BISC220/S14: Mod 1 Clustal Omega Tutorial

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Multiple Sequence Alignment Using Clustal Omega

The alignment and subsequent analysis of protein amino acid sequences can provide potential insights into their structure, function and evolutionary relationships. As a complement to your molecular modeling work with RasMol, you will be using a software program called Clustal Omega to compare an E. coli β-galactosidase sequence with β-galactosidase sequences obtained from other bacterial species. This is called a multiple sequence alignment. The primary sequences were obtained at the National Center for Biotechnology Information (NCBI): http://www.ncbi.nlm.nih.gov/. We will use the Clustal Omega software available at the EMBL-EBI website: http://www.ebi.ac.uk/Tools/msa/clustalo/. The sequences from the various β-galactosidases are in the RasMol folder (as a word document) on the lab’s computers.

When you access the EMBL-EBI web site you will find a form to fill out. Make sure Protein is in the drop down list in 'STEP 1.' Copy and paste the entire word file of sequences into the box. They can all be pasted at the same time. Click the 'More options...' button in 'STEP 2,' and select 'OUTPUT ALIGNMENT FORMAT' 'Clustal w/ numbers.' Then hit the submit button. Once you see the output, press the button that will toggle between “Show Colors/Hide Colors”. Click it to color code the alignment (the key below shows the meaning of the colors). Excerpts found below from the Help & Documentation page from Clustal Omega should help in interpreting the output.

For your assignment, determine the degree of conservation among the amino acids you have located in the active site using the RasMol program. Jacobsen et al. (1994) and Roth et al. (1998) conducted similar analyses. What are the potential implications if an amino acid is highly conserved across all of the species tested? See the introduction in the paper by Juers et al. (2001) for additional information on amino acids that have been previously shown to be important for catalysis.

Color-Coding:
AVFPMILW-Red: Small (small + hydrophobic [includes armomatic –Y])
DE-Blue: Acidic
RHK-Magenta: Basic -H
STYHCNGQ -Green: Hydroxly + sulfhydryl + amine + G
Others-Gray: Unusual amino/imino acids etc

Consensus Symbols: The following symbols denote the degree of residue conservation.
* (asterix) indicates positions which have a single, fully conserved residue.
: (colon) indicates conservation between groups of strongly similar properties.
. (period) indicates conservation between groups of weakly similar properties.

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