Pitambar,
the resonance in your structure is not reflected in the BOND section of
the mol2 file, but is taken into account by the choice of atom types.
At a first glance, I do not see anything obvious strange in your mol2
file, apart from the large difference in atomic charge of the two
guanidinium nitrogen atoms, -0.5978 and -1.0190.
In standard Amber force fields, the two nitrogen atoms of the
guanidinium group in Arginine have the same (symmetrized) atomic charge.
But the situation is not that straightforward:
For the free ligand in solution a description with symmetrized charges
could be sufficient. When bound to a protein target, however,
polarization takes place that might not be described well by fixed
atomic charges stemming from the isolated structure.
Since you found that the ligand dissociates from the protein, "wrong"
atomic charges could be the reason. Have a look at the binding pocket
and the protein's ligand interaction there. Maybe simply using
symmetrized nitrogen charges solves your problem (if your initial
structure is an experimentally determined one).
Maybe that helps.
Best,
Anselm
Bioinformatik | NHR.FAU
Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU)
Germany
Am 23.06.2025 um 19:48 schrieb Pitambar Poudel:
> Thank you very much for the reply,
>
> As suggested, I'm currently reviewing the parameter files for a ligand
> that were generated by Antechamber. One thing I'm confused about is how
> AMBER or Antechamber handles resonance, especially in cases involving
> delocalized double bonds. For example, my ligand is a zwitterion when
> bound to the protein. One end contains a carboxylate group (COO⁻), and
> the other end has two NH₂⁺ groups attached to a single carbon, forming a
> guanidinium-like structure. The double bond between the central carbon
> and one of the nitrogen atoms can resonate between the two nitrogens,
> just like the two C–O bonds in the carboxylate group are also
> delocalized. I have attached the image of the structure and the mol2
> file created.
>
> My question is: how is this resonance represented in the MOL2 file? Does
> it specify one C=N and one C–N, or both as C=N C=N? And for the
> carboxylate group, is it written as one C=O and one C–O⁻ or C=O for
> both, or is the resonance captured differently? An incorrect or
> incomplete representation of these resonance structures be the reason
> why the ligand flies away during molecular dynamics simulations since
> this double bond is very important to maintain the zwitterion form
>
> *Pitambar Poudel*
> Graduate Research Assistant
> Computational Biophysics and Bioinformatics Lab
> Department of Physics and Astronomy, Clemson University
> /Lab: http:/compbio.clemson.edu/ <http://compbio.clemson.edu/lab/>/
>
>
> On Fri, Jun 20, 2025 at 5:42 AM Dr. Anselm Horn via AMBER
> <amber.ambermd.org <mailto:amber.ambermd.org>> wrote:
>
> Pitambar,
>
> your guess about a ligand parameterization issue seems reasonable to me,
> if you start from a known complex structure.
> I'd suggest to check the ligand parameters, i.e. atom types and charges,
> as well as the structural elements (planarity vs. non-planarity) of the
> ligand (=> minimization/simulation of the free ligand).
> Additionally, ensure that the ligand has the correct molecular charge
> and protonation state when simulating the bound state.
> Maybe key polar interactions between ligand and protein are not
> correctly modelled and you want to try a different charge generation
> method.
>
> If you do not start from a known complex structure, then the protein
> environment of the binding pocket might disfavor ligand binding: your
> protein could have different conformations in bound and unbound state.
>
> Maybe that helps.
>
> Best,
>
> Anselm
>
> Bioinformatik | NHR.FAU
> Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU)
> Germany
>
>
> Am 19.06.2025 um 22:57 schrieb Pitambar Poudel via AMBER:
> > Hello all,
> > I’m working on a system consisting of a transporter protein with a
> ligand
> > positioned at a binding pocket and the whole system embedded in a
> lipid
> > bilayer. The bilipid layer was constructed using packmol-memgen.
> Before
> > that, I used Antechamber for ligand parameterization with the
> following
> > script:
> >
> >
> > *antechamber -i ligand.mol2 -fi mol2 -o UNL.mol2 -fo mol2 -c abcg2
> -s 2 -pf
> > y -j 5 -at gaff2 -nc 0 antechamber -i UNL.mol2 -fi mol2 -o
> UNL.prep -fo
> > prepi -c abcg2 -s 2 -pf y -j 5 -at gaff2 -nc 0 parmchk2 -i UNL.prep -f
> > prepi -o UNL.frcmod -s 2*
> > During equilibration, with gradually decreasing positional
> restraints (~100
> > ns), the system remains stable. However, once restraints are fully
> lifted
> > in the production run, the ligand instantly dissociates and flies
> away.
> > Initially, I had used -j 4, but some double bonds were incorrectly
> assigned
> > during parametrization, so I switched to -j 5 as it allows to read the
> > connectivity table from the input and then run ’bondtype’ and
> ’atomtype’
> > sequentially. My current guess is that the issue lies in ligand
> > parametrization—possibly incorrect charges or missing parameters.
> I don't
> > see issues after running parmchk2, however. Any suggestions or
> insights
> > would be appreciated.
> > *Pitambar Poudel*
> > Graduate Research Assistant
> > Computational Biophysics and Bioinformatics Lab
> > Department of Physics and Astronomy, Clemson University
> > *Lab: http:/compbio.clemson.edu/ <http://compbio.clemson.edu/>
> <http://compbio.clemson.edu/lab/>*
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> > http://lists.ambermd.org/mailman/listinfo/amber
> >
>
>
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Received on Tue Jun 24 2025 - 03:30:02 PDT
