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