MCB 432 Name key Assignment 8 30 pt total Multiple Sequence Alignment on EBI Clustal Servers Due Apr 17 2014 http www ebi ac uk Tools msa There are several WWW servers for multiple sequence alignment We will use two of those at EBI The data files for this assignment 2seq txt 3seq txt are available in from the course web site We are going to compare the results of ClustalW2 and Clustal Omega Go to the WWW site listed above and follow the link to Launch ClustalW2 Begin your analysis by pasting a copy of the sequences in the file 2seq txt into the text box in STEP 1 We will analyze proteins so the default sequence type works We will also use the default values in the STEP 2 and STEP 3 sections of the form Click the Submit button in STEP 4 After messages about validating and waiting the program will display an alignment of the sequences 1 The EBI tools provide a job ID that will allow you to go back to the results for seven days last year it was 21 days What is the job ID it is the rather long text following Results for job It is a very long Universally Unique ID that only a computers scientist could love The alignment itself should begin CLUSTAL 2 1 multiple sequence alignment seq1 seq2 MRILVTGGAGFIGSHLIDRLMSEGHEVICLDNFYTGRKDNIKHWLNNPYFEAIRHDIT 58 MLMKILISGGAGFLGSHLTEALLEKGEEITIVDDLSTAKYFNIR KDVEFIKKKVE 55 Many of the questions in this assignment will ask which residues in one of the sequences are aligned with particular residues in another sequence For example the residues in seq2 aligned with VICLDN in seq1 are ITIVDD the residues in seq2 aligned with the PY of NPYF in seq2 are KD the residues in seq2 aligned with HWLN in seq1 are none they fall in a gap between NIR and KDV in seq2 What residues in seq2 align with IKHWLNNPY in seq1 IRKD Get it If I ask about residues I do not want the gaps Also aligning a residue with a gap does not count as an identity 2 Locate the residues ARFIYTSSSEVYG in seq2 2a What residues in seq1 align with the ARF ARF 2b What residues in seq1 align with the VYG VYG 2c Of these 13 residues in seq2 how many are aligned with identical residues in seq1 9 3a What residues in seq1 align with the residues GIRSCYD in seq2 GIRSCYD 3b Of these 7 residues in seq2 how many are aligned with identical residues in seq1 7 4a What residues in seq2 align with the residues EGKRIAE in seq1 ESKRFSE 4b Of these 7 residues in seq1 how many are aligned with identical residues in seq2 4 This all looks pretty good You might have noticed that on the entry page to clustalW2 it suggested trying Clustal Omega Go back to www ebi ac uk Tools msa and follow the link to Launch Clustal Omega Paste a copy of the sequences in the file 2seq txt into the text box in STEP 1 We will analyze proteins so the default sequence type works We will also use the default values in the STEP 2 section of the form Click the Submit button in STEP 3 After messages about validating and waiting the program will display an alignment of the sequences starting with CLUSTAL O 1 2 1 multiple sequence alignment seq1 seq2 MRILVTGGAGFIGSHLIDRLMSEGHEVICLDNFYTGRKDNIKHWLNNPYFEAIRHDITEP 5 Locate the residues ARFIYTSSSEVYG in seq2 5a What residues in seq1 align with the ARF RD 5b What residues in seq1 align with the VYG PIE Page 2 Assignment 8 Name 5c Of these 13 residues in seq2 how many are aligned with identical residues in seq1 2 6a What residues in seq1 align with the residues GIRSCYD in seq2 LCGNCYD 6b Of these 7 residues in seq2 how many are aligned with identical residues in seq1 3 7a What residues in seq2 align with the residues EGKRIAE in seq1 ESKRFSE 7b Of these 7 residues in seq1 how many are aligned with identical residues in seq2 4 With these data ClustalW2 clearly provides a better alignment than Clustal Omega It is frequently better to include more sequences in the alignment Go back to the Clustal Omega launch page but this time paste in the 3 sequences from 3seq txt and click Submit The output begins CLUSTAL O 1 2 1 multiple sequence alignment seq1 seq2 seq3 MRILVTGGAGFIGSHLIDRLMSEGHEVICLDNFYTGRKDNIKHWLNNPYFEA MLMKILISGGAGFLGSHLTEALLEKGEEITIVDDLSTAKYFNIRK DVEF MGEQYFQEKNILVTGGAGFLGSWICDALIAQGANVVCVDNLSSGLISNISHLLDADRFEF 8 Locate the residues ARFIYTSSSEVYG in seq2 8a What residues in seq1 align with the ARF ARF 8b What residues in seq1 align with the VYG VYG 8c Of these 13 residues in seq2 how many are aligned with identical residues in seq1 10 9a What residues in seq1 align with the residues GIRSCYD in seq2 GIRSCYD 9b Of these 7 residues in seq2 how many are aligned with identical residues in seq1 7 10a What residues in seq2 align with the residues EGKRIAE in seq1 KRFSE 10b Of these 7 residues in seq1 how many are aligned with identical residues in seq2 3 This looks much better What about ClustalW2 Go back to the ClustalW2 launch page but this time paste in the 3 sequences from 3seq txt and click Submit Be careful the order of sequences is changed The output begins CLUSTAL 2 1 multiple sequence alignment seq2 seq3 seq1 MLMKILISGGAGFLGSHLTEALLEKGEEITIVDDLSTAKYFNIR KDVEF 49 MGEQYFQEKNILVTGGAGFLGSWICDALIAQGANVVCVDNLSSGLISNISHLLDADRFEF 60 MRILVTGGAGFIGSHLIDRLMSEGHEVICLDNFYTGRKDNIKHWLNNPYFEA 52 11 Locate the residues ARFIYTSSSEVYG in seq2 11a What residues in seq1 align with the ARF ARF 11b What residues in seq1 align with the VYG VYG 11c Of these 13 residues in seq2 how many are aligned with identical residues in seq1 9 12a What residues in seq1 align with the residues GIRSCYD in seq2 GIRSCYD 12b Of these 7 residues in seq2 how many are aligned with identical residues in seq1 7 13a What residues in seq2 align with the residues EGKRIAE in seq1 ESKRFSE 13b Of these 7 residues in seq1 how many are aligned with identical residues in seq2 4 Note the large gap regions in the alignments of seq2 and seq3 are now at nearly but not quite the same positions in seq1 These gaps span an intein in seq1 The ClustalW2 alignment of 3 sequences missed the actual location of the insertion by 3 amino acids but there is a precise repeat of the sequence CYD in seq1 making it impossible to do better with these data 14a From what organism is seq1 derived include strain Halothece sp PCC 7418 14b What is your logic for answering 14a A BLASTP search of the nr protein database gives a 100 identical match to a protein coded by the genome this organism