<div class="gmail_quote">
<div>Thanks for the reply Justin,</div>
<div> </div>
<blockquote style="BORDER-LEFT: #ccc 1px solid; MARGIN: 0px 0px 0px 0.8ex; PADDING-LEFT: 1ex" class="gmail_quote">> In theory, that should work. Please post the entirety of your .mdp file.<br><br>Have you done any prior equilibration, or have you moved straight into<br>
annealing? I would suggest a restrained NVT before applying NPT or annealing<br>when using the restraints.<br></blockquote>
<div>
<p style="MARGIN: 0in 0in 0pt" class="MsoNormal"><font size="3"><font face="Times New Roman">Yes I did the restrained NVT before but the same problem came, morover there was a hole in the upper part of the system with the SOL molecules displaced. So then I tried to run the annealing but in this case the SOL molecules were intact but similar problem came with the lipid membrane. though, just to check then I tried to equilibration with restrained NVT where I restrained the movement of the lipid in all the x, y, z axis the problem did not occur.</font></font></p>
<p style="MARGIN: 0in 0in 0pt" class="MsoNormal"> </p>
<p style="MARGIN: 0in 0in 0pt" class="MsoNormal"><font size="3"><font face="Times New Roman">Here is my mdp file </font></font></p>
<p style="MARGIN: 0in 0in 0pt" class="MsoNormal"><span style="mso-spacerun: yes"> </span>--------------------------------------------------------------------------</p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">title<span style="mso-tab-count: 2"> </span>= NVT equilibration for B3-DPPC </span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">define<span style="mso-tab-count: 2"> </span>= -DPOSRES -DPOSRES_LIPID<span style="mso-tab-count: 1"> </span>; position restrain the protein and lipid</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt"> </span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">; Run parameters</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">integrator<span style="mso-tab-count: 1"> </span>= md<span style="mso-tab-count: 2"> </span>; leap-frog integrator</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">nsteps<span style="mso-tab-count: 2"> </span>= 50000<span style="mso-tab-count: 2"> </span>; 2 * 50000 = 100 ps</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">dt<span style="mso-tab-count: 2"> </span><span style="mso-spacerun: yes"> </span>= 0.002<span style="mso-tab-count: 2"> </span>; 2 fs</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">; Output control</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">nstxout<span style="mso-tab-count: 2"> </span>= 100<span style="mso-tab-count: 2"> </span>; save coordinates every 0.2 ps</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">nstvout<span style="mso-tab-count: 2"> </span>= 100<span style="mso-tab-count: 2"> </span>; save velocities every 0.2 ps</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">nstenergy<span style="mso-tab-count: 1"> </span>= 100<span style="mso-tab-count: 2"> </span>; save energies every 0.2 ps</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">nstlog<span style="mso-tab-count: 2"> </span>= 100<span style="mso-tab-count: 2"> </span>; update log file every 0.2 ps</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">; Bond parameters</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">continuation<span style="mso-tab-count: 1"> </span>= no<span style="mso-tab-count: 2"> </span><span style="mso-spacerun: yes"> </span>; first dynamics run</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">constraint_algorithm = lincs<span style="mso-tab-count: 1"> </span>; holonomic constraints </span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">constraints<span style="mso-tab-count: 1"> </span>= all-bonds<span style="mso-tab-count: 1"> </span><span style="mso-spacerun: yes"> </span>; all bonds (even heavy atom-H bonds) constrained</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">lincs_iter<span style="mso-tab-count: 1"> </span>= 1<span style="mso-tab-count: 2"> </span><span style="mso-spacerun: yes"> </span>; accuracy of LINCS</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">lincs_order<span style="mso-tab-count: 1"> </span>= 4<span style="mso-tab-count: 2"> </span><span style="mso-spacerun: yes"> </span>; also related to accuracy</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">; Neighborsearching</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">ns_type<span style="mso-tab-count: 2"> </span>= grid<span style="mso-tab-count: 2"> </span>; search neighboring grid cels</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">nstlist<span style="mso-tab-count: 2"> </span>= 5<span style="mso-tab-count: 2"> </span><span style="mso-spacerun: yes"> </span>; 10 fs</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">rlist<span style="mso-tab-count: 2"> </span>= 1.2<span style="mso-tab-count: 2"> </span>; short-range neighborlist cutoff (in nm)</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">rcoulomb<span style="mso-tab-count: 1"> </span>= 1.2<span style="mso-tab-count: 2"> </span>; short-range electrostatic cutoff (in nm)</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">rvdw<span style="mso-tab-count: 2"> </span>= 1.2<span style="mso-tab-count: 2"> </span>; short-range van der Waals cutoff (in nm)</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">; Electrostatics</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">coulombtype<span style="mso-tab-count: 1"> </span>= PME<span style="mso-tab-count: 2"> </span>; Particle Mesh Ewald for long-range electrostatics</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">pme_order<span style="mso-tab-count: 1"> </span>= 4<span style="mso-tab-count: 2"> </span><span style="mso-spacerun: yes"> </span>; cubic interpolation</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">fourierspacing<span style="mso-tab-count: 1"> </span>= 0.16<span style="mso-tab-count: 2"> </span>; grid spacing for FFT</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">; Temperature coupling is on</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">tcoupl<span style="mso-tab-count: 2"> </span>= V-rescale<span style="mso-tab-count: 1"> </span><span style="mso-spacerun: yes"> </span>; modified Berendsen thermostat</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">tc-grps<span style="mso-tab-count: 2"> </span>= Protein DPPC SOL_CL-<span style="mso-tab-count: 1"> </span>; three coupling groups - more accurate</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">tau_t<span style="mso-tab-count: 2"> </span>= 0.1<span style="mso-tab-count: 1"> </span>0.1<span style="mso-tab-count: 1"> </span>0.1<span style="mso-tab-count: 1"> </span><span style="mso-spacerun: yes"> </span>; time constant, in ps</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">ref_t<span style="mso-tab-count: 2"> </span>= 323 <span style="mso-tab-count: 1"> </span>323<span style="mso-tab-count: 1"> </span>323<span style="mso-tab-count: 1"> </span><span style="mso-spacerun: yes"> </span>; reference temperature, one for each group, in K</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">; Pressure coupling is off</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">pcoupl<span style="mso-tab-count: 2"> </span>= no <span style="mso-tab-count: 2"> </span>; no pressure coupling in NVT</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">; Periodic boundary conditions</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">pbc<span style="mso-tab-count: 2"> </span><span style="mso-spacerun: yes"> </span>= xyz<span style="mso-tab-count: 2"> </span>; 3-D PBC</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">; Dispersion correction</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">DispCorr<span style="mso-tab-count: 1"> </span>= EnerPres<span style="mso-tab-count: 1"> </span>; account for cut-off vdW scheme</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">; Velocity generation</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">gen_vel<span style="mso-tab-count: 2"> </span>= yes<span style="mso-tab-count: 2"> </span>; assign velocities from Maxwell distribution</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">gen_temp<span style="mso-tab-count: 1"> </span>= 323<span style="mso-tab-count: 2"> </span>; temperature for Maxwell distribution</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">gen_seed<span style="mso-tab-count: 1"> </span>= -1<span style="mso-tab-count: 2"> </span>; generate a random seed</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">; COM motion removal</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">; These options remove motion of the protein/bilayer relative to the solvent/ions</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">nstcomm<span style="mso-tab-count: 2"> </span>= 1</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">comm-mode<span style="mso-tab-count: 1"> </span>= Linear</span></p>
<p style="MARGIN: 0in 0in 0pt; mso-layout-grid-align: none" class="MsoNormal"><span style="FONT-FAMILY: 'Courier New'; FONT-SIZE: 10pt">comm-grps<span style="mso-tab-count: 1"> </span>= Protein_DPPC SOL_CL-</span></p>
<p style="MARGIN: 0in 0in 0pt" class="MsoNormal">--------------------------------------------------------------------------</p>
<p style="MARGIN: 0in 0in 0pt" class="MsoNormal">This is the topology file:</p>
<p style="MARGIN: 0in 0in 0pt" class="MsoNormal"><span style="mso-spacerun: yes"> </span>--------------------------------------------------------------------------</p><span lang="EN">
<p>;Include DPPC chain topology</p>
<p>#include "dppc.itp"</p>
<p>#ifdef POSRES_LIPID</p>
<p>; Position restraint for each lipid</p>
<p>#include "lipid_posre.itp"</p>
<p>#endif</p>
<p> </p>
<p>; Include water topology</p>
<p>#include "spc.itp"</p>
<p>#ifdef POSRES_WATER</p>
<p>; Position restraint for each water oxygen</p>
<p>[ position_restraints ]</p>
<p>; i funct fcx fcy fcz</p>
<p>1 1 1000 1000 1000</p>
<p>#endif</p>
<p>; Include generic topology for ions</p>
<p>#include "ions.itp"</p>
<p> </p>
<p>[ system ]</p>
<p>; Name</p>
<p>protein</p>
<p>128-Lipid DPPC Bilayer </p>
<p>[ molecules ]</p>
<p>; Compound #mols</p>
<p>Protein_A 1</p>
<p>DPPC 121</p>
<p> </p>
<p>SOL 9867</p>
<p>CL- 14</p>
<p>--------------------------------------------------------------------------</p>
<p>thanks </p>
<p>Parul Tewatia</p>
<p> </p></span></div></div>