1. ©CMBI 2008 Data and Databases Your questions: –Lookup –Compare –Predict

2. ©CMBI 2008 Your questions Lookup Is the gene known for my protein (or vice versa)? What sequence patterns are present in my protein? Are the mutations known which cause this disease? To what class or family does my protein belong? Compare Are there protein sequences in the database which resemble the protein I cloned? How can I optimally align the members of this protein family? Are these two sequences similar? Predict Can I predict the active site residues of this enzyme? Can I make a 3D model for my protein? Can I predict a (better) drug for this target? How can I improve the thermostability of this protein? (protein engineering) How can I predict the genes located on this genome?

3. ©CMBI 2008 Sequence similarity MVVSGAPPAL GGGCLGTFTS LLLLASTAIL NAARIPVPPA CGKPQQLNRV VGGEDSTDSE WPWIVSIQKN GTHHCAGSLL TSRWVITAAH CFKDNLNKPY LFSVLLGAWQ LGNPGSRSQK VGVAWVEPHP VYSWKEGACA DIALVRLERS IQFSERVLPI CLPDASIHLP PNTHCWISGW GSIQDGVPLP HPQTLQKLKV PIIDSEVCSH LYWRGAGQGP ITEDMLCAGY LEGERDACLG DSGGPLMCQV DGAWLLAGII SWGEGCAERN RPGVYISLSA HRSWVEKIVQ GVQLRGRAQG Image, you sequenced this human protein. You know it is a serine protease. Which residues belong to the active site? Is its sequence similar to the mouse serine protease?

4. ©CMBI 2008 Alignment MVVSGAPPAL GGGCLGTFTS LLLLASTAIL NAARIPVPPA CGKPQQLNRV VGGEDSTDSE MMISRPPPAL GGDQFSILIL LVLLTSTAPI SAATIRVSPD CGKPQQLNRI VGGEDSMDAQ *::*.**** **. :. : *:**:*** :.** * *.* *********: ****** *:: WPWIVSIQKN GTHHCAGSLL TSRWVITAAH CFKDNLNKPY LFSVLLGAWQ LGNPGSRSQK WPWIVSILKN GSHHCAGSLL TNRWVVTAAH CFKSNMDKPS LFSVLLGAWK LGSPGPRSQK ******* ** *:******** *.***:**** ***.*::** *********: **.**.**** VGVAWVEPHP VYSWKEGACA DIALVRLERS IQFSERVLPI CLPDASIHLP PNTHCWISGW VGIAWVLPHP RYSWKEGTHA DIALVRLEHS IQFSERILPI CLPDSSVRLP PKTDCWIAGW **:*** *** ******: * ********:* ******:*** ****:*::** *:*.***:** GSIQDGVPLP HPQTLQKLKV PIIDSEVCSH LYWRGAGQGP ITEDMLCAGY LEGERDACLG GSIQDGVPLP HPQTLQKLKV PIIDSELCKS LYWRGAGQEA ITEGMLCAGY LEGERDACLG ********** ********** ******:*. ********. ***.****** ********** DSGGPLMCQV DGAWLLAGII SWGEGCAERN RPGVYISLSA HRSWVEKIVQ GVQLRGRAQG DSGGPLMCQV DDHWLLTGII SWGEGCAD-D RPGVYTSLLA HRSWVQRIVQ GVQLRG---- ********** *. ***:*** *******: : ***** ** * *****::*** ****** => Transfer of information

5. ©CMBI 2008 Data & Databases Data in databases what the data looks like Programs (tools) to search these databases how it can be accessed

6. ©CMBI 2008 Biological Databases The number of databases - DBCAT lists over 1200 databases (2006) The size of databases - Grows exponentially - EMBL database: New entries entered at 5 sec/seq!

7. ©CMBI 2008 Database Size

8. ©CMBI 2008 Primary and Secondary Databases Primary databases REAL EXPERIMENTAL DATA Biomolecular sequences or structures and associated annotation information (organism, function, mutation linked to disease, functional/structural patterns, bibliographic etc.) Secondary databases DERIVED INFORMATION Fruits of analyses of sequences in the primary sources (patterns, blocks, profiles etc., that represent the most conserved features of multiple alignments)

9. ©CMBI 2008 Primary Databases Sequence Information DNA: EMBL, Genbank, DDBJ Protein: SwissProt, TREMBL, PIR Genome Information EnsEMBL, TIGR rice genome, Celera, SNP databases Structure Information PDB, NDB, CCDB/CSD

10. ©CMBI 2008 Secondary Databases Sequence-related Information -ProSite, Enzyme, REBase Genome-related Information -OMIM, TransFac Structure-related Information -DSSP, HSSP, FSSP, PDBFinder Pathway Information -KEGG, Pathways

11. ©CMBI 2008 Prosite example

12. ©CMBI 2008 KEGG example

13. ©CMBI 2008 Databases Data must be in a certain format for software to recognize Every database can have its own format but some data elements are essential for every database: 1. Unique identifier, or accession code 2. Name of depositor 3. Literature references 4. Deposition date 5. The real data

14. ©CMBI 2008 Quality of Data SwissProt Data is only entered by annotation experts EMBL, PDB Everybody can submit data No human intervention when submitted; some automatic checks

15. ©CMBI 2008 SwissProt database Database of protein sequences derived from: - translations of DNA (from EMBL Database) - adapted from the PIR collection - extracted from the literature - and directly submitted by researchers 385721 entries (may 2008) Ca. 200 Annotation experts worldwide Keyword-organised flatfile Obligatory deposit of in SwissProt before publication Presently, databases are being merged into UniProt.

16. ©CMBI 2008 SwissProt records ID identification line ID ENTRY_NAME DATA_CLASS; MOLECULE_TYPE; SEQUENCE_LENGTH. ID CRAM_CRAAB STANDARD; PRT; 46 AA. Format for the ENTRY_NAME: NAME_SPECIES (  12 characters) For number of organisms (16) recognizable names: HUMAN, MOUSE, CHICK, BOVIN, YEAST, ECOLI…. N.B. The ID can change, e.g. serotonine receptors have got a new nomenclature

17. ©CMBI 2008 SwissProt records AC accession number AC P01542; AC is unique: Name, sequence, everything can change but AC stays the same DT deposition date DT 21-JUL-1986 (Rel. 01, Created) DT 30-MAY-2000 (Rel. 39, Last sequence update) DT 30-MAY-2000 (Rel. 39, Last annotation update) 1) You can not see what the last annotation update was 2) No depositor record (Implicit: author of first reference)

18. ©CMBI 2008 SwissProt records DE description DE CRAMBIN. DE 6-phosphofructo-2-kinase 1 (EC 2.7.1.105) (Phosphofructokinase 2 I) General descriptive information GN gene name GN THI2. OS & OC & OG OS Crambe abyssinica (Abyssinian crambe). OC Eukaryota; Viridiplantae; Embryophyta; Tracheophyta; OC Magnoliophyta; eudicotyledons; Rosidae; Brassicales; OC Brassicaceae; Crambe. Organism Species; Organism Classification; Organelle

19. ©CMBI 2008 SwissProt records RN References RN [1] RP SEQUENCE. RX MEDLINE; 82046542. RA Teeter M.M., Mazer J.A., L'Italien J.J.; RT "Primary structure of the plant protein crambin."; RL Biochemistry 20:5437-5443(1981). CC Comments or notes CC -!- FUNCTION: THE FUNCTION OF THIS HYDROPHOBIC PLANT SEED PROTEIN CC IS NOT KNOWN. CC -!- MISCELLANEOUS: TWO ISOFORMS EXISTS (MAJOR ONE SHOWN HERE) CC AND A MINOR FORM SI. CC -!- SIMILARITY: BELONGS TO THE PLANT THIONIN FAMILY.

20. ©CMBI 2008 SwissProt records DR Database Cross Reference DR PIR; A01805; KECX. DR PDB; 1AB1; X-ray; @=1-46. DR PDB; 1CBN; X-ray; @=1-46. (…) DR PDB; 1JXY; X-ray; A=1-46. DR InterPro; IPR001010; Thionin. DR Pfam; PF00321; Thionin; 1. DR PRINTS; PR00287; THIONIN. DR PROSITE; PS00271; THIONIN; 1. KW Keyword Not standardized (under control of depositor) KW Thionin; 3D-structure.

21. ©CMBI 2008 SwissProt records FT Feature table data FT DISULFID 3 40 FT DISULFID 4 32 FT DISULFID 16 26 FT VARIANT 22 22 P -> S (IN ISOFORM SI). FT VARIANT 25 25 L -> I (IN ISOFORM SI). FT STRAND 2 3 FT HELIX 7 16 FT TURN 17 19 FT HELIX 23 30 FT TURN 31 31 FT STRAND 33 34 FT TURN 42 43

22. ©CMBI 2008 Feature table ( continued ) Other features: post-translational modifications, binding sites, enzyme active sites, local secondary structure or other characteristics reported in the cited references. Sequence conflicts between references are also included. FT CONFLICT 33 33 MISSING (IN REF. 2). FT MUTAGEN 123 123 G->R,L,M: DNA BINDING LOST. FT MOD_RES 11 11 PHOSPHORYLATION (BY PKC). FT LIPID 1 1 MYRISTATE. FT CARBOHYD 103 103 GLUCOSYLGALACTOSE. FT METAL 87 87 COPPER (POTENTIAL). FT BINDING 14 14 HEME (COVALENT). FT PROPEP 27 28 ACTIVATION PEPTIDE. FT DOMAIN 22 788 EXTRACELLULAR (POTENTIAL). FT ACT_SITE 193 193 ACCEPTS A PROTON DURING CATALYSIS.

23. ©CMBI 2008 SwissProt records SQ sequence header SQ SEQUENCE 46 AA; 4736 MW; 919E68AF159EF722 CRC64; Sequence data TTCCPSIVAR SNFNVCRLPG TPEALCATYT GCIIIPGATC PGDYAN Termination line //

24. ©CMBI 2008 SwissProt entry in MRS

25. ©CMBI 2008 SwissProt entry in MRS

26. ©CMBI 2008 SwissProt entry in MRS

27. ©CMBI 2008 EMBL database Nucleotide database –EMBL: 114 million sequence entries comprising 215 billion nucleotides (March 2008) –Of which EMEST: 50 million sequence entries comprising 27 billion nucleotides (March 2008) –EMBL records follows roughly same scheme as SwissProt –Obligatory deposit of sequence in EMBL before publication –Most EMBL sequences never seen by a human

28. ©CMBI 2008 Protein Data Bank (PDB) Databank for macromolecular structure data (3-dimensional coordinates). Started ca. 30 years ago (on punched cards!) Obligatory deposit of coordinates in the PDB before publication ~ 44000 entries (Aug 2007) ( ~2500 “unique” structures) PDB file is a keyword-organised flat-file (80 column) 1) human readable 2) every line starts with a keyword (3-6 letters) 3) platform independent

29. ©CMBI 2008 PDB records Filename= accession number= PDB Code Filename is 4 positions (often 1 digit & 3 letters, e.g. 1CRN) HEADER describes molecule & gives deposition date HEADER PLANT SEED PROTEIN 30-APR-81 1CRN CMPND name of molecule COMPND CRAMBIN SOURCE organism SOURCE ABYSSINIAN CABBAGE (CRAMBE ABYSSINICA) SEED

30. ©CMBI 2008 PDB records AUTHOR AUTHOR W.A.HENDRICKSON,M.M.TEETER 1CRN 6 Revision date REVDAT 5 16-APR-87 1CRND 1 HEADER 1CRND 2 REVDAT 4 04-MAR-85 1CRNC 1 REMARK 1CRNC 1 REVDAT 3 30-SEP-83 1CRNB 1 REVDAT 1CRNB 1 REVDAT 2 03-DEC-81 1CRNA 1 SHEET 1CRNB 2 REVDAT 1 28-JUL-81 1CRN 0 REMARK There are very many different REMARK records & subrecords! Not standardized. REMARK 1 REFERENCE 3 1CRNC 10 REMARK 1 AUTH M.M.TEETER,W.A.HENDRICKSON 1CRN 16 REMARK 1 TITL HIGHLY ORDERED CRYSTALS OF THE PLANT SEED PROTEIN 1CRN 17 REMARK 1 TITL 2 CRAMBIN 1CRN 18 REMARK 1 REF J.MOL.BIOL. V. 127 219 1979 1CRN 19 REMARK 1 REFN ASTM JMOBAK UK ISSN 0022-2836 070 1CRN 20 REMARK 2 1CRN 21 REMARK 2 RESOLUTION. 1.5 ANGSTROMS. 1CRN 22

31. ©CMBI 2008 PDB records SEQRES Sequence of protein; Be aware: Not always all 3d-coordinates are present for all the amino acids in SEQRES!! SEQRES 1 46 THR THR CYS CYS PRO SER ILE VAL ALA ARG SER ASN PHE 1CRN 51 SEQRES 2 46 ASN VAL CYS ARG LEU PRO GLY THR PRO GLU ALA ILE CYS 1CRN 52 SEQRES 3 46 ALA THR TYR THR GLY CYS ILE ILE ILE PRO GLY ALA THR 1CRN 53 SEQRES 4 46 CYS PRO GLY ASP TYR ALA ASN 1CRN 54 HET & FORMUL HET NAD A 1 44 NAD CO-ENZYME 4MDH 219 HET SUL A 2 5 SULFATE 4MDH 220 HET NAD B 1 44 NAD CO-ENZYME 4MDH 221 HET SUL B 2 5 SULFATE 4MDH 222 FORMUL 3 NAD 2(C21 H28 N7 O14 P2) 4MDH 223 FORMUL 4 SUL 2(O4 S1) 4MDH 224 FORMUL 5 HOH *471(H2 O1) 4MDH 225

32. ©CMBI 2008 PDB records HELIX/SHEET/TURN Secondary structure elements provided by the crystallographer (subjective). HELIX 1 H1 ILE 7 PRO 19 1 3/10 CONFORMATION RES 17,19 SHEET 2 S1 2 CYS 32 ILE 35 -1 TURN 1 T1 PRO 41 TYR 44 SSBOND disulfide bridges SSBOND 1 CYS 3 CYS 40 SSBOND 2 CYS 4 CYS 32 CRYST1, ORIG, SCALE crystallographic parameters CRYST1 40.960 18.650 22.520 90.00 90.77 90.00 P 21 2 1CRN 63 SCALE1.024414 0.000000 -.000328 0.00000 1CRN 67 SCALE2 0.000000.053619 0.000000 0.00000 1CRN 68 SCALE3 0.000000 0.000000.044409 0.00000 1CRN 69

33. ©CMBI 2008 PDB records ATOM One line per atom with, unique name and x,y,z coordinates ATOM 1 N THR 1 17.047 14.099 3.625 1.00 13.79 1CRN 70 ATOM 2 CA THR 1 16.967 12.784 4.338 1.00 10.80 1CRN 71 ATOM 3 C THR 1 15.685 12.755 5.133 1.00 9.19 1CRN 72 ATOM 4 O THR 1 15.268 13.825 5.594 1.00 9.85 1CRN 73 ATOM 5 CB THR 1 18.170 12.703 5.337 1.00 13.02 1CRN 74 ATOM 6 OG1 THR 1 19.334 12.829 4.463 1.00 15.06 1CRN 75 ATOM 7 CG2 THR 1 18.150 11.546 6.304 1.00 14.23 1CRN 76 ATOM 8 N THR 2 15.115 11.555 5.265 1.00 7.81 1CRN 77 ATOM 9 CA THR 2 13.856 11.469 6.066 1.00 8.31 1CRN 78 ATOM 10 C THR 2 14.164 10.785 7.379 1.00 5.80 1CRN 79 ATOM 11 O THR 2 14.993 9.862 7.443 1.00 6.94 1CRN 80 TER The TER record terminates the amino acid chain ATOM 325 OD1 ASN 46 11.982 4.849 15.886 1.00 11.00 1CRN 394 ATOM 326 ND2 ASN 46 13.407 3.298 15.015 1.00 10.32 1CRN 395 ATOM 327 OXT ASN 46 12.703 4.973 10.746 1.00 7.86 1CRN 396 TER 328 ASN 46 1CRN 397

34. ©CMBI 2008 PDB records HETATM atomic coordinates for atoms within "non-standard" groups (cofactors, ions, …) and for water molecules HETATM 5158 AP NAD B 1 42.641 30.361 41.284 1.00 26.73 4MDH5495 HETATM 5159 AO1 NAD B 1 43.440 31.570 40.868 1.00 20.69 4MDH5496 HETATM 5160 AO2 NAD B 1 41.161 30.484 41.376 1.00 33.73 4MDH5497 HETATM 5207 O HOH 0 15.379 1.907 3.295 1.00 58.12 4MDH5544 HETATM 5208 O HOH 1 58.861 0.984 17.024 1.00 37.58 4MDH5545 HETATM 5209 O HOH 2 24.384 1.184 74.398 1.00 35.92 4MDH5546 CONECT connection records (not obligatory) indicate which atoms are connected (mainly for HETATM)