1. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 1 Practical exercises Answers…

2. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 2

3. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 3 - Nucleic acid database in Japan: DDBJ: http://www.ddbj.nig.ac.jp/http://www.ddbj.nig.ac.jp/ - Microarrays data: Arrayexpress: http://www.ebi.ac.uk/microarray/ - Mass spectrometry data: PRIDE: http://www.ebi.ac.uk/pride/,http://www.ebi.ac.uk/pride/ OPD http://www.ebi.ac.uk/pride/http://www.ebi.ac.uk/pride/ - Protein-protein interaction: INTACT: http://www.ebi.ac.uk/intact/site/http://www.ebi.ac.uk/intact/site/ DIP: http://dip.doe-mbi.ucla.edu/ JCB: http://www.imb-jenade/jcb/ppi/http://dip.doe-mbi.ucla.edu/http://www.imb-jenade/jcb/ppi/ - rat enamel 2D gel electrophoresis: http://biocadmin.otago.ac.nz/fmi/xsl/toothprint/home.xsl http://biocadmin.otago.ac.nz/fmi/xsl/toothprint/home.xsl (Last revision August 2006) - CFTR mutation http://www.genet.sickkids.on.ca/cftr/; This web site was last updated March 2007http://www.genet.sickkids.on.ca/cftr/

4. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 4 Exercise 2 E.coli K12 recombinase A (recA) in different protein sequence databases Find, if it exists, the entry corresponding to the E.coli (strain K12) recA protein sequence in the following protein sequence databases - EMBL http://www.ebi.ac.uk/embl/http://www.ebi.ac.uk/embl/ - RefSeq http://www.ncbi.nlm.nih.gov/RefSeq/http://www.ncbi.nlm.nih.gov/RefSeq/ - UniProtKB http://www.expasy.org/sprot/ or http://beta.uniprot.org/ find sequence(s) inhttp://www.expasy.org/sprot/http://beta.uniprot.org/ UniProtKB/Swiss-Prot and sequence(s) in UniProtKB/TrEMBL - PIR-PSD http://pir.georgetown.edu/pirwww/dbinfo/pir_psd.shtmlhttp://pir.georgetown.edu/pirwww/dbinfo/pir_psd.shtml - PDB http://www.rcsb.org/pdb/home/home.dohttp://www.rcsb.org/pdb/home/home.do - UniParc (use SRS ) or the UniParc query tool.SRS UniParc - EnsEMBL http://www.ensembl.org/index.htmlhttp://www.ensembl.org/index.html -Find the UniProtKB/Swiss-Prot entry corresponding to the RefSeq entry NP_036231 Hints: You can use the query tool provided by each database. You can use SRSSRS You can use the crosslinks (if they exist) to go from one database to another... You can use the mapping tool on the new UniProt web site http://beta.uniprot.org/http://beta.uniprot.org/

5. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 5 EMBL: U00096 RefSeq: NC_000913 UniProtKB: P0A7G6, Swiss-Prot only (there are 2 fragments in TrEMBL, but they are not from K12) PIR-PSD: G65049; RQECA. Retrieved from UniProtRQECA UniParc: UPI0000112C1C PDB: 1AA3,1N03,1REA,1U94 etc, easily retrieved from UniProt. EnsEMBL: Not possible, bacteria are not in EnsEMBL!

7. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 7 Exercise 3 -Find the human erythropoietin protein sequence in UniProt. - BLASTp it at ExPASy (http://www.expasy.org/tools/blast/); restrict the BLAST to human sequences (Homo sapiens). - Look at the Blast results and guess from which database(s) the protein sequences are derived. How many distinct human erythropoietin protein sequences do you get? -Do the same, but at (http://www.ncbi.nlm.nih.gov/BLAST/) - How many distinct human erythropoietin protein sequences do you get?

8. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 8 BLASTp at ExPASy against UniProtKB Only 2 entries; one annotated in Swiss-Prot, the other unannotated in TrEMBL. Looking at the Swiss-Prot entry you see a lot of rich information.

9. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 9 BLASTp at NCBI against nr At least 9 entries; RefSeq (ref, 1), GenPept (embl, gb, 6) and PDB (pdb, 2). The Swiss-Prot entry has most of these cross-references, and more besides.

10. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 10 Exercise 4: Understanding BLAST output Compare the results of BLASTp for entry O05891 -against UniProtKB (http://www.expasy.org/tools/blast/) -against NCBI-nr (http://www.ncbi.nlm.nih.gov/BLAST/) Look for the same best hits and compare the scores, why are they different? Keep the UniProtKB output, we will use it again in a minute.

11. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 11 BLASTp at NCBI against nr BLASTp at ExPASy against UniProtKB

12. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 12 NCBI BLAST FAQ: http://www.ncbi.nlm.nih.gov/blast/blast_FAQs.shtml Q: What is the Expect (E) value? The Expect value (E) is a parameter that describes the number of hits one can "expect" to see just by chance when searching a database of a particular size. It decreases exponentially with the Score (S) that is assigned to a match between two sequences. Essentially, the E value describes the random background noise that exists for matches between sequences. For example, an E value of 1 assigned to a hit can be interpreted as meaning that in a database of the current size one might expect to see 1 match with a similar score simply by chance. This means that the lower the E-value, or the closer it is to "0" the more "significant" the match is.

13. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 13 http://www.ncbi.nlm.nih.gov/BLAST/tutorial/Altschul-1.html The Statistics of Sequence Similarity Scores The E-value of equation (1) applies to the comparison of two proteins of lengths m and n. How does one assess the significance of an alignment that arises from the comparison of a protein of length m to a database containing many different proteins, of varying lengths? One view is that all proteins in the database are a priori equally likely to be related to the query. This implies that a low E-value for an alignment involving a short database sequence should carry the same weight as a low E-value for an alignment involving a long database sequence. To calculate a "database search" E-value, one simply multiplies the pairwise-comparison E-value by the number of sequences in the database. The E value depends on the size of the database.

14. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 14 Exercise 5: Start site issues: bacteria Take the UniProtKB BLASTp output for O05891. Align the first 9 sequences using ClustalW (tool on the BLAST output page). What do you see, what is one possible interpretation? Look at the entry in UniProt, what can you see to strengthen this interpretation?

15. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 15

16. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 16 MYCTF = Mycobacterium tuberculosis strain F11. It is not clear if it is a WGS or a fully finished genome… In either case there has probably been an error in the start codon prediction. In bacteria there are several other codons beside ATG that can start a protein (Val (GTG) and Leu (TTG)). That is probably what happened here, and the fact that there is another potential start a few residues upstream, that corresponded to predictions for other Mycobacteria, was not noticed… O05891 has the KW Direct protein sequencing, and one reference has the descriptor “Protein sequence of N- terminus…”

17. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 17 Exercise 6: BLASTp and UniRef Compare the results of BLASTing P04150 against UniProtKB, UniRef100, UniRef90 and UniRef50 (use BLAST at ExPASy). Compare the results. In which cluster(s) do you find the alternatively spliced sequences (how many are there)?

18. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 18 The UniProt Non-redundant Reference (UniRef) databases combine closely related sequences (including some from UniParc) into a single record to speed searches.UniProt Non-redundant Reference (UniRef) databases One UniRef100 entry -> all identical sequences (including fragments) – reduction of 12% of DB. One UniRef90 entry -> sequences that have at least 90% identity – reduction of 45% of DB. One UniRef50 entry-> sequences that are at least 50% identical – reduction of 69% ofDB. Species independent!! UniRef is useful for comprehensive BLAST sequence searches by providing sets of representative sequences.

19. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 19 First BLAST against UniProtKB

20. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 20 UniRef100: + more further down the output They are not all in the same cluster (remember 12% reduction in DB size)

21. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 21 UniRef90: Still not all in the same cluster (remember 45% reduction in DB size)

22. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 22 UniRef50: All in the same cluster (remember 69% reduction in DB size)

23. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 23 Exercise 7 Different looks and tools for a same entry depending on the server... Starting with the new UniProt server (http://beta.uniprot.org/):http://beta.uniprot.org/ a.Look for the amino acid sequence of human carbonic anhydrase 2. b.Get the corresponding nucleic acid entries in EMBL and GenBank: try to find a nucleic acid sequence derived from genomic DNA sequencing and another one derived from cDNA sequencing. c.From the UniProtKB/Swiss-Prot entry, look at the data available for the variant Pro-92 and in particular its position in the 3D structure (Use the “Astex viewer”). Starting with the NCBI server (http://www.ncbi.nlm.nih.gov/):http://www.ncbi.nlm.nih.gov/ a.Look for the amino acid sequence of human carbonic anhydrase 2 using ENTREZ protein at the NCBI server. b.b. Find the UniProtKB/Swiss-Prot entry and as above: - Get the corresponding nucleic acid entries in EMBL and GenBank. - Find the data available for the variant Pro-92.

24. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 24 UniProt: P00918, follow links

25. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 25 NCBI, Entrez protein, can also just type in P00918

26. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 26 Note differences in UniProt cross-reference presentation, and in information present about a given cross- reference

27. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 27 Feature table ordering is very different here, numerical only

28. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 28 Exercise 8 Environmental sequences: how to check the quality of a protein sequence... a.Look at DQ284920 at EMBL (http://srs.ebi.ac.uk/srs6bin/cgi- bin/wgetz?-page+top+-newId): where does the sequence come from? How reliable is the translated CoDing Sequences (CDS)? b.How many environmental sequences are found in the acid nucleic databases (use SRS (ENV)?SRS c.Look at DQ380558: can you find the protein sequence in UniProtKB? Where does the annotation come from (from which type of analysis)?

29. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 29 a.

30. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 30 b. (March 11, 22:00)

31. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 31 c.

32. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 32

33. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 33

34. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 34 Exercise 9 Genomic databases (I) a.Look for the Swiss-Prot entry of the E.coli gene gutQ (http://beta.uniprot.org/). b.Follow the link to EcoGene (EcoGene Database of Escherichia coli sequence and function) and find the chromosomal location. c.Get the next E.coli gene on the same strand. d.Follow the link to Swiss-Prot. e.Find the subcellular localisation of the protein. f.What regions and domains does the protein contain, visualize them. g.Have a look at the domain structure in the different domain databases. In PROSITE, get the list of proteins with at least one common domain.

35. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 35

36. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 36 EcoGene page

37. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 37 Or in this pull down list

38. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 38 Note: Currently the NiceProt view

39. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 39 Flavodoxin-like Zinc metallo-hydrolase Rubredoxin-like

40. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 40

41. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 41 From InterPro or

42. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 42 …

43. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 43 From PROSITE

44. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 44 Exercise 10 Protein domain / family databases a.How many different databases are used by InterPro? b.Do an InterPro scan with the sequence on the next page. c.How many different domains does the protein contain? d.How many phosphopantetheine-binding domain does the protein contain? e.How many different protein domain databases have a discriminator for the phosphopantetheine-binding domain? Are they using patterns, profiles or HMMs? What are the most frequent domains found in Mycobacterium tuberculosis H37Rv? (Go to the integr8 site complete proteome: http://www.ebi.ac.uk/integr8/ProteomeAnalysisAction.do?orgProteo meId=30). What percentage of proteins in M.tuberculosis have a phosphopantetheine-binding domain? http://www.ebi.ac.uk/integr8/ProteomeAnalysisAction.do?orgProteo meId=30

45. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 45 MVHATACSEI IRAEVAELLG VRADALHPGA NLVGQGLDSI RMMSLVGRWR RKGIAVDFAT LAATPTIEAW SQLVSAGTGV APTAVAAPGD AGLSQEGEPF PLAPMQHAMW VGRHDHQQLG GVAGHLYVEF DGARVDPDRL RAAATRLALR HPMLRVQFLP DGTQRIPPAA GSRDFPISVA DLRHVAPDVV DQRLAGIRDA KSHQQLDGAV FELALTLLPG ERTRLHVDLD MQAADAMSYR ILLADLAALY DGREPPALGY TYREYRQAIE AEETLPQPVR DADRDWWAQR IPQLPDPPAL PTRAGGERDR RRSTRRWHWL DPQTRDALFA RARARGITPA MTLAAAFANV LARWSASSRF LLNLPLFSRQ ALHPDVDLLV GDFTSSLLLD VDLTGARTAA ARAQAVQEAL RSAAGHSAYP GLSVLRDLSR HRGTQVLAPV VFTSALGLGD LFCPDVTEQF GTPGWIISQG PQVLLDAQVT EFDGGVLVNW DVREGVFAPG VIDAMFTHQV DELLRLAAGD DAWDAPSPSA LPAAQRAVRA ALNGRTAAPS TEALHDGFFR QAQQQPDAPA VFASSGDLSY AQLRDQASAV AAALRAAGLR VGDTVAVLGP KTGEQVAAVL GILAAGGVYL PIGVDQPRDR AERILATGSV NLALVCGPPC QVRVPVPTLL LADVLAAAPA EFVPGPSDPT ALAYVLFTSG STGEPKGVEV AHDAAMNTVE TFIRHFELGA ADRWLALATL ECDMSVLDIF AALRSGGAIV VVDEAQRRDP DAWARLIDTY EVTALNFMPG WLDMLLEVGG GRLSSLRAVA VGGDWVRPDL ARRLQVQAPS ARFAGLGGAT ETAVHATIFE VQDAANLPPD WASVPYGVPF PNNACRVVAD SGDDCPDWVA GELWVSGRGI ARGYRGRPEL TAERFVEHDG RTWYRTGDLA RYWHDGTLEF VGRADHRVKI SGYRVELGEI EAALQRLPGV HAAAATVLPG GSDVLAAAVC VDDAGVTAES IRQQLADLVP AHMIPRHVTL LDRIPFTDSG KIDRAEVGAL LAAEVERSGD RSAPYAAPRT VLQRALRRIV ADILGRANDA VGVHDDFFAL GGDSVLATQV VAGIRRWLDS PSLMVADMFA ARTIAALAQL LTGREANADR LELVAEVYLE IANMTSADVM AALDPIEQPA QPAFKPWVKR FTGTDKPGAV LVFPHAGGAA AAYRWLAKSL VANDVDTFVV QYPQRADRRS HPAADSIEAL ALELFEAGDW HLTAPLTLFG HCMGAIVAFE FARLAERNGV PVRALWASSG QAPSTVAASG PLPTADRDVL ADMVDLGGTD PVLLEDEEFV ELLVPAVKAD YRALSGYSCP PDVRIRANIH AVGGNRDHRI SREMLTSWET HTSGRFTLSH FDGGHFYLND HLDAVARMVS ADVR

46. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 46 c. 6 domains, 1 PTM, 1 family detected d. 2 phosphopantetheine -binding domains

47. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 47 Pfam: HMM, PROSITE; Profile

48. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 48

49. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 49

50. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 50 Exercise 11 Use of UniProtKB/Swiss-Prot for creating dataset and prediction tools. -Find proteins with the following EC numbers; 3.5.1.1, 3.5.1.38 -Look for proteins which have been experimentally proven to have an active site. - Alignment the sequences. -From the alignment suggest a pattern based around the active threonine (do this manually). -Scan your pattern against UniProtKB/Swiss-Prot (http://expasy.org/tools/scanprosite/). How many matches do you find?http://expasy.org/tools/scanprosite/ - Compare your pattern with that found in the PROSITE database PS00144, (http://www.expasy.org/cgi-bin/prosite-search-ac?PDOC00132). How many matches in UniProtKB/Swiss-Prot are there with PS00144? - Can you do the same with the NCBInr data ?

51. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 51 PS00144, have to give the EC numbers ATGGTIAG Scan against SP, get 13 hits PROSITE pattern gives 517 hits against UniProt, 45 against SP

52. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 52 Done March 2007, ANA

53. Tunis, March 2007 A. Auchincloss UniProtKB and ExPASy 53