[gmx-users] disulfide bond missing even with -ss

Justin Lemkul jalemkul at vt.edu
Sun Jan 21 17:48:54 CET 2018 


On 1/21/18 11:42 AM, MD wrote:
> On Sun, Jan 21, 2018 at 11:33 AM, Justin Lemkul <jalemkul at vt.edu> wrote:
>
>>
>> On 1/21/18 11:30 AM, MD wrote:
>>
>>> On Sun, Jan 21, 2018 at 11:22 AM, Justin Lemkul <jalemkul at vt.edu> wrote:
>>>
>>>
>>>> On 1/21/18 11:20 AM, MD wrote:
>>>>
>>>> On Sun, Jan 21, 2018 at 11:05 AM, Justin Lemkul <jalemkul at vt.edu> wrote:
>>>>>
>>>>> On 1/21/18 10:59 AM, MD wrote:
>>>>>> On Sun, Jan 21, 2018 at 10:49 AM, Justin Lemkul <jalemkul at vt.edu>
>>>>>> wrote:
>>>>>>
>>>>>>> On 1/21/18 10:46 AM, MD wrote:
>>>>>>>
>>>>>>>> On Sun, Jan 21, 2018 at 10:37 AM, Justin Lemkul <jalemkul at vt.edu>
>>>>>>>> wrote:
>>>>>>>>
>>>>>>>> On 1/21/18 10:29 AM, MD wrote:
>>>>>>>>> I modified the specbond.dat and change the cutoff to be 2.04A, still
>>>>>>>>>> nothing...
>>>>>>>>>>
>>>>>>>>>>> Please don't spam the list with minute-by-minute updates.
>>>>>>>>>>>
>>>>>>>>>>> ​
>>>>>>>>>>>
>>>>>>>>>> sorry
>>>>>>>>>>
>>>>>>>>> ​for the scattered information, ​
>>>>>>>>> I didn't mean to spam the thread..
>>>>>>>>>
>>>>>>>>>
>>>>>>>>>
>>>>>>>>> Provide your pdb2gmx command, full screen output, and whatever
>>>>>>>>> evidence
>>>>>>>>>
>>>>>>>>> you have that the bond wasn't formed. The definitive answer is in
>>>>>>>>> your
>>>>>>>>>
>>>>>>>>>> topology. If it's not there, we can diagnose.
>>>>>>>>>>
>>>>>>>>>> ​
>>>>>>>>>>
>>>>>>>>>> command line: gmx pdb2gmx -f ncat.pdb -o ncat.gro -water spc -ignh
>>>>>>>>>>
>>>>>>>>> -ss
>>>>>>>>>
>>>>>>>>>
>>>>>>>>>
>>>>>>>>> Link CYS-335 SG-2487 and CYS-338 SG-2507 (y/n) ?y
>>>>>>>>>
>>>>>>>>> There is no indication that anything went wrong, and this should
>>>>>>>>> have
>>>>>>>>>
>>>>>>>>> been
>>>>>>>> done. Are you saying that there is no line in the topology
>>>>>>>> specifying a
>>>>>>>> bond between atoms 2487 and 2507?
>>>>>>>>
>>>>>>>> ​Right. I didn't see anything said about the bond. I went forward to
>>>>>>>>
>>>>>>> the
>>>>>>> step of em.mdp and got a minimized gro, which was converted to pdb.
>>>>>>> And
>>>>>>> I
>>>>>>> found no disulfide bond in the pdb file.​
>>>>>>>
>>>>>>> So, to be clear, please answer these two questions directly:
>>>>>>>
>>>>>> 1. You find no line in [bonds] in topol_Protein_chain_B.itp file
>>>>>> between
>>>>>> atoms 2487 and 2507?
>>>>>>
>>>>>> ​Correct.​
>>>>>>
>>>>>
>>>>> 2. When you visualize the structure, both Cys335 and Cys338 are in their
>>>>>
>>>>>> thiol (-SH) form?
>>>>>>
>>>>>> T
>>>>> ​hey are not in thiol form. ​
>>>>> ​
>>>>> https://docs.google.com/document/d/1HOuMWAylWZOp1KP1cHRp82Y_
>>>>> EiM5xAc8QM5KCAOOkSo/edit?usp=sharing
>>>>> ​
>>>>>
>>>>> This is not consistent with your above assertion. If your structure,
>>>> after
>>>> pdb2gmx or any point thereafter, does not have -SH, then you are
>>>> modeling a
>>>> disulfide. You can't simultaneously have no bond defined in the topology
>>>> and also have no thiols. These are mutually exclusive. I suspect the bond
>>>> was formed and you're perhaps looking in the wrong place.
>>>>
>>> ​Then how can we explain there are no bond formed in the
>>> topol_Protein_chain_B.itp?​
>>>
>> Is the image you showed before or after pdb2gmx? I'm trying to help you
>> figure this out, but the information at hand is not consistent unless this
>> structure is pre-pdb2gmx. What I was asking about was a structure *after*
>> processing with pdb2gmx.
>>
> ​Yes I am very thankful for the help here and was just trying to discuss :)
>
> The structure picture I showed is the one after pdb2gmx. I double checked
> the original pdb which has disulfide bond. Then I processed the pdb with
> pdb2gmx. I checked the topol_Protein_chain_B.itp and there was no disulfide
> bond. I know I should stop here but I want to look at the pdb file so I
> then moved forward to em.mdp and convert the em.gro to em.pdb with ​trjconv
> and looked at the pdb file (the one shown in the picture), which only has
> two sulfurs in close distance. The distance is about 2.0A but no disulfide
> bond shown. I thought it might be chimera which didn't detect the bond but
> I thought the topol_Protein_chain_B.itp or any gmx log should have
> confirmed the bond formation. Does it make sense?

Yes and no.

NEVER rely on visualization software to tell you if a bond has been 
formed. They are simply guessing based on assumptions coded into them. 
The only thing that is definitive is the topology and its listing of the 
actual bonds.

Is the disulfide actually in chain B? Your screen output is incomplete 
so maybe something was lost in copy and paste and you should be looking 
in the chain A topology if it exists. There's not much more I or anyone 
can say about this because all indications are that pdb2gmx did its job 
based on (1) the lack of any error or warning and (2) the fact that Cys 
is being modeled in its oxidized form in the output coordinate file.

-Justin

>> If pdb2gmx has converted these Cys to -SH form despite being told
>> otherwise, please upload the original PDB file somewhere and provide a link.
>>
>> -Justin
>>
>>
>>>> -Justin
>>>>
>>>>
>>>>
>>>> -Justin
>>>>>>
>>>>>> -Justin
>>>>>>
>>>>>>>> Start terminus THR-24: NH3+
>>>>>>>>
>>>>>>>> End terminus LEU-385: COO-
>>>>>>>>
>>>>>>>>> Opening force field file ./charmm36-jul2017.ff/merged.arn
>>>>>>>>> Checking for duplicate atoms....
>>>>>>>>> Generating any missing hydrogen atoms and/or adding termini.
>>>>>>>>> Now there are 362 residues with 5724 atoms
>>>>>>>>> Chain time...
>>>>>>>>>
>>>>>>>>> Back Off! I just backed up topol_Protein_chain_B.itp to
>>>>>>>>> ./#topol_Protein_chain_B.itp.5#
>>>>>>>>> Making bonds...
>>>>>>>>> Warning: Long Bond (3206-3204 = 0.339276 nm)
>>>>>>>>> Number of bonds was 5800, now 5800
>>>>>>>>> Generating angles, dihedrals and pairs...
>>>>>>>>> Before cleaning: 15256 pairs
>>>>>>>>> Before cleaning: 15446 dihedrals
>>>>>>>>> Keeping all generated dihedrals
>>>>>>>>> Making cmap torsions...
>>>>>>>>> There are  359 cmap torsion pairs
>>>>>>>>> There are 15446 dihedrals,  949 impropers, 10509 angles
>>>>>>>>>                15142 pairs,     5800 bonds and     0 virtual sites
>>>>>>>>> Total mass 40928.695 a.m.u.
>>>>>>>>> Total charge -13.000 e
>>>>>>>>> Writing topology
>>>>>>>>>
>>>>>>>>> Back Off! I just backed up posre_Protein_chain_B.itp to
>>>>>>>>> ./#posre_Protein_chain_B.itp.5#
>>>>>>>>> Processing chain 2 'B' (1 atoms, 1 residues)
>>>>>>>>> Warning: Starting residue MG430 in chain not identified as
>>>>>>>>> Protein/RNA/DNA.
>>>>>>>>> Problem with chain definition, or missing terminal residues.
>>>>>>>>> This chain does not appear to contain a recognized chain molecule.
>>>>>>>>> If this is incorrect, you can edit residuetypes.dat to modify the
>>>>>>>>> behavior.
>>>>>>>>> 8 out of 8 lines of specbond.dat converted successfully
>>>>>>>>> Opening force field file ./charmm36-jul2017.ff/merged.arn
>>>>>>>>> Checking for duplicate atoms....
>>>>>>>>> Generating any missing hydrogen atoms and/or adding termini.
>>>>>>>>> Now there are 1 residues with 1 atoms
>>>>>>>>> Chain time...
>>>>>>>>>
>>>>>>>>> Back Off! I just backed up topol_Ion_chain_B2.itp to
>>>>>>>>> ./#topol_Ion_chain_B2.itp.5#
>>>>>>>>> Making bonds...
>>>>>>>>> No bonds
>>>>>>>>> Generating angles, dihedrals and pairs...
>>>>>>>>> Making cmap torsions...
>>>>>>>>> There are    0 dihedrals,    0 impropers,    0 angles
>>>>>>>>>                   0 pairs,        0 bonds and     0 virtual sites
>>>>>>>>> Total mass 24.305 a.m.u.
>>>>>>>>> Total charge 2.000 e
>>>>>>>>> Writing topology
>>>>>>>>>
>>>>>>>>> Back Off! I just backed up posre_Ion_chain_B2.itp to
>>>>>>>>> ./#posre_Ion_chain_B2.itp.5#
>>>>>>>>> Including chain 1 in system: 5724 atoms 362 residues
>>>>>>>>> Including chain 2 in system: 1 atoms 1 residues
>>>>>>>>> Now there are 5725 atoms and 363 residues
>>>>>>>>> Total mass in system 40953.000 a.m.u.
>>>>>>>>> Total charge in system -11.000 e
>>>>>>>>>
>>>>>>>>> Writing coordinate file...
>>>>>>>>>
>>>>>>>>> Back Off! I just backed up ncat.gro to ./#ncat.gro.5#
>>>>>>>>>
>>>>>>>>>
>>>>>>>>>
>>>>>>>>> https://docs.google.com/document/d/1HOuMWAylWZOp1KP1cHRp82Y_
>>>>>>>>> EiM5xAc8QM5KCAOOkSo/edit?usp=sharing
>>>>>>>>> ​
>>>>>>>>>       ​
>>>>>>>>>
>>>>>>>>>
>>>>>>>>> -Justin
>>>>>>>>>
>>>>>>>>> On Sun, Jan 21, 2018 at 10:23 AM, MD
>>>>>>>>> <https://maps.google.com/?q=at+10:23+AM,+MD+wro&entry=gmail&source=g>
>>>>>>>>> <refmac5 at gmail.com> wro
>>>>>>>>> <https://maps.google.com/?q=at+10:23+AM,+MD+wro&entry=gmail&source=g>
>>>>>>>>> te:
>>>>>>>>>
>>>>>>>>>> I have also tried renaming my two CYS to be CYS2 but no luck
>>>>>>>>>> either.
>>>>>>>>>>
>>>>>>>>>> On Sun, Jan 21, 2018 at 10:22 AM, MD
>>>>>>>>>>> <https://maps.google.com/?q=n,+Jan+21,+2018+at+10:22+AM,+MD&
>>>>>>>>>>> entry=gmail&source=g>
>>>>>>>>>>>
>>>>>>>>>>> <refmac5 at gmail.com> wrote:
>>>>>>>>>>>
>>>>>>>>>>> gromacs did ask me to confirm linking the two cys before the run,
>>>>>>>>>>>> but I
>>>>>>>>>>>>
>>>>>>>>>>>> didn't see any log saying the bond is formed.
>>>>>>>>>>>>
>>>>>>>>>>>> On Sun, Jan 21, 2018 at 10:19 AM, MD <refmac5 at gmail.com> wrote:
>>>>>>>>>>>>> Hi Gromacs folks,
>>>>>>>>>>>>>
>>>>>>>>>>>>> I realized I kept losing disulfide bond after gmx. The length is
>>>>>>>>>>>>>
>>>>>>>>>>>>> 2.01A
>>>>>>>>>>>>>> but I did use -ss when pdb2gmx. Any thoughts?
>>>>>>>>>>>>>> Thanks,
>>>>>>>>>>>>>> Ming
>>>>>>>>>>>>>>
>>>>>>>>>>>>>>
>>>>>>>>>>>>>> --
>>>>>>>>>>>>>>
>>>>>>>>>>>>>> ==================================================
>>>>>>>>>>>>>>
>>>>>>>>>>>>> Justin A. Lemkul, Ph.D.
>>>>>>>>>>> Assistant Professor
>>>>>>>>>> Virginia Tech Department of Biochemistry
>>>>>>>>>>
>>>>>>>>>> 303 Engel Hall
>>>>>>>>>> 340 West Campus Dr.
>>>>>>>>>> Blacksburg, VA 24061
>>>>>>>>>>
>>>>>>>>>> jalemkul at vt.edu | (540) 231-3129
>>>>>>>>>> http://www.biochem.vt.edu/people/faculty/JustinLemkul.html
>>>>>>>>>>
>>>>>>>>>> ==================================================
>>>>>>>>>>
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>>>>>>>>>>
>>>>>>>>>>
>>>>>>>>>> --
>>>>>>>>>>
>>>>>>>>>> ==================================================
>>>>>>>> Justin A. Lemkul, Ph.D.
>>>>>>>> Assistant Professor
>>>>>>>> Virginia Tech Department of Biochemistry
>>>>>>>>
>>>>>>>> 303 Engel Hall
>>>>>>>> 340 West Campus Dr.
>>>>>>>> Blacksburg, VA 24061
>>>>>>>>
>>>>>>>> jalemkul at vt.edu | (540) 231-3129
>>>>>>>> http://www.biochem.vt.edu/people/faculty/JustinLemkul.html
>>>>>>>>
>>>>>>>> ==================================================
>>>>>>>>
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>>>>>>>>
>>>>>>>> --
>>>>>>>>
>>>>>>> ==================================================
>>>>>> Justin A. Lemkul, Ph.D.
>>>>>> Assistant Professor
>>>>>> Virginia Tech Department of Biochemistry
>>>>>>
>>>>>> 303 Engel Hall
>>>>>> 340 West Campus Dr.
>>>>>> Blacksburg, VA 24061
>>>>>>
>>>>>> jalemkul at vt.edu | (540) 231-3129
>>>>>> http://www.biochem.vt.edu/people/faculty/JustinLemkul.html
>>>>>>
>>>>>> ==================================================
>>>>>>
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>>>>>> Gromacs Users mailing list
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>>>>>>
>>>>>>
>>>>>> --
>>>> ==================================================
>>>>
>>>> Justin A. Lemkul, Ph.D.
>>>> Assistant Professor
>>>> Virginia Tech Department of Biochemistry
>>>>
>>>> 303 Engel Hall
>>>> 340 West Campus Dr.
>>>> Blacksburg, VA 24061
>>>>
>>>> jalemkul at vt.edu | (540) 231-3129
>>>> http://www.biochem.vt.edu/people/faculty/JustinLemkul.html
>>>>
>>>> ==================================================
>>>>
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>>>>
>> --
>> ==================================================
>>
>> Justin A. Lemkul, Ph.D.
>> Assistant Professor
>> Virginia Tech Department of Biochemistry
>>
>> 303 Engel Hall
>> 340 West Campus Dr.
>> Blacksburg, VA 24061
>>
>> jalemkul at vt.edu | (540) 231-3129
>> http://www.biochem.vt.edu/people/faculty/JustinLemkul.html
>>
>> ==================================================
>>
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>> Gromacs Users mailing list
>>
>> * Please search the archive at http://www.gromacs.org/Support
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-- 
==================================================

Justin A. Lemkul, Ph.D.
Assistant Professor
Virginia Tech Department of Biochemistry

303 Engel Hall
340 West Campus Dr.
Blacksburg, VA 24061

jalemkul at vt.edu | (540) 231-3129
http://www.biochem.vt.edu/people/faculty/JustinLemkul.html

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

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