I feel that it might be worth looking into the literature at published work
on DNA quadruplexes, and see if this is reasonable behavior compared to
those or not. It seems to me that the simulation has reached a fairly
stable structure. The remaining question is how this differs from your
initial model, and if those differences are expected in your force field or
are reasonable for G-quadruplexes (depending on the quality of your initial
structure). We can help here with software technical issues, but for
feedback on whether your simulations are scientifically reasonable you'll
probably want to see what experts in the field of DNA simulations have
previously reported, and to perform the types of analyses that are typical
for describing G-quadruplex structure. You'll want to know that before
writing up your own data of course.
Also as a side note I don't think the ligand rmsd plot is telling you much
other than the internal motion of the ligand is not large. As I
mentioned before, the rmsd of a ligand is fairly unhelpful unless you first
fit the binding site and then calculate the ligand rmsd without redoing the
best-fit process.
On Mon, Feb 22, 2021 at 12:27 PM Delwakkada Liyanage, Senal Dinuka <
sdd313.msstate.edu> wrote:
> Dear Amber Users and Developers,
>
> Thank you all for your wonderful ideas. My system is a G-Quadraplex (24
> residues) intercalated ligand complex. As Dr. Simmerling and Dr. Chalmers
> mentioned, I have further investigated the contact distance profiles and
> visualized the trajectories. I noticed that there was a lot of
> conformational changes in the DNA although the ligand was stable within the
> binding site throughout the period. On further note, I obtained a RMSD
> profile for the second half of the trajectory reference to the final frame
> (ligand in green and complex in red). It seems that the ligand was
> conformationally stable although the complex show lot of fluctuations. I
> must mention I have been running the equilibration inputs on the GPU code.
> I feel like these rapid fluctuations could be result of that as well. I
> appreciate your thoughts on this as well.
>
> Following is the cpptraj input for calculations.
>
> # 02/17/21 11:05:24
> parm ../complex_solv.prmtop
>
>
> trajin equil-12.nc 5000 10000 1
> trajin equil-13.nc
>
> reference equil-13.rst7 [last]
>
> center :7-27
> autoimage
>
> hbond :6-27 out nhb.dat avgout avghb.dat nointramol
>
> rms Last ref [last] :7-27&!.H= out rmsd1.dat mass time 0.01
> rms ToLigand ref [last] :26&!.H= out rmsdTMP.dat mass time 0.01
>
> run
>
>
> Thank you
>
> Senal Liyanage
> [cid:e8039c97-38b2-4b77-94d8-d201472a586f]
>
> ________________________________
> From: Gordon Richard Chalmers <gordoncs.uga.edu>
> Sent: Sunday, February 21, 2021 11:00 AM
> To: AMBER Mailing List <amber.ambermd.org>
> Subject: Re: [AMBER] Sufficiently equilibrated or not
>
>
> Yeah, 2.1 cents. Also, I forgot to mention that a single hydrogen
> bond of about 1kCal (e.g. protein to ligand) can cause an RMSD
> change by an 1A. That may not be common knowledge.
>
> BTW I do agree with the referencing and tumbling comment in the
> RMSD, but was thinking about H-bonds and overall motion. Looks
> like RMSD is protein-ligand complex and just ligand in the green line?
>
> Gordon Chalmers
>
> ________________________________
> From: Gordon Richard Chalmers <gordoncs.uga.edu>
> Sent: Sunday, February 21, 2021 11:12 AM
> To: AMBER Mailing List <amber.ambermd.org>
> Subject: Re: [AMBER] Sufficiently equilibrated or not
>
>
> Checking protein-ligand contact distances in the simulation is probably
> the best move. Couple more quick points with my 2 cents:
>
> Energy fluctuations like the discrete one found in your plot
> can be explained by the presence of distance thresholds of an interaction
> in the force field forcing the interaction to turn off or on. If the
> ligand
> is in fact in the process of disassociating from the protein or rotating
> very slowly then this could explain the discrete jump.
>
> The RMSD fluctuations are expected from internal motion and also from
> real-world non-isotropic rigid body tumbling and compared to the initial
> state.
>
> I would expect an equilibration or simulation time of 25 to 50 the
> tumbling time of your system (or just the ligand) to accurately use the
> trajectory in a physical calculation.
> At that order. 100 ns would be appropriate for a molecule of about 8 to
> 4 kDa, or more depending on what the purpose of the simulation and
> what observable you are estimating from MD, not taking into account
> any conformational changes.
>
> The simulation has not plateaued halfway it appears. It is still linearly
> increasing linearly from ~4 about to ~5 in 60, slower than ~1 to ~4 in
> the 1st 60.
>
> Gordon Chalmers
>
> ________________________________
> From: Carlos Simmerling <carlos.simmerling.gmail.com>
> Sent: Sunday, February 21, 2021 8:58 AM
> To: AMBER Mailing List <amber.ambermd.org>
> Subject: Re: [AMBER] Sufficiently equilibrated or not
>
> [EXTERNAL SENDER - PROCEED CAUTIOUSLY]
>
>
> I think it's important to visually examine your trajectories, not just
> calculate rmsd. is that the rmsd of the ligand fit to itself? if so it can
> stay low even if the ligand dissociates. It might be better to calculate
> distances for the key ligand-DNA contacts and make sure they are staying
> close.
>
> for your DNA rmsd, 5A seems quite high, but it really depends strongly on
> the system you have. Is the DNA long? Normal bending motions can give large
> RMSD, as can fraying of ends. These might be fine and are unlikely to
> affect your free energy if they are not close to the ligand.Also, the RMSD
> for the complex does reach a plateau around 4-5A about halfway through. You
> cannot tell, though, if the structure has stabilized because it may still
> be chaning conformation but staying 5A from the reference. I prefer taking
> RMSD to the final structure (or better, to a cluster representative, if you
> can do that analysis). Then use that as the reference structure for RMSD
> and see if the RMSD values are low for the second half with respect to that
> structure.
>
> Overall the issue is that these RMSD plots do not give enough information
> to know if the system is behaving well or not, since we don't know much
> about what you are simulating and how you are calculating the RMSD values.
> I think it's best not to use broad measures like overall RMSD, but to use
> your knowledge of the system to analyze specific properties and see if they
> are reasonable (for example, DNA helical properties, ligand-DNA hydrogen
> bonds, and so on).
>
> On Sat, Feb 20, 2021 at 9:34 PM Delwakkada Liyanage, Senal Dinuka <
> sdd313.msstate.edu> wrote:
>
> > Hello Dr. Foley,
> >
> > Yes, the simulations were done in the NPT ensemble. I looked at the
> energy
> > plots and it seemed to me that the energy convergence is attained. Below
> is
> > the attached snapshot of total energy vs simulation progress plot. Also,
> as
> > Dr. Simmerling suggested I looked in to the rmsd of the ligand, and the
> > ligand was stable throughout the last 100 ns of simulation time (image
> > attached). Nevertheless, I thought of extending my simulation time to
> > another 100 ns due to previous fluctuations in the complex rmsd.
> >
> > [cid:355520f2-ea0b-4298-8278-ed731b561041]
> >
> > [cid:785d16f5-99fc-4d41-b210-bcd7636d70c1]
> >
> > Thank you
> >
> > Senal Liyanage
> > ________________________________
> > From: Lachele Foley <lf.list.gmail.com>
> > Sent: Saturday, February 20, 2021 3:30 PM
> > To: AMBER Mailing List <amber.ambermd.org>
> > Subject: Re: [AMBER] Sufficiently equilibrated or not
> >
> > With RMSD, the issue might be more about convergence than
> > equilibration. But, you're certainly not ready for TI.
> >
> > Which ensemble are you in? Is it NPT? If so, what does your total
> > energy plot vs simulation progress look like?
> >
> > On Sat, Feb 20, 2021 at 2:58 PM Delwakkada Liyanage, Senal Dinuka
> > <sdd313.msstate.edu> wrote:
> > >
> > > Hello Dr. Foley,
> > >
> > > Thank you very much for your valuable input.
> > >
> > > Senal Liyange
> > > ________________________________
> > > From: Lachele Foley <lf.list.gmail.com>
> > > Sent: Saturday, February 20, 2021 1:55 PM
> > > To: AMBER Mailing List <amber.ambermd.org>
> > > Subject: Re: [AMBER] Sufficiently equilibrated or not
> > >
> > > It's due to the rising nature, mostly. But, you also need stable
> > > fluctuations. That is, if you have a significant change in the
> > > amplitude of the fluctuation or if there is a trend (rising or
> > > falling) in the data, then the system is not equlibrated.
> > >
> > > On Sat, Feb 20, 2021 at 2:46 PM Delwakkada Liyanage, Senal Dinuka
> > > <sdd313.msstate.edu> wrote:
> > > >
> > > > Hello Dr. Foley,
> > > >
> > > > Thank you for your feedback. If I may ask, is that due to the rising
> > nature of rmsd or rapid fluctuations of the points?
> > > >
> > > > Senal Liyanage
> > > > ________________________________
> > > > From: Lachele Foley <lf.list.gmail.com>
> > > > Sent: Saturday, February 20, 2021 1:41 PM
> > > > To: AMBER Mailing List <amber.ambermd.org>
> > > > Subject: Re: [AMBER] Sufficiently equilibrated or not
> > > >
> > > > I would not consider the system in that image to be equilibrated.
> > > >
> > > > On Sat, Feb 20, 2021 at 2:17 PM Delwakkada Liyanage, Senal Dinuka
> > > > <sdd313.msstate.edu> wrote:
> > > > >
> > > > > Dear Amber Users and Developers,
> > > > >
> > > > > Here I have attached is a rmsd plot obtained for a ~100 ns
> > equilibrated trajectory (saved at every 10 ps) of a DNA/ligand system. I
> am
> > attempting to calculate absolute binding free energy through a
> > thermodynamic integration (TI) approach. I was hoping if you could
> clarify
> > to me whether the system is equilibrated enough to proceed with the TI
> > calculations. I was under doubt due to the fluctuations shown throughout
> > the last 6000 frames (60 ns). I understand there isn't an exact answer to
> > this. But I appreciate your opinion on this.
> > > > > [cid:f8200194-9003-4107-accf-086dd0f15353]
> > > > >
> > > > > Thank you
> > > > >
> > > > > Senal Liyanage
> > > > > _______________________________________________
> > > > > AMBER mailing list
> > > > > AMBER.ambermd.org
> > > > >
> >
> http://secure-web.cisco.com/116hL3r26c60Pja8rn_aYFWEm7xUTHXaZuKnMzMO7EeTnyrUrIjWOChCXCV4tzsu9b670bcf4lJMFhcJx8DtrtoCKNvzMA9U_1l3OQnAE5ZIbVojX2uNG9PGBLKlm6I7sswRsWwO337AMzdK7070amGhn7CsrHDVUQ3YPBcZAArNxm2mTaKbv3RyFFvKnWiEwDB5nF3HJ3CI79UIRTKh25CT1MfW86LhFw4CuiH0VK3nPYRhUGs8ZiV1P4TdJhMnO9C75M4yctjzmP9fKYnd_lJS-Q4czdX9Rsj4VaS_meUiQLk6GIsLImCMY9dFfwO6e/http%3A%2F%2Flists.ambermd.org%2Fmailman%2Flistinfo%2Famber
> > > >
> > > >
> > > >
> > > > --
> > > > :-) Lachele
> > > > Lachele Foley
> > > > CCRC/UGA
> > > > Athens, GA USA
> > > >
> > > > _______________________________________________
> > > > AMBER mailing list
> > > > AMBER.ambermd.org
> > > >
> >
> http://secure-web.cisco.com/116hL3r26c60Pja8rn_aYFWEm7xUTHXaZuKnMzMO7EeTnyrUrIjWOChCXCV4tzsu9b670bcf4lJMFhcJx8DtrtoCKNvzMA9U_1l3OQnAE5ZIbVojX2uNG9PGBLKlm6I7sswRsWwO337AMzdK7070amGhn7CsrHDVUQ3YPBcZAArNxm2mTaKbv3RyFFvKnWiEwDB5nF3HJ3CI79UIRTKh25CT1MfW86LhFw4CuiH0VK3nPYRhUGs8ZiV1P4TdJhMnO9C75M4yctjzmP9fKYnd_lJS-Q4czdX9Rsj4VaS_meUiQLk6GIsLImCMY9dFfwO6e/http%3A%2F%2Flists.ambermd.org%2Fmailman%2Flistinfo%2Famber
> > > >
> > > > _______________________________________________
> > > > AMBER mailing list
> > > > AMBER.ambermd.org
> > > >
> >
> http://secure-web.cisco.com/1nYcGarqXbx_X59IqL9sLgdETxc9asWQp1ph2TDPhoZNtf8MLpuKstvTAL3tvlduRQFHc6_7TAB038IzCcxgqGOFzbmviUVmN9Xjt_-sx1zlCDnDRw-QVRRam3kes3N2lfV4NUlqAESRpBP7Dm9hmtBPtrFCH3NGddauxNM0PpVwkX_5gnhBSPeF3JICpfuYIXQv_AdP2CeU4Y0IOfhUavyGnFg1mOuI6YyvLP7CaqZIE4b9x1t2ubP3qpFahX_T8NBkXFcZPGTF0m8ZDRlSe7XydLkO0IfCNv5WQaCYq-OnMHNWk8Kg8kqYm_RcP6gVR/http%3A%2F%2Flists.ambermd.org%2Fmailman%2Flistinfo%2Famber
> > >
> > >
> > >
> > > --
> > > :-) Lachele
> > > Lachele Foley
> > > CCRC/UGA
> > > Athens, GA USA
> > >
> > > _______________________________________________
> > > AMBER mailing list
> > > AMBER.ambermd.org
> > >
> >
> http://secure-web.cisco.com/1nYcGarqXbx_X59IqL9sLgdETxc9asWQp1ph2TDPhoZNtf8MLpuKstvTAL3tvlduRQFHc6_7TAB038IzCcxgqGOFzbmviUVmN9Xjt_-sx1zlCDnDRw-QVRRam3kes3N2lfV4NUlqAESRpBP7Dm9hmtBPtrFCH3NGddauxNM0PpVwkX_5gnhBSPeF3JICpfuYIXQv_AdP2CeU4Y0IOfhUavyGnFg1mOuI6YyvLP7CaqZIE4b9x1t2ubP3qpFahX_T8NBkXFcZPGTF0m8ZDRlSe7XydLkO0IfCNv5WQaCYq-OnMHNWk8Kg8kqYm_RcP6gVR/http%3A%2F%2Flists.ambermd.org%2Fmailman%2Flistinfo%2Famber
> > >
> > > _______________________________________________
> > > AMBER mailing list
> > > AMBER.ambermd.org
> > >
> >
> http://secure-web.cisco.com/1pH31Gockmoi_e7miSy2KRwpLsRBlztgTj93vqsvHQemb67xixbWlEQfMIKdjxeNu0iq9wlFYcWR6FwI7XRZWRAnPSTLYL17ZBx56Jxkt63SNVS9elmmOOhFLinhCrkwpQOMbfC-HOs8T-DHUv8-xOV5w6v_zvwvCBecjHPHKa4Wu-wOmPr8ZMSdi67dR9vFe3NJjWi8SFAgLrDvOGRJlu-nEXOMYQTgwfcANdCCCacZhrwLaG1GrFHg6qfyCwV0ODZaCbV6Z0ocIimcNw6fXx61CqspvzdiRdpaZvjDeeGkEQ-uyYywr_NjrrT8eF0lF/http%3A%2F%2Flists.ambermd.org%2Fmailman%2Flistinfo%2Famber
> >
> >
> >
> > --
> > :-) Lachele
> > Lachele Foley
> > CCRC/UGA
> > Athens, GA USA
> >
> > _______________________________________________
> > AMBER mailing list
> > AMBER.ambermd.org
> >
> >
> http://secure-web.cisco.com/1pH31Gockmoi_e7miSy2KRwpLsRBlztgTj93vqsvHQemb67xixbWlEQfMIKdjxeNu0iq9wlFYcWR6FwI7XRZWRAnPSTLYL17ZBx56Jxkt63SNVS9elmmOOhFLinhCrkwpQOMbfC-HOs8T-DHUv8-xOV5w6v_zvwvCBecjHPHKa4Wu-wOmPr8ZMSdi67dR9vFe3NJjWi8SFAgLrDvOGRJlu-nEXOMYQTgwfcANdCCCacZhrwLaG1GrFHg6qfyCwV0ODZaCbV6Z0ocIimcNw6fXx61CqspvzdiRdpaZvjDeeGkEQ-uyYywr_NjrrT8eF0lF/http%3A%2F%2Flists.ambermd.org%2Fmailman%2Flistinfo%2Famber
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Received on Mon Feb 22 2021 - 10:30:02 PST
