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Corrections
- The cacao genome is currently being sequenced
http://www.ncbi.nlm.nih.gov/genomes/leuks.cgi
- Human Chromosome 1 sequence
Search ‘Genome’ with ‘homo sapiens chromosome 1’
RefSeq has an entry for each human chromosome (genome reference):
these entries are numbered NC_000001 to NC_000024
Mapviewer view: http://www.ncbi.nlm.nih.gov/mapview/maps.cgi?taxid=9606&chr=1
Entrez nucleotide view: http://www.ncbi.nlm.nih.gov/nuccore/224589800
- Measles virus complete genome (RefSeq)
http://www.ncbi.nlm.nih.gov/nuccore/NC_001498
- Nucleic acid sequences available for human erythropoietin (EPO) (GenBank and RefSeq)
http://www.ncbi.nlm.nih.gov/nucleotide/?term=homo+sapiens+erythropoietin+EPO
mRNA coding for human EPO (in RefSeq)
http://www.ncbi.nlm.nih.gov/nuccore/NM_000799.2
Look for Escherichia coli strain K-12 substrain W3110 complete genome
(Query @ NCBI 'Genome')
Query ‘Genome’ with ‘Escherichia coli strain K-12 substrain
W3110’
http://www.ncbi.nlm.nih.gov/sites/entrez?Db=genome&Cmd=ShowDetailView&TermToSearch=19221
CU466930.1
• http://www.ncbi.nlm.nih.gov/nuccore/167729007
Corresponding proteins in UniProtKB (query with CU466930):
http://www.uniprot.org/uniprot/?query=CU466930&sort=score
Corresponding proteins in NCBInr (query ‘nucleotide’ with CU466930 and follow the link to
‘protein’
http://www.ncbi.nlm.nih.gov/nuccore?Db=protein&DbFrom=nuccore&Cmd=Link&LinkNam
e=nuccore_protein&IdsFromResult=167729007
AAFZ00000000.1
• http://www.ncbi.nlm.nih.gov/nuccore/60175893
No protein yet : no annotated CDS available (or not yet submitted by the authors)!
EPO protein in different databases
• UniProtKB/Swiss-Prot (reviewed)
http://www.uniprot.org/uniprot/P01588
• UniProtKB/TrEMBL (unreviewed)
http://www.uniprot.org/uniprot/B7ZKK5
• UniParc (follow the link from the UniProtKB sequence section)
http://www.uniprot.org/uniparc/UPI0000033477
•
NCBInr (see next slide)
http://www.ncbi.nlm.nih.gov/protein/?term=homo+sapiens+erythropoietin+EPO
• RefSeq (see next slide)
http://www.ncbi.nlm.nih.gov/protein/NP_000790.2?
•
Ensembl (follow the link from the UniProt entry)
http://www.ensembl.org/Homo_sapiens/Transcript/ProteinSummary?g=ENSG00000130427;r=7:100318423-100321323;t=ENST00000252723
http://www.uniprot.org/uniprot/P04150
- Which server? UniProt
- Which database? UniProtKB/Swiss-Prot
- What is the function of the protein?
Receptor for glucocorticoids (GC).
- How many different post-translational modifications (PTMs) ?
Phosphorylated, Sumoylated, Ubiquitinated
- How many phosphorylation sites? 10 sites
- What are the associated GO terms?
http://www.uniprot.org/uniprot/P04150#section_terms
- What is the evidence for the existence of the protein(s)?
http://www.uniprot.org/uniprot/P04150#section_attribute: at protein level
- What is the corresponding mRNA?
X03225 for example
- How many different protein sequences are available for the corresponding gene?
http://www.uniprot.org/uniprot/P04150#section_alternative: 9 isoforms
http://www.ncbi.nlm.nih.gov/protein/NP_000167.1?
- Which server? NCBI
- Which database? RefSeq
- What is the function of the protein?
receptor for glucocorticoids
- How many different post-translational modifications (PTMs) ?
Phosphorylated, Sumoylated, Ubiquitinated
- How many phosphorylation sites? 14 sites
- What are the associated GO terms?
Not directly available
- What is the evidence for the existence of the protein(s)?
This information is not available
- What is the corresponding mRNA?
‘The reference sequence was derived from AC091925.3, X03225.1 and AC004782.1. ‘
- How many different protein sequences are available for the corresponding gene?
This information is not directly available; go to Entrez Gene.
http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=Retrieve&dopt=full_report&list_uids=2908
Find the UniProt entry corresponding to:
- RefSeq NP_036231
- GI:584682
- Find the GI numbers corresponding to
UniProtKB P04150
- Why are there so many GI numbers ?
Find the UniProt entry corresponding to:
- GI:584682
- Find the GI numbers corresponding to
UniProtKB P04150
- Why are there so many GI numbers ?
Find the GI numbers corresponding to
UniProtKB P04150
Why are there so many GI numbers ?
- Because there are several protein sequences
corresponding to this gene due to alternative
splicing events etc.; sequences have also been
updated/modified;
- The merging policy at NCBI (one sequence one
entry) is not same as the one at UniProt (one
entry one gene one species)
Look for the URL corresponding to the following queries:
- Mouse proteins localized in the nucleus
Query: taxonomy:"Mus musculus [10090]" AND annotation:(type:location nucleus)
URL:http://www.uniprot.org/uniprot/?query=taxonomy%3A%22Mus+musculus+[10090]%22+AND+annotation%3A%28type%3Alocation+nucleus%29&sort=score
- Proteins for which a 3D structure is known (Hint: they have a cross-reference to PDB)
Query: database:(type:pdb)
URL: http://www.uniprot.org/uniprot/?query=database%3A%28type%3Apdb%29&sort=score
- What is the query corresponding to this URL:
http://www.uniprot.org/uniprot/?query=taxonomy%3A9606+AND+keyword%3A"complete+proteome“
Query: taxonomy:9606 AND keyword:"complete proteome“
-> human complete proteome
- Modify the URL to get the same result, but for Escherichia coli (strain K12)
Query: taxonomy:83333 AND keyword:"complete proteome“
URL: http://www.uniprot.org/uniprot/?query=taxonomy%3A83333+AND+keyword%3A"complete+proteome“
- Yeast (Saccharomyces cerevisiae) proteins found in the nucleus in UniProtKB/Swiss-Prot.
Query: organism:4932 AND annotation:(type:location AND nucleus) AND reviewed:yes
- How many of them have a nuclear localization which is 'experimentally proven' ?
Query: organism:"Saccharomyces cerevisiae [4932]" AND
annotation:(type:location nucleus confidence:experimental) AND reviewed:yes
1655 and 1392 entries respectively (query done in January, the 19th)
- Download the list of corresponding accession numbers / protein names / gene names
(use 'customize display').
- The set might not be complete:
not all proteins have been tested to be localized in the nucleus ! And UniProtKB might not
have annotated all the experiments showing that the yeast proteins are nuclear.
P00001 @ UniProtKB
P00001 @ NCBInr
An 'old' publication cites a protein sequence with accession number (AC) o00597: Could you find it ?
You did a proteomics analysis in December 2007 without any match.
You repeat the analysis in April 2008 and get entry with AC P0C6S9 as the best match. Why ?
P0C6S9 was first created in April 2008 in UniProtKB
Compare the GO terms associated with mouse and human erythropoeitin (EPO)
Have a look to the different GO evidence tags
How many GO terms have been 'inferred by direct assay (IDA)' to the human EPO gene ?
hierarchy of the GO term 'apoptosis'
These GO terms are associated with
insulin !
Several proteins have been identified in a proteomic experiment.
Which GO terms do they share?
(GI numbers of the identified proteins: 16130093, 20664033, 1789812, 89110178, 85677033, 27574045, 89111003, 229597766).
GO terms in common…
Searching databases with Blast
The ‘alternative’ sequence(s)
not ‘directly available’ for a lot of tools, including
protein identification tools, Blast, depending on
the server !….
Murcia, February, 2011
Protein Sequence Databases
Blast P04150 against Swiss-Prot / homo sapiens @ UniProt
Isoform
sequences
Murcia, February, 2011
Protein Sequence Databases
Blast P04150 against Swiss-Prot / homo sapiens @ NCBI
The isoform sequences (from Swiss-Prot) are not present in
the NCBI protein database !
The .x number (P06401.4) correspond to the version number
of the sequence…not to an alternatively spliced sequence !
Murcia, February, 2011
Protein Sequence Databases
Blast
A tool associated with the standard
options to search sequences
in UniProt databases
The metal binding (Fe) site is conserved
between HBB human and pea
leghemoglobin!
Detailed BLAST results
Murcia, February, 2011
Protein Sequence Databases
InterPro : other shema (Graphical view from UniProtKB)
InterPro shema
PFAM Graphical view
Prosite Graphical view
Not kepted in the InterPro overview !
Do a Blast with the sequence of the domain 'C-type lectin' of protein P28175 against UniProtKB/Swiss-Prot.
Discuss the overview of your results. Look at the other domains present in the most similar sequences.
Blast p28175 (complete seq) @ Swiss-Prot
UniProt: Color code for identity scores (not alignment !)
UniProt: Color code for identity scores (not alignment !)
Blast p28175 (complete seq) @ 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)….
N-linked glycosylation (GlcNac):
Look at the Swiss-Prot annotation (in a random ‘glycosylated’ entry)
Query UniProtKB
Taxonomic distribution
TPNLINDTME
Multiple alignment (ClustalW)
-[LAPIQ]-N-[HAYRCS]-[ST]-[KLESGM]
Logo
N-glycosylation does not occur in Bacteria: …false positive !
28 protein (within the set of 1000 proteins) are glycosylated according
to the UniProtKB annotation…!
Not easy to find that there is never a P after the N glycvosylation site
(needs a lot of sequences….) !
C-x(2,4)-C-x(3)-[LIVMFYWC]-x(8)-H-x(3,5)-H
Pattern scan
The pattern missed a second Zn finger in the same protein
i.e. Q24174
Pattern
Profile
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.
But 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)
Doublecortin
Kinase
The doublecortin domain is
associated with many different
domains (not only kinase)
Seq 1
Seq 2
Patient with cardiovascular disease
Lost of the protein kinase ATP binding site !
Step 1: scan UniProtKB/Swiss-Prot with the pattern
Use the ‘scanprosite’ tool at http://www.expasy.org/tools/scanprosite/
At the bottom of the Scan prosite result page:
Step 2: Retrieve the 103 human entries @ UniProt
(go at the bottom of the Scan Prosite result page; Matched UniProtKB entries)
Step 3: Retrieve the sequences annotated as being
‘phosphorylated on a Thr’
-> 19 candidates to be manually checked ….
The end