From the saved simulation trajectories, C-H bond order parameters S CH were calculated with the calcOrderParameters.py Python program (available on the NMRlipids Databank GitHub repository) that uses the MD-Analysis library; the program calculates S CH based on equation (1) for each lipid over time separately, and then the average and the standard error of mean over different lipids. To evaluate equilibration of simulations we used area per lipid (see Fig. ) calculated as the xy-area of the simulation box divided by the number of lipids per leaflet.