1. Sequence similarity search using BLAST
query sequence:
APVKSQESIN
QKLALVIKSG KYTLGYKSTV KSLRQGKSKL IIIAANTPVL
RKSELEYYAM
LSKTKVYYFQ GGNNELGTAV GKLFRVGVVS ILEAGDSDIL
TTLA
The first thing you might want to do is to search if there
is any similar sequences in the databases. One of the database searching
program available on the web is BLAST (Basic Local Alignment Search Tool).
1. Go to the BLAST home page at the National Center for Biotechnology Information (NCBI):
http://www.ncbi.nlm.nih.gov/blast/
2. Choose Standard protein-protein BLAST [blastp]
3. Select database. Two commonly used databases for protein sequence searching:
- swissprot - SWISS-PROT is a high quality protein sequence database with excellent annotations. Minimal redundancy but poorer sequence coverage.
- nr - Non-Redundant database. It is a composite protein sequence database consisted of non-identical sequences derived from several databases: GenPept, PDB, Swiss-Prot, PIR, PRT. Although its name is called 'non-redundant', it may contain redundant sequences as a result of polymorphisms, sequencing errors and sequences of protein fragment. Comprehensive sequence coverage but poorer quality of the database.
We will use swissprot in this example:
4. Copy the query sequence and paste it into the window:
APVKSQESIN
QKLALVIKSG KYTLGYKSTV KSLRQGKSKL IIIAANTPVL
RKSELEYYAM
LSKTKVYYFQ GGNNELGTAV GKLFRVGVVS ILEAGDSDIL
TTLA
Then press the 'BLAST!'
button.
5. You will receive a notification saying your search has been submitted and put into a queue. Press the 'Format results' button to check your results.
6. You should now see a new window of your BLAST search results. Scroll down the window until you see the following:
7. Check if the matches are significant or not.
The BLAST results report a list of sequences that may be similar to your query sequence. The statistical significance of the matches is measured by E-value (Expect value). Highly significant matches should have E-values very close to zero.
By default, BLAST reports all sequences that have an E-value
<= 10.
8. Firstly, we use a conservative threshold E-value. Take a look at the proteins with E-value < 0.001. What generalization can you make?
9. We can obtain more information about the matches by clicking on the bits score. Try click on the highest bits score '177':
Notice the query sequence exactly matched (identities=100%) the sequence of
RL30_YEAST. What does it tell you about the query sequence?
10. Now look at sequences that have E-values > 0.001. The next several matches are L7Ae ribsosomal proteins. The ribsomoal protein L30 may be also related to the ribosomal protein L7Ae.
11. A summary of what you have found for your query sequence:
- It is the yeast ribosomal protein L30.
- Ribosomal protein L30 is conserved among different organisms.
- Ribosomal protein L30 may be related to ribosomal protein L7Ae.