# Biphasic tutorial

**URL:** <https://gromacs.bioexcel.eu/t/biphasic-tutorial/8857>\
**Category:** User discussions\
**Created:** [April 12, 2024, 1:05am UTC](https://gromacs.bioexcel.eu/t/biphasic-tutorial/8857 "2024-04-12T01:05:45Z")\
**Posts on this page:** 1\
**Showing post:** 22

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**Author:** ![SP06](https://avatars.discourse-cdn.com/v4/letter/s/ecb155/32.png) [@SP06](https://gromacs.bioexcel.eu/u/SP06)\
**Post date:** [April 24, 2024, 8:26am UTC](https://gromacs.bioexcel.eu/t/biphasic-tutorial/8857/22 "2024-04-24T08:26:25Z")

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Ok. Got this. For my study, after building the biphasic system, I need to center my protein in the system, then go for the subsequent steps of MD simulation.

After NVT equilibration of 500 ps on the cyclohexane box, still I am getting unstable system-

 ![IMG_20240424_134720](https://europe1.discourse-cdn.com/flex017/uploads/bioexcel1/original/2X/4/4d9e7ce7409d6eeb0a3dcb82aa2cca74134ba09c.jpeg)

nvt.mdp parameters are as follows-

title = charmm36 CYHE NVT equilibration  
; Run parameters  
integrator = md ; leap-frog integrator  
nsteps = 250000 ; 2 \* 250000 = 500 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 = no ; first dynamics run  
constraint\_algorithm = lincs ; holonomic constraints  
constraints = h-bonds ; bonds to H are constrained  
lincs\_iter = 1 ; accuracy of LINCS  
lincs\_order = 4 ; 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 ; short-range electrostatic cutoff (in nm)  
pme\_order = 4 ; cubic interpolation  
fourierspacing = 0.16 ; grid spacing for FFT  
; Temperature coupling  
tcoupl = V-rescale ; modified Berendsen thermostat  
tc-grps = system ; two coupling groups - more accurate  
tau\_t = 0.1 ; time constant, in ps  
ref\_t = 300 ; reference temperature, one for each group, in K  
; Pressure coupling  
pcoupl = no ; no pressure coupling in NVT  
; 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 = yes ; assign velocities from Maxwell distribution  
gen\_temp = 300 ; temperature for Maxwell distribution  
gen\_seed = -1 ; generate a random seed

I am using charmm36 ff and the box is cubic (4.9 nm) with 218 cyclohexane molecules in it. Can you please tell me how much more equilibration needed so that my system can be stable?

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