Your name: Your email address:
1. (2 Minutes) 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). Select "Accession/GI number" from the drop-down box that will by default have "FASTA format" selected.
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 ("PSI-BLAST Threshold") to 0.0001 and change the maximum target sequences to 10000. 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?
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) Do a PSI-BLAST (use SwissProt as the database) 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?
Here is a FASTA formatted file containing annotated IS605 transposase protein sequences from Frankia genomes.
We will use it to build a PSSM for this protein family, and then compare (mainly quantitatively) two searches:
To do this, we will use the cluster.
A FASTA file of the proteins in all 3 Frankia genomes is here - ThreeFrankia.faa
A FASTA file of the nucleotide sequences of all 3 Frankia genomes is here - ThreeFrankia.fna
Note that these (currently 3) Frankia genomes were retreived from Entrez Genome (using the F links in the right-hand column). In this case, we retreived the *.faa (protein) files, and the *.fna (genome nucleotide) files.
blastpgp -d nr -i ThreeFrankiaIS605.faa -j 2 -C IS605Check.chk -e 1e-5 -o blast.out -a 2
This takes quite a while, so grab the checkpoint file from here - IS605Check.chk
Options for blastpgp here
formatdb -p T -i ThreeFrankia.faa -o T
formatdb -p F -i ThreeFrankia.fna -o T
blastall -p blastp -i ThreeFrankiaIS605.faa -d ThreeFrankia.faa -e 1e-5 -m 8 -o blastp.out -a 2
blastpgp -d ThreeFrankia.faa -i ThreeFrankiaIS605.faa -a 2 -R IS605Check.chk -o PSIblastP.out
blastall -p psitblastn -i ThreeFrankiaIS605.faa -d ThreeFrankia.fna -R IS605Check.chk -e 1e-5 -m 8 -o psitblastn.out -a 2
Counting the number of lines in a file:
wc -l blastp.out
wc -l psitblastn.out
How does the number of blastp matches compare to the number of PSI-blast and PSI-tblastn matches?
If there is a significant difference in the number of matches, can you think of a reason why this could happen?
Also see MCB 372 PSI-Blast exercise
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