Dr. Anselm,
It worked! Thank you for the help!
*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 27, 2025 at 6:32 AM Dr. Anselm Horn <anselm.horn.fau.de> wrote:
> 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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Received on Fri Jun 27 2025 - 08:30:02 PDT
