These pre-equilibrated systems were the basis for the REUS campaign. Exchanges between adjacent replicas during REUS were carried out every picosecond, with exchange ratios near 13 the 20% optimum. To sample interleaflet exchange (Figure ), the sorgoleone precursors were pulled via steered molecular dynamics (SMD) to prepare the REUS ladder over 5 ns using a 5 kcal mol -1 Å-2 force constant, with each precursor sampling from the membrane midplane to the membrane edge, 25 Å away. The membrane midplane was determined by selecting the C16 atoms of the lipid acyl chains. All non hydrogen atoms on sorgoleone and its precursors along with the even carbon atoms on the acyl chain were used in colvars for REUS. The REUS simulation used 48 equi-spaced replicas taken from this initial SMD trajectory to cover this span with 99 ns of sampling in each umbrella, or 4.7µs in aggregate per compound.