Transcript The pattern
Corrections N-linked glycosylation (GlcNac): Look at the Swiss-Prot annotation (in a random ‘glycosylated’ entry) Query: annotation:(type:carbohyd "N-linked (GlcNAc...)" confidence:experimental) reviewed:yes Taxonomic distribution TPNLINDTME Multiple alignment (ClustalW) -[LAPIQ]-N-[HAYRCS]-[ST]-[KLESGM] N-glycosylation does not occur in Bacteria: …false positive ! 301 protein (within the set of 1000 proteins) are N-glycosylated according to the UniProtKB annotation…! Scan Prosite with the official pattern The official pattern also match with bacteria sequences (false positives) PRATT pattern with 20 sequences D-K-T-G-T-[IL]-T-x(3)-[ILMV]-x-[FILV] AT31_HUMAN: SIMILARITY: Belongs to the cation transport ATPase (P-type) family. Type V subfamily. The pattern is a discriminator for ATP ase family (Cation-transporting ) C-x(2,4)-C-x(3)-[LIVMFYWC]-x(8)-H-x(3,5)-H Pattern scan The pattern missed some Zn finger in the same protein i.e. Q24174 Pattern Profile Not found with the pattern The pattern: C - X(2,4) - C - X(3) - [LIVMFYWC] - X(8) - H - X(3,5) – H Should includes: YRCVLCGTVAKSRNSLHSHMSrQHRGIST C-X(2,4)-C-X(3)-[LIVMFYWCA]-X(8)-H-X(3,5)-H Yes ! But: The pattern becomes less restrictive. You get more sequences which should not be here. (As the results are limited to 1000, the number of hits is not the same…) Discriminators (Signatures, descriptors) for the Zinc finger C2H2 type domain can be found in Prosite (Pattern and Profile) and Pfam (HMM) Step 1: scan UniProtKB/Swiss-Prot with the pattern Use the ‘scanprosite’ tool at http://www.expasy.org/tools/scanprosite/ Step 2: Retrieve the matched human entries @ UniProt (go at the end of the Scan Prosite result page: click on ‘Matched UniProtKB entries’) Step 3: Retrieve the sequences annotated as being ‘phosphorylated on a Thr’ Step 3: Retrieve the sequences annotated as being ‘phosphorylated on a Thr’ -> 19 candidates to be manually checked …. InterPro scan results InterPro : other shema (Graphical view from UniProtKB) InterPro shema PFAM Graphical view Prosite Graphical view Blast @ NCBI against Swiss-Prot NCBI: Color key for alignment scores NCBI Swiss-Prot does not contain the alternative sequences (i.e. P28175-2) – !! NCBI gives the ‘version number’ of the Swiss-Prot sequence (i.e. Q8BU25.2)…. UniProt: Color code for identity scores (not alignment !) UniProt: Color code for identity scores (not alignment !) ProDom database List of proteins sharing at least a common domain… 1) BLAST at www.uniprot.org 2) PROSITE tools You are lucky: domains are rarely not annotated in the different domain/family databases ! 3) Construct a profile with My hits at SIB Use PSI Blast Do a PSI BLAST against UniProtKB Select sequence with a E value > 0.001 and do a second cycle Look at the MSA Construct a profile with the MSA The profile The profile hits Construct a HMM with the MSA The HMM The HMM hits - Look at the Goloco data in InterPro. How many proteins (and/or hits) are found by the different methods ? http://www.ebi.ac.uk/interpro/ According to InterPro: Goloco domain is described by at least one of the different methods (PFAM, Prosite, Smart) PFAM: 167 proteins Prosite: 192 proteins SMART: 1 proteins These different numbers are the consequence of the interval between the different releases of the different databases (including the sequence databases (UniProtKB). It may also be due to the different methods used (HMM, profile…) Look for the HMM for the Goloco domain in PFAM Look for the HMM for the Goloco domain in PFAM Download the HMM matrix the HMM matrix