The bending modulus (kc) quantifies the amount of energy that is required to deform a bilayer membrane from its spontaneous curvature and is an important physical constant for characterizing membranes mechanical properties. Two different methods were used to estimate kc: Real-space fluctuation (RSF) of lipid tilt and splays and simulated buckling . As for the RSF method, we used a published Python implementation . Bilayer patches of 169 lipids per leaflet were built with insane 9 and simulated using GROMACS 2024.0 for 10 µs, with the 5-10 µs portions utilized for analysis. The DOPG system contained one sodium ion per lipid to achieve charge neutrality.