- Direct your browser to the NCBI BLAST (http://blast.ncbi.nlm.nih.gov). Choose Protein BLAST.
- Enter the NP_ number for S. cerevisiae protein (Chapter 5) that your group is studying.
- Choose the records to be searched. For the database, select reference proteins. For the
organism, type Schizosaccharomyces pombe. (This is taxid 4896 from the dropdown box.)
- Expand the algorithm parameters at the bottom of the page. We will use the default values for the word size (=3), threshold value (=10) and a gap penalty (=11). The search could be made
more stringent by increasing the word size, threshold value or gap penalty. The search could
be made less stringent by decreasing these values.
- Click BLAST and wait for the results to appear.
- Analyze the results page:
- The graphic summary at the top gives you an instant overview about the extent and strength of the match with S. pombe sequences. Colors are used to distinguish alignments with different ranges of bit scores. The top line represents a match between the S. cerevisiaeMetp protein and its closest S. pombe ortholog. There may be shorter and less significant matches with other S. pombe protein sequences.
- The summary table provides the numerical data. Matches with an E-value of 1E-10 or less and total scores above 100 are likely to be significant.
- Cursor down to see the actual alignment between the sequences. Dashes have been introduced to either the S. cerevisiae or S. pombe sequence where gaps interrupt the alignments. The center row summarizes the homology between the protein sequences. If an amino acid is conserved between the two species, its 1-letter code is shown. Plus signs indicate conservative substitutions, i.e. substitutions with BLOSUM values of 1 or more.
- Click on the link to the NP_ record for the S. pombe ortholog. Find the EC number. Is the EC number of the S. pombe protein the same as its S. cerevisiae ortholog? If so, the two proteins catalyze the same reaction.