©CMBI 2001 What are we looking for? Data & databases

©CMBI 2002 Your questions Lookup Compare Predict

©CMBI 2002 Your questions Lookup Is the gene known for my protein (or vice versa)? On which chromosome is the gene located? 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? What is known about this family?

©CMBI 2002 Your questions 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 proteins similar?

©CMBI 2002 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?

©CMBI 2002 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

©CMBI 2000 J Leunissen Are these structures similar?

©CMBI 2002 Your questions Predict Can I predict the active site residues of this enzyme? Why are these patients ill? 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?

©CMBI 2002 How to find the answers to these questions? Outline Morning Data in databases Afternoon Programs (tools) to search these databases Knowledge how to search the databases with these tools (hands-on)

©CMBI 2002 Biological Databases The number of databases - DBCAT currently lists over 500 databases The size of databases - Grows exponentially - EMBL database: New entries entered at 6.3 sec/seq! (July 2001)

©CMBI 2001 J Leunissen (July 2001)

©CMBI 2002 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. which represent the most conserved features of multiple alignments)

©CMBI 2002 Primary Databases Sequence Information –DNA: EMBL, Genbank, DDBJ –Protein: SwissProt, TREMBL, PIR, OWL Genome Information – GDB, MGD, ACeDB, ENSEMBL Structure Information – PDB, NDB, CCDB/CSD

©CMBI 2002 Secondary Databases Sequence-related Information –ProSite, REBase Genome-related Information –OMIM, TransFac Structure-related Information –DSSP, HSSP, FSSP, PDBFinder Pathway Information –KEGG, Pathways Function-related –Enzyme, GO

©CMBI 2002 Databases Data must be in certain format for the programs to recognize them. 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

©CMBI examples 1. SwissProt 2. EMBL 3. PDB

©CMBI 2002 Quality of databases SwissProt Data is only entered by annotation experts EMBL, PDB Everybody can submit data Data are accepted the way they are submitted

©CMBI 2002 SwissProt database Database of protein sequences Produced by Amos Bairoch (University of Geneva) and the EMBL Data Library Data derived from: –translations of DNA sequences (from EMBL Database) –adapted from the PIR collection –extracted from the literature –and directly submitted by researchers SwissProt & SwissNew July 2001: –~86,600 entries, ~15,000 new entries / year –Swissnew: 53,000 entries Ca. 200 Annotation experts worldwide Keyword-organised flatfile

©CMBI 2002 SwissProt records (1) 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 (  10 characters) For number of organisms (16) SPECIES has a recognizable name: HUMAN, MOUSE, CHICK, BOVIN, YEAST, ECOLI…. N.B. The ID can change, e.g. serotonine receptors have got a new nomenclature

©CMBI 2002 SwissProt records (2) 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)

©CMBI 2002 SwissProt records (3) DE description DE CRAMBIN. DE 6-phosphofructo-2-kinase 1 (EC ) (Phosphofructokinase 2 I) 1) General descriptive information 2) Free-format GN gene name GN THI2. OS & OC & OG OS Crambe abyssinica (Abyssinian crambe). OC Eukaryota; Viridiplantae; Embryophyta;Tracheophyta;Spermatophyta; OC Magnoliophyta; eudicotyledons; Rosidae; eurosids II; Brassicales; OC Brassicaceae; Crambe. Organism Species; Organism Classification; OrGanelle

©CMBI 2002 SwissProt records (4) RN References RN [1] RP SEQUENCE. RX MEDLINE; RA Teeter M.M., Mazer J.A., L'Italien J.J.; RT "Primary structure of the hydrophobic plant protein crambin."; RL Biochemistry 20: (1981). CC Comments or notes CC -!- FUNCTION: THE FUNCTION OF THIS HYDROPHOBIC PLANT SEED PROTEIN CC IS NOT KNOWN. CC -!- MISCELLANEOUS: TWO ISOFORMS EXISTS, A MAJOR FORM PL (SHOWN HERE) CC AND A MINOR FORM SI. CC -!- SIMILARITY: BELONGS TO THE PLANT THIONIN FAMILY.

©CMBI 2002 SwissProt records (5) DR Database Cross Reference DR PIR; A01805; KECX. DR PDB; 1CRN; 16-APR-87. DR PDB; 1CBN; 31-JAN-94. DR PDB; 1CCM; 31-OCT-93. DR PDB; 1CCN; 31-JAN-94. DR PDB; 1CNR; 31-AUG-94. DR PDB; 1AB1; 12-AUG-97. DR INTERPRO; IPR001010; -. DR PFAM; PF00321; plant_thionins; 1. DR PRINTS; PR00287; THIONIN. DR PROSITE; PS00271; THIONIN; 1. KW Keyword Not standardized (under control of depositor) KW Thionin; 3D-structure.

©CMBI 2002 SwissProt records (6) FT Feature table data FT DISULFID 3 40 FT DISULFID 4 32 FT DISULFID FT VARIANT P -> S (IN ISOFORM SI). FT VARIANT L -> I (IN ISOFORM SI). FT STRAND 2 3 FT HELIX 7 16 FT TURN FT HELIX FT TURN FT STRAND FT TURN 42 43

©CMBI 2002 Feature table 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 MISSING (IN REF. 2). FT MUTAGEN G->R,L,M: DNA BINDING LOST. FT MOD_RES PHOSPHORYLATION (BY PKC). FT LIPID 1 1 MYRISTATE. FT CARBOHYD GLUCOSYLGALACTOSE. FT METAL COPPER (POTENTIAL). FT BINDING HEME (COVALENT). FT PROPEP ACTIVATION PEPTIDE. FT DOMAIN EXTRACELLULAR (POTENTIAL). FT ACT_SITE ACCEPTS A PROTON DURING CATALYSIS.

©CMBI 2002 SwissProt records (7) SQ sequence header SQ SEQUENCE 46 AA; 4736 MW; 919E68AF159EF722 CRC64; Sequence data TTCCPSIVAR SNFNVCRLPG TPEALCATYT GCIIIPGATC PGDYAN // Termination line

©CMBI 2002 EMBL database Nucleotide database EMBL & EMNEW July 2001: EMBL: 3,951,820 entries, EMNEW: 323,703 EMEST*: 8,092,600, EMNEWEST*: 619,777 *) EMEST/EMNEWEST = EST-section of EMBL, EST = expressed sequence tag EMBL records follows roughly same scheme as SwissProt Obligatory deposit of sequence in EMBL (or SwissProt) before publication

©CMBI 2002 Protein Data Bank (PDB) Databank for macromolecular structure data (3- dimensional coordinates) Obligatory deposit of coordinates in the PDB before publication ~16,000 entries (October 2001) 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 Started ca. 25 years ago (on punche cards!)

©CMBI 2002 PDB records (1) Filename= accession number= PDB Code 1) Filename is 4 positions (often 1 digit & 3 letters, e.g. 1CRN) 2) Be aware: 0HYK means entry HYK does not contain coordinates HEADER describes molecule & gives deposition date HEADER PLANT SEED PROTEIN 30-APR-81 1CRN 1CRND 1 CMPND name of molecule COMPND CRAMBIN 1CRN 4 SOURCE organism SOURCE ABYSSINIAN CABBAGE (CRAMBE ABYSSINICA) SEED 1CRN 5

©CMBI 2002 PDB records (2) AUTHOR AUTHOR W.A.HENDRICKSON,M.M.TEETER 1CRN 6 The depositor JRNL JRNL AUTH M.BLABER,X.-J.ZHANG,B.W.MATTHEWS 111L 10 JRNL TITL STRUCTURAL BASIS OF ALPHA-HELIX PROPENSITY AT TWO 111L 11 JRNL TITL 2 SITES IN T4 LYSOZYME 111L 12 JRNL REF SCIENCE V L 13 JRNL REFN ASTM SCIEAS US ISSN L 14 REMARK Not standardized: many different REMARK records & subrecords! 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 CRN 19 REMARK 1 REFN ASTM JMOBAK UK ISSN CRN 20 REMARK 2 1CRN 21 REMARK 2 RESOLUTION. 1.5 ANGSTROMS. 1CRN 22

©CMBI 2002 PDB records (3) 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 metals, cofactors, ions, etc. 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

©CMBI 2002 PDB records (4) HELIX/SHEET/TURN Secondary structure elements as provided by the crystallographer (subjective) HELIX 1 H1 ILE 7 PRO /10 CONFORMATION RES 17,19 1CRN 55 SHEET 2 S1 2 CYS 32 ILE CRN 58 TURN 1 T1 PRO 41 TYR 44 1CRN 59 SSBOND disulfide bridges SSBOND 1 CYS 3 CYS 40 1CRN 60 SSBOND 2 CYS 4 CYS 32 1CRN 61 CRYST1, ORIGX1, ORIGX2, ORIGX3, SCALE1, SCALE2, SCALE3 crystallographic parameters CRYST P CRN 63 ORIGX CRN 64 ORIGX CRN 65 ORIGX CRN 66 SCALE CRN 67 SCALE CRN 68 SCALE CRN 69

©CMBI 2002 PDB records (5) ATOM one line for each atom with its unique name and its x,y,z coordinates ATOM 1 N THR CRN 70 ATOM 2 CA THR CRN 71 ATOM 3 C THR CRN 72 ATOM 4 O THR CRN 73 ATOM 5 CB THR CRN 74 ATOM 6 OG1 THR CRN 75 ATOM 7 CG2 THR CRN 76 ATOM 8 N THR CRN 77 ATOM 9 CA THR CRN 78 ATOM 10 C THR CRN 79 ATOM 11 O THR CRN 80 TER record terminates the amino acid chain ATOM 325 OD1 ASN CRN 394 ATOM 326 ND2 ASN CRN 395 ATOM 327 OXT ASN CRN 396 TER 328 ASN 46 1CRN 397

©CMBI 2002 PDB records (6) HETATM atomic coordinate records for atoms within “HET & FORMUL”-lines (metals, cofactors, ions, …) and for water molecules HETATM 5158 AP NAD B MDH5495 HETATM 5159 AO1 NAD B MDH5496 HETATM 5160 AO2 NAD B MDH5497 HETATM 5207 O HOH MDH5544 HETATM 5208 O HOH MDH5545 HETATM 5209 O HOH MDH5546

©CMBI 2002