In this case, the window closest to the surface should be large enough so as to avoid that the tail remains intermingled in the first maximum of the solvent layering while the colvar is being explored. Otherwise all the samples acquired for the first window would correspond to the same tail position, biasing the exploration of the phase space. Due to these considerations, the calculations for SA and GMS were run with three windows: from 1.8-2.3 to 20 Å, 20 to 30 Å and 30 to 40 Å, since their saturated linear tailgroups are more extended than the kinked Z-unsaturated tailgroups in GMO. Therefore, four windows were used for GMO: 2.3 to 10 Å, 10 to 20 Å, 20 to 30 Å and 30 to 40 Å. The initial configurations for each window were obtained using steered molecular dynamics (SMD), as implemented in the LAMMPS colvars package. The force constant of the applied harmonic biasing potential was 10 kcal mol -1 Å-2 , and the equilibrium distance was moved at a velocity of 4 m s -1 from the initial position of the molecule towards the surface. One initial condition was saved for each window and was equilibrated for 2 ns before applying the ABF algorithm to each window. The chosen width of the colvar bins was ∆ξ = 0.05 Å.