# Analysis of AWH simulations

**URL:** <https://gromacs.bioexcel.eu/t/analysis-of-awh-simulations/10517>\
**Category:** User discussions\
**Tags:** awh, free-energy\
**Created:** [November 11, 2024, 11:31am UTC](https://gromacs.bioexcel.eu/t/analysis-of-awh-simulations/10517 "2024-11-11T11:31:41Z")\
**Posts on this page:** 3\
**Page:** 1

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**Author:** ![felippe\_colombari](https://avatars.discourse-cdn.com/v4/letter/f/41988e/32.png) [@felippe\_colombari](https://gromacs.bioexcel.eu/u/felippe_colombari)\
**Post date:** [November 11, 2024, 11:31am UTC](https://gromacs.bioexcel.eu/t/analysis-of-awh-simulations/10517/1 "2024-11-11T11:31:41Z")

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GROMACS version: 2024.1  
GROMACS modification: Yes/No  
Dear all,

I just learned about the AWH method, and found it very interesting and powerful (I am simulating the binding of small ligands into protein active sites). I read a lot of tutorials, papers and most of the posts here which helped to optimize my simulation parameters, but I still have some specific doubts.

As an initial test case, after equilibration steps I ran a a multi-walker awh simulation with 2 walkers (t = 100 ns) and observed the following in the log files:

1. equilibrated histogram at t = 28927.8 ps
2. awh1: covering at t = 36003 ps. Decreased the update size; awh1: out of the initial stage at t = 36003.

Question A:  
If I wish to perform other analysis (such as extract relevant frames close to each PMF minima), those should be performed considering the simulation after ~36 ns? Since there are two trajectories, could I skip the initial 36 ns of each and concatenate them for further structural analysis?

Question B:  
although these 100 ns test simulations are very short (most papers I read simulated more than 500 ns for each walker), PMF profiles between 90-100 ns seemed “converged”, with differences below 0.2 kT. Is this a sufficient criteria?

Question C:  
To streghten tha results, I plan to run two more “replicas” for each protein-ligand system, in which the initial structures are different (different protein conformation and ligand poses) prior to minimization/equilibration steps and mw-awh simulations. Is this OK?

Kind regards,  
Felippe

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**Author:** ![hess](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/hess/32/416_2.png) [@hess](https://gromacs.bioexcel.eu/u/hess)\
**Post date:** [November 11, 2024, 7:53pm UTC](https://gromacs.bioexcel.eu/t/analysis-of-awh-simulations/10517/2 "2024-11-11T19:53:52Z")

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A: Yes. But note that you will be analyzing a biased ensemble, not a Boltzmann ensemble.  
B: Yes, if you run a sufficient number of simulations, at least 3 or 4.  
C: Yes.

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**Author:** ![felippe\_colombari](https://avatars.discourse-cdn.com/v4/letter/f/41988e/32.png) [@felippe\_colombari](https://gromacs.bioexcel.eu/u/felippe_colombari)\
**Post date:** [November 18, 2024, 5:57pm UTC](https://gromacs.bioexcel.eu/t/analysis-of-awh-simulations/10517/3 "2024-11-18T17:57:55Z")

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Thanks, Hess!