Re: Overestimated PMF

From: Aron Broom (
Date: Mon Jun 17 2013 - 11:51:57 CDT

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

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.


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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