[gmx-users] Questions about position restraint for charged proteins

[Koki Yokoi]

Hello. I am currently using GROMACS4.6.5 and trying to simulate a fluorescent protein(GFP,1EMB.pdb) fused to a muscarinic acetylcholine receptor(M2,3UON.pdb). PDB file of M2(3UON.pdb) included a lysozyme part(res#1002-1161), so I removed it to make the simulation consistent with our experimental data. As a result, there is a gap in M2 structure, which means M2 is divided into two chains(res#20-217, res#377-456), and when in NVT equilibration process, these chains move apart from each other because both are positively charged. I would like to keep the conformation of M2 and immobilize it in simulation to see only the motion of the fluorescent protein, so I applied position restrains to M2, but two chains still move away from each other and eventually I get many LINCS warnings during NVT equilibration process and simulation collapses.

My question is:
Position restrain is a proper way to keep conformations of proteins which have the same charge? Or is there any other way to do so?

I am using all-bonds constraint and 2fs time step to save the simulation time. Other fundamental parameters are default.
Any help would be appreciated. Thank you very much.

Koki


Below is the error messages during NVT equilibration process:
-----------------------------------------------------------------------
relative constraint deviation after LINCS:
rms 0.050664, max 3.739141 (between atoms 962 and 963)
bonds that rotated more than 30 degrees:
 atom 1 atom 2  angle  previous, current, constraint length
    962    963   89.9    0.1109   0.5118      0.1080
    964    965   90.1    0.1106   0.4336      0.1111
    964    966   31.9    0.1104   0.1199      0.1111
Wrote pdb files with previous and current coordinates

Step 575382, time 575.382 (ps)  LINCS WARNING
relative constraint deviation after LINCS:
rms 611.466194, max 51433.625000 (between atoms 570 and 573)
bonds that rotated more than 30 degrees:
 atom 1 atom 2  angle  previous, current, constraint length
    555    557   46.2    0.1345   0.1917      0.1345
    557    558   49.3    0.0997   0.1394      0.0997
    557    559   82.2    0.1430   0.4301      0.1430
    559    560   78.3    0.1080   0.3101      0.1080
    559    561   84.3    0.1538   1.6306      0.1538
    559    574   80.8    0.1490   0.3913      0.1490
    561    562   85.8    0.1111   1.4345      0.1111
    561    563   85.0    0.1111   1.3860      0.1111
    561    564   88.6    0.1538   7.0559      0.1538
    564    565   88.3    0.1111   6.1646      0.1111
    564    566   88.8    0.1538   6.5956      0.1538
    564    570  145.8    0.1538  18.8304      0.1538
    566    567   87.6    0.1111   1.7574      0.1111
    566    568   87.6    0.1111   1.7576      0.1111
    566    569   88.0    0.1111   1.7916      0.1111
    570    571   90.0    0.1111  10.2121      0.1111
    570    572  125.4    0.1111  10.2996      0.1111
    570    573   90.0    0.1111 5714.3872      0.1111
    574    575   42.6    0.1230   0.1646      0.1230
    574    576   42.1    0.1345   0.1834      0.1345
    916    917   33.8    0.0997   0.1182      0.0997
    916    918  115.0    0.1428   2.7045      0.1430
    918    919   92.6    0.1079   2.7119      0.1080
    918    920  149.6    0.1533   2.6632      0.1538
    920    921   36.5    0.1111   0.1355      0.1111
    920    922   35.7    0.1111   0.1401      0.1111
    933    934  106.0    0.1226   2.8814      0.1230
    933    960   86.3    0.1372   9.0101      0.1345
    935    962  121.3    0.1295  70.4373      0.1490
    936    940  172.6    0.1365   9.4523      0.1400
    937    955  150.4    0.1411   9.9921      0.1450
    938    939   43.2    0.1241   4.6305      0.1220
    938    940   79.6    0.1482   1.2946      0.1490
    940    941   48.9    0.1378   3.7713      0.1360
    941    942   42.8    0.1098   0.1650      0.1100
    941    943   33.2    0.1444   0.2096      0.1450
    955    956   73.3    0.1086   4.4738      0.1080
    955    957   68.8    0.1085   3.8836      0.1080
    955    958   43.4    0.1509   4.5246      0.1490
    958    959   33.6    0.1226   0.1674      0.1230
    958    969   36.3    0.1343   0.1844      0.1345
    962    963  103.5    0.5118  19.1058      0.1080
    962    964  131.9    0.1441 141.6376      0.1538
    964    965   88.9    0.4336 2773.4438      0.1111
    964    966   58.9    0.1199  82.5709      0.1111
    964    967   55.1    0.1502 133.7226      0.1420
    967    968  105.8    0.0947  47.8773      0.0960
Wrote pdb files with previous and current coordinates

WARNING: Listed nonbonded interaction between particles 557 and 564
at distance 3f which is larger than the table limit 3f nm.

This is likely either a 1,4 interaction, or a listed interaction inside
a smaller molecule you are decoupling during a free energy calculation.
Since interactions at distances beyond the table cannot be computed,
they are skipped until they are inside the table limit again. You will
only see this message once, even if it occurs for several interactions.

IMPORTANT: This should not happen in a stable simulation, so there is
probably something wrong with your system. Only change the table-extension
distance in the mdp file if you are really sure that is the reason.
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