# Flat-bottomed position restraints on lipid **URL:** \ **Category:** User discussions\ **Created:** [September 5, 2024, 10:01pm UTC](https://gromacs.bioexcel.eu/t/flat-bottomed-position-restraints-on-lipid/10109 "2024-09-05T22:01:57Z")\ **Posts on this page:** 1\ **Page:** 1
**Author:** ![EOrfanakis](https://avatars.discourse-cdn.com/v4/letter/e/7cd45c/32.png) [@EOrfanakis](https://gromacs.bioexcel.eu/u/EOrfanakis)\ **Post date:** [September 5, 2024, 10:01pm UTC](https://gromacs.bioexcel.eu/t/flat-bottomed-position-restraints-on-lipid/10109/1 "2024-09-05T22:01:57Z")
GROMACS version: 2023.1 GROMACS modification: No I am applying an umbrella biasing potential to a ligand that is at the center of a DOPC membrane (consisting of 72 lipids) in order to pull it out and use the Weighted Histogram Analysis Method (WHAM) to extract the PMF. The membrane system is surrounded by TIP3P water molecules, and it was made through CHARMM-GUI website using the charmm36 force field. During the time of my initial NVT equilibration and then at my NPT equilibration i observe the hydrophobic mismatch that causes lipid tails to move apart giving unrealistic results. I thought of applying flat-bottomed position restraints after using this [script](https://drive.google.com/file/d/1RKV2caOWmG9BbDIdnThEtwybHe4r7xP2/view?usp=drive_link) in order to measure the length of the lipids at z axis and also the length of the ligand. I get DOPC lipids length = 4.905 and ligand length = 0.8 which i divide by 2 and add some length. I create .itp files like that: ; position restraint file for DOPC [position\_restraints] ; id funct g r k ; Atom number Function (2 = flatbottomed) Direction (8 = cylindrical) Radius Force constant 1 2 8 -3.1 500 2 2 8 -3.1 500 3 2 8 -3.1 500 4 2 8 -3.1 500 5 2 8 -3.1 500 6 2 8 -3.1 500 7 2 8 -3.1 500 I then add the essential lines at topol.top file and i define the restraints at my .mdp files for equilibration. This is my mdp file for nvt equilibration: NVT equilibration define= -DPOSRES\_LIG\_flat\_bottomed -DPOSRES\_LIPID\_flat\_bottomed ; Run control integrator = md dt = 0.001 ; Decreased it to avoid instability errors nsteps = 50000 nstcomm = 1 nstcalcenergy = 1 comm-mode = Linear comm-grps = DOPC POT CLA TIP3 ATE ; Output control nstxout = 1000 nstvout = 1000 nstenergy = 1000 nstlog = 1000 ; Bond parameters continuation = no ; first dynamics run constraint\_algorithm = lincs constraints = all-bonds lincs\_iter = 2 ; Increased it to run my eq with flat bottomed restraints lincs\_order = 6 ; Increased it to run my eq with flat bottomed restraints ; Neighborsearching ns\_type = grid nstlist = 5 rlist = 1.2 rcoulomb = 1.2 rvdw = 1.2 ; Electrostatics coulombtype = PME pme\_order = 4 fourierspacing = 0.16 ; van der Waals vdwtype = cutoff ; Temperature coupling tcoupl = V-rescale tc-grps = system tau\_t = 0.1 ; reduced to avoid errors with big variance and instability ref\_t = 323 ; Pressure coupling pcoupl = no ; Periodic boundary conditions pbc = xyz ; Dispersion correction DispCorr = EnerPres ; Velocity generation gen\_vel = yes gen\_temp = 323 gen\_seed = -1 But when i try to run my simulation, i get messages like these: starting mdrun ‘Title’ 50000 steps, 50.0 ps. WARNING: Listed nonbonded interaction between particles 7840 and 7847 at distance 2.284 which is larger than the table limit 2.268 nm. This is likely either a 1,4 interaction, or a listed interaction inside a smaller molecule you are decoupling during a free energy calculation. Since interactions at distances beyond the table cannot be computed, they are skipped until they are inside the table limit again. You will only see this message once, even if it occurs for several interactions. IMPORTANT: This should not happen in a stable simulation, so there is probably something wrong with your system. Only change the table-extension distance in the mdp file if you are really sure that is the reason. Is there any way to fix it or avoid the hydrophobic mismatch? I have also thought of applying position restraints at the phosphorous atoms or the lipids headgroups. Thank you for your time!