EBI is an Outstation of the European Molecular Biology Laboratory. InterPro Database Protein Functional Analysis Jennifer McDowall, Ph.D. Senior InterPro.

EBI is an Outstation of the European Molecular Biology Laboratory. InterPro Database Protein Functional Analysis Jennifer McDowall, Ph.D. Senior InterPro Curator

http://www.ebi.ac.uk/interpro EBI Sequence Databases UniProtKB Swiss-Prot manual annotation UniProtKB TrEMBL protein sequence translate (GenBank, DDBJ) nucleotide sequence EMBL CGCGCCTGTACGC TGAACGCTCGTGA CGTGTAGTGCGCG >7M >400,000

http://www.ebi.ac.uk/interpro EBI Sequence Databases UniProtKB Swiss-Prot manual annotation UniProtKB TrEMBL protein sequence translate InterPro Protein signatures protein annotation (GenBank, DDBJ) nucleotide sequence EMBL CGCGCCTGTACGC TGAACGCTCGTGA CGTGTAGTGCGCG groups of related proteins (same family or share domains)

http://www.ebi.ac.uk/interpro UniProtKB UniProt/ SwissProt proteins InterPro ~370,000 ~400,000 Signature matches InterPro ~80% Protein Coverage UniMESS Metagenomic proteins >6M Available 2009 UniProt/ TrEMBL proteins >5.3M >7M

http://www.ebi.ac.uk/interpro What are protein signatures? Multiple sequence alignment A signature describes the pattern of a set of conserved residues in a group of proteins  Define a protein family  Define a protein feature (domain or conserved site)

http://www.ebi.ac.uk/interpro More sensitive homology searches  Find more distant homologues than BLAST What value are signatures?

http://www.ebi.ac.uk/interpro More sensitive homology searches What value are signatures? Classification of proteins  Associate proteins that share: Function Domains Sequence Structure

http://www.ebi.ac.uk/interpro What value are signatures? Annotation of protein sequences  Define conserved regions of a protein -e.g. location and type of domains key structural or functional sites Classification of proteins More sensitive homology searches

http://www.ebi.ac.uk/interpro What value are signatures? Transfer additional (automatic) annotation  Associate TrEMBL proteins with well- annotated SwissProt proteins Transfer annotation More sensitive homology searches Classification of proteins Annotation of protein sequences

http://www.ebi.ac.uk/interpro Signature methods Pattern Fingerprint Sequence clustering HMM SAM

http://www.ebi.ac.uk/interpro Patterns Pattern/motif in sequence  regular expression Can define important sites Enzyme catalytic site Prosthetic group attachment Metal ion binding site Cysteines for disulphide bonds Protein or molecule binding B chain xxxxxxCxxxxxxxxxxxxCxxxxxxxxx A chain xxxxxCCxxxCxxxxxxxxCx | | EXAMPLE: Insulin

http://www.ebi.ac.uk/interpro Patterns Pattern/motif in sequence  regular expression Can define important sites MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLV EALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPG AGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN B chain xxxxxxCxxxxxxxxxxxxCxxxxxxxxx A chain xxxxxCCxxxCxxxxxxxxCx | | EXAMPLE: PS00262 Insulin family signature

http://www.ebi.ac.uk/interpro Patterns Pattern/motif in sequence  regular expression Can define important sites B chain xxxxxxCxxxxxxxxxxxxCxxxxxxxxx A chain xxxxxCCxxxCxxxxxxxxCx | | EXAMPLE: PS00262 Insulin family signature MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLV EALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPG AGSLQPLALEGSLQKRGIVEQ CCTSICSLYQLENYC N

http://www.ebi.ac.uk/interpro Patterns Pattern/motif in sequence  regular expression Can define important sites B chain xxxxxxCxxxxxxxxxxxxCxxxxxxxxx A chain xxxxxCCxxxCxxxxxxxxCx | | EXAMPLE: PS00262 Insulin family signature C-C-{P}-x(2)-C-[STDNEKPI]-x(3)-[LIVMFS]-x(3)-C Regular expression MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLV EALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPG AGSLQPLALEGSLQKRGIVEQ CCTSICSLYQLENYC N

http://www.ebi.ac.uk/interpro Patterns – understanding a regular expression C - C - {P} - x(2) - C - [STDNEKPI] - x(3) - [LIVMFS] - x(3) - C Strictly conserved site; only one amino acid is accepted at this position Curly brackets denote amino acids that cannot occur at a single position x denotes any amino acid can occur at a single position There are dashes between each position

http://www.ebi.ac.uk/interpro Patterns – understanding a regular expression C - C - {P} - x(2) - C - [STDNEKPI] - x(3) - [LIVMFS] - x(3) - C X(2) – therefore any amino acid can occur at the next two position Square brackets denote range of amino acids that occur at a single position

http://www.ebi.ac.uk/interpro Patterns Extract pattern sequences xxxxxx Sequence alignment Insulin family motif Define pattern Pattern signature C-C-{P}-x(2)-C-[STDNEKPI]-x(3)-[LIVMFS]-x(3)-C Build regular expression PS 00000

http://www.ebi.ac.uk/interpro Fingerprints Several motifs  characterise family Different combinations of motifs describe subfamilies Identify small conserved regions in divergent proteins EXAMPLE: PR00107 Phosphocarrier HPr signature PTHP_ENTFA: MEKKEFHIVAETGIHARPATLLVQTASKFNSDINLEYKGKSVNLK SIMGVMSLGVGQGSDVTITVDGADEAEGMAAIVETLQKEGLAE

http://www.ebi.ac.uk/interpro Fingerprints Several motifs  characterise family Different combinations of motifs describe subfamilies Identify small conserved regions in divergent proteins EXAMPLE: PR00107 Phosphocarrier HPr signature PTHP_ENTFA: MEKKEFHIVAET GIHARPATLLVQTASKF NSDINLEYKGKSVNLK SIMGVMSLGVGQGSDVTITVDGADEAEGMAAIVETLQKEGLAE His phosphorylation site

http://www.ebi.ac.uk/interpro Fingerprints Several motifs  characterise family Different combinations of motifs describe subfamilies Identify small conserved regions in divergent proteins EXAMPLE: PR00107 Phosphocarrier HPr signature PTHP_ENTFA: His phosphorylation site Ser phosphorylation site MEKKEFHIVAET GIHARPATLLVQTASKF NSDINLEY KGKSVNLK SIMGVMSL GVGQGSDVTITVDGADEAEGMAAIVETLQKEGLAE

http://www.ebi.ac.uk/interpro Fingerprints Several motifs  characterise family Different combinations of motifs describe subfamilies Identify small conserved regions in divergent proteins EXAMPLE: PR00107 Phosphocarrier HPr signature PTHP_ENTFA: His phosphorylation site Ser phosphorylation site Conserved site MEKKEFHIVAET GIHARPATLLVQTASK FNSDINLEY KGKSVNLK SIMGVMSL GVGQGSDVTITVDGADE AEGMAAIVETLQKEGLAE

http://www.ebi.ac.uk/interpro Fingerprints Several motifs  characterise family Different combinations of motifs describe subfamilies Identify small conserved regions in divergent proteins EXAMPLE: PR00107 Phosphocarrier HPr signature PTHP_ENTFA: MEKKEFHIVAET GIHARPATLLVQTASK FNSDINLEY KGKSVNLK SIMGVMSL GVGQGSDVTITVDGADE AEGMAAIVETLQKEGLAE 1) GIHARPATLLVQTASKF 2) KGKSVNLKSIMGVMSL 3) LGVGQGSDVTITVDGADE 3-motif fingerprint

http://www.ebi.ac.uk/interpro Fingerprints Extract motif sequences xxxxxx Sequence alignment Correct order Correct spacing Ser phosphorylation site Conserved site His phosphorylation site Define motifs Fingerprint signature 123 PR 00000

http://www.ebi.ac.uk/interpro Sequence clustering Automatic clustering of homologous domains **Rarely covers entire domain (conserved core) **Signature size can change with release Known domain families Recruit homologous domains PSI-BLAST MKDOM2 Automatic clustering ProDomAlign Align domain families

http://www.ebi.ac.uk/interpro Hidden Markov Models (HMM) Can characterise protein over entire length Models conserved and divergent regions (position-specific scoring) Models insertions and deletions  Outperform in sensitivity and specificity  More flexible (can use partial alignments)

Sequence 1: Sequence 2: Sequence 3: Sequence 4: Sequence 5: Sequence 6: Sequence 7: Sequence alignment Scoring matrix (residue frequency at each position in alignment) Profile Hidden Markov Models (HMM) Bayesian statistics probability scoring

Sequence 1: Sequence 2: Sequence 3: Sequence 4: Sequence 5: Sequence 6: Sequence 7: M = match state M1 Hidden Markov Models (HMM)

Sequence 1: Sequence 2: Sequence 3: Sequence 4: Sequence 5: Sequence 6: Sequence 7: M1 Hidden Markov Models (HMM) M2 M = match state

Sequence 1: Sequence 2: Sequence 3: Sequence 4: Sequence 5: Sequence 6: Sequence 7: M1 Hidden Markov Models (HMM) M2M3 M = match state

Sequence 1: Sequence 2: Sequence 3: Sequence 4: Sequence 5: Sequence 6: Sequence 7: M1 Hidden Markov Models (HMM) M2M3M4M5M6M7M8M9M10M4M5M6M7M8M9M10 M = match state

http://www.ebi.ac.uk/interpro M1M2M3M4M5M6M7M8M9M10M4M5M6M7M8M9M10 I = insert state I1I2I3I4I5I6I7I8I9 D = delete state D2D3D4D5D6D7D8D9 Hidden Markov Models (HMM)

http://www.ebi.ac.uk/interpro Hidden Markov Models (HMM) HMM databases: PIR SUPERFAMILY PANTHER TIGRFAM PFAM SMART SUPERFAMILY GENE3D Domains conserved in sequence Families conserved in sequence Domains conserved in structure

http://www.ebi.ac.uk/interpro SAM Profile HMMs Homologous structural superfamilies Start with single seed sequence Proteins in superfamily may have low sequence identity Few proteins in family have PDB structures Create 1 model for every protein in superfamily  combine results

http://www.ebi.ac.uk/interpro SAM Profile models T99 script: Low identity matches Close homologues WU-BLASTP search Final model Single seed sequence GIHARPATLLVQTASKF Initial model GIHARPATLLVQTASKF GIHARPATLLVQTASKF GIHARPATLLVQTASKF New larger alignment GIHARPATLLVQTASKF GIHARPATLLVQTASKF GIHARPATLLVQTASKF

http://www.ebi.ac.uk/interpro Signatures Methods Pattern Fingerprint Sequence clustering HMM SAM Describe protein features: active sites, binding sites… Describe families and sibling subfamilies Predicts conserved domains

http://www.ebi.ac.uk/interpro Signature Methods Pattern Fingerprint Sequence clustering HMM SAM Functional classification of families Functional domain annotation Structural domain annotation

http://www.ebi.ac.uk/interpro Comprehensive annotation InterPro removes redundancy SWIB/MDM2 domain RanBP2-type zinc finger RING-type zinc finger Domain annotation

http://www.ebi.ac.uk/interpro Comprehensive annotation Conserved site within zinc finger Annotate features

http://www.ebi.ac.uk/interpro Comprehensive annotation Mdm2/Mdm4 family Mdm4 subfamily Parent Child Family classification

http://www.ebi.ac.uk/interpro Domain Boundaries Gene3D (and SSF) determines domain structural boundaries Pfam trims domains to regions of good sequence conservation ProDom displays shortest conserved sequence

http://www.ebi.ac.uk/interpro Fragmented Signatures 4) Non-contiguous domains 3) Repeated elements 2) Duplicated domains 1) Signature method

http://www.ebi.ac.uk/interpro Fragmented Signatures e.g. PRINTS – discrete motifs Signature method 1) Signature method 3) Repeated elements 2) Duplicated domains 4) Non-contiguous domains

http://www.ebi.ac.uk/interpro Fragmented Signatures 1) Signature method Duplicated domains 2) Duplicated domains 3) Repeated elements 4) Non-contiguous domains e.g. SSF - duplication consisting of 2 domains with same fold

http://www.ebi.ac.uk/interpro Fragmented Signatures Repeated elements 3) Repeated elements 2) Duplicated domains e.g. Kringle,WD40 4) Non-contiguous domains 1) Signature method

http://www.ebi.ac.uk/interpro Fragmented Signatures 3) Repeats Non-contiguous domains 4) Non-contiguous domains 2) Duplicated domains 1) Signature method Structural domains can consist of non-contiguous sequence

http://www.ebi.ac.uk/interpro Fragmented Signatures 4) Non-contiguous domains 3) Repeats 2) Duplicated domains 1) Signature method

http://www.ebi.ac.uk/interpro Complementary Annotation  Sequence-based signature (Pfam) shows that the domain is made up of repeating sequence elements Beta-propeller repeat  Structural-based signature (SSF) shows boundaries of structural domain 7-blade beta-propeller

http://www.ebi.ac.uk/interpro Complementary Annotation PFAM shows domain is composed of two types of repeated sequence motifs SUPERFAMILY shows the potential domain boundaries

http://www.ebi.ac.uk/interpro Complementary Annotation GENE3D shows that these domains share homologous structure PFAM/SMART show 2 domains from distinct sequence families

http://www.ebi.ac.uk/interpro Searching InterPro: InterProScan sequence search

http://www.ebi.ac.uk/interpro Searching InterPro http://www.ebi.ac.uk/interpro/ Search tools include: Text Search InterProScan (sequence search)

http://www.ebi.ac.uk/interpro InterPro Text Search Text search box Search using: text protein ID InterPro ID GO term Search results Direct links to entry

http://www.ebi.ac.uk/interpro InterProScan Search Use ftp site to run multiple sequences simultaneously Member database search engines Paste in sequence (protein/nucleotide)

http://www.ebi.ac.uk/interpro InterProScan Search Results single InterPro entry Direct links to entry Direct links to signature databases

http://www.ebi.ac.uk/interpro EXERCISE 1

http://www.ebi.ac.uk/interpro Exploring InterPro entries

http://www.ebi.ac.uk/interpro InterPro Entry Groups similar signatures together Adds extensive annotation Linked to other databases Structural information and viewers Links related signatures

http://www.ebi.ac.uk/interpro Grouping Signatures Together Same positions Different protein hits 2) PFAM PROSITE (100) (50) PFAM PROSITE 1) (100) Same positions Same protein hits IPR000001 IPR000002 IPR000001 IPR000002 IPR000001 IPR000002 Different positions 4) PFAM PROSITE (100) PROSITE PFAM 3)(100) Different positions Same protein hits

http://www.ebi.ac.uk/interpro Extensive Annotation Annotation Fields in InterPro Name and short name List of signatures (links to member databases) Entry type (family, domain, site) Relationships (links related signatures) GO mapping (  large scale classification) Abstract Taxonomy (search/download using taxonomy) Examples Publications

http://www.ebi.ac.uk/interpro Extensive Annotation Annotation Fields in InterPro Name and short name List of signatures (links to member databases) Entry type (family, domain, site) Relationships (links related signatures) GO mapping (  large scale classification) Abstract Taxonomy (search/download using taxonomy) Examples Publications Short names appear in UniProt entries

http://www.ebi.ac.uk/interpro Extensive Annotation Annotation Fields in InterPro Name and short name List of signatures (links to member databases) Entry type (family, domain, site) Relationships (links related signatures) GO mapping (  large scale classification) Abstract Taxonomy (search/download using taxonomy) Examples Publications DomainBiological units with defined boundaries Full-length signatures grouping related proteinsFamily RegionAny signature that doesn’t fit the above Repeat Site Signature repeated as a series of short motifs Protein feature described by a Prosite pattern

http://www.ebi.ac.uk/interpro Links to Other Databases Additional annotation from databases: Blocks (family alignments) IntEnz (enzymes) Prosite documents COME (bioinorganic motifs) CAZy (carbohydrate-active enzymes) IUPHAR (GPCR receptors) CluS-Tr (protein clusters) Pandit (phylogenetic trees of PFAMs) Merops (peptidases & inhibitors)

http://www.ebi.ac.uk/interpro Links to Structural Databases SCOP (structural classification of proteins) CATH (structural classification of proteins) PDB (protein structure databank) List of proteins with structural data PDB database of structures

http://www.ebi.ac.uk/interpro Links to Structural Databases SCOP (structural classification of proteins) CATH (structural classification of proteins) PDB (protein structure databank) Links to structural classification

http://www.ebi.ac.uk/interpro Links to Interaction Databases IntAct (protein-protein interactions) Lists proteins in entry known to be involved in protein-protein interactions IntAct database of interactions

http://www.ebi.ac.uk/interpro Exploring InterPro relationships

http://www.ebi.ac.uk/interpro InterPro Relationships Parent/Child Contains/Found in Hierarchical subdivision into more closely related groups Domain/subdomain composition OverlappingRemaining relationships

http://www.ebi.ac.uk/interpro Link related signatures - relationships 1) Parent - Child (subgroup of more closely related proteins) PFAM (75) (100) SMART Protein kinase Serine kinase PROSITE (25) Tyrosine kinase * PFAM (100)Protein kinase * No proteins in common SMARTPROSITE Parent Children PFAM Protein kinase SMARTPROSITE Serine kinase Tyrosine kinase (IPR000001) (IPR000002)(IPR000003)

http://www.ebi.ac.uk/interpro Relationships – evolutionary context GENE3D Grandparent Parents Children InterPro Relationship Criteria for Signature Structural family PFAM Sequence families TIGRFAM Functional families Unique to InterPro

http://www.ebi.ac.uk/interpro IPR011009 Protein kinase-like IPR000403 PI 3/4 kinase IPR000719 Protein kinase IPR001245 Tyr kinase IPR017442 Ser/Thr kinase-rel IPR015772 TNK1 kin IPR015783 ATMRK kin IPR002575 APH kinase IPR004147 ABC-1 IPR004166 EF2 kinase IPR015275 Actin-fragmin kin IPR015897 CHK kinase IPR002290 Ser/Thr kin IPR015515 GCN2 IPR015771 Hrmn Rcpt IPR015768 Activin Rcpt IPR015769 TGFb2 Rcpt IPR015770 BMPRII IPR015785 MAPK3 kin IPR015787 IL1 kin IPR008350 ERK3 MAPK IPR015732 PSKH kin IPR015733 Ca-dep kin4 IPR015734 Ca-dep kin1 IPR015739 Leu zip kin IPR015740 Plant kin IPR015747 MAPKKK4 IPR015748 MAPKKK3 IPR015749 MAPKKK1 IPR015750 Pak kin IPR015730 Myosin kin IPR008351 JNK kin Example hierarchy: IPR018934 RIO-like kin IPR000687 RIO kin IPR002573 Choline kinase IPR008349 ERK1 kin IPR006748 Hydroxyurea kin IPR009212 MethylTR kin IPR014093 Thiamine kin IPR009330 Lipopoly syn IPR004119 DUF IPR012877 Put kinase

http://www.ebi.ac.uk/interpro Different entries  not redundant Parent/child – evolutionary context

http://www.ebi.ac.uk/interpro Most specific subfamily classification Superfamily classification Parent/child – evolutionary context

http://www.ebi.ac.uk/interpro 2) Contains – Found in PROSITE C-terminal domain SMART N-terminal domain PFAM Receptor family PFAM Receptor Family SMARTPROSITE N-terminal domainC-terminal domain Found in (Pfam) Contains (Smart and Prosite) Link related signatures - relationships (Describes domain composition)

http://www.ebi.ac.uk/interpro 2) Contains – Found in Link related signatures - relationships CoverageSignature must cover the entire (>90%) sequence of contained signature PFAM SMART Contains Found in PFAM SMART Contains Found in

http://www.ebi.ac.uk/interpro 3) Overlapping Link related signatures - relationships All remaining relationships PROSITE SMART Overlapping

http://www.ebi.ac.uk/interpro Exploring InterPro taxonomy

http://www.ebi.ac.uk/interpro InterPro taxonomy Select species- specific protein sets

http://www.ebi.ac.uk/interpro InterPro taxonomy

http://www.ebi.ac.uk/interpro Exploring protein structure in InterPro

http://www.ebi.ac.uk/interpro Structural information PDB Classification Structures CATH SCOP Homology Models Swiss-Model ModBase

http://www.ebi.ac.uk/interpro Structural information CATH and SCOP divide PDB structures into domains Swiss-Model and ModBase predict structure for regions not covered by PDB Note that one domain is discontiguous

http://www.ebi.ac.uk/interpro Sequence-Structure Display Signatures predictive of protein annotation Structural data for specific proteins AstexViewer® for structure

http://www.ebi.ac.uk/interpro Structure Viewer Navigate between structure and sequence Manipulate structures

http://www.ebi.ac.uk/interpro Exploring splice variants in InterPro

http://www.ebi.ac.uk/interpro Other Features – splice variants Splice variants

http://www.ebi.ac.uk/interpro Exploring InterPro Domain Architecture

http://www.ebi.ac.uk/interpro Other Features – domain architecture Select data set of these proteins Each ‘balloon’ represents a linked InterPro domain

http://www.ebi.ac.uk/interpro Protein Sequence Coverage InterPro signatures cover: 95% of UniProt/Swiss-Prot proteins 79% of UniProt/TrEMBL proteins >5 million matches in InterPro ~17,000 InterPro entries >57,500 signature methods

http://www.ebi.ac.uk InterPro Team: InterPro Consortium: Team leader: Sarah Hunter Acknowledgements David Lonsdale Louise Daugherty Jennifer McDowall Craig McAnulla David Binns Ujjwal Das Anthony Quinn John Maslen Manjula Thimma Phil Jones

EBI is an Outstation of the European Molecular Biology Laboratory. InterPro Database Protein Functional Analysis Jennifer McDowall, Ph.D. Senior InterPro.

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EBI is an Outstation of the European Molecular Biology Laboratory. InterPro Database Protein Functional Analysis Jennifer McDowall, Ph.D. Senior InterPro.

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Presentation on theme: "EBI is an Outstation of the European Molecular Biology Laboratory. InterPro Database Protein Functional Analysis Jennifer McDowall, Ph.D. Senior InterPro."— Presentation transcript:

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