ebi is an outstation of the european molecular biology laboratory. alex mitchell interpro team...
TRANSCRIPT
EBI is an Outstation of the European Molecular Biology Laboratory.
Alex MitchellInterPro [email protected]
Using InterPro for functional analysis of protein sequences
• 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?
• Good at recognising similarity between closely related sequences
• Perform less well at detecting divergent homologues
Pairwise alignment approaches (e.g., BLAST)
The protein signature approach
• We can then use these models to infer relationships with previously characterised sequences
• This is the approach taken by protein signature databases
• They go about this in 3 different ways...
• Alternatively, we can model the conservation of amino acids at specific positions within a multiple sequence alignment, seeking ‘patterns’ across closely related proteins
What are protein signatures?
Multiple sequence alignment
Protein family/domainBuild model
Search
Mature model
ITWKGPVCGLDGKTYRNECALL
AVPRSPVCGSDDVTYANECELK
UniProt
Significant match
Protein analysis
Full domain alignment methods
Single motif methods
Multiple motif methods
Regex patterns (PROSITE)
Profiles (Profile Library)
HMMs (Pfam)
Identity matrices (PRINTS)
Diagnostic approaches (sequence-based)
Patterns
Extract pattern sequencesxxxxxxxxxxxxxxxxxxxxxxxx
Sequence alignment
MotifDefine pattern
Pattern signature
C-C-{P}-x(2)-C-[STDNEKPI]-x(3)-[LIVMFS]-x(3)-CBuild regular expression
PS00000
Patterns
Patterns are mostly directed against functional residues: active sites, PTM, disulfide bridges, binding sites
• Anchoring the match to the extremity of a sequence<M-R-[DE]-x(2,4)-[ALT]-{AM}
• 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
• Simple but less powerful
Advantages
Fingerprints
Sequence alignment
Correct order
Correct spacing
Motif 2 Motif 3Motif 1Define motifs
Fingerprint signature 1 2 3
PR00000
Extract motif sequences
xxxxxxxxxxxxxxxxxxxxxxxx
xxxxxxxxxxxxxxxxxxxxxxxx
xxxxxxxxxxxxxxxxxxxxxxxx
Weight matrices
The significance of motif context
order
interval
• 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
Profiles & HMMs
Sequence alignment
Entire domainDefine coverage
Whole protein
Use entire alignment for domain or protein xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx
xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx
xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx
Build model Models insertions and deletions
Profile or HMM signature
• PROSITE domains: high quality manually curated seeds (using biologically characterized UniProtKB/Swiss-Prot entries), documentation and annotation rules. Oriented toward functional domain discrimination.
• HAMAP families: manually curated bacterial, archaeal and plastid protein families (represented by profiles and associated rules), covering some highly conserved proteins and functions.
PROSITE and HAMAP profiles:a functional annotation perspective
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
Structuraldomains
Hidden Markov Models Finger-Prints
Profiles Patterns
Functional annotation of families/domains Protein features
(sites)
InterPro Entry
Adds extensive annotation
Links to other databases
Structural information and viewers
Groups similar signatures together
Adds extensive annotation
Links to other databases
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 Entry
Adds extensive annotation
Links to other databases
Structural information and viewers
Groups similar signatures together
Adds extensive annotation
Links to other databases
InterPro Entry
Adds extensive annotation
Links to other databases
Structural information and viewers
Groups similar signatures together
Adds extensive annotation
Links to other databases
The Gene Ontology project provides a controlled vocabulary of terms for
describing gene product characteristics
InterPro Entry
Adds extensive annotation
Links to other databases
Structural information and viewers
Groups similar signatures together
Adds extensive annotation
Links to other databases
UniProt
KEGG ... Reactome ... IntAct ...
UniProt taxonomy
PANDIT ... MEROPS ... Pfam clans ...
Pubmed
InterPro Entry
Adds extensive annotation
Links to other databases
Structural information and viewers
Groups similar signatures together
Adds extensive annotation
Links to other databases
PDB 3-D Structures
SCOP Structural domains
CATH Structural domain classification
Interactive:http://www.ebi.ac.uk/Tools/pfa/iprscan/
Webservice (SOAP and REST):http://www.ebi.ac.uk/Tools/webservices/services/pfa/iprscan_resthttp://www.ebi.ac.uk/Tools/webservices/services/pfa/iprscan_soap
Downloadable:ftp://ftp.ebi.ac.uk/pub/software/unix/iprscan/
InterProScan access
• Large volumes of data can be queried efficiently
• The interface is shared with many other bioinformatics resources
• It allows federation with other databases:
PRIDE (mass spectrometry-derived proteins and peptidesREACTOME (biological pathways)
BioMart Search
BioMart allows more powerful and flexible queries
BioMart Search
1) Choose Dataseta. Choose InterPro BioMart
b. Choose InterPro entries or protein matches
BioMart Search
4) Choose additional Dataset (optional) This is where you link results to Pride and Reactome