GROMACS version: 2020

GROMACS modification: Yes/No

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I have a protein + water system.

I am trying to do simulated annealing and following the below protocol:

First did the NVT equilibration using below mdp file:

title = OPLS Lysozyme NVT equilibration

define = -DPOSRES ; position restrain the protein

; Run parameters

integrator = md ; leap-frog integrator

nsteps = 500000 ; 2 * 500000 = 1000 ps

dt = 0.002 ; 2 fs

; Output control

nstxout = 500 ; save coordinates every 1.0 ps

nstvout = 500 ; save velocities every 1.0 ps

nstenergy = 500 ; save energies every 1.0 ps

nstlog = 500 ; update log file every 1.0 ps

; Bond parameters

continuation = no ; first dynamics run

constraint_algorithm = lincs ; holonomic constraints

constraints = h-bonds ; bonds involving H are constrained

lincs_iter = 1 ; accuracy of LINCS

lincs_order = 4 ; also related to accuracy

; Nonbonded settings

cutoff-scheme = Verlet ; Buffered neighbor searching

ns_type = grid ; search neighboring grid cells

nstlist = 10 ; 20 fs, largely irrelevant with Verlet

rcoulomb = 1.0 ; short-range electrostatic cutoff (in nm)

rvdw = 1.0 ; short-range van der Waals cutoff (in nm)

DispCorr = EnerPres ; account for cut-off vdW scheme

; Electrostatics

coulombtype = PME ; Particle Mesh Ewald for long-range electrostatics

pme_order = 4 ; cubic interpolation

fourierspacing = 0.16 ; grid spacing for FFT

; Temperature coupling is on

tcoupl = V-rescale ; modified Berendsen thermostat

tc-grps = Protein Non-Protein ; 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 is off

pcoupl = no ; no pressure coupling in NVT

; Periodic boundary conditions

pbc = xyz ; 3-D PBC

; Velocity generation

gen_vel = yes ; assign velocities from Maxwell distribution

gen_temp = 300 ; temperature for Maxwell distribution

gen_seed = -1 ; generate a random seed

Then I did NPT Equilibration where I introduced Simulated Annealing parameters:

title = OPLS Lysozyme NPT equilibration

define = -DPOSRES ; position restrain the protein

; Run parameters

integrator = md ; leap-frog integrator

nsteps = 50000 ; 2 * 50000 = 100 ps

dt = 0.002 ; 2 fs

; Output control

nstxout = 500 ; save coordinates every 1.0 ps

nstvout = 500 ; save velocities every 1.0 ps

nstenergy = 500 ; save energies every 1.0 ps

nstlog = 500 ; update log file every 1.0 ps

; Bond parameters

continuation = yes ; Restarting after NVT

constraint_algorithm = lincs ; holonomic constraints

constraints = h-bonds ; bonds involving H are constrained

lincs_iter = 1 ; accuracy of LINCS

lincs_order = 4 ; also related to accuracy

; Nonbonded settings

cutoff-scheme = Verlet ; Buffered neighbor searching

ns_type = grid ; search neighboring grid cells

nstlist = 10 ; 20 fs, largely irrelevant with Verlet scheme

rcoulomb = 1.0 ; short-range electrostatic cutoff (in nm)

rvdw = 1.0 ; short-range van der Waals cutoff (in nm)

DispCorr = EnerPres ; account for cut-off vdW scheme

; Electrostatics

coulombtype = PME ; Particle Mesh Ewald for long-range electrostatics

pme_order = 4 ; cubic interpolation

fourierspacing = 0.16 ; grid spacing for FFT

; Temperature coupling is on

tcoupl = V-rescale ; modified Berendsen thermostat

tc-grps = Protein Non-Protein ; 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 is on

pcoupl = Parrinello-Rahman ; Pressure coupling on in NPT

pcoupltype = isotropic ; uniform scaling of box vectors

tau_p = 2.0 ; 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

; Velocity generation

gen_vel = no ; Velocity generation is off

; SIMULATED ANNEALING

; Type of annealing for each temperature group (no/single/periodic)

annealing = single single

; Number of time points to use for specifying annealing in each group

annealing_npoints = 2 2

; List of times at the annealing points for each group

annealing_time = 0 50000 0 50000

; Temp. at each annealing point, for each group.

annealing_temp = 300 500 300 500

But still there is no change in temperature profile. Avg. temperature is 300K.

What mistake am I doing?

I have 10 protein molecules and around 500 molecules of water in my system along with ions added in ionization step.