ebi is an outstation of the european molecular biology laboratory. uniprotkb sandra orchard

EBI is an Outstation of the European Molecular Biology Laboratory.

UniProtKB

Sandra Orchard

Importance of reference protein sequence databases

• Completeness and minimal redundancy

A non redundant protein sequence database, with maximal coverage including splice isoforms, disease variant and PTMs.

Low degree of redundancy for facilitating peptide assignments

• Stability and consistency Stable identifiers and consistent nomenclature

Databases are in constant change due to a substantial amount of work to improve their completeness and the quality of sequence annotation

• High quality protein annotation

Detailed information on protein function, biological processes, molecular interactions and pathways cross-referenced to external source

Summary of protein sequence databases

Database Description Species

UniProtKB Expertly curated section (UniProtKB/Swiss-Prot) and computer-annotated section (UniProtKB/TrEMBL); minimum level of redundancy; high level of integration with other databases; stable identifiers; diversity of sources including large scale genomics, small scale cloning and sequencing, protein sequencing, PDB, predicted sequences from Ensembl and RefSeq

Many

UniRef100 Assembled from UniProtKB, Ensembl and RefSeq; merges 100% identical sequences; stable identifiers

Many

Ensembl Predictions using automated genome annotation pipeline; explicitly linked to nucleotide and protein sequences; stable reference; merge their annotations with Vega annotations at transcript level; extensive quality checks to remove erroneous gene models ; high level of integration with other databases

Over 50 Eukaryotic genomesEnsembl Genomes: Metazoa, Plants and Fungi, Protists, Bacteria and Archaea

RefSeq NCBI creates from existing data; ongoing curation; non-redundant; explicitly linked nucleotide and protein sequences; stable reference; high level of integration with other databases

Limited to fully sequenced organisms

Entrez protein (NCBInr) Assembled from GenBank and RefSeq coding sequence translations and UniProt KB ; annotations extracted from source curated databases; high degree of sequence redundancy

Many

Updated from Nesvizhskii, A. I., and Aebersold, R. (2005) Interpretation of shotgun proteomic data: the protein inference problem. Mol. Cell. Proteomics. 4,1419–1440l

UniProtKB

Master headline

UniProt Knowledgebase: 2 sections

1. UniProtKB/Swiss-Prot Non-redundant, high-quality manual annotation - reviewed

2. UniProtKB/TrEMBL Redundant, automatically annotated - unreviewed

www.uniprot.org

Sequence Sequence features

Ontologies

ReferencesNomenclature

Splice variants

Annotations

UniProtKB

Manual annotation of UniProtKB/Swiss-Prot

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Sequence curation, stable identifiers, versioning and archiving

For example – erroneous gene model predictions, frameshifts….

..premature stop codons, read-throughs, erroneous initiator methionines…..

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Splice variants

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Identification of amino acid variants

..and of PTMs

… and also

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Domain annotation

Binding sites

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Protein nomenclature

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Controlled vocabularies used whenever possible…

Annotation - >30 defined fields

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..and also imported from external resources

Binary interactions taken from the IntAct database

Interactors of human p53

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Controlled vocabulary usage increasing – for example from the Gene Ontology

Annotation for human Rhodopsin

1 Evidence at protein levelThere is experimental evidence of the existence of a protein

(e.g. Edman sequencing, MS, X-ray/NMR structure, good quality protein-protein interaction , detection by antibodies)

2 Evidence at transcript levelThe existence of a protein has not been proven but there is expression data (e.g. existence of cDNAs, RT-PCR or Northern blots)

that indicates the existence of a transcript.

3 Inferred from homologyThe existence of a protein is likely because orthologs exist in closely related species

4 Predicted

5 Uncertain

Sequence evidence

Type of evidence that supports the existence of a protein

Manual annotation of the human proteome(UniProtKB/Swiss-Prot)

• A draft of the complete human proteome has been available in UniProtKB/Swiss-Prot since 2008

• Manually annotated representation of 20,231 protein coding genes with 36,865 protein sequences - an additional 33,243 UniProtKB/TrEMBL form the complete proteome set

• Approximately 67,600 single amino acid polymorphisms (SAPs), mostly disease-linked

• ~75,500 post-translational modifications (PTMs)• Close collaboration with NCBI, Ensembl, Sanger Institute

and UCSC to provide the authoritative set to the user community

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Searching UniProt – Simple Search

• Text-based searching• Logical operators ‘&’ (and), ‘|’

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Searching UniProt – Advanced Search

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Searching UniProt – Search Results

Each linked to the UniProt entry

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Searching UniProt – Search Results

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Searching UniProt – Search Results

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Searching UniProt – Blast Search

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Searching UniProt – Blast Search

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Searching UniProt – Blast Results

Alignment with query sequence

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Searching UniProt – Blast Results

UniProtKB/TrEMBL

Multiple entries for the same protein (redundancy) can arise in UniProtKB/TrEMBL due to:

o Erroneous gene model predictionso Sequence errors (Frame shifts)o Polymorphismso Alternative start siteso Isoforms

Apart from 100% identical sequences all merged sequences are analysed by a curator so they can be annotated accordingly.

Why do we need predictive annotation tools?

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UniProtKB

UniProtKB/Swiss-Prot

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• Automated clean-up of annotation from original nucleotide sequence entry

• Additional value added by using automatic annotation

• Recognises common annotation belonging to a

closely related family within UniProtKB/Swiss-Prot

• Identifies all members of this family using pattern/motif/HMMs in InterPro

• Transfers common annotation to related family members in TrEMBL

Automatic Annotation

← Name (non-standard)

← Taxonomy

← Publication

← Sequence

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InterPro

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Finding a complete proteome in UniProtKB

Complete Proteomes

MS Proteomics

• Require each sequence (inc isoforms) to be present in the dataset as an separate entity for search engines to access

• For higher organisms, with isoforms, expanded set made available on ftp site

• Fasta files by FTP• One file per species containing canonical + isoform sequences

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