Psi-blast  (Page 2/3)

In the first round, PSI-BLAST is just like a normal BLAST; it finds sequence homologues. In the second round or "iteration" of PSI-BLAST,it figures out which residues tend to be conserved by creating a custom profile for each position of the sequence from a multiple alignment. Then another BLASTis performed, using the profile to produce a position-specific scoring matrix based on which positions evolution has conserved vs. which positions evolutionhas allowed to vary. The sequences found after the first round are added to the profile, allowing PSI-BLAST to detect more distant homologues in eachiteration.

One of the known weaknesses of PSI-BLAST is that its ability to detect distantrelationships between proteins is critically dependent on the choice of the query sequence. For this reason, a recommended strategy with PSI-BLAST is toquery using individual functional domains. PSI-BLAST will then find other proteins that share this domain, even if they do not possess overall homology.To acquaint the new user with PSI-BLAST, this tutorial mimics an investigation performed by Aravind and Koonin (3) in 1999, wherein new members of the HSP70/actin protein family were identified, exceptthe analysis in our tutorial will be on a much smaller scale than that presented in the paper. The HSP70/actin family members were originallyrecognized to have a common evolutionary origin as a result of a study performed by Bork et al. (4) in 1992. A structural superposition of the structures of actin, hexokinase, and themolecular chaperonin hsp70, and alignment of many sequences in each of the three families, uncovered a set of common conserved residues, distributed infive sequence motifs, that are involved in ATP binding and in a flexible interdomain hinge. Although each of these proteins performs very different functions, and their sequences are quite divergent, the similarity in the foldof the ATP-binding domain is visually recognizable. These are all ATP-dependent enzymes and the patterns discovered by Bork and associates could not bedetected by traditional BLAST-type sequence searches. Therefore, Aravind and Koonin chose this family as a test of PSI-BLAST's ability to detect distantevolutionary relationships.

Aravind and Koonin chose actin from the PDB file with accession code 1atn as one of their query sequences. Begin the query by retrieving this sequencefrom the PDB . Check the box for searching the PDB archive that says "PDB ID", then enter theaccession code 1atn as the query. Notice that the crystal structure deposited in this entry contained DNase I complexed with actin. There will be a link inthe menu in the blue border on the left entitled "FASTA Sequence", select this link to download the sequence file. This file will containtwo sequences 1ATN:A (actin) and 1ATN:D (DNase I). Copy the sequence for 1ATN:A and paste it into the BLAST query box that arises from choosing the Protein BLAST, then selecting "PSI-BLAST" under the algorithm section. Change the database from "nr" to "swissprot",but accept the default values for everything else. Click on BLAST, then view the results. NOTE THAT each time another iteration of PSI-BLAST runs, the results page will indicate the iteration number. This is very helpful for keeping track of the stage of the results.