As part of the validation of the sterols, the melting temperature was calculated using a simulated annealing approach, where the simulations starts at 373 K, followed by a quenching of the temperature at a rate of 1 K/100 ns, until reaching 273 K, followed by a heating of 1 K/100 ns returning to 373 K. The Lindemann index was then calculated for all lipid beads in blocks of 100 ns, such that each block corresponds to a change of approximately 1 K (Eq. 1, 2). Overheating (T+) and under-cooling (T-) temperatures were determined using the largest gradient in the Lindemann index. The mean T+ and T-were calculated from 5 repeats each of 2 𝜇s. The simulation Tm was the obtained through Equation , known ad the "hysteresis method" .