Residue Selection

In this section, you will use the Select Residues tool for finding structural features of aquaporins that have been conserved throughout species and are crucial for their function.
 
The Select Residues features analysis of structural similarity and sequence conservation. It makes use of different measures to highlight residues in the Sequence Display and Structure Display simultaneously. It allows you to examine the conservation and similarity on a per residue basis, using two different measures, Qres value measuring structure similarity and Sequence Identity measuring sequence conservation.

Structure conservation

You can use Multiseq to look at the residues that are structurally similar above or below a certain value of Qres.

Figure: Residue Selection

1

Choose the Search $\rightarrow$ Select Residues tool. A side menu will appear (Fig. 17). Within the menu, you can select residues based on their Sequence Identity or Qres.

2

In box following Qres, select $\geq$ and key in 0.5 for the value.

3

Click on the Select button.

Note the changes in the display of the Open GL (structure) Display window (Fig. 18, left) and in the Sequence Display of the Multiseq program window (Fig. 19).

4

Look in the Sequence Display (Fig. 19). Since you selected the Qres value to be greater or equal to 0.5, most of the structural elements of the aligned molecules will be shown as conserved (similar), and will be displayed in yellow.

Figure: View of the Residue Selection for Qres$\geq$0.5 in the Open GL Display window. Highlight style set to Licorice (left) and NewCartoon (right)

5

Now, look at the OpenGL Display window (Fig. 18, left). In this case also most parts of the molecules are highlighted, in particular, the transmembrane segments.

Figure: View of the Residue Selection for Q$\geq$0.5 in the Sequence Display window.

6

Due to the high level of conservation, the Licorice highlighting style is not very informative. To improve the view, chose the View $\rightarrow$ Highlight Style menu item, and click on the New Cartoon style (Fig. 18, right). You can recognize immediately that high Qres values indicating conservation of neighborhood relationships for amino acids are seen in the transmembrane region throughout.

Now you will investigate what happens for higher values of Qres.

7

In the Select Residues window, select $\geq$ after the Qres and key in 0.75. Click on the Select button.

8

Look at the OpenGL Display window (Fig. 20). Notice that most of the structurally similar regions are still in the transmembrane region, but the highlighted regions are more confined than before.

Figure: View of the Residue Selection for Qres$\geq$0.75 in the OpenGL Display window

9

Look in the Sequence Display window (Fig. 21). You can still distinguish blocks of structurally similar residues, which correspond to the helices. They are not as well defined as before, but the structural similarity is still noticeable when probing for high values of Qres.

Figure: View of the Residue Selection for Qres$\geq$0.75 in the Sequence Display window

10

Repeat steps 7 to 9 and set the Qres to $\geq$0.9. You will notice that almost all the structure conservation is lost.

What does this mean? The aligned aquaporins have a moderate to high structure conservation. Structurally, the most similar areas are the transmembrane helices.

Sequence conservation

You can also use Select Residues to look at the residues that are conserved on the sequence level.

1

Go back to the main Multiseq program window and select Search $\rightarrow$ Select Residues.

Figure: View of the Residue Selection by Sequence Identity in the OpenGL Display window.

2

In the Select Residues window, following Sequence Identity select $\geq$.

3

You can then key in a value between 0 and 100. Try 70, which will select residues that were assigned a sequence identity measure of 70% or more.

4

Click on the Select button.

Figure: View of the Residue Selection by Sequence Identity in the Sequence Display window

5

Look at the Sequence Display in the Multiseq program window. You will notice very few residues are selected. If you look carefuly, you will locate some of the residues that correspond to key structural features of aquaporins, for which you made representations in section 1 of this tutorial. Search for the NPA motifs, highlighted in yellow (Fig. 23).

6

In the OpenGL window, look at the areas highlighted in yellow. Notice that many residues in the reentrant loops (Fig. 3) conserved their sequence over the course of evolution. This is a clear indication that the reentrant loops are functionally significant.

The conserved amino acids in the sequence are mostly located in the pore, and in the reentrant loops. You will now compare these residues to the representations you created in section 1 of this tutorial, which correspond to residues that are relevant to the function of aquaporins.

7

In the Multiseq program window, select View $\rightarrow$ Highlight Style $\rightarrow$ Licorice.

8

In the VMD Main window, select Graphics $\rightarrow$ Representations....

9

In the Graphical Representations window, in the Selected Molecules pull-down menu, select the molecule 1j4n.

10

In the Graphical Representations window, double click on the representations you created for the NPA motif, the glutamates and the arginine. What is the sequence identity for these amino acids? Why do you think the NPA motif, the lysine and the glutamates are conserved throughout species?

When you are done examining the NPA motif, close the Graphical Representations window to proceed to the next section.