CLASS 14. PSI-BLAST

INSTRUCTIONS:

For each exercise, provide search query used and keep the answers brief. Email me the answers by Sunday 11:59PM AST at the latest.

Use "CLASS 14 EXERCISE" as a message subject, and type answers directly to email body (i.e., no document attachments please). Make sure that first line of your message is your NAME.
LINKS

  1. Using PRSS determine if there is significant similarity between proteins with the following GI numbers 145722 (D-Ala D-Ala ligase) and 121663 (Glutathione synthetase). What are the results of the comparison? [Recall the story of these two enzymes]

  2. Do a PSI-BLAST search with the glutathione synthetase as a query (use SwissProt as database). In the "Algorithm Parameters" section at the end of the page, set the E-value cut-off for inclusion in the next round ("PSI-BLAST Threshold") to 0.0001, change the maximum target sequences to 10000, tun "low complexity" filter ON and turn "compositional adjustments" OFF. 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?

  3. Do a PSI-BLAST search with the D-Ala D-Ala ligase as a query (use SwissProt as database and the same parameters as in previous question). After how many iterations (do not do more than 5 iterations!) do you start to pick up carbamoyl phosphate synthetases? 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. Do a CDD search with the D-Ala D-Ala ligase as a query? Do you see carbamoyl phosphate synthetases among the results? Do you see glutathione synthetase?

  5. Do a PSI-BLAST search (use SwissProt as the database and parameters as in previous searches) for 3 iterations with the sequence here. What types of enzymes do you get as hits? Do you notice anything strange about the search results? Save PSSM matrix from this search (on top of the PSI-BLAST results page, click "Download" and choose "PssmWithParameters". Save the file on your computer (we will use it for the next exercise). Do not close the browser window with results of this exercise (see below).

  6. Click on "Edit and Resubmit" on top of the PSI-BLAST search results page from the previous exercise. Choose refseq_protein as the database. Choose Saccharomyces cerevisiae as organism. After that scroll down to the end of the page and "Upload PSSM" (using the PSSM file you saved in previous exercise). Submit the search. 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? Investigate if the hits are indeed inteins.