[gmx-users] help with chromophore of a GFP

Thu Mar 21 21:43:16 CET 2013

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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