To validate the cholesterol model, CG simulations were performed of either a single cholesterol molecule in water or several inserted in lipid bilayer systems. 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 built and solvated using the insane.py CG building tool 79 by arranging the lipids and cholesterol on a regular array in the bilayer (xy) plane to obtain approximately 2000 lipids per leaflet, solvated by roughly 27,000 water beads. Hexagonal periodic boundary conditions were used when necessary to avoid packing defects upon gel-phase formation. Lipid membrane compositions tested are summarized in the supplementary material (Table ). All CG systems were solvated with regular Martini water beads. Counterions were added to neutralize the systems as necessary, plus 140 mM NaCl ionic strength. Additionally, CG simulations of two cholesterol crystal structures were performed. The atomistic crystal structures of cholesterol (CCDC code: CHOEST20 80 ) and cholesterol monohydrate (CCDC code: CHOLES20 81 ) were coarse-grained and used to construct supercells of size 3 × 2 × 4 and 3 × 3 × 2 nm 3 , respectively. Three different proteins were also studied. Protein structures were obtained from the Protein Data Bank 82 (PDB). The G Protein-Coupled Receptor Smoothened (SMO) model used in our simulations was based on the near full-length structure (PDB: 5L7D 83 ) and prepared as described elsewhere . The voltage-dependent anion channel 2 (VDAC2) model used in our simulations was based on a well-resolved structure of mouse VDAC1 (PDB: 4C69 85 ) and prepared as described elsewhere . The β2-adrenergic receptor (β2AR) model used in our simulations was based on the full-length structure (PDB: 3D4S ) and prepared similarly to SMO. All CG protein models were constructed using Martinize2 , with an applied elastic network with a bond force constant of 700 kJ/mol and a cutoff distance of 0.8 nm. Side chain corrections were also applied . Both SMO and VDAC2 models were positioned in the membrane roughly as described in previous simulation studies . All proteins were simulated in POPC:CHOL membranes at 85:15 mol% ratios. Nonbonded interactions were cut off at 1.1 nm, and Coulombic interactions were treated using reaction-field electrostatics with a dielectric constant of 15 and an infinite reaction-field dielectric constant. The particle neighbor list was updated using the Verlet list scheme. A v-rescale thermostat was used with a coupling time of 4.0 ps to maintain the temperature at 300 K (unless other temperatures are specified). A constant pressure of 1.0 bar was established by isotropic or semi-isotropic (in the case of membranes) coupling to a Parrinello-Rahman barostat with a relaxation time of 16.0 ps. After initial energy minimization and pressure/temperature equilibration runs, simulations were run at a 20 fs time step. Stability runs were carried out with either a 30 or 40 fs time step. To avoid numerical stability issues, ions were not added in the stability runs . The LINCS algorithm was used to solve bonded constraints using lincs_order = 4 and lincs_iter = 1. For Martini 2 simulations, lincs_order = 12 and lincs_iter = 2 were used to avoid artificial temperature gradients due to non converged constraints . All CG systems were run for at least 10 μs, and unless otherwise stated, the last 2 μs of each simulation was used for subsequent analysis. Simulations containing proteins were run for at least 30 μs. For the crystal structure simulations, the supercells were minimized and equilibrated in temperature steps of 50 K beginning at 50 K until the desired temperature for production was reached. Production simulations were performed at 200 K and 295 K with the latter being the temperature of the experimental measurements. The time step of the production simulations was 1 fs, equilibrations were performed at 0.5 fs (50 K, 100 K) and 1 fs (150 K, 200 K, 250 K), respectively. All simulations were performed without shear stress . For the simulations of cholesterol monohydrate, the water molecules were represented by tiny water beads.