Hi everyone,

I’m simulating the interaction between a DNA duplex and an asymmetric lipid bilayer using steered molecular dynamics (SMD). The goal is to pull the DNA through the bilayer along the Z-axis (membrane normal), but I’ve encountered some unexpected behavior — both physical and possibly artifact-related — and would greatly appreciate feedback.

I'm using GROMACS2024.4 and the CHARMM36m force field.

System Overview

• DNA duplex placed ~5 nm above the bilayer center of mass (COM), roughly parallel to the membrane plane.
• Built in VMD, simulated using the CHARMM36m force field.
• The membrane is asymmetric and composed of multiple lipid types.

Equilibration Protocol Performed a multi-step equilibration with decreasing position restraints:

• EM: Energy minimization Restraints — DNA: 1000 | Lipid: 1000 | Dihedral: 1000 (kJ/mol/nm²)
• NVT: 1 ns Restraints — DNA: 1000 | Lipid: 1000 | Dihedral: 1000
• NPT1: 1 ns Restraints — DNA: 1000 | Lipid: 1000 | Dihedral: 1000
• NPT2: 1 ns Restraints — DNA: 500 | Lipid: 400 | Dihedral: 400
• NPT3: 1 ns Restraints — DNA: 200 | Lipid: 200 | Dihedral: 100
• NPT4: 5 ns No restraints
• Thermostat groups: tc-grps = DNA Membrane Water_and_ions
• Semiisotropic pressure coupling with compressibility = 4.5e-5 0.0

Pulling Setup (SMD)

pull = yes pull_ncoords = 1 pull_ngroups = 2 pull_group1_name = Membrane pull_group2_name = DNA pull_coord1_geometry = direction-periodic pull_coord1_dim = N N Y pull_coord1_vec = 0 0 -1 ; Pulling DNA downward pull_coord1_rate = 0.001 ; 1 nm/ns pull_coord1_k = 2000 pull_coord1_start = yes pull-group1-pbcatom = 11114 pull-group2-pbcatom = 43856 pull-pbc-ref-prev-step-com = yes pull-nstxout = 50 pull-nstfout = 50

• Pulling duration: 10 ns (No restraints)

Key Observations

• DNA does not enter the membrane.
• The pulling distance (pullx) starts from a negative value, despite the DNA starting above the membrane.
• Pulling force (pullf) reaches ~400–500 kJ/mol/nm, indicating a high energy barrier.
• The DNA drifts in the XY plane by the end of the simulation.
• The membrane rotates during simulation — starts in the xy plane (z-axis normal) and ends in the xz plane (y-axis normal).
• Water molecules reappear in the membrane core during equilibration and production, even after manual removal post-solvation.

Questions

Any suggestions, experiences, or relevant references would be extremely helpful! I'm happy to provide additional files or details if needed.

Thanks in advance!