# Low density after NPT step (methanol/water mixture solvent) **URL:** \ **Category:** User discussions\ **Created:** [July 4, 2025, 9:10am UTC](https://gromacs.bioexcel.eu/t/low-density-after-npt-step-methanol-water-mixture-solvent/12325 "2025-07-04T09:10:48Z")\ **Posts on this page:** 2\ **Page:** 1
**Author:** ![phamedhue](https://avatars.discourse-cdn.com/v4/letter/p/e480ec/32.png) [@phamedhue](https://gromacs.bioexcel.eu/u/phamedhue)\ **Post date:** [July 4, 2025, 9:10am UTC](https://gromacs.bioexcel.eu/t/low-density-after-npt-step-methanol-water-mixture-solvent/12325/1 "2025-07-04T09:10:48Z")
GROMACS version: 2024.3 GROMACS modification: No I’m running a GROMACS simulation of a mixed methanol/water solvent system. I added **2000 methanol molecules** using `insert-molecules` and **2500 water molecules** using `solvate`. The initial box density seems reasonable. However, after the NPT equilibration step, the box size increases and the density drops significantly to around **620 kg/m³** , while the expected density of a methanol/water mixture should be above **791 kg/m³**. 🔹 _What could be causing this drop in density, and how can I fix it?_ Should I revisit how I insert the solvent molecules, or adjust my NPT equilibration settings (e.g., force field choice, barostat parameters, compressibility)? ---
**Author:** ![phamedhue](https://avatars.discourse-cdn.com/v4/letter/p/e480ec/32.png) [@phamedhue](https://gromacs.bioexcel.eu/u/phamedhue)\ **Post date:** [July 4, 2025, 11:29am UTC](https://gromacs.bioexcel.eu/t/low-density-after-npt-step-methanol-water-mixture-solvent/12325/2 "2025-07-04T11:29:20Z")
My protocol is: gmx editconf -f TFA.gro -o box.gro -c -box 6.0 6.0 6.0 gmx insert-molecules -f box.gro -ci methanol.gro -nmol 2000 -o box\_methanol.gro gmx solvate -cp box\_methanol.gro -cs spc216.gro -o solvated.gro -p topol.top gmx grompp -f EM.mdp -c solvated.gro -p topol.top -o em.tpr -n index.ndx gmx mdrun -deffnm em -v gmx grompp -f NVT.mdp -c em.gro -r em.gro -p topol.top -o nvt.tpr -n index.ndx gmx mdrun -deffnm nvt -v gmx grompp -f NPT.mdp -c nvt.gro -r nvt.gro -t nvt.cpt -p topol.top -o npt.tpr -n index.ndx gmx mdrun -deffnm npt -v NPT.mdp file: title = TFA in methanol NPT equilibration ; Run parameters integrator = md ; leap-frog integrator nsteps = 500000 ; 2 \* 500000 = 1000 ps dt = 0.002 ; 2 fs ; Output control nstenergy = 500 ; save energies every 1.0 ps nstlog = 500 ; update log file every 1.0 ps nstxout-compressed = 500 ; save coordinates every 1.0 ps ; Bond parameters continuation = yes ; continuing from NVT constraint\_algorithm = lincs ; holonomic constraints constraints = h-bonds ; bonds to H are constrained lincs\_iter = 1 ; accuracy of LINCS lincs\_order = 2 ; also related to accuracy ; Neighbor searching and vdW cutoff-scheme = Verlet ns\_type = grid ; search neighboring grid cells nstlist = 20 ; largely irrelevant with Verlet rlist = 1.2 vdwtype = cutoff vdw-modifier = force-switch rvdw-switch = 1.0 rvdw = 1.2 ; short-range van der Waals cutoff (in nm) ; Electrostatics coulombtype = PME ; Particle Mesh Ewald for long-range electrostatics rcoulomb = 1.2 pme\_order = 4 ; cubic interpolation fourierspacing = 0.16 ; grid spacing for FFT ; Temperature coupling tcoupl = V-rescale ; modified Berendsen thermostat tc-grps = BER Solvent\_and\_ions ; two coupling groups - more accurate tau\_t = 0.1 0.1 ; time constant, in ps ref\_t = 300 300 ; reference temperature, one for each group, in K ; Pressure coupling pcoupl = C-rescale ; pressure coupling is on for NPT pcoupltype = isotropic ; uniform scaling of box vectors tau\_p = 5.0 ; time constant, in ps ref\_p = 1.0 ; reference pressure, in bar compressibility = 4.5e-5 ; isothermal compressibility of water, bar^-1 refcoord\_scaling = com ; Periodic boundary conditions pbc = xyz ; 3-D PBC ; Dispersion correction is not used for proteins with the C36 additive FF DispCorr = no ; Velocity generation gen\_vel = no ; velocity generation off after NVT After NPT step, box size increase from 6x6x6 to 6.8x6.8x6.8 and density decrease to 620 kg/m³ What wrong with my protocol?