Hi generally such a warning comes when the system has not been properly minimized. First solution is to minimize the system for a higher number of steps. However a good protocol is, after minimization, to perform a NVT simulation with the protein (or macromolecule you have in) constrained (the best would be a fast annealing however it is not necessary), than pass to a NPT simulation contrained and finally release the contraints on the protein. This should be enough. The simulation time for each step can be quite short 50-100 ps, while for the last NPT with the protein constrained you can use a longer simulation say 200-300 ps.

Hope this helps

Thanks Marco, I will try to minimize the system again with the higher number of steps.

In my MD protokol after energy min. I run NVT simulation fallowed by NPT.

Dear Gromacs users,

I am still having the pressure scaling warning. I increased the number of steps in the energy minimization part, then I run NVT and that was fine. However the npt simulations end up with an error. Below I pasted my input files ( energy min., NVT and NPT), please have a look maybe there is something wrong.

EM:

define = -DFLEXIBLE ; defines to pass to the preprocessor

integrator = steep ; steepest descents energy minimization

nsteps = 8000 ; maximum number of steps to integrate

emtol = 1000 ; [kJ/mol/nm] minimization is converged when max force is < emtol

emstep = 0.01 ; [nm] initial step-size

nstxout = 100 ; [steps] freq to write coordinates to trajectory

nstvout = 100 ; [steps] freq to write velocities to trajectory

nstfout = 100 ; [steps] freq to write forces to trajectory

nstlog = 1 ; [steps] freq to write energies to log file

nstenergy = 1 ; [steps] freq to write energies to energy file

energygrps = System ; group(s) to write to energy file

nstlist = 1 ; [steps] freq to update neighbor list

ns_type = grid ; method of updating neighbor list

pbc = xyz ; periodic boundary conditions in all directions

rlist = 0.8 ; [nm] cut-off distance for the short-range neighbor list

coulombtype = PME ; Particle-Mesh Ewald electrostatics

rcoulomb = 0.8 ; [nm] distance for Coulomb cut-off

vdwtype = cut-off ; twin-range cut-off with rlist where rvdw >= rlist

rvdw = 0.8 ; [nm] distance for LJ cut-off

DispCorr = Ener ; apply long range dispersion corrections for energy

fourierspacing = 0.12 ; [nm] grid spacing for FFT grid when using PME

pme_order = 4 ; interpolation order for PME, 4 = cubic

ewald_rtol = 1e-5 ; relative strength of Ewald-shifted potential at rcoulomb

NVT:

; 7.3.2 Preprocessing

define = -DPOSRES ; defines to pass to the preprocessor

; 7.3.3 Run Control

integrator = md ; md integrator

tinit = 0 ; [ps] starting time for run

dt = 0.002 ; [ps] time step for integration

nsteps = 100000 ; maximum number of steps to integrate, 0.002 * 25,000 = 50 ps

comm_mode = Linear ; remove center of mass translation

nstcomm = 1 ; [steps] frequency of mass motion removal

comm_grps = Protein Non-Protein ; group(s) for center of mass motion removal

; 7.3.8 Output Control

nstxout = 25000 ; [steps] freq to write coordinates to trajectory

nstvout = 25000 ; [steps] freq to write velocities to trajectory

nstfout = 25000 ; [steps] freq to write forces to trajectory

nstlog = 100 ; [steps] freq to write energies to log file

nstenergy = 100 ; [steps] freq to write energies to energy file

nstxtcout = 100 ; [steps] freq to write coordinates to xtc trajectory

xtc_precision = 1000 ; [real] precision to write xtc trajectory

xtc_grps = System ; group(s) to write to xtc trajectory

energygrps = System ; group(s) to write to energy file

; 7.3.9 Neighbor Searching

nstlist = 1 ; [steps] freq to update neighbor list

ns_type = grid ; method of updating neighbor list

pbc = xyz ; periodic boundary conditions in all directions

rlist = 0.8 ; [nm] cut-off distance for the short-range neighbor list

; 7.3.10 Electrostatics

coulombtype = PME ; Particle-Mesh Ewald electrostatics

rcoulomb = 0.8 ; [nm] distance for Coulomb cut-off

; 7.3.11 VdW

vdwtype = cut-off ; twin-range cut-off with rlist where rvdw >= rlist

rvdw = 0.8 ; [nm] distance for LJ cut-off

DispCorr = EnerPres ; apply long range dispersion corrections

; 7.3.13 Ewald

fourierspacing = 0.12 ; [nm] grid spacing for FFT grid when using PME

pme_order = 4 ; interpolation order for PME, 4 = cubic

ewald_rtol = 1e-5 ; relative strength of Ewald-shifted potential at rcoulomb

; 7.3.14 Temperature Coupling

tcoupl = berendsen ; temperature coupling with Berendsen-thermostat

tc_grps = Protein Non-Protein ; groups to couple seperately to temperature bath

tau_t = 0.1 0.1 ; [ps] time constant for coupling

ref_t = 310 310 ; [K] reference temperature for coupling

; 7.3.17 Velocity Generation

gen_vel = yes ; generate velocities according to Maxwell distribution of temperature

gen_temp = 310 ; [K] temperature for Maxwell distribution

gen_seed = -1 ; [integer] used to initialize random generator for random velocities

; 7.3.18 Bonds

constraints = all-bonds ; convert all bonds to constraints

constraint_algorithm = LINCS ; LINear Constraint Solver

continuation = no ; no = apply constraints to the start configuration

lincs_order = 4 ; highest order in the expansion of the contraint coupling matrix

lincs_iter = 1 ; number of iterations to correct for rotational lengthening

lincs_warnangle = 30 ; [degrees] maximum angle that a bond can rotate before LINCS will complain

(END)

NPT:

; 7.3.2 Preprocessing

define = -DPOSRES ; defines to pass to the preprocessor

; 7.3.3 Run Control

integrator = md ; md integrator

tinit = 0 ; [ps] starting time for run

dt = 0.002 ; [ps] time step for integration

nsteps = 250000 ; maximum number of steps to integrate, 0.002 * 250,000 = 500 ps

comm_mode = Linear ; remove center of mass translation

nstcomm = 1 ; [steps] frequency of mass motion removal

comm_grps = Protein Non-Protein ; group(s) for center of mass motion removal

; 7.3.8 Output Control

nstxout = 250000 ; [steps] freq to write coordinates to trajectory

nstvout = 250000 ; [steps] freq to write velocities to trajectory

nstfout = 250000 ; [steps] freq to write forces to trajectory

nstlog = 100 ; [steps] freq to write energies to log file

nstenergy = 500 ; [steps] freq to write energies to energy file

nstxtcout = 500 ; [steps] freq to write coordinates to xtc trajectory

xtc_precision = 1000 ; [real] precision to write xtc trajectory

xtc_grps = System ; group(s) to write to xtc trajectory

energygrps = System ; group(s) to write to energy file

; 7.3.9 Neighbor Searching

nstlist = 1 ; [steps] freq to update neighbor list

ns_type = grid ; method of updating neighbor list

pbc = xyz ; periodic boundary conditions in all directions

rlist = 0.8 ; [nm] cut-off distance for the short-range neighbor list

; 7.3.10 Electrostatics

coulombtype = PME ; Particle-Mesh Ewald electrostatics

rcoulomb = 0.8 ; [nm] distance for Coulomb cut-off

; 7.3.11 VdW

vdwtype = cut-off ; twin-range cut-off with rlist where rvdw >= rlist

rvdw = 0.8 ; [nm] distance for LJ cut-off

DispCorr = EnerPres ; apply long range dispersion corrections for energy

; 7.3.13 Ewald

fourierspacing = 0.12 ; [nm] grid spacing for FFT grid when using PME

pme_order = 4 ; interpolation order for PME, 4 = cubic

ewald_rtol = 1e-5 ; relative strength of Ewald-shifted potential at rcoulomb

; 7.3.14 Temperature Coupling

tcoupl = nose-hoover ; temperature coupling with Nose-Hoover ensemble

tc_grps = Protein Non-Protein ; groups to couple seperately to temperature bath

tau_t = 0.1 0.1 ; [ps] time constant for coupling

ref_t = 310 310 ; [K] reference temperature for coupling

; 7.3.15 Pressure Coupling

pcoupl = parrinello-rahman ; pressure coupling where box vectors are variable

pcoupltype = isotropic ; pressure coupling in x-y-z directions

tau_p = 2.0 ; [ps] time constant for coupling

compressibility = 4.5e-5 ; [bar^-1] compressibility

ref_p = 1.0 ; [bar] reference pressure for coupling

; 7.3.17 Velocity Generation

gen_vel = no ; velocity generation turned off

; 7.3.18 Bonds

constraints = all-bonds ; convert all bonds to constraints

constraint_algorithm = LINCS ; LINear Constraint Solver

continuation = yes ; apply constraints to the start configuration

lincs_order = 4 ; highest order in the expansion of the contraint coupling matrix

lincs_iter = 1 ; number of iterations to correct for rotational lengthening

lincs_warnangle = 30 ; [degrees] maximum angle that a bond can rotate before LINCS will complain

Can anyone suggest me something, I am new to GROMACS.

My protein contains 784 AA.

best regards

Urszula

It´s true.This can have several causes. The most common is that your system is not properly minimized

Another cause can be on couple pressure. Use another coupling.

I have changed the couple pressure to Berendsen. However after running grompp I am getting an fatal error:

Back Off! I just backed up mdout.mdp to ./#mdout.mdp.14#

NOTE 1 [file npt.mdp]:

leapfrog does not yet support Nose-Hoover chains, nhchainlength reset to 1

WARNING 1 [file npt.mdp]:

Using Berendsen pressure coupling invalidates the true ensemble for the

thermostat

NOTE 2 [file npt.mdp]:

This run will generate roughly 4716 Mb of data

There were 2 notes

There was 1 warning

-------------------------------------------------------

Program grompp, VERSION 4.5.3

Source code file: grompp.c, line: 1563

Fatal error:

Too many warnings (1), grompp terminated.

If you are sure all warnings are harmless, use the -maxwarn option.

For more information and tips for troubleshooting, please check the GROMACS

website at http://www.gromacs.org/Documentation/Errors

-------------------------------------------------------

"Cut It Deep and Cut It Wide" (The Walkabouts)

My npt.mdb file:

; 7.3.15 Pressure Coupling

pcoupl = berendsen ; pressure coupling where box vectors are variable

pcoupltype = isotropic ; pressure coupling in x-y-z directions

tau_p = 1.0 ; [ps] time constant for coupling

compressibility = 4.5e-5 ; [bar^-1] compressibility

ref_p = 1.0 ; [bar] reference pressure for coupling

How many steps should I use for energy minimization? In my first run I sued

nsteps = 10000 ; maximum number of steps to integrate

Thank you in advance for any suggestions.

/Urszula

Hi Urszula! This is only a simple idea: I mean, the fatal error is only caused by a single warning. If you want to use the configuration you have chosen, why do you not just use the -maxwarn option, just as suggested by the output?

However, a simple google search provides an answer from Justin Lemkul, who I am just citing here:

"The Berendsen method does not produce a proper ensemble for either temperature or pressure. If you want true NPT, use Parrinello-Rahman. This is all documented there are numerous threads about it in the literature and in the list archive."

You can run 1st NPT with thermostat = v - rescale and barostat = Berendsen for say 100 ps and then run 2ND NPT with thermostat = Nose-Hooover and barostat = Parinello -Rahman. If there is "leap frog" warning during grompp run then change integrator = md into integrator = md-vv

Hello Urszula Sylwia Uciechowska,

I got similar error, I resolved it by adding  -t previous_run input in my run command. It is suggested in new version of Gromacs (5.0+) that, when we change ensemble, add -t command in run input. For example, change from NVT to NPT or form NPT (with Berendsen) to NPT (Noose-Hoover) add -t previous_run. Here are some example commands.

1. Minimization:

gmx grompp -f minimize.mdp -c structure.gro -p topol.top -o  min_structure.tpr

gmx mdrun -deffnm min_structure -v -c

2. Then we do NVT

gmx grompp -f nvt.mdp -c min_structure.gro -p topol.top -o nvt.tpr -t min_structure

3. Then we do NPT (Berendsen thermostat)

gmx grompp -f npt-berend.mdp -c nvt.gro -p topol.top -o npt-berend.tpr -t nvt

gmx mdrun -deffnm npt-berend -v -c

4. Then we do NPT (Noose-Hoover)

gmx grompp -f npt-noose-hoover.mdp -c npt-berend.gro -p topol.top -o npt-noose-hoover.tpr -t npt-berend

gmx mdrun -deffnm npt-noose-hoover -v -c

I hope, you will understand what you need to do!

-Yogesh