Your name: Your email address:
1. (2 Minutes) From NCBI, download proteins with the following gi numbers 145722 (D-Ala D-Ala ligase) and 121663 (Glutathione synthetase) [Hint: you can paste multiple space/comma separated gi numbers to retrieve proteins simultaneously]. Using PRSS determine if there is significant similarity between these proteins.
What is the P-Value of the comparison?
2. (10 minutes) Do a PSI-BLAST search with the Glutathione synthetase as a query (use swissprot as database). On the Format page, set the E-value cut-off for inclusion in the next round to 0.0001. Note : By default PSI-Blast switches back and forth between the format and the result window. DO NOT CLICK the "Run PSI_Blast iteration X" button repeatedly. Click it once and open the Format window!.
After how many iterations (do not more than 5 iterations!) do you start to pick up carbamoyl phosphate synthetases and D-Ala D-Ala ligase?
Which other types of enzyme are included among the hits?
Notice! : If this takes a long time, collaborate with your neighbor. One of you could do task #2, the other #3.
3. (10 minutes) Do a PSI-BLAST search with the D-Ala D-Ala ligase as a query (swissprot as database). After how many iterations (do not more than 5 iterations!)do you start to pick up carbamoyl phosphate synthetases?
After how many iterations do you start to pick up Glutathione synthetase?
Which other types of enzyme are included among the hits? What might be the reason for the different results obtained in tasks 2 and 3?
4. (14 Minutes) Use Safari for this exercise (sorry, netscape does not put in the line breaks into the PSSM). Do a PSI-BLAST search for 3 iterations with the following sequence:
>Pab_VMA intein from gi|7436316|pir||D75028 CVDGDTLVLTKEFGLIKIKDLYKILDGKGKKTVNGNEEWTELERPITLYGYKDGKIVEIKATHVYKGFS AGMIEIRTRTGRKIKVTPIHKLFTGRVTKNGLEIREVMAKDLKKGDRIIVAKKIDGGERVKLNIRVEQKR GKKIRIPDVLDEKLAEFLGYLIADGTLKPRTVAIYNNDESLLRRANELANELFNIEGKIVKGRTVKALLI HSKALVEFFSKLGVPRNKKARTWKVPKELLISEPEVVKAFIKAYIMCDGYYDENKGEIEIVTASEEAAYG FSYLLAKLGIYAIIREKIIGDKVYYRVVISGESNLEKLGIERVGRGYTSYDIVPVEVEELYNALGRPYAE LKRAGIEIHNYLSGENMSYEMFRKFAKFVGMEEIAENHLTHVLFDEIVEIRYISEGQEVYDVTTETHNFIGG NMPTLLHNT
What types of enzymes do you get as hits?
Do you notice anything strange about the search results?
Save the PSSM (Position Specific Scoring Matrix, or profile) from your search on the 4th iteration. To do that choose PSSM from pull-down menu under Format options (in the SHOW line) and THEN click the "Format!" button. After the search is done, you should get strangely looking alphanumerical symbol mixture in your browser window. This is a PSSM. Save the PSSM matrix to disk as text file, and keep this browser window open. We are going to use this profile in the task #5.
5. (14 Minutes) Now we will use the PSSM to BLAST the completed genomes. Go to Microbial Genomes Genomic BLAST page ( Let it load completely before choosing any options! ). Paste intein sequence into query sequence box, change Query and Database entries to "Protein" and select blast program BLASTP . Choose one or more of the following genomes as database:
After that click "Adv. BLAST" button . This will redirect you to the advanced BLAST search window. Paste your PSSM from Question #4 into PSSM box (under Options). click BLAST, then FORMAT (keep your fingers crossed). What are the results of your search? Did you get any significant matches? What are they? If you have significant matches, does the match occur over the full lengths of both query and subject sequences?
Use Blink to investigate if the hits are indeed inteins. What is your conclusion?
In your answer indicate - genomes searched, - number of significant matches found, - the E-values of these matches, and - the identity of these matches (i.e., are these probable inteins, or are they likely to be something else?).
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