For cholesterol simulations in water, a single cholesterol molecule was inserted in a 3 × 3 × 3 nm 3 simulation box, which was then fully solvated. For the lipid membrane systems, initial structures were generated using the CHARMM-GUI membrane builder module by arranging the lipids on a regular array in the bilayer (xy) plane. Membranes were built with roughly 260 lipids per leaflet, which were then solvated by ∼22,000 water molecules. Lipid membrane compositions tested are summarized in the supplementary material (Table ). Every system was neutralized and an additional 140 mM NaCl was added.