Dear Pitambar,
the bond order of the mol2 file does not play any role during MD, but
the atom types do: these tell leap which parameters to take for the
bond, the angle etc.
Thus, you may give your changed parameter file a try.
Good luck,
Anselm
Bioinformatik | NHR.FAU
Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU)
Germany
Am 26.06.2025 um 20:09 schrieb Pitambar Poudel:
> Dr Anselm,
>
> Thank you for the suggestion. As per your suggestion, I resolved the
> charge symmetry issue using the following script.
>
> antechamber -i ligand.mol2 -fi mol2 -o UNL.mol2 -fo mol2 -c abcg2 -s 2
> -pf y -at gaff2 -nc 0 -j 4
>
> However, I noticed something in the resulting MOL2 (UNL.mol2 is
> attached) file: the bond order. The MOL2 file generated by Antechamber
> assigns a double bond only to C1=N.
>
> While this structure is not wrong, the most probable resonance structure
> for my ligand would involve a double bond between C1 and either N1 or
> N2—the terminal nitrogens. The same applies to the terminal oxygens:
> ideally, the bond between C11 and either O12 or O13 should be a double bond.
>
> Should I leave the MOL2 file as it is (even though the bond information
> may not be chemically ideal), or should I manually adjust the bond
> orders before proceeding with the simulations? Specifically, should I
> modify the structure to reflect either C1–N (single), C1=N1 (double), or
> C1–N2, and similarly for the oxygen as C11=O12 or C11–O13?
>
> Thanks!
>
> *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 Tue, Jun 24, 2025 at 6:35 AM Pitambar Poudel <pitambp.g.clemson.edu
> <mailto:pitambp.g.clemson.edu>> wrote:
>
> Hello Anslem,
>
> Thanks for the clarification.
>
> The experimental structure is not available. The structure for
> protein was found via homology modeling and then I used Autodock to
> dock the ligand into the binding pocket. I opend the docked complex
> in Chimera and saved the mol2 of ligand from chimera and used
> antechamber to generate a new mol2, prep, lib, frcmod files.
>
>
> *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 Tue, Jun 24, 2025 at 6:16 AM Dr. Anselm Horn via AMBER
> <amber.ambermd.org <mailto:amber.ambermd.org>> wrote:
>
> 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/> <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>
> <mailto: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/>
> > <http://compbio.clemson.edu/lab/>*
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> > >
> >
> >
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Received on Fri Jun 27 2025 - 04:00:02 PDT
