[gmx-users] help with chromophore of a GFP

[Anna Marabotti]

Dear Justin,
there is not a "unique" GFP chromophore, the chromophore I am dealing 
with is not the same for which CHARMM parameters have been published (I 
am aware of CHARMM parameters for p-hydroxybenzylideneimidazolinone 
chromophore of green fluorescent protein published by Reuter et al 2002, 
of OPLS-AA parameters for DsRed fluorescent protein chromophore as a 
residue of [(4
cis)
2
[(1
cis)
4
 
amino
4
oxobutanimidoyl]
4
(4
hydroxybenzylidene)
5
oxo
4,5
dihydro
1H
imidazol
1
yl]acetic 
acid, of other parameters of other GFP chromophores), but mine is 
different: is of the same family of proteins, but different residues are 
involved and different heterocycles are generated.

Since I have to recalculate parameters, I chose Amber ff because I 
already used it and I have tools to calculate Amber parameters, whereas 
I have no tools to calculate CHARMM parameters. In a preliminary assay, 
I tried to do the same parameterization using Gromos ff and PRODRG to 
obtain parameters and topology (apart from the fact that charges are 
probably wrong), but I experimented the same problem.

I am talking not only about the problem of obtaining parameters for this 
particular chromophore, mine is a more general question: how to deal 
with a "HETATM" entry which is not a ligand, but it's a part of the 
protein chain? I tried to follow indications to make a new .rtp entry in 
the GROMACS HowTo's, probably my problem would be solved if I would be 
able to modify the aminoacids.hdb file, but this is not a simple 
modification of a residue (eg. an oxidised Met or a methylation of a 
Lys), this is a profound modification of four residues, so how can I 
deal with this? I had a look at the .hdb file, but hydrogens I can see 
are typical for amino acids residues and I cannot find any suggestions 
on how to treat hydrogens that are bound to a "residue" which is so 
different from classic standard residues. Has anyone made this before (I 
am sure yes)? Could you please give some suggestions?

Thank you very much
Anna

______________________________________________
Anna Marabotti, Ph.D.
Assistant Professor
Department of Chemistry and Biology
University of Salerno
Via Ponte don Melillo
84084 Fisciano (SA)
Italy
Phone: +39 089 969583
Fax: +39 089 969603
E-mail: amarabotti at unisa.it
Skype: annam1972

"When a man with a gun meets a man with a pen, the man with the gun is a dead man"
(Roberto Benigni, about Roberto Saviano)

Il 21/03/2013 06:37, gmx-users-request at gromacs.org ha scritto:
> Message: 3 Date: Wed, 20 Mar 2013 13:05:08 -0400 From: Justin Lemkul 
> <jalemkul at vt.edu> Subject: Re: [gmx-users] help with chromophore of a 
> GFP To: Discussion list for GROMACS users <gmx-users at gromacs.org> 
> Message-ID: 
> <CADUqwc5C+2NZWrwvzHh11Wnd=ShwnhyQtTVophzKWwEOeG9p6Q at mail.gmail.com> 
> Content-Type: text/plain; charset=ISO-8859-1 On Wed, Mar 20, 2013 at 
> 1:01 PM, Anna MARABOTTI <amarabotti at unisa.it> wrote:
>> >
>> >
>> >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. 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]
>> >
>> >>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.
>> >         *
>> >
>> >If I add
>> >-ignh, all the H of CFY will be ignored too, and I will not be able to
>> >add them since I did not modify aminoacids.hdb
>> >         *
>> >
>> >since I made
>> >calculations on CFY with H added by PyMol, probably also my codes for H
>> >will be wrong.
>> >         *
>> >
>> >If I use "reduce" (the Amber tool to add H, as
>> >suggested by the tutorial) to add H to my protein, it does not add H to
>> >CFY because it complaints that the residue is not in HETATM connection
>> >database (but the record CONECT is present in .pdb file). If I add H to
>> >CFY alone, I have problems with the terminals.
>> >
>> >My question is,
>> >obviously: how can I parameterize this chromophore correctly? Please
>> >give me, if possible, some step-by-step indications on what to do. I
>> >made dozens of trials, ALL with errors, and I really do not know how to
>> >do.
>> >
>> >
> There are parameters published for the GFP chromophore under the CHARMM
> force field; is there some reason those are unsuitable?  No need to
> reinvent the wheel.  With the published parameters, one simply needs to
> create an .rtp entry and pdb2gmx runs fine, no need to mess with
> antechamber, PyMOL, etc.
>
> -Justin