Normal Mode Analysis of ARSA

Introduction

Normal Mode Analysis (NMA) is a very useful tool to analyse large-scale motions in proteins. There are two approaches. In the first approach, all-atoms were used to calculate the harmonic motions. As this procedure needs a lot of memory, the elastic network model was developed, which does not take all interactions into account. Like this the memory needed can be dramatically reduced and the method can be applied to larger proteins.
In this TASK, we apply different NMA methods to our protein ARSA to investigate the flexibility and motions of the structure.
For most methods, we were able generate animated pictures to visualise the results. We used gifsicle to generate the animated gifs:

gifsicle --delay=5 --loop --colors 256 *.gif > anim.gif

WEBnm@

WEBnm@ was developed by Hollup et al. in 2005 <ref> Hollup SM, Sælensminde G, Reuter N. (2005) WEBnm@: a web application for normal mode analysis of proteins BMC Bioinformatics </ref>. On the server, there are two types of analyses possible:

• Single Analysis calculates the lowest frequency normal modes of the protein.
• Comparative Analysis calculates and compares the normal modes of a set of aligned protein structures.

We chose the single analysis for our protein with default settings. after a short calculation time, the interface directly guides the user to a Jmol applet were the mode is dynamically visualized. We used this appled to generated images for each frame and then used gifsicle as described above to generate the animated gifs, shown below.
Furthermore we saved plots of the initial protein structure together with vectors of the movements, as well as a figure which visualizes the extent of the molecular displacement along the protein.

Mode	Motion	Vectors	Displacement
mode 7
mode 8
mode 9
mode 10
mode 11
mode 12

Mode 7, 8, 9, 10 and 12 show a huge displacement at the end of the protein. If we have a look at the animated gifs, we can see that a helix is involved in this movement.
Int mode 11, the movement is distributed more along the whole protein. Also here, the helix is involved in the movement, but not as strong as in the other modes.

The helix extends from position 450 to 469. The question is, if this movement is functional or not, i.e. if it is for example involved during substrate binding.

ElNemo

Mode	Pymol animation	picture 1 from ElNemo	picture 2 from ElNemo	picture 3 from ElNemo	Fluctuations
Mode 7
Mode 8
Mode 9
Mode 10
Mode 11

Like in the analysis with WEBnm@, all modes computed by Elnemo show a large movement of the terminal helix of the protein. In these results, the movements look even larger than in the previous analysis. It even looks like a "closure-movement". Interestingly, the substrate binding sites are located at the bottom of the protein (below the yellow colored beta-sheets). Unfortunately, PDB does not contain any resolved structures where the substrate is bound to the protein. So we can not verify this hypothesis.

Anisotropic Network Model web server

for file in *.png; do convert "$file" "$(basename $file .png).gif";done

	mode 1	mode 2	mode 3	mode 4	mode 5
Motion
Distance
Fluctuations

oGNM – Gaussian network model

http://ignm.ccbb.pitt.edu/ognm/1960654514/temp/index.htm

	mode 7	mode 8	mode 9	mode 10	mode 11
Motion
Fluctuations

Fluctuations of mode 7-11

Correlation of mode 1-20

Correlation of mode 7-11

NOMAD-Ref

NMA of ARSA

	mode 7	mode 8	mode 9	mode 10	mode 11
Motion
Distance

NMA of 1BPT

Temperature	mode	mode 8	mode 9	mode 10	mode 11
600K (all-atom)
2000K (all-atom)
Elastic Network