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Subsections


Examining the Aquaporin Tetramer

Aquaporins form homotetramers in cell membranes.The monomers are arranged side by side in a tight cluster. Each monomer conducts water. A fifth pore is formed in the center of the tetramer, with a yet unknown function. In the following we have a closer look at the aqua-glyceroporin GlpF (PDB code: 1lda), an aquaporin that conducts both water and glycerol.

Loading Tetramer

In this section we will load the tetramer structure of GlpF and align it with the other four AQP molecules. A pdb file with the atom coordinates of the tetramer is provided with the tutorial (see Getting Started).

1
Go to the VMD Main Window

2
Choose File $\rightarrow$ New Molecule. Another window, the Molecule File Browser, will appear.

3
Use the Browse button to find the file glpf_tetramer.pdb in the directory
aqp_tutorial_files $\rightarrow$ PDB, in the tutorial directory.

4
Once you have selected the pdb file, press the Load button in the Molecule File Browser window to load the molecule. It will take a while for the tetramer to load

5
The GlpF tetramer is loaded in Lines representation. To have a better view of the molecule after alignment, you need to change the molecule's graphical representation to Tube. From the VMD Main window, choose Graphics $\rightarrow$ Representations. In the Graphical Representation window, change the molecule's Coloring Method to SegName, and its Drawing Method to Tube.

Now that you have the tetramer loaded, go back to the Multiseq program window and select Tools $\rightarrow$ STAMP structural alignment to align this molecule with the other four. Note that for this section you have to have the aquaporins 1j4n, 1fqy, 1lda and 1rc2 (Table 1.2) loaded. If you don't have them, go back and load the four pdb files, as explained in section 1.

Examining channel lining and tetrameric contacts

In the previous section, you have identified the conserved residues among the aquaporin molecules. Here we look where these residues are located in the tetrameric structure.

Figure: Select Residue Dialog
\begin{figure}\begin{center}
\par
\par
\latex{
\includegraphics[width=2.8 in]{pictures/select-conserved}
}
\end{center}\vspace{-0.7cm}\end{figure}

1
Go to the Multiseq program window.

2
Choose Search $\rightarrow$ Select Residues.

3
The Select Residue dialog will appear (Fig. 29). Select All Sequences.

4
In the following box, select Where Sequence Identity is, $\geq $, and type in 100 for the value.

5
Click on the Select button. Now you will see the conserved residues highlighted in the OpenGL Display window as well as in the Multiseq program window.

6
Take some time to examine the highlighted parts as shown in Figure 30. You recognize that most conserved residues line the channel interior. Locate the conserved residues that are outside the water conducting pore. Note that these residues are indeed located at the subunit-subunit interfaces of the tetrameric GlpF.

Figure: GlpF tetramer aligned with other aquaporin molecules. Conserved residues are highlighted in bond representation (yellow).
\begin{figure}\begin{center}
\par
\par
\latex{
\includegraphics[width=2.4 in]{pictures/tetramer-overlap}
}
\end{center}\vspace{-0.7cm}\end{figure}

7
Before starting the next section, go to the VMD Multiseq program window, choose the tetramer molecule by clicking on it (It will turn yellow), and delete the molecule by using the Delete key on your keyboard.


next up previous contents
Next: Phylogenetic Tree Up: Aquaporin Tutorial Previous: Investigating Structural Alignment   Contents
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