InterPro

1. Sandra Orchard InterPro

2. Why do we need predictive annotation tools?

3. Given a set of uncharacterised sequences, we usually want to know: • what are these proteins; to what family do they belong? • what is their function; how can we explain this in structural terms?

4. 2. The protein signature approach 1. Pairwise alignment approaches (e.g. BLAST) • Good at recognising similarity between closely related sequences • Perform less well at detecting divergent homologues • Alternatively, we can model the conservation of amino acids at specific positions within a multiple sequence alignment, seeking ‘patterns’ across closely related proteins • We can then use these models to infer relationshipswith previously characterised sequences • This is the approach taken by protein signature databases

5. Multiple sequence alignment What are protein signatures? Protein family/domain Build model Search UniProt Protein analysis Significant match ITWKGPVCGLDGKTYRNECALL Mature model AVPRSPVCGSDDVTYANECELK

6. Diagnostic approaches (sequence-based) Single motif methods Regex patterns (PROSITE) Full domain alignment methods Profiles (Profile Library) HMMs (Pfam) Multiple motif methods Identity matrices (PRINTS)

7. Motif Define pattern xxxxxx xxxxxx xxxxxx xxxxxx Extract pattern sequences Build regular expression C-C-{P}-x(2)-C-[STDNEKPI]-x(3)-[LIVMFS]-x(3)-C Pattern signature PS00000 Patterns Sequence alignment

8. Patterns Advantages • Some aa can be forbidden at some specific positions which can help to distinguish closely related subfamilies • Short motifs handling - a pattern with very few variability and forbidden positions, can produce significant matches e.g. conotoxins: very short toxins with few conserved cysteinesC-{C}(6)-C-{C}(5)-C-C-x(1,3)-C-C-x(2,4)-C-x(3,10)- C Drawbacks • High False Positive/False Negative rate Patterns are mostly directed against functional residues: active sites, PTM, disulfide bridges, binding sites

9. Motif 1 Motif 2 Motif 3 Define motifs xxxxxx xxxxxx xxxxxx xxxxxx xxxxxx xxxxxx xxxxxx xxxxxx xxxxxx xxxxxx xxxxxx xxxxxx Extract motif sequences Correct order Fingerprint signature 1 2 3 Correct spacing PR00000 Fingerprints Sequence alignment Weight matrices

10. 1 2 3 4 5 The significance of motif context • Identify small conserved regions in proteins • Several motifs  characterise family • Offer improved diagnostic reliability over single motifs by virtue of the biological context provided by motif neighbours order interval

11. Profiles & HMMs Whole protein Sequence alignment Entire domain Define coverage xxxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxxxxx Use entire alignment for domain or protein xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx Models insertions and deletions Build model Profile or HMM signature

12. HMM databases • Sequence-based • PIR SUPERFAMILY: families/subfamilies reflect the evolutionary relationship • PANTHER: families/subfamilies model the divergence of specific functions • TIGRFAM: microbial functional family classification • PFAM : families & domains based on conserved sequence • SMART: functional domain annotation • Structure-based • SUPERFAMILY : models correspond to SCOP domains • GENE3D: models correspond to CATH domains

13. Why we created InterPro • By uniting the member databases, InterPro capitalises on their individual strengths, producing a powerful diagnostic tool & integrated database • to simplify & rationalise protein analysis • to facilitate automatic functional annotation of uncharacterised proteins • to provide concise information about the signatures and the proteins they match, including consistent names, abstracts (with links to original publications), GO terms and cross-references to other databases

14. InterPro Entry Groups similar signatures together Adds extensive annotation Adds extensive annotation Links to other databases Links to other databases Structural information and viewers • Hierarchical classification

15. InterProhierarchies: Families FAMILIES can have parent/child relationships with other Families • Parent/Child relationships are based on: • Comparison of protein hits • child should be a subset of parent • siblings should not have matches in common • Existing hierarchies in member databases • Biological knowledge of curators

16. InterProhierarchies: Domains DOMAINS can have parent/child relationships with other domains

17. Domains and Families may be linked through Domain Organisation Hierarchy

18. InterPro Entry Groups similar signatures together Adds extensive annotation Adds extensive annotation Links to other databases Links to other databases Structural information and viewers

19. InterPro Entry Groups similar signatures together Adds extensive annotation Adds extensive annotation Links to other databases Links to other databases Structural information and viewers The Gene Ontology project provides a controlled vocabulary of terms for describing gene product characteristics

20. InterPro Entry Groups similar signatures together Adds extensive annotation Adds extensive annotation Links to other databases Links to other databases Structural information and viewers UniProt KEGG ... Reactome ... IntAct ... UniProt taxonomy PANDIT ... MEROPS ... Pfam clans ... Pubmed

21. InterPro Entry Groups similar signatures together Adds extensive annotation Adds extensive annotation Links to other databases Links to other databases Structural information and viewers PDB 3-D Structures SCOP Structural domains CATH Structural domain classification

22. Searching InterPro

23. Searching InterPro Protein family membership Domain organisation Domains, repeats & sites GO terms

24. Searching InterPro

26. InterProScan access Interactive: http://www.ebi.ac.uk/Tools/pfa/iprscan/ Webservice (SOAP and REST): http://www.ebi.ac.uk/Tools/webservices/services/pfa/iprscan_rest http://www.ebi.ac.uk/Tools/webservices/services/pfa/iprscan_soap Download: ftp://ftp.ebi.ac.uk/pub/software/unix/iprscan/

27. ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? Master headline