Next: Viewing conserved domains of Up: Bioinformatics Tutorial Previous: Sequence Alignment Algorithms

Subsections

Sequence and structural alignments in MOE

In this section we will investigate two catalytic domain pairs of class II tRNA synthetases, by examining their relatedness by means of sequence and structural alignments.

This will show the limitations of simple pairwise sequence alignment methods for sequences with low sequence identity.

More related:
Pair 1 $\rightarrow$
d1eqrb3 vs. d1efwa3 (Aspartyl Eubacteria 1EQR:B vs. Aspartyl Eubacteria 1EFW:A)

More divergent:
Pair 2 $\rightarrow$
d1eqrb3 vs. d1adjc2 (Aspartyl Eubacteria 1EQR:B vs. Histidyl Archaea 1ADJ:C)

All our alignments will be carried out with the BLOSUM40 matrix , a gap start penalty of 12 and an extended gap penalty of 2 (you can try other substitution matrices and gap penalties). You will carry out your alignments in the sequence editor of Moe. The sequences will be structurally aligned first by sequence and then using structure-based methods.

**Figure 2:** A pair of aligned domains displayed in MOE.
$\begin{figure}\begin{center} \par\par\latex{ \includegraphics[scale=0.5]{pictures/align_struct_70} } \end{center} \end{figure}$

Align Pair 1 by sequence

Go to the directory ~/tbss.work/Bioinformatics/moeData
Start Moe by typing moe at the command line.
Load the two structures of Pair 1 into Moe by clicking on: File:Open in the main Moe menu. Select d1eqrb3.ent. Click OK when the "Read PDB file" option box appears". Repeat for d1ewfa3.ent.
After loading the pair, center both of them by clicking on View on the right button bar.

Sequence alignment

We will work from now on only in the Sequence Editor. Select Window:Sequence editor from the main Moe menu. The first alignment will be a sequence alignment with the following setup:

Click on: Homology:Align in the Sequence Editor menu.
Following Figure 3, change the substitution matrix to BLOSUM40, the gap start value to 12 and the gap extend value to 2. Uncheck round-robin, iterative refinement, and structural alignment and superpose chains.

Figure 3: Sequence alignment in MOE.
$\begin{figure}\begin{center} \par\par\latex{ \includegraphics[scale=0.5]{pictures/seq_aln} } \end{center} \end{figure}$
Hit the OK button and Moe will carry out a sequence alignment.
$\framebox[\textwidth]{ \begin{minipage}{.2\textwidth} \includegraphics[width=2... ...program {\tt pair} compare to the sequence alignments in MOE?} \end{minipage} }$

**Figure 3:** Sequence alignment in MOE.
$\begin{figure}\begin{center} \par\par\latex{ \includegraphics[scale=0.5]{pictures/seq_aln} } \end{center} \end{figure}$

Superposition

In the next steps you will superpose all CA atoms of both structures according to your sequence alignment

**Figure 4:** Superpostion options in MOE.
$\begin{figure}\begin{center} \par\par\latex{ \includegraphics[scale=0.5]{pictures/superpose} } \end{center} \end{figure}$

Click on Homology:Superpose in the Sequence Editor menu.
Press Superpose.
Make a note of the RMSD value.
For better visualization of the superposition render the structures in Cartoon representation and hide all other atom representations. Select Render:Backbone:Cartoon and Render:Hide All from the main menu.
For rendering and selection commands refer to the ``Determining Force Fields'' tutorial.

Align Pair 1 by structure

Select chain 1 and chain 2 in the sequence window by shift-clicking the square buttons at the left of each sequence. Reset the sequence alignment by clicking on: Homology:Reset Alignment
Carry out the same steps as for the sequence alignment except this time be sure to check the Structural Alignment boxes as depicted in Figure 5.
Superimpose the two aligned structures and make a note of the RMSD value.

Figure 5: Structural alignment in MOE.
$\begin{figure}\begin{center} \par\par\latex{ \includegraphics[scale=0.5]{pictures/struct_aln} } \end{center} \end{figure}$

**Figure 5:** Structural alignment in MOE.
$\begin{figure}\begin{center} \par\par\latex{ \includegraphics[scale=0.5]{pictures/struct_aln} } \end{center} \end{figure}$

Repeat sequence and structure alignments for more divergent Pair 2

Delete the currently loaded proteins. Select chain 1 and chain 2 in the sequence window by shift-clicking the square buttons at the left of each sequence. Select Edit:Delete selected chains
Repeat, for Pair 2 (d1eqrb3.ent and d1adjc2.ent, the same steps for loading and performing sequence and structural alignments listed above in section 6.1 and 6.2.

$\framebox[\textwidth]{ \begin{minipage}{.2\textwidth} \includegraphics[width=2... ...lignment method is better: sequence-based or structure-based?} \end{minipage} }$

Next: Viewing conserved domains of Up: Bioinformatics Tutorial Previous: Sequence Alignment Algorithms

zan@uiuc.edu