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

Justin Lemkul jalemkul at vt.edu
Mon May 19 23:24:58 CEST 2014



On 5/19/14, 12:28 PM, Koki Yokoi wrote:
> Dear Massimo,
>
> Thank you for your help. I have never used MODELLER, but I will try.
> I corrected some amino acids which were different from our experiments. Because I am a beginner of biophysics, I actually don't understand the meaning of "stabilizing mutation". I will try to figure it out and check the structure again. Thank you very much anyway.
>

Some proteins (especially membrane proteins) are very unstable in non-native 
environments, so they are engineered to be more stable (i.e. "stabilizing 
mutations").  The paper associated with the structure should detail such things 
fairly obviously.

-Justin

> Koki
>
>> Actually, simulating it with no loop at all is much less realistic than
>> modeling your own loop. Just use MODELLER to fill the gap in the loop
>> regions and use the result.
>
>> Also, did you check for stabilizing mutations in the crystallized protein
>> and did you correct them?
>
>> Cheers,
>> Massimo
>> On 19 May 2014 03:07, "Koki Yokoi" <kyokoi at uwm.edu> wrote:
>
>> Dear Mark,
>>
>> Thank you for the reply. The muscarinic acetylcholine receptor consists of
>> seven coils(
>> http://en.wikipedia.org/wiki/File:Muscarinic_acetylcholine_receptor_M2-3UON.png).
>> These coils are connected by intra/extracellular loops in vivo. However, in
>> the PDB file of M2(3UON.pdb), one loop is replaced by lysozyme, which I
>> don't need in my system, so I removed it. I couldn't replace it with an
>> original loop because I don't know its structure. Above link shows M2
>> without lysozyme. You see two coils on near side are not connected to other
>> coils. These two and the remaining five coils are positively charged. I
>> would like to maintain this structure of M2(without lysozyme) as it is and
>> immobilize it during my simulation, but position restraint doesn't seem to
>> work..
>> Any help would be appreciated. Thank you very much.
>>
>> Koki
>>
>>
>> On May 16, 2014 11:36 PM, "Koki Yokoi" <kyokoi at uwm.edu> wrote:
>>>
>>> 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.
>>
>> Why do these positively charged parts stay together in the system you are
>> modeling?
>>
>> Mark
>>
>>> 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.
>>>

-- 
==================================================

Justin A. Lemkul, Ph.D.
Ruth L. Kirschstein NRSA Postdoctoral Fellow

Department of Pharmaceutical Sciences
School of Pharmacy
Health Sciences Facility II, Room 601
University of Maryland, Baltimore
20 Penn St.
Baltimore, MD 21201

jalemkul at outerbanks.umaryland.edu | (410) 706-7441
http://mackerell.umaryland.edu/~jalemkul

==================================================


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