensure reliable measurement of Δd. The introduction of restraints, although practical for RAMD, may alter the original free energy landscape. This underlines the importance of the following step where a restraint-free, near-equilibrium Metadynamics allows recovery of the correct absolute residence time.