Bigger isn't always better: Comparing system size, hydration, and software for lipid membrane analysis

The structural and dynamic properties of lipid membranes vary with bilayer size and hydration. While Molecular Dynamics (MD) simulations are a powerful tool for studying cellular membranes, results can be sensitive to the analysis workflow and software. In this study, all-atom MD simulations were conducted on 128, 256, 512, and 1024 POPC lipids at 40, 80, and 160 water molecules per lipid. A two-fold study was performed: (1) to assess the convergence of structural and dynamic properties of POPC bilayers as a function of membrane size and hydration level, including area per lipid (APL), bilayer thickness, order parameter, headgroup orientation, and lateral diffusion, and (2) to compare the performance of four software packages: CPPTRAJ (CPP), GROMACS (GRO), MDAnalysis (MDA), and LiPyphilic (LiP). For the first objective, the average APL, bilayer thickness, order parameter, and headgroup orientation were largely independent of size and hydration, whereas the lateral diffusion coefficient was sensitive to both. Notably, 128-lipid systems were susceptible to artificially inflated lateral diffusion due to finite-size artifacts at higher hydration levels. Increasing system size primarily decreased the statistical variance of APL and thickness. For the second objective, all four packages produced consistent results for APL and thickness, with the main discrepancy being a known artifact of the gmx order tool applied to unsaturated carbons. CPP was the fastest serial tool, whereas parallelization benefited MDA and LiP for some metrics. These findings demonstrate that moderately sized systems (e.g., 256L), combined with CPP, offer an efficient workflow for membrane structural property analysis.