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Practical exercises

Practical exercises. Answers…. Nucleic acid database in Japan: DDBJ: 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/ , OPD http://www.ebi.ac.uk/pride/

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Practical exercises

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  1. Practical exercises Answers… A. Auchincloss UniProtKB and ExPASy

  2. A. Auchincloss UniProtKB and ExPASy

  3. Nucleic acid database in Japan: DDBJ: 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/, • OPD http://www.ebi.ac.uk/pride/ • Protein-protein interaction: INTACT: http://www.ebi.ac.uk/intact/site/ • DIP: http://dip.doe-mbi.ucla.edu/ JCB: http://www.imb-jenade/jcb/ppi/ • rat enamel 2D gel electrophoresis: 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 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/ • RefSeq http://www.ncbi.nlm.nih.gov/RefSeq/ • UniProtKB http://www.expasy.org/sprot/ or http://beta.uniprot.org/ find sequence(s) in • UniProtKB/Swiss-Prot and sequence(s) in UniProtKB/TrEMBL • - PIR-PSD http://pir.georgetown.edu/pirwww/dbinfo/pir_psd.shtml • - PDB http://www.rcsb.org/pdb/home/home.do • - UniParc (use SRS ) or the UniParc query tool. • EnsEMBL http://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 SRS • 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/ 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 UniProt UniParc: UPI0000112C1C PDB: 1AA3,1N03,1REA,1U94 etc, easily retrieved from UniProt. EnsEMBL: Not possible, bacteria are not in EnsEMBL! A. Auchincloss UniProtKB and ExPASy

  6. 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? A. Auchincloss UniProtKB and ExPASy

  7. 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. A. Auchincloss UniProtKB and ExPASy

  8. 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. A. Auchincloss UniProtKB and ExPASy

  9. 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. A. Auchincloss UniProtKB and ExPASy

  10. BLASTp at ExPASy against UniProtKB BLASTp at NCBI against nr A. Auchincloss UniProtKB and ExPASy

  11. 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. A. Auchincloss UniProtKB and ExPASy

  12. The Statistics of Sequence Similarity Scores http://www.ncbi.nlm.nih.gov/BLAST/tutorial/Altschul-1.html 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. A. Auchincloss UniProtKB and ExPASy

  13. 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? A. Auchincloss UniProtKB and ExPASy

  14. A. Auchincloss UniProtKB and ExPASy

  15. 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…” A. Auchincloss UniProtKB and ExPASy

  16. 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)? A. Auchincloss UniProtKB and ExPASy

  17. The UniProt Non-redundant Reference (UniRef) databases combine closely related sequences (including some from UniParc) into a single record to speed searches. One UniRef100 entry -> all identical sequences (including fragments) – reduction of 12% of DB. One UniRef90 entry -> sequences that have at least90% 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. A. Auchincloss UniProtKB and ExPASy

  18. First BLAST against UniProtKB A. Auchincloss UniProtKB and ExPASy

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

  20. UniRef90: Still not all in the same cluster (remember 45% reduction in DB size) A. Auchincloss UniProtKB and ExPASy

  21. UniRef50: All in the same cluster (remember 69% reduction in DB size) A. Auchincloss UniProtKB and ExPASy

  22. Exercise 7 • Different looks and tools for a same entry depending on the server... • Starting with the new UniProt server (http://beta.uniprot.org/): • Look for the amino acid sequence of human carbonic anhydrase 2. • 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. • 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/): • Look for the amino acid sequence of human carbonic anhydrase 2 using ENTREZ protein at the NCBI server. • 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. A. Auchincloss UniProtKB and ExPASy

  23. UniProt: P00918, follow links A. Auchincloss UniProtKB and ExPASy

  24. NCBI, Entrez protein, can also just type in P00918 A. Auchincloss UniProtKB and ExPASy

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

  26. Feature table ordering is very different here, numerical only A. Auchincloss UniProtKB and ExPASy

  27. Exercise 8 • Environmental sequences: how to check the quality of a protein sequence... • 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)? • How many environmental sequences are found in the acid nucleic databases (use SRS(ENV)? • Look at DQ380558: can you find the protein sequence in UniProtKB? Where does the annotation come from (from which type of analysis)? A. Auchincloss UniProtKB and ExPASy

  28. a. A. Auchincloss UniProtKB and ExPASy

  29. b. (March 11, 22:00) A. Auchincloss UniProtKB and ExPASy

  30. c. A. Auchincloss UniProtKB and ExPASy

  31. A. Auchincloss UniProtKB and ExPASy

  32. A. Auchincloss UniProtKB and ExPASy

  33. Exercise 9 • Genomic databases (I) • Look for the Swiss-Prot entry of the E.coli gene gutQ (http://beta.uniprot.org/). • Follow the link to EcoGene (EcoGene Database of Escherichia coli sequence and function) and find the chromosomal location. • Get the next E.coli gene on the same strand. • Follow the link to Swiss-Prot. • Find the subcellular localisation of the protein. • What regions and domains does the protein contain, visualize them. • 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. A. Auchincloss UniProtKB and ExPASy

  34. A. Auchincloss UniProtKB and ExPASy

  35. EcoGene page A. Auchincloss UniProtKB and ExPASy

  36. Or in this pull down list A. Auchincloss UniProtKB and ExPASy

  37. Note: Currently the NiceProt view A. Auchincloss UniProtKB and ExPASy

  38. Flavodoxin-like Zinc metallo-hydrolase Rubredoxin-like A. Auchincloss UniProtKB and ExPASy

  39. A. Auchincloss UniProtKB and ExPASy

  40. From InterPro or A. Auchincloss UniProtKB and ExPASy

  41. … A. Auchincloss UniProtKB and ExPASy

  42. From PROSITE A. Auchincloss UniProtKB and ExPASy

  43. Exercise 10 • Protein domain / family databases • How many different databases are used by InterPro? • Do an InterPro scan with the sequence on the next page. • How many different domains does the protein contain? • How many phosphopantetheine-binding domain does the protein contain? • 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?orgProteomeId=30). What percentage of proteins in M.tuberculosis have a phosphopantetheine-binding domain? A. Auchincloss UniProtKB and ExPASy

  44. 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 A. Auchincloss UniProtKB and ExPASy

  45. c. 6 domains, 1 PTM, 1 family detected d. 2 phosphopantetheine-binding domains A. Auchincloss UniProtKB and ExPASy

  46. Pfam: HMM, PROSITE; Profile A. Auchincloss UniProtKB and ExPASy

  47. A. Auchincloss UniProtKB and ExPASy

  48. A. Auchincloss UniProtKB and ExPASy

  49. 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? • 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 ? A. Auchincloss UniProtKB and ExPASy

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