29 'Single-transformation' means that the Van der Waals and the charge transformations are carried out in one step using periodic boundary conditions. To avoid the boundary singularity effects close to the input and final structures, softcore versions of both the Lennard Jones (LJ) and the Coulomb potentials were used. The softness of the LJ potential (scalpha) and the electrostatic potential (scbeta) were kept at their default values. Thus, the interactions among the 'disappearing' atoms are unchanged, and any interaction involving at least one '(dis)appearing' atom did not contribute to the average free energy change. Any shake constraint applying to bonds between common and unique atoms is being removed but kept for bonds between unique atoms.