[gmx-users] help with chromophore of a GFP

[lloyd riggs]

I had problems having not used gromacs in years a couple years ago.  Try running it through with the output as a pdb from pdb2gmx, cut off all headers, and you can then just compare the two files in gedit emacs or word and see differences.  That might help.  I routinely just keep everything in pdb format as its easier than jumping back and forth.


-------- Original-Nachricht --------
> Datum: Thu, 21 Mar 2013 21:43:16 +0100
> Von: Mark Abraham <mark.j.abraham at gmail.com>
> An: Discussion list for GROMACS users <gmx-users at gromacs.org>
> Betreff: Re: [gmx-users] help with chromophore of a GFP

> On Thu, Mar 21, 2013 at 4:30 PM, Anna MARABOTTI <amarabotti at unisa.it>
> wrote:
> 
> >
> >
> > Dear Mark,
> >
> > thank you for your message. I'm happy to be on the
> > right track; unfortunately the end point seems to be very far away...
> >
> >
> > I tried to obtain that CFY hydrogens and protein hydrogens are all
> > matching the aminoacids.rtp entry, in order to avoid dealing with
> > aminoacids.hdb. This is what I did:
> >
> > - starting from the pdb file of
> > the protein, I removed CFY entry (prot_noCFY.pdb)
> >
> > - I used pdb2gmx to
> > add H to the protein only: pdb2gmx -f prot_noCFY.pdb -o prot_noCFY_H.pdb
> > -p topol.top
> >
> > - I inserted CFY_H.pdb (obtained with Pymol in a previous
> > passage in which I added H with Pymol to the protein, including CFY)
> > into prot_noCFY_H.pdb, obtaining prot_CFY_H.pdb.
> >
> > In this way, H atoms
> > bound to "regular" residues have been added using Amber99SB, therefore
> > they are compatible with this ff, and atoms of CFY (previously added
> > with Pymol) have the same naming convention in aminoacids.rtp (that I
> > edited using atom types, charges etc. calculated with Antechamber on
> > this molecule coming from Pymol). Obviously, the atom numbering is not
> > sequential: the last atom of V63 (the last "regular" residue before CFY)
> > is numbered 938, the first atom of H68 (the first "regular" residue
> > after CFY) is numbered 939, and the atoms of CFY66 are numbered from 1
> > to 70. Moreover, since the sequence of atoms in aminoacids.rtp is not
> > the same as in the coordinates of CFY (I adapted the sequence of atoms
> > following the format of other residues in aminoacids.rtp), the numbering
> > of CFY in the prot_CFY_H.pdb is not ordered (1-2-3-....-69-70) but
> > disordered (19-54-20-55...49-50-24-25).
> >
> 
> Seems fine. pdb2gmx is mostly about atom/residue naming. grompp is mostly
> about atom/residue/moleculetype ordering.
> 
> - At this stage, I used
> > pdb2gmx again to create the topol.top file with all coordinates correct:
> >
> >
> > pdb2gmx -f prot_CFY_H.pdb -o prot_complete.gro -p topol.top
> >
> >
> > (selecting amber99sb forcefield and tip3p for water, as recommended
> > option)
> >
> > This is the message error from pdb2gmx:
> >
> > Read 'FLUORESCENT
> > PROTEIN', 3346 atoms
> > Analyzing pdb file
> > Splitting PDB chains based on
> > TER records or changing chain id.
> > There are 1 chains and 0 blocks of
> > water and 218 residues with 3346 atoms
> >
> >  chain #res #atoms
> >  1 'A' 213
> > 3346
> >
> 
> I'd be concerned about the difference in residue count here, but 4.5.4 is
> so old I've no idea whose fault this is.
> 
> 
> > All occupancies are one
> > Opening force field file
> > ./amber99sb.ff/atomtypes.atp
> > Atomtype 1
> > Reading residue database...
> > (amber99sb)
> > Opening force field file
> > ./amber99sb.ff/aminoacids.rtp
> > Residue 94
> > Sorting it all out...
> > Opening
> > force field file ./amber99sb.ff/dna.rtp
> > Residue 110
> > Sorting it all
> > out...
> > Opening force field file ./amber99sb.ff/rna.rtp
> > Residue
> > 126
> > Sorting it all out...
> > Opening force field file
> > ./amber99sb.ff/aminoacids.hdb
> > Opening force field file
> > ./amber99sb.ff/dna.hdb
> > Opening force field file
> > ./amber99sb.ff/rna.hdb
> > Opening force field file
> > ./amber99sb.ff/aminoacids.n.tdb
> > Opening force field file
> > ./amber99sb.ff/aminoacids.c.tdb
> >
> > Processing chain 1 'A' (3346 atoms, 213
> > residues)
> > There are 327 donors and 319 acceptors
> > There are 539 hydrogen
> > bonds
> > Will use HISE for residue 22
> > Will use HISD for residue 38
> > Will use
> > HISE for residue 62
> > Will use HISE for residue 68
> > Will use HISD for
> > residue 109
> > Will use HISE for residue 119
> > Will use HISE for residue
> > 172
> > Will use HISH for residue 193
> > Will use HISH for residue 197
> > Will use
> > HISE for residue 217
> > Identified residue SER3 as a starting
> > terminus.
> > Identified residue SER218 as a ending terminus.
> > 8 out of 8
> > lines of specbond.dat converted successfully
> > Special Atom Distance
> > matrix:
> >  MET9 MET11 MET15 HIS22 HIS38 MET41 MET47
> >  SD110 SD149 SD232
> > NE2317 NE2549 SD596 SD700
> >  MET11 SD149 0.807
> >  MET15 SD232 2.279 1.627
> >
> > HIS22 NE2317 3.707 2.983 1.466
> >  HIS38 NE2549 1.401 0.928 2.127 3.254
> >
> > MET41 SD596 1.458 0.665 1.144 2.384 1.001
> >  MET47 SD700 3.059 2.324 0.995
> > 0.801 2.656 1.761
> >  MET53 SD777 2.786 1.999 0.990 1.171 2.160 1.373
> > 0.603
> >  HIS62 NE2917 2.340 1.733 0.833 1.797 1.988 1.236 1.583
> >  HIS68
> > NE21002 0.884 0.597 1.466 2.916 1.356 0.885 2.347
> >  HIS109 NE21638 2.061
> > 1.886 1.380 2.614 2.661 1.862 2.279
> >  HIS119 NE21803 1.459 0.967 0.923
> > 2.372 1.617 0.812 1.870
> >  MET135 SD2041 3.480 2.751 1.316 0.606 2.919
> > 2.121 0.993
> >  MET162 SD2439 2.521 1.976 1.656 2.412 1.855 1.543 2.264
> >
> > HIS172 NE22588 3.632 2.949 1.894 1.657 2.872 2.338 1.945
> >  CYS174 SG2623
> > 2.968 2.372 1.452 1.861 2.428 1.848 1.924
> >  MET189 SD2891 2.167 2.379
> > 2.736 4.000 2.754 2.569 3.722
> >  HIS193 NE22942 2.003 2.001 2.490 3.686
> > 2.049 2.075 3.396
> >  HIS197 NE23011 2.012 1.634 1.830 2.896 1.554 1.426
> > 2.614
> >  HIS217 NE23329 2.545 2.376 2.831 3.805 2.039 2.305 3.575
> >  MET53
> > HIS62 HIS68 HIS109 HIS119 MET135 MET162
> >  SD777 NE2917 NE21002 NE21638
> > NE21803 SD2041 SD2439
> >  HIS62 NE2917 1.363
> >  HIS68 NE21002 2.107 1.482
> >
> > HIS109 NE21638 2.365 1.568 1.372
> >  HIS119 NE21803 1.688 0.976 0.584
> > 1.078
> >  MET135 SD2041 1.057 1.365 2.661 2.490 2.119
> >  MET162 SD2439 1.878
> > 0.871 1.805 2.246 1.520 1.861
> >  HIS172 NE22588 1.721 1.401 2.829 2.860
> > 2.359 1.067 1.342
> >  CYS174 SG2623 1.694 0.725 2.140 2.152 1.681 1.297
> > 0.745
> >  MET189 SD2891 3.547 2.310 1.858 1.893 1.980 3.627 2.290
> >  HIS193
> > NE22942 3.076 1.890 1.639 2.197 1.760 3.221 1.547
> >  HIS197 NE23011 2.229
> > 1.149 1.407 2.078 1.323 2.401 0.676
> >  HIS217 NE23329 3.146 2.112 2.205
> > 2.935 2.272 3.263 1.402
> >  HIS172 CYS174 MET189 HIS193 HIS197
> >  NE22588
> > SG2623 SD2891 NE22942 NE23011
> >  CYS174 SG2623 0.826
> >  MET189 SD2891 3.417
> > 2.599
> >  HIS193 NE22942 2.831 2.079 1.020
> >  HIS197 NE23011 2.011 1.324
> > 1.766 0.939
> >  HIS217 NE23329 2.629 2.068 1.936 0.946 1.003
> > Opening force
> > field file ./amber99sb.ff/aminoacids.arn
> > Opening force field file
> > ./amber99sb.ff/dna.arn
> > Opening force field file
> > ./amber99sb.ff/rna.arn
> > Checking for duplicate atoms....
> > Now there are
> > 3345 atoms. Deleted 1 duplicates.
> >
> 
> That also looks suspicious.
> 
> 
> > Now there are 213 residues with 3345
> > atoms
> > Making bonds...
> > Warning: Long Bond (988-989 = 0.453624
> > nm)
> >
> 
> That seems like it might be a peptide bond bridging a "gap" where pdb2gmx
> was unable to recognize the intervening content as a peptide residue.
> 
> 
> >
> > WARNING: atom O1 is missing in residue CFY 66 in the pdb
> > file
> >
> > -------------------------------------------------------
> > Program
> > pdb2gmx_d, VERSION 4.5.4
> > Source code file: pdb2top.c, line: 1463
> >
> > Fatal
> > error:
> > There were 1 missing atoms in molecule Protein_chain_A, if you
> > want to use this incomplete topology anyhow, use the option -missing
> > For
> > more information and tips for troubleshooting, please check the
> > GROMACS
> > website at http://www.gromacs.org/Documentation/Errors
> >
> > The
> > strange thing is that I checked for this error, but atom O1 in residue
> > CFY66 is present BOTH in the starting .pdb file (the one I used for
> > pdb2gmx) AND in the aminoacids.rtp file!!!! I checked 4 or 5 times,
> > every time erasing the old file, checking the file IMMEDIATELY BEFORE
> > submitting it to pdb2gmx. All atoms present in aminoacids.rtp for CFY
> > residue are also present in the .pdb file and vice versa, and I am sure
> > I did not make the stupid error of naming the atom 01 (zero-one) instead
> > of O1 (o-one).
> >
> > I suspect that this atom is the one which is deleted
> > because recognized as duplicated, but I'm not sure about it and I don't
> > know how to check it. I am sure there are no duplicated atoms in CFY.
> >
> >
> > I feel like this is a "fake" error message (i.e.: there is an error in
> > my files, but it is not the one that is reported in the message:
> > probably a problem occur around this atom, but it is not exactly ON this
> > atom). However, I am not able to find errors.
> >
> 
> Hmm that seems weird. Justin's theory sounds plausible, but I haven't seen
> someone stumble on that before. Also plausible is that pdb2gmx thinks your
> CFY is a disconnected part of the chain and needs terminating (which might
> happen with an oxygen named O1?).
> 
> It's possible there's buggy behaviour here that has been fixed in the two
> years since that code was released. There certainly has been an upgrade of
> the "is this really a new chain" machinery. Unless you have a strong
> scientific reason to keep 4.5.4, I'd switch to 4.6.1 (or 4.5.6 if you
> really have to keep 4.5). If Justin's fix doesn't work, and you have
> problems with a more recent version, then we can look closer.
> 
> 
> > BTW the "long bond" of
> > the other warning message is not involving residue CFY.
> >
> 
> Yeah, but my bet is those atoms are the C-terminus and N-terminus of the
> fragments that should form peptide bonds to CFY.
> 
> Mark
> 
> 
> > Any help is
> > welcome
> >
> > Thank you so much.
> >
> > Anna
> >
> > Il 21.03.2013 12:00
> > gmx-users-request at gromacs.org ha scritto:
> >
> > >> Dear gmx-users, it's
> > about two weeks that I'm trying to solve this problem, and I can't, so
> > I'm asking your help. I want to do some MD simulations on a protein of
> > the family of green fluorescent protein. This protein, as you know, has
> > a chromophore (CFY) derived from four residues of the protein
> > (F64-C65-Y66-G67) and covalently bound to the rest of the protein chain.
> > How to parametrize this object, since it is not recognized by pdb2gmx? I
> > looked at the gmx-users list and the suggestion was to create a new
> > entry in the .rtp file of the selected forcefield.
> > >
> > > Indeed, this
> > kind of problem is most easily solved by making a new
> > > "residue" that
> > contains the whole chromophore, such that it links to its
> > > neighbours
> > with normal peptide links.
> > > ------------------------------ Message: 5
> > Date: Thu, 21 Mar 2013 11:46:12 +0100 From: Mark Abraham
> > <mark.j.abraham at gmail.com [2]> Subject: Re: [gmx-users] help with
> > chromophore of a GFP To: Discussion list for GROMACS users
> > <gmx-users at gromacs.org [3]> Message-ID:
> > <CAMNuMASicyMGiVb_x5sY1YB44th8VKNioQVhzDqq-tAm9TnRqQ at mail.gmail.com [4]>
> > Content-Type: text/plain; charset=ISO-8859-1 On Wed, Mar 20, 2013 at
> > 6:01 PM, Anna MARABOTTI <amarabotti at unisa.it [5]> wrote:
> > >
> > >> I
> > decided to use Amber99SB since it seemed the better for my scope, then I
> > start trying to parameterize it. This is what I did: * I used Pymol to
> > add H to my pdb file, since I want to use an all H forcefield and since
> > Antechamber (see below) does not work without H * I extracted the
> > segment V63-CFY-H68 from my .pdb file. I did this since, when I
> > extracted CFY only, I had problems with the terminals * Following the
> > Antechamber tutorial, I used Antechamber (using the traditional Amber
> > force field, not GAFF) to calculate charges and to assign atom types to
> > this segment. * I used these calculated parameters in order to add the
> > CFY residue to aminoacids.rtp in amber99sb.ff directory. * I tried to
> > modify also aminoacids.hdb, but since it seemed too complicated to me, I
> > decided to keep it unchanged, and to give pdb2gmx the protein with H
> > already present * No need to add new atom/bond types to ffbonded.itp and
> > ffnonbonded.itp: they seem all present. Since CFY is bound to the rest
> > of protein with common peptide bonds, I did not change specbond.dat
> > either. * I added CFY in residuetypes.dat with the specification
> > "Protein" In my opinion, all was ready to go, instead... When I launched
> > pdb2gmx to my protein with H added by PyMol, I got immediately an error:
> > Fatal error: Atom H01 in residue SER 3 was not found in rtp entry NSER
> > with 13 atoms while sorting atoms. For a hydrogen, this can be a
> > different protonation state, or it might have had a different number in
> > the PDB file and was rebuilt (it might for instance have been H3, and we
> > only expected H1 & H2). Note that hydrogens might have been added to the
> > entry for the N-terminus. Remove this hydrogen or choose a different
> > protonation state to solve it. Option -ignh will ignore all hydrogens in
> > the input. For more information and tips for troubleshooting, please
> > check the GROMACS website at http://www.gromacs.org/Documentation/Errors
> > [1][1]
> > >>
> > >>> From this error I
> > >> understand that: * the code for H
> > in PyMol is different from the code for H in Amber (read from
> > aminoacids.rtp); in order to correct this error, I should add -ignh in
> > order to ignore H in input.
> > >
> > > pdb2gmx has to be able to make sense of
> > the atom naming. There are lots of
> > > different conventions for how to
> > name atoms, particularly hydrogen atoms.
> > > pdb2gmx can't possibly encode
> > the logic to convert all of those
> > > conventions. So the path of least
> > resistance can be to ignore hydrogens and
> > > regenerate them according to
> > the generation rules.
> > >
> > > However, you can just rename them in the
> > input file so that pdb2gmx
> > > understands your meaning. The NSER entry in
> > the .rtp file shows you the
> > > names pdb2gmx expects. If you edit the
> > names of those hydrogen atoms
> > > (probably H01, H02, H03) in your input
> > coordinate file accordingly (to H1,
> > > H2, H3), things will be fine. Be
> > sure you don't break the required column
> > > formatting of the coordinate
> > file!
> > >
> > > *
> >
> >
> >
> > Links:
> > ------
> > [1]
> > http://www.gromacs.org/Documentation/Errors
> > [2]
> > mailto:mark.j.abraham at gmail.com
> > [3] mailto:gmx-users at gromacs.org
> > [4]
> >
> mailto:CAMNuMASicyMGiVb_x5sY1YB44th8VKNioQVhzDqq-tAm9TnRqQ at mail.gmail.com
> > [5]
> > mailto:amarabotti at unisa.it
> > --
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