[gmx-users] PMF Transmembrane proteins

Justin Lemkul jalemkul at vt.edu
Sun Dec 30 20:25:54 CET 2012

On 12/30/12 12:28 PM, Nash, Anthony wrote:
> Dear gmx users,
> I posted a couple of weeks ago with regards to correctly using umbrella
> sampling and the WHAM on atomistic transmembrane proteins with a reaction
> coordinate as a function of interhelical distance. I have a single TM dimer,
> but with a different transmembrane domain face at the helix-helix interface.

So you have proteins with identical sequences in each case, but different 

> What did I conclude from the replies? Correct calculation of the differences
> in free energy is taken by normalizing to zero at the plateau (flat) of the
> graph i.e., where your PMF graph (after g_wham) flattens out you apply the
> -zprof0 at this point, before calculating the difference between of each
> system.
> The problem I face is that this occurs around 6.8 - 7.5nm along the reaction
> coordinate. And, applying a umbrella window every half angstrom has made this
> extremely computationally expensive. However, I have persisted, and I have

Every half Angstrom?  That sounds like massive overkill for almost any system. 
Do you mean half nanometer instead?

> all my graphs, and I have a plateau on all of them. Yet the PMF graphs
> between 4 - 7.5 nm are not yet converging or close to the same sampled
> energy, even though all four systems are identical in amino acid composition.
> Each window has ran for up to 40 ns.

Are there any differences at all in these systems, or are they simply intended 
to be replicates of one another?  I'm just curious to know what you're comparing 

> So when I normalise, depending on where I normalise I will get massively
> different difference in free energies.

Real numbers would help us understand what's going on here, as would a report of 
the error estimate that g_wham cna provide.

> I have tried the strategy of taking the windows of a particular systems at
> 4.5 nm to 7.5 nm along the reaction coordinate (where there is no short range
> interaction), and applying those windows into the other three systems. This
> brings their region of plateau a lot closer together, whilst preserving the
> windows and the convergence of the region along the reaction coordinate where
> there are close-range forces in play (converges 0.8 nm - 4.5 nm).
> Alternatively, could I make the assumption that as their amino acid
> composition is identical across all four systems, I can normalise where each
> of the four graphs finish converging (around 4.5 nm)?

In theory, two peptides that are at a given distance from one another can sample 
basically an infinite number of configurations, so at some point you should see 
some similarities between the different systems.  During the length of a 40-ns 
MD simulation, such an assumption likely is not valid, so picking and choosing 
configurations that you think will make the results better is questionable, at 
best.  Since you're dealing with a lipid bilayer, you can't consider only the 
protein dynamics; you have lipid reorientation times that may be at play, 
especially in the presence of artificial restraints between the proteins.  There 
is an interplay between all elements of the system, and 40 ns may not be long 
enough to assure that you really have sufficient sampling (heartbreaking to 
hear, I know, especially for expensive simulations like umbrella sampling).

On a related note, how large are your boxes?  Is there adequate space between 
periodic images at such a large COM separation?  I would imagine that your 
systems would have to be quite large to avoid spurious PBC interactions.  Are 
you pulling along a single dimension or along a diagonal?  Can you post any 
images to demonstrate two systems that give very different results, but visually 
seem that they should not?  There is so much to guess at here that it's very 
difficult to make conclusive recommendations.

> I would really appreciate any advice from someone who has insight on this
> issue. I have looked through many journals and founds lots of entries where
> they just stop at 2.0 nm with no plateau, yet I haven't found a single WHAM
> of reconstructing umbrella windows where they normalise at the plateau. I

Shameless plug:


> have also contacted a few authors of journals with regards to why they cut
> off at 2 nm. None of them took into consideration that their PMF graphs were
> still raising.

Typical ;)  There are a fair number of very questionable PMF curves in the 
literature, I'm afraid.



Justin A. Lemkul, Ph.D.
Research Scientist
Department of Biochemistry
Virginia Tech
Blacksburg, VA
jalemkul[at]vt.edu | (540) 231-9080


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