Part I: Multiple Alignment A. Go to the EBI multiple sequence alignment tool page: (http://www.ebi.ac.uk/Tools/msa/) B. Click the "Launch Clustal Omega" link and at step 1 paste the CLUSTAL query from

the Week 4 links page into the sequences in any supported format window. C. At step 2, click on "output format" and change it to Pearson/FASTA. Now click "submit" and wait for your job to finish. D. When your alignment job finishes, take a picture of your output and insert it here. E. Now click on the "Phylogenetic Tree" tab. This shows the tree used to construct the alignment. Take a picture of your tree and insert it here. F. You can create much nicer and more useful outputs using Mview. Click on the "Results Viewer" tab and then "View in MView" button. At Step 2 select input format: automatic. At step 3 on the MView submission page you can customize how your alignment is colored and formatted. Take a picture of your Mview output and insert it here. G. Return to the EBI multiple sequence alignment tool page: (http://www.ebi.ac.uk/Tools/msa) Repeat steps B-D, except click "Launch T-Coffee" for part B. H. Click on the "Phylogenetic Tree" tab. This shows the tree used to construct the T-Coffee alignment. Take a picture of your tree and insert it here. I. Now send your alignment to MView. At step 3 on the MView submission page be sure to use the same settings as for the Clustal Omega alignment. Take a picture of your Mview T- Coffee output and insert it here. J. Return to the EBI multiple sequence alignment tool page: (http://www.ebi.ac.uk/Tools/msa) Repeat steps B-D, except click "Launch MAFFT" for part B./nB. K. Click on the "Phylogenetic Tree" tab. This shows the tree used to construct the MAFFT alignment. Take a picture of your tree and insert it here. L. Now send your alignment to MView At step 3 on the MView submission page be sure to use the same settings as for the Clustal Omega alignment. Take a picture of your Mview MAFFT output and insert it here. M. Do you see any differences between the three alignments in the order in which the sequences are listed? N. Do you see any differences between the three alignments in the consensus? O. Do you see any other differences between the three alignments? P. Which alignment program would be most appropriate according to the table included in this week's lecture?/nQ. What other alignment methods might have been appropriate? R. What settings (if any) may have increased the quality of the alignment for ClustalOmega or MAFFT? Part II: Finding motifs A. Why is it useful to find motifs? B. Why might motifs be missed by global alignment programs? C. Why can motifs tell us about a group of proteins? D. Copy the "Clustal query" sequences, then go to the meme website http://meme- suite.org/tools/meme a. Select "classic mode" b. Under "Input the primary sequences" select "type in sequences" and then paste the CLUSTAL query from the Week 4 links page into the sequences under input primary sequences "10" for Maximum number of motifs to find then click the "start search" button. When your job finishes use the link at the top of the page to see your results. 1. How many motifs did it find? 2. Did each sequence have the same motifs in the same order?/nB. Now copy the sequence for gi|1084385 then analyze it with Motif Scan at prosite: http://myhits.isb-sib.ch/cgi-bin/motif scan (make sure you check all of the databases below the sequence window) 1. How many motifs did it find? 2. How many were significant (i.e. had a blue exclamation point)? 3. Were there any differences between the two sites? C. Now copy the sequence for gi|1084385 then analyze it with interproscan http://www.ebi.ac.uk/Tools/pfa/iprscan/ 1. What homologous families does this protein belong to? 2. What motifs did it identify? Were any not previously identified?