1. InterPro
Sandra Orchard

2. Why do we need predictive annotation tools?

3. 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

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
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

5. What are protein signatures?
Protein family/domain
Build model
Multiple sequence alignment
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. 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

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 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

9. 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

10. 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

11. 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

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. 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

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

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

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
TALK MORE ABOUT HOW WE DO GO MAPPING IN INTERPRO

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:
Webservice (SOAP and REST):
Download:
ftp://ftp.ebi.ac.uk/pub/software/unix/iprscan/

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