Re: Overestimated PMF

From: Felipe Merino (
Date: Mon Jun 17 2013 - 13:33:13 CDT

Hi Aron,

It is like this. We run ~60 ns of unbiased MD and then we started the
Pull. The windows have a very high constant and are very close together.
Every window is only 6.75 ns (60 windows) but the centers are spaced by
only .05 A and every window is started with after 4.5 ns of the last
window so i do not think the problem is really about convergence (we are
really pulling slowly). To test it i was trying to discard different
lengths as equilibration and while the PMF gets lower it definitely does
not go to "reasonable" values.



On 17.06.2013 18:51, Aron Broom wrote:
> Hi Felipe,
> A few questions:
> 1) How long are your windows?
> 2) What analysis have you done to see that all the windows have
> reached equilibrium?
> In general, assuming your initial coordinates were of the bound state,
> then you will overestimate the dG unless everything has come to
> equilibrium. If you start from an unbound state, you'll underestimate
> the dG. If you have done 400ns per window, then I would think you
> should have a lot equilibrated, but if 400ns is the total of all
> windows, I suspect equilibration may be an issue. As a reference, I
> have a system of a protein binding a lipid, and the average window
> length before that window becomes equilibrated is ~60ns (that is with
> explicit solvent).
> If you are in doubt about equilibration, the easiest thing to do is to
> rerun a couple of windows, from a different starting coordinate and
> with a different langevin seed. Then compare the trajectories in
> terms of your reaction coordinate, you may be surprised by how
> non-equilibrated many of the windows really are. In fact, running
> pairs of each window in this manner, while obviously costly, is a
> really good way to ensure you're results are meaningful. You can also
> be very extreme and run one set of windows starting from the unbound
> state, but that can be EXTREMELY time consuming, so I would suggest
> just a different bound set of coordinates (e.g. equilibrated for an
> extra 10ns) with a different langevin seed.
> ~Aron
> On Mon, Jun 17, 2013 at 11:15 AM, Felipe Merino
> <
> <>> wrote:
> Hi Giacomo,
> Thanks for the reply. No, both domains separately have dG's around
> 20 something. The calculations are a bit expensive and that is why
> we did not go further separating the other domain. In total it is
> like 400 ns per domain so extending it is not particularly cheap.
> Best
> Felipe
> On 06/17/2013 05:10 PM, Giacomo Fiorin wrote:
>> Hello Felipe, are you summing the free energies from two separate
>> calculations where you detach only one domain while keeping the
>> other domain still bound?
>> If you did that, this would explain why the discrepancy: those
>> two terms are not necessarily additive.
>> The correction would be to finish either simulation by also
>> detaching the bound domain, and letting the entire protein go far
>> enough from the DNA for all interactions to vanish.
>> Giacomo
>> On Mon, Jun 17, 2013 at 10:43 AM, Felipe Merino
>> <
>> <>> wrote:
>> Dear all,
>> I know this is a little bit off topic but i think somebody
>> could help us here. We have been doing some umbrella sampling
>> simulations to calculate the binding free energy of a
>> protein-DNA complex. We are using the minimal interatomic
>> distance as reaction coordinate. The thing is that the
>> protein has two domains and we are only pulling them
>> separately, so in the end we have always an endpoint with the
>> other domain still bound (that was planned). In the end, the
>> binding free energies are much higher than expected (around
>> 25 kcal/mol). The point is that it sounds to me that there
>> should be a correction on the PMF to account for the
>> "confinement" of being bound to the other (still attached)
>> domain (very much line in the case where you restrain the
>> ligand when doing FEP annihilation) but i am not sure if this
>> is correct. Any insights for this will be highly appreciated.
>> Best
>> Felipe
> --
> Aron Broom M.Sc
> PhD Student
> Department of Chemistry
> University of Waterloo

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