[gmx-users] help with chromophore of a GFP

[Anna MARABOTTI]

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. 

Many thanks in advance and best regards 

Anna 



Links:
------
[1] http://www.gromacs.org/Documentation/Errors