# Error while using position restarints in MD run **URL:** \ **Category:** User discussions\ **Tags:** mdp-parameters, mdrun\ **Created:** [April 16, 2024, 9:33am UTC](https://gromacs.bioexcel.eu/t/error-while-using-position-restarints-in-md-run/8895 "2024-04-16T09:33:29Z")\ **Posts on this page:** 20\ **Page:** 1
**Author:** ![mnabanita](https://avatars.discourse-cdn.com/v4/letter/m/57b2e6/32.png) [@mnabanita](https://gromacs.bioexcel.eu/u/mnabanita)\ **Post date:** [April 16, 2024, 9:33am UTC](https://gromacs.bioexcel.eu/t/error-while-using-position-restarints-in-md-run/8895/1 "2024-04-16T09:33:29Z")
GROMACS version: 2023.1 GROMACS modification: Yes/No Here post your question Hello all, I am trying to simulate a DNA-membrane system where I want to apply position restraints only on DNA in the production run. But while using the command: gmx mdrun -v -deffnm md\_0\_1, it is showing LINCS error and the run is stopped. Here is the error: Step 876, time 0.876 (ps) LINCS WARNING relative constraint deviation after LINCS: rms 31216132.000000, max 3863014656.000000 (between atoms 88473 and 88474) bonds that rotated more than 30 degrees: atom 1 atom 2 angle previous, current, constraint length 39161 39162 112.1 0.1111 0.2950 0.1111 39164 39165 91.2 4.6748 5.1557 0.1111 39164 39166 103.2 0.3457 0.4882 0.1111 39167 39168 92.2 5.0048 2.9605 0.1111 39732 39733 73.3 0.4217 0.1111 0.1111 39734 39735 110.0 0.1011 0.3246 0.1111 39734 39736 98.5 0.3566 0.1376 0.1111 39734 39737 54.0 0.1172 0.1173 0.1111 85119 85121 98.0 0.1111 0.8011 0.1111 85134 85135 36.9 0.1115 0.1153 0.1111 85134 85136 39.3 0.1116 0.1149 0.1111 85134 85137 100.9 0.1104 0.5852 0.1111 85144 85145 40.8 0.1111 0.1111 0.1111 85178 85180 94.0 0.9634 1.5993 0.1111 85181 85182 91.9 0.1187 3.4375 0.1111 85181 85183 123.8 0.6689 0.1997 0.1111 85187 85188 111.7 0.2891 0.3004 0.1111 85189 85190 95.0 0.2718 1.2723 0.1111 85189 85192 101.0 0.5227 0.5831 0.1111 85193 85194 99.5 0.1111 0.6733 0.1111 85193 85195 97.9 0.1111 86.4671 0.1111 85199 85200 52.0 0.5286 0.1013 0.1111 85199 85201 94.2 0.1503 1.5152 0.1111 87736 87737 92.0 0.1111 3.2267 0.1111 87736 87738 90.9 0.1111 7.2435 0.1111 87739 87740 122.3 0.1111 429180832.0000 0.1111 87739 87741 77.0 0.1111 429180864.0000 0.1111 87773 87774 65.4 0.1129 0.1141 0.1111 87773 87775 83.4 0.6898 0.1087 0.1111 87776 87777 102.1 0.2135 701.3235 0.1111 87776 87778 100.0 0.1114 0.6405 0.1111 87779 87780 91.3 0.1116 4.7411 0.1111 87779 87781 95.1 0.3253 1.2405 0.1111 88324 88325 161.3 0.2327 5031.8369 0.1111 88324 88326 144.7 100.1726 4813.1401 0.1111 88324 88327 77.7 0.3314 5032.0215 0.1111 88464 88466 91.7 0.5818 3.6829 0.1111 88469 88470 35.3 1.0136 8601994.0000 0.1111 88473 88474 82.3 143.8495 429180928.0000 0.1111 88473 88475 63.0 0.9098 429180768.0000 0.1111 88476 88477 114.8 0.1111 0.2646 0.1111 88476 88478 102.1 0.1111 40.5930 0.1111 88482 88483 98.8 5.9134 0.1294 0.1111 89192 89193 115.1 0.1111 0.2624 0.1111 89198 89199 39.3 0.1111 48.9639 0.1111 89198 89200 73.6 0.1111 42.9575 0.1111 89207 89208 90.9 0.3581 6.7295 0.1111 89209 89210 96.1 0.1111 0.1240 0.1111 89209 89211 31.3 0.1111 0.1121 0.1111 89209 89212 100.9 0.1111 0.1337 0.1111 89259 89261 92.0 0.1111 3.1863 0.1111 89262 89263 84.2 0.1111 721879.1875 0.1111 89392 89393 54.2 0.2772 0.1111 0.1111 91854 91855 110.1 1.4639 0.3228 0.1111 91854 91856 115.0 0.2031 0.2625 0.1111 91857 91858 44.2 0.1144 231.9760 0.1111 91859 91860 105.3 0.1111 0.1538 0.1111 91859 91861 119.6 0.1111 0.1853 0.1111 91859 91862 92.0 0.1111 3.1109 0.1111 91863 91864 101.1 1.2794 0.5751 0.1111 91866 91867 93.5 0.1376 1.8082 0.1111 91866 91868 91.9 1.6835 3.2789 0.1111 91869 91870 92.5 0.4026 2.5030 0.1111 91869 91871 99.0 1.3699 0.7099 0.1111 91872 91873 90.8 0.1111 7.8312 0.1111 91874 91875 92.1 0.2672 2.9756 0.1111 91874 91876 99.5 0.2225 0.6703 0.1111 91874 91877 99.9 0.2612 0.6491 0.1111 91878 91880 92.9 0.5104 2.2284 0.1111 91878 91881 93.3 0.3811 1.9263 0.1111 93246 93247 90.7 0.3242 9.6808 0.1111 93246 93248 93.6 0.7115 1.7602 0.1111 93252 93253 91.3 2.6884 0.1137 0.1111 93261 93262 92.2 0.1111 2.8810 0.1111 93261 93263 44.2 0.1111 0.1026 0.1111 93269 93272 93.9 0.1111 1.6159 0.1111 96219 96220 99.1 0.1111 0.7003 0.1111 96219 96221 91.5 0.1111 4.2736 0.1111 96284 96286 98.0 0.1111 5222.8994 0.1111 96284 96287 100.0 0.1111 5223.0332 0.1111 97388 97391 103.1 0.1111 0.4917 0.1111 99889 99890 64.4 3.0742 13.3486 0.1111 99889 99891 98.3 0.4694 0.7683 0.1111 99904 99905 91.4 0.2280 4.4763 0.1111 99904 99906 108.6 0.6482 0.3491 0.1111 99964 99965 62.5 0.1986 0.1111 0.1111 99971 99973 112.9 0.2093 0.2860 0.1111 Back Off! I just backed up step876b.pdb to ./#step876b.pdb.1# Back Off! I just backed up step876c.pdb to ./#step876c.pdb.1# ^CWrote pdb files with previous and current coordinates ^C^CAborted (core dumped) I am also attaching the topol.top and md.mdp file which I am using. topol.top File ‘topol.top’ was generated ; By user: nabanita (1001) ; On host: kashi ; At date: Mon Apr 15 13:47:07 2024 ; ; This is a standalone topology file ; ; Created by: ; :-) GROMACS - gmx pdb2gmx, 2023.3 (-: ; ; Executable: /usr/local/gromacs/bin/gmx ; Data prefix: /usr/local/gromacs ; Working dir: /data/nabanita/13th\_demo/hj\_test/15thapr\_demo ; Command line: ; gmx pdb2gmx -f new.pdb -o dna\_mem.gro -ter ; Force field was read from current directory or a relative path - path added. ; ; Include forcefield parameters #include “./charmm36-jul2022.ff/forcefield.itp” ; Include chain topologies #include “topol\_DNA\_chain\_D.itp” #include “topol\_DNA\_chain\_D2.itp” #include “topol\_DNA\_chain\_D3.itp” #include “topol\_DNA\_chain\_D4.itp” #include “topol\_Other\_chain\_P.itp” #include “topol\_Other\_chain\_P2.itp” #include “topol\_Other\_chain\_P3.itp” #include “topol\_Other\_chain\_P4.itp” #include “topol\_Other\_chain\_P5.itp” #include “topol\_Other\_chain\_P6.itp” #ifdef POSRES\_DNAP #include “posre\_dnaP.itp” #endif ; Include water topology #include “./charmm36-jul2022.ff/tip3p.itp” #ifdef POSRES\_WATER ; Position restraint for each water oxygen [position\_restraints] ; i funct fcx fcy fcz 1 1 1000 1000 1000 #endif ; Include topology for ions #include “./charmm36-jul2022.ff/ions.itp” [system] ; Name Protein in water [molecules] ; Compound #mols DNA\_chain\_D 1 DNA\_chain\_D2 1 DNA\_chain\_D3 1 DNA\_chain\_D4 1 Other\_chain\_P 1 Other\_chain\_P2 1 Other\_chain\_P3 1 Other\_chain\_P4 1 Other\_chain\_P5 1 Other\_chain\_P6 1 SOL 87531 CL 47 MG 47 CL 47 NA 155 CL 63 md.mdp title = KALP15-DPPC Production MD define = -DPOSRES -DPOSRES\_DNAP ; position restarint the protein ; Run parameters integrator = sd ; leap-frog integrator nsteps = 1000000 ; 1 \* 1000000 = 1000 ps (1 ns) dt = 0.001 ; 1 fs ; Output control nstxout = 100 ; save coordinates every 2 ps nstvout = 100 ; save velocities every 2 ps nstxtcout = 100 ; xtc compressed trajectory output every 2 ps nstenergy = 100 ; save energies every 2 ps nstlog = 100 ; update log file every 2 ps ; Bond parameters continuation = yes ; Restarting after NPT constraint\_algorithm = lincs ; holonomic constraints constraints = h-bonds ; all bonds (even heavy atom-H bonds) constrained lincs\_iter = 2 ; accuracy of LINCS lincs\_order = 4 ; also related to accuracy ; Neighborsearching ns\_type = grid ; search neighboring grid cels nstlist = 5 ; 10 fs rlist = 1.2 ; short-range neighborlist cutoff (in nm) rcoulomb = 1.2 ; short-range electrostatic cutoff (in nm) rvdw = 1.2 ; short-range van der Waals cutoff (in nm) ; 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 ; More accurate thermostat tc-grps = DNA\_DFPE Water\_and\_ions ; two coupling groups - more accurate tau\_t = 0.5 0.5 ; time constant, in ps ref\_t = 300 300 ; reference temperature, one for each group, in K ; Pressure coupling is on pcoupl = C-rescale ; Pressure coupling on in NPT pcoupltype = semiisotropic ; uniform scaling of x-y box vectors, independent z tau\_p = 2.0 ; time constant, in ps ref\_p = 1.0 1.0 ; reference pressure, x-y, z (in bar) compressibility = 4.5e-5 4.5e-5 ; isothermal compressibility, 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 ; Velocity generation is off ; COM motion removal ; These options remove motion of the protein/bilayer relative to the solvent/ions nstcomm = 100 comm-mode = Linear comm-grps = DNA\_DFPE Water\_and\_ions ; Scale COM of reference coordinates refcoord\_scaling = com ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [April 16, 2024, 9:54am UTC](https://gromacs.bioexcel.eu/t/error-while-using-position-restarints-in-md-run/8895/2 "2024-04-16T09:54:07Z")
It seems like you need to run longer equilibration with more restraints before turning them off. There are also a few things that spring to my mind when I see your parameters, but they are not related to the crash. - Why are you using the sd integrator for this simulation? - If you want to use the sd integrator, why are you also specifying the v-rescale thermostat? - If you want to use the sd integrator I would suggest tau-t of 2. - Why are you writing your output files so often, every 100 fs? Do you really need 10,000 frames? Do you really need uncompressed output at all? `nstenergy = 10000` should be enough. ---
**Author:** ![mnabanita](https://avatars.discourse-cdn.com/v4/letter/m/57b2e6/32.png) [@mnabanita](https://gromacs.bioexcel.eu/u/mnabanita)\ **Post date:** [April 16, 2024, 11:14am UTC](https://gromacs.bioexcel.eu/t/error-while-using-position-restarints-in-md-run/8895/3 "2024-04-16T11:14:51Z")
Thank you @MagnusL for your valuable suggestion. Regarding your questions: 1. There are atoms at both ends of an angle, connected by constraints and with masses that differ by more than a factor of 13. This means that there are likely dynamic modes that are only very weakly coupled. To ensure good equipartitioning, you need to either not use constraints on all bonds (but, if possible, only on bonds involving hydrogens) or use integrator = sd or decrease one or more tolerances: verlet-buffer-tolerance \<= 0.0001, LINCS iterations \>= 2, LINCS order \>= 4 or SHAKE tolerance \<= 1e-05 2. The bond in molecule-type Protein\_chain\_A between atoms 173 O1 and 174 HO1 has an estimated oscillational period of 9.0e-03 ps, which is less than 10 times the time step of 1.0e-03 ps. Maybe you forgot to change the constraints mdp option”. For that reason I am using 1 fs timestep. ---
**Author:** ![mnabanita](https://avatars.discourse-cdn.com/v4/letter/m/57b2e6/32.png) [@mnabanita](https://gromacs.bioexcel.eu/u/mnabanita)\ **Post date:** [April 16, 2024, 11:17am UTC](https://gromacs.bioexcel.eu/t/error-while-using-position-restarints-in-md-run/8895/4 "2024-04-16T11:17:37Z")
Regarding the second question: The bond in molecule-type Protein\_chain\_A between atoms 79 C2 and 80 O2 has an estimated oscillational period of 1.9.0e-03 ps, which is less than 10 times the time step of 2.0e-03 ps. Maybe you forgot to change the constraints mdp option”. For that reason I am using 1 fs timestep. For that reason I am using 1 fs timestep. ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [April 16, 2024, 11:59am UTC](https://gromacs.bioexcel.eu/t/error-while-using-position-restarints-in-md-run/8895/5 "2024-04-16T11:59:55Z")
Thanks for the explanation. I think you misunderstood my questions a little. But I’ll follow-up on your answers. 1. You are currently only constraining bonds to hydrogens - and that is usually the best choice. But still, if you want to use the sd integrator it is confusing to set parameters for the v-rescale thermostat. A tau-t of 2 is better for sd. 2. I didn’t question your choice of timestep. I’m wondering about the output intervals. Why are `nstxout`, `nstvout` etc so low? And do you really need uncompressed data at all? ---
**Author:** ![mnabanita](https://avatars.discourse-cdn.com/v4/letter/m/57b2e6/32.png) [@mnabanita](https://gromacs.bioexcel.eu/u/mnabanita)\ **Post date:** [April 16, 2024, 12:34pm UTC](https://gromacs.bioexcel.eu/t/error-while-using-position-restarints-in-md-run/8895/6 "2024-04-16T12:34:53Z")
Regrading the answer 2 I don’t need output for such small intervals. I kept it because I assumed that the crash is happening for keeping large nstxout, nstvout etc. ---
**Author:** ![mnabanita](https://avatars.discourse-cdn.com/v4/letter/m/57b2e6/32.png) [@mnabanita](https://gromacs.bioexcel.eu/u/mnabanita)\ **Post date:** [April 16, 2024, 12:42pm UTC](https://gromacs.bioexcel.eu/t/error-while-using-position-restarints-in-md-run/8895/7 "2024-04-16T12:42:19Z")
Regarding the timestep, if I keep 2 fs instead of 1 fs will it pose a problem alongwith the restraints. ---
**Author:** ![mnabanita](https://avatars.discourse-cdn.com/v4/letter/m/57b2e6/32.png) [@mnabanita](https://gromacs.bioexcel.eu/u/mnabanita)\ **Post date:** [April 16, 2024, 12:45pm UTC](https://gromacs.bioexcel.eu/t/error-while-using-position-restarints-in-md-run/8895/8 "2024-04-16T12:45:23Z")
And also if we use sd, should we not set any parameters for v-rescale? Should we off the pressure coupling Pressure coupling is on pcoupl = C-rescale ; Pressure coupling on in NPT pcoupltype = semiisotropic ; uniform scaling of x-y box vectors, independent z tau\_p = 2.0 ; time constant, in ps ref\_p = 1.0 1.0 ; reference pressure, x-y, z (in bar) ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [April 16, 2024, 1:20pm UTC](https://gromacs.bioexcel.eu/t/error-while-using-position-restarints-in-md-run/8895/9 "2024-04-16T13:20:23Z")
The things is that when you are using the sd integrator that takes over as a thermostat, and uses tau-t as the inverse friction constant. When running `gmx grompp` you also get a note that says that v-rescale is inactivated (or something along those lines) when you are using the sd integrator. So, it’s less confusing to remove v-rescale from the mdp file. I’d suggest something like: ```auto tcoupl = no ; handled by the sd integrator tc-grps = DNA_DFPE Water_and_ions ; two coupling groups - more accurate tau_t = 2 2 ; inverse friction for the sd integrator ref_t = 300 300 ; reference temperature, one for each group, in K ``` or even ```auto tcoupl = no ; handled by the sd integrator tc-grps = system tau_t = 2 ; inverse friction for the sd integrator ref_t = 300 ; reference temperature, one for each group, in K ``` The pressure coupling is not affected by the sd integrator. So you can keep your current settings. ---
**Author:** ![mnabanita](https://avatars.discourse-cdn.com/v4/letter/m/57b2e6/32.png) [@mnabanita](https://gromacs.bioexcel.eu/u/mnabanita)\ **Post date:** [April 16, 2024, 1:24pm UTC](https://gromacs.bioexcel.eu/t/error-while-using-position-restarints-in-md-run/8895/10 "2024-04-16T13:24:41Z")
Thank you so much for your suggestions. I have one more question, can I keep a time of 2 fs despite the error; “The bond in molecule-type Protein\_chain\_A between atoms 79 C2 and 80 O2 has an estimated oscillational period of 1.9.0e-03 ps, which is less than 10 times the time step of 2.0e-03 ps. Maybe you forgot to change the constraints mdp option”. For that reason I am using 1 fs timestep. For that reason I am using 1 fs timestep.” ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [April 16, 2024, 1:34pm UTC](https://gromacs.bioexcel.eu/t/error-while-using-position-restarints-in-md-run/8895/11 "2024-04-16T13:34:24Z")
I would not use a 2 fs time step when you get that warning. But I’m a bit surprised that you get such a long oscillation period for a bond in a protein. What force field is this? ---
**Author:** ![mnabanita](https://avatars.discourse-cdn.com/v4/letter/m/57b2e6/32.png) [@mnabanita](https://gromacs.bioexcel.eu/u/mnabanita)\ **Post date:** [April 16, 2024, 1:46pm UTC](https://gromacs.bioexcel.eu/t/error-while-using-position-restarints-in-md-run/8895/12 "2024-04-16T13:46:51Z")
I am using Charmm36 force-field and sorry for not changing the name, I am simulating DNA with membrane ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [April 16, 2024, 2:01pm UTC](https://gromacs.bioexcel.eu/t/error-while-using-position-restarints-in-md-run/8895/13 "2024-04-16T14:01:35Z")
I’m not used to simulating DNA, but I’m surprised if the Charmm36 force field would not be able to run with a 2 fs time step when you have constrained the bonds to hydrogens. I assume you haven’t modified any of the force field parameters, right? ---
**Author:** ![mnabanita](https://avatars.discourse-cdn.com/v4/letter/m/57b2e6/32.png) [@mnabanita](https://gromacs.bioexcel.eu/u/mnabanita)\ **Post date:** [April 16, 2024, 2:04pm UTC](https://gromacs.bioexcel.eu/t/error-while-using-position-restarints-in-md-run/8895/14 "2024-04-16T14:04:39Z")
No I have not modified any of the force-field parameters. But from nvt step, this error is coming up ---
**Author:** ![mnabanita](https://avatars.discourse-cdn.com/v4/letter/m/57b2e6/32.png) [@mnabanita](https://gromacs.bioexcel.eu/u/mnabanita)\ **Post date:** [April 16, 2024, 2:05pm UTC](https://gromacs.bioexcel.eu/t/error-while-using-position-restarints-in-md-run/8895/15 "2024-04-16T14:05:05Z")
Sorry it’s a note which pops up ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [April 16, 2024, 2:28pm UTC](https://gromacs.bioexcel.eu/t/error-while-using-position-restarints-in-md-run/8895/16 "2024-04-16T14:28:40Z")
Your settings are very close to the threshold for getting a note (and with reasonable margin from getting a warning. From `src/gromacs/gmxpreprocess/grompp.cpp` ```auto The stability limit of leap-frog or velocity verlet is 4.44 steps per oscillational period. But accurate bonds distributions are lost far before that limit. To allow relatively common schemes (although not common with Gromacs) of dt=1 fs without constraints and dt=2 fs with only H-bond constraints we set the note limit to 10. ``` You are currently at 9 steps per oscillational period. You could test if it works OK with a time step of 2 ns. Run a few nanoseconds in an NVE ensemble and check the energy conservation. If the energy and the system itself seems stable, it should be OK. ---
**Author:** ![mnabanita](https://avatars.discourse-cdn.com/v4/letter/m/57b2e6/32.png) [@mnabanita](https://gromacs.bioexcel.eu/u/mnabanita)\ **Post date:** [May 7, 2024, 5:08am UTC](https://gromacs.bioexcel.eu/t/error-while-using-position-restarints-in-md-run/8895/17 "2024-05-07T05:08:26Z")
Hello, I want to restraint only the DNA in my case. This is the md.mdp file which I am using. But after running for 100 ns I don’t see any bending in the membrane as well. Does it mean my lipids are also restrained. title = KALP15-DPPC Production MD define = -DPOSRES -DPOSRES\_DNA ; Run parameters integrator = md ; leap-frog integrator nsteps = 500000000 ; 1 \* 500000000 = 500000 ps (500 ns) dt = 0.001 ; 1 fs ; Output control nstxout = 1000000 ; save coordinates every 2 ps nstvout = 1000000 ; save velocities every 2 ps nstxtcout = 1000000 ; xtc compressed trajectory output every 2 ps nstenergy = 1000000 ; save energies every 2 ps nstlog = 1000000 ; update log file every 2 ps ; Bond parameters continuation = yes ; Restarting after NPT constraint\_algorithm = lincs ; holonomic constraints constraints = h-bonds ; all bonds (even heavy atom-H bonds) constrained lincs\_iter = 1 ; accuracy of LINCS lincs\_order = 4 ; also related to accuracy ; Neighborsearching ns\_type = grid ; search neighboring grid cels nstlist = 5 ; 10 fs rlist = 1.2 ; short-range neighborlist cutoff (in nm) rcoulomb = 1.2 ; short-range electrostatic cutoff (in nm) rvdw = 1.2 ; short-range van der Waals cutoff (in nm) ; 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 = Nose-Hoover ; More accurate thermostat tc-grps = DNA\_DFPE Water\_and\_ions ; two coupling groups - more accurate tau\_t = 0.5 0.5 ; time constant, in ps ref\_t = 300 300 ; reference temperature, one for each group, in K ; Pressure coupling is on pcoupl = C-rescale ; Pressure coupling on in NPT pcoupltype = semiisotropic ; uniform scaling of x-y box vectors, independent z tau\_p = 2.0 ; time constant, in ps ref\_p = 1.0 1.0 ; reference pressure, x-y, z (in bar) compressibility = 4.5e-5 4.5e-5 ; isothermal compressibility, 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 ; Velocity generation is off ; COM motion removal ; These options remove motion of the protein/bilayer relative to the solvent/ions nstcomm = 100 comm-mode = Linear comm-grps = DNA\_DFPE Water\_and\_ions ; Scale COM of reference coordinates refcoord\_scaling = all ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [May 7, 2024, 6:12am UTC](https://gromacs.bioexcel.eu/t/error-while-using-position-restarints-in-md-run/8895/18 "2024-05-07T06:12:54Z")
If you look for `#ifdef POSRES` in your topology (and all files that are included from it) you will see which molecules/atoms are restrained by that define statement. Without knowing your force field or topology, I would presume that `-DPOSRES` would restrain proteins and possibly lipids, whereas `-DPOSRES_DNA` would restrain your DNA molecules. If you only want to restrain the DNA you can almost certainly remove `-DPOSRES`. ---
**Author:** ![mnabanita](https://avatars.discourse-cdn.com/v4/letter/m/57b2e6/32.png) [@mnabanita](https://gromacs.bioexcel.eu/u/mnabanita)\ **Post date:** [May 8, 2024, 7:23am UTC](https://gromacs.bioexcel.eu/t/error-while-using-position-restarints-in-md-run/8895/19 "2024-05-08T07:23:36Z")
@MagnusL thank you so much for your suggetions. I am using Charmm 36 force- field and below mentioned is the topol.top file. File ‘topol.top’ was generated ; By user: nabanita (1001) ; On host: kashi ; At date: Wed Apr 17 10:32:08 2024 ; ; This is a standalone topology file ; ; Created by: ; :-) GROMACS - gmx pdb2gmx, 2023.3 (-: ; ; Executable: /usr/local/gromacs/bin/gmx ; Data prefix: /usr/local/gromacs ; Working dir: /data/nabanita/13th\_demo/hj\_test/17th\_apr ; Command line: ; gmx pdb2gmx -f new.pdb -o dna\_mem.gro -ter ; Force field was read from current directory or a relative path - path added. ; ; Include forcefield parameters #include “./charmm36-jul2022.ff/forcefield.itp” ; Include chain topologies #include “topol\_DNA\_chain\_D.itp” #include “topol\_DNA\_chain\_D2.itp” #include “topol\_DNA\_chain\_D3.itp” #include “topol\_DNA\_chain\_D4.itp” #include “topol\_Other\_chain\_P.itp” #include “topol\_Other\_chain\_P2.itp” #include “topol\_Other\_chain\_P3.itp” #include “topol\_Other\_chain\_P4.itp” #include “topol\_Other\_chain\_P5.itp” #include “topol\_Other\_chain\_P6.itp” ; Include position restrain file #ifdef POSRES\_DNA #include “posre\_dna.itp” #endif ; Include water topology #include “./charmm36-jul2022.ff/tip3p.itp” #ifdef POSRES\_WATER ; Position restraint for each water oxygen [position\_restraints] ; i funct fcx fcy fcz 1 1 1000 1000 1000 #endif ; Include topology for ions #include “./charmm36-jul2022.ff/ions.itp” [system] ; Name Protein in water [molecules] ; Compound #mols DNA\_chain\_D 1 DNA\_chain\_D2 1 DNA\_chain\_D3 1 DNA\_chain\_D4 1 Other\_chain\_P 1 Other\_chain\_P2 1 Other\_chain\_P3 1 Other\_chain\_P4 1 Other\_chain\_P5 1 Other\_chain\_P6 1 SOL 87531 CL 47 MG 47 CL 47 NA 155 ---
**Author:** ![MagnusL](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/magnusl/32/2380_2.png) [@MagnusL](https://gromacs.bioexcel.eu/u/MagnusL)\ **Post date:** [May 8, 2024, 7:30am UTC](https://gromacs.bioexcel.eu/t/error-while-using-position-restarints-in-md-run/8895/20 "2024-05-08T07:30:35Z")
Just based on the contents you copied, `-DPOSRES` does not have any effect at all. However, I guess there might be sections similar to: ```auto #ifdef POSRES #include "posre_topol_Other_chain_P.itp" #endif ``` in the included .itp files. Still, if you only want to restrain the DNA you can definitely set: `define = -DPOSRES_DNA` in the mdp file. [Next page](https://gromacs.bioexcel.eu/t/error-while-using-position-restarints-in-md-run/8895.md?page=2)