InterPro Sandra Orchard

Why do we need predictive annotation tools?

what are these proteins; to what family do they belong? 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? 3

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 2. The protein signature approach 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 relationships with previously characterised sequences This is the approach taken by protein signature databases 4

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

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)

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

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 cysteines C-{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

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

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 1 2 3 4 5 order interval

Models insertions and deletions Profiles & HMMs Entire domain Define coverage Whole protein Sequence alignment Use entire alignment for domain or protein xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxxxxx Build model Models insertions and deletions This is a good summary of how Profiles and HMMs are made. Take a multiple sequence alignment and either use the entire alignment (family model) or define the domain of interest (domain model). If a domain model, then extract the sequence from the alignment defining the domain. Use the alignment to build a Profile matrix or an HMM. A signature match is either non-positional and defines family membership, or it defines the position of the domain on the protein. The view of a Profile or HMM hit in InterPro. Profile or HMM signature 11

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

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

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

InterPro hierarchies: 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

InterPro hierarchies: Domains DOMAINS can have parent/child relationships with other domains

Domains and Families may be linked through Domain Organisation Hierarchy 17

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

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 TALK MORE ABOUT HOW WE DO GO MAPPING IN INTERPRO

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

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

Searching InterPro

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

Searching InterPro

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/

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