Dear Gmx Users, Dear Justin, I pulled my ligand away from my protein. Ligand was attached to lower part of my protein, I pulled in Z coordinate it using:
; Run parameters integrator = md ; leap-frog integrator nsteps = 5000000 ; 2 * 5000000 = 10 ns dt = 0.002 ; 2 fs tinit = 0 nstcomm = 10 ; Output control nstxout = 50000 ; save coordinates every 100 ps nstvout = 50000 ; save velocities every nstfout = 5000 nstxtcout = 5000 ; every 10 ps nstenergy = 5000 ; Bond parameters continuation = yes ; first dynamics run constraint_algorithm = lincs ; holonomic constraints constraints = all-bonds ; all bonds (even heavy atom-H bonds) constrained ; Neighborsearching ns_type = grid ; search neighboring grid cells nstlist = 5 ; 10 fs rlist = 0.9 ; short-range neighborlist cutoff (in nm) rcoulomb = 0.9 ; short-range electrostatic cutoff (in nm) rvdw = 0.9 ; short-range van der Waals cutoff (in nm) ewald_rtol = 1e-5 ; relative strenght of the Ewald-shifted potential rcoulomb ; Electrostatics coulombtype = PME ; Particle Mesh Ewald for long-range electrostatics pme_order = 4 ; cubic interpolation fourierspacing = 0.12 ; grid spacing for FFT fourier_nx = 0 fourier_ny = 0 fourier_nz = 0 optimize_fft = yes ; Temperature coupling is on tcoupl = V-rescale ; modified Berendsen thermostat tc_grps = Protein_LIG Water_and_ions ; two coupling groups - more accurate tau_t = 0.1 0.1 ; time constant, in ps ref_t = 298 298 ; reference temperature, one for each group, in K ; Pressure coupling is on pcoupl = Parrinello-Rahman ; pressure coupling is on for NPT pcoupltype = isotropic ; uniform scaling of box vectors tau_p = 1.0 ; time constant, in ps ref_p = 1.0 ; reference pressure, in bar compressibility = 4.5e-5 ; isothermal compressibility of water, bar^-1 ; Periodic boundary conditions pbc = xyz ; 3-D PBC ; Dispersion correction DispCorr = EnerPres ; account for cut-off vdW scheme ; Velocity generation gen_vel = no ; assign velocities from Maxwell distribution ; These options remove COM motion of the system ; Pull code pull = umbrella pull_geometry = distance pull_dim = N N Y pull_start = yes pull_ngroups = 1 pull_group0 = Protein pull_group1 = LIG182 pull_init1 = 0 pull_rate1 = 0.0 pull_k1 = 200 ; kJ mol^-1 nm^-2 pull_nstxout = 1000 ; every 2 ps pull_nstfout = 1000 ; every 2 ps Following Justin's tutorial I used perl script to extract coordinate for each window. 0 2.4595039 1 2.4745028 ... 500 8.74 My ligand at the begining was at such distance as it was in the lower part of the protein. Then I used 0.1 nm spacing at the begining (till 4 nm) and 0.2 nm later on. And following equilibration in each window I run umbrella sampling for 10ns in app 49 windows: Run parameters integrator = md ; leap-frog integrator nsteps = 5000000 ; 2 * 5000000 = 10 ns dt = 0.002 ; 2 fs tinit = 0 nstcomm = 10 ; Output control nstxout = 50000 ; save coordinates every 100 ps nstvout = 50000 ; save velocities every nstfout = 5000 nstxtcout = 5000 ; every 10 ps nstenergy = 5000 ; Bond parameters continuation = yes ; first dynamics run constraint_algorithm = lincs ; holonomic constraints constraints = all-bonds ; all bonds (even heavy atom-H bonds) constrained ; Neighborsearching ns_type = grid ; search neighboring grid cells nstlist = 5 ; 10 fs rlist = 0.9 ; short-range neighborlist cutoff (in nm) rcoulomb = 0.9 ; short-range electrostatic cutoff (in nm) rvdw = 0.9 ; short-range van der Waals cutoff (in nm) ewald_rtol = 1e-5 ; relative strenght of the Ewald-shifted potential rcoulomb ; Electrostatics coulombtype = PME ; Particle Mesh Ewald for long-range electrostatics pme_order = 4 ; cubic interpolation fourierspacing = 0.12 ; grid spacing for FFT fourier_nx = 0 fourier_ny = 0 fourier_nz = 0 optimize_fft = yes ; Temperature coupling is on tcoupl = V-rescale ; modified Berendsen thermostat tc_grps = Protein_LIG Water_and_ions ; two coupling groups - more accurate tau_t = 0.1 0.1 ; time constant, in ps ref_t = 298 298 ; reference temperature, one for each group, in K ; Pressure coupling is on pcoupl = Parrinello-Rahman ; pressure coupling is on for NPT pcoupltype = isotropic ; uniform scaling of box vectors tau_p = 1.0 ; time constant, in ps ref_p = 1.0 ; reference pressure, in bar compressibility = 4.5e-5 ; isothermal compressibility of water, bar^-1 ; Periodic boundary conditions pbc = xyz ; 3-D PBC ; Dispersion correction DispCorr = EnerPres ; account for cut-off vdW scheme ; Velocity generation gen_vel = no ; assign velocities from Maxwell distribution ; These options remove COM motion of the system ; Pull code pull = umbrella pull_geometry = distance pull_dim = N N Y pull_start = yes pull_ngroups = 1 pull_group0 = Protein pull_group1 = LIG182 pull_init1 = 0 pull_rate1 = 0.0 pull_k1 = 200 ; kJ mol^-1 nm^-2 pull_nstxout = 1000 ; every 2 ps pull_nstfout = 1000 ; every 2 ps My PMF profile: http://speedy.sh/zerqZ/profile.JPG My histogram: http://speedy.sh/PyhnN/Histo.JPG Why g_wham takes into account distances below 2.45 nm as the 1st structure was at 2.45. If I get rid of the distances below 2.45 (those weird values PMF values) I obtain beautiful profile: http://speedy.sh/TUXGC/profile1.JPG Please, explain! Steven
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