Additionally, the mapping was kept as close as possible to that used for cholesterol. In short, three beads (typically R1, R2 and C1) were used to define a rigid triangular frame assembled using linear constraints. The beads that make up this triangular frame were then used as the constructing particles from which the remaining beads (other than aliphatic chain beads) were defined as out-of-plane virtual sites. Parameters for virtual site construction were obtained as described in detail elsewhere , from CHARMM36 simulations of each sterol inserted in POPC bilayers. Briefly, the atomistic references were CG-mapped and the positions of the particles were then averaged after rotationally and translationally fitting the entire molecule to the virtual-site frame. The placement of the virtual site beads relative to the frame 𝑖 𝑗 𝑘 was then defined as a function of 𝑖 𝑗, 𝑖𝑘 and of their cross product. Nonbonded interactions between virtual site particles were excluded. Martini bead masses were then redistributed from the virtual site beads to the beads making up the virtual site frame. The corresponding aliphatic chains were then attached to each core as described above.