# Questions about RMSD calculations using the medoid of the largest cluster as the reference

**URL:** <https://gromacs.bioexcel.eu/t/questions-about-rmsd-calculations-using-the-medoid-of-the-largest-cluster-as-the-reference/2629>\
**Category:** User discussions\
**Created:** [August 3, 2021, 8:41am UTC](https://gromacs.bioexcel.eu/t/questions-about-rmsd-calculations-using-the-medoid-of-the-largest-cluster-as-the-reference/2629 "2021-08-03T08:41:20Z")\
**Posts on this page:** 1\
**Page:** 1

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**Author:** ![wehs7661](https://avatars.discourse-cdn.com/v4/letter/w/5e9695/32.png) [@wehs7661](https://gromacs.bioexcel.eu/u/wehs7661)\
**Post date:** [August 3, 2021, 8:41am UTC](https://gromacs.bioexcel.eu/t/questions-about-rmsd-calculations-using-the-medoid-of-the-largest-cluster-as-the-reference/2629/1 "2021-08-03T08:41:21Z")

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GROMACS version: 2020.4  
GROMACS modification: No  
I’m trying to use `gmx cluster` to find the centroid of the biggest cluster and use it as the reference structure for RMSD calculations. I wanted to see if there is a transition in the RMSD value at the end of the simulation to justify the length of a long simulation (if the RMSD has reached an equilibrium).  
Below are the steps of my method:

- Use `gmx cluster` to perform clustering analysis. For my protein, with a cutoff distance of 0.2 nm, 31 clusters were identified. The biggest cluster had 5782 members (72% of the total number of frames ) and its medoid was the configuration at 1412.25 ns (as shown in `cluster.log`).
- Copy the first PDB frame from `clusters.pdb` and save it as `protein_cluster_medoid.pdb`. I assumed that the first PDB frame should correspond to the medoid of the biggest cluster (please let me know if this is wrong).
- Use `gmx rms` to take in `protein_cluster_medoid.pdb` as the reference structure and calculate the RMSD value.  
As a result, there was no transition shown in the RMSD values, but it was shown that the RMSD value could be up to 3.5 nm, which is pretty large. I assumed that the large values were due to the fact that there were a fair amount of structures from other clusters. (Although I still think that 3.5 nm is still too large.) I further plotted the distribution of the RMSD values and I was then confused. I thought that the distribution should be left-skewed and at least 72% of the data should be below 0.2 nm, but this is apparently not the case shown by the figure. Regarding this method, I’m wondering if there is something I misunderstood.  
 ![4eyd_rmsd](https://europe1.discourse-cdn.com/flex017/uploads/bioexcel1/original/1X/4cb54d33ee7c8f7e03fd103a9120a50af3128417.png)

Specifically, my questions can be summarized as follows:

- What is the reason for having such large RMSD values when using the medoid of the largest cluster as the reference?
- Why the histogram showed that the majority of the samples had RMSD more than 2.0 nm? Ideally, I thought that at least 72% of the data should be below 0.2 nm.
- Is there another way to justify the simulation length except for looking at the RMSD value?