Nucleic Acids Research Advance Access published online on November 11, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp987
© The Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Gene3D: merging structure and function for a Thousand genomes
Jonathan Lees,
Corin Yeats*,
Oliver Redfern,
Andrew Clegg and
Christine Orengo
Department of Biochemistry and Molecular Biology, University College London, Gower St. London, WC1 6BT, UK
*To whom correspondence should be addressed. Tel: +207 679 3890; Fax: +207 679 7193; Email: yeats{at}biochem.ucl.ac.uk
Received September 15, 2009. Revised October 15, 2009. Accepted October 16, 2009.
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ABSTRACT
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Over the last 2 years the Gene3D resource has been significantly
improved, and is now more accurate and with a much richer interactive
display via the Gene3D website (
http://gene3d.biochem.ucl.ac.uk/).
Gene3D provides accurate structural domain family assignments
for over 1100 genomes and nearly 10 000 000 proteins. A hidden
Markov model library, constructed from the manually curated
CATH structural domain hierarchy, is used to search UniProt,
RefSeq and Ensembl protein sequences. The resulting matches
are refined into simple multi-domain architectures using a recently
developed in-house algorithm, DomainFinder 3 (available at:
ftp://ftp.biochem.ucl.ac.uk/pub/gene3d_data/DomainFinder3/).
The domain assignments are integrated with multiple external
protein function descriptions (e.g. Gene Ontology and KEGG),
structural annotations (e.g. coiled coils, disordered regions
and sequence polymorphisms) and family resources (e.g. Pfam
and eggNog) and displayed on the Gene3D website. The website
allows users to view descriptions for both single proteins and
genes and large protein sets, such as superfamilies or genomes.
Subsets can then be selected for detailed investigation or associated
functions and interactions can be used to expand explorations
to new proteins. Gene3D also provides a set of services, including
an interactive genome coverage graph visualizer, DAS annotation
resources, sequence search facilities and SOAP services.
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INTRODUCTION
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Gene3D uses the manually curated protein domain assignments
from CATH (
1) to generate accurate protein domain architecture
predictions for over 1000 genomes, as well as the UniProt (
2)
and RefSeq (
3) protein sequence databases. These assignments
can be queried, and the results visualized via the Gene3D website.
Also displayed are other structural features, such as active
sites and disordered regions from Disopred2 (
4), and functional
terms from UniProt, InterPro (
5), the Gene Ontology (
6), KEGG
(
7) and others (
Table 1). The website enables complex sub-querying
with these terms, giving the user the ability to retrieve information
both for global studies and detailed analyses of function by
molecular biologists. Gene3D also provides multiple web services,
including DAS feature annotation sources and a set of SOAP XML
services.
The CATH domain database provides a compendium of domains found
in the protein structures deposited in the wwPDB (
8), grouping
them into a hierarchy based on homology relationships and structural
features. Domain boundaries and superfamily assignments are
determined using manual curation and automated evidence-gathering
tools. For each superfamily (the CATH ‘H(omology)-level’)
one or more representatives are selected and a set of sequence
profiles generated. From these hidden Markov model (Hmmer)3
(
http://hmmer.janelia.org/) models are built and, in combination
with a recently developed in-house method for resolving conflicting
matches (DomainFinder 3), accurate domain architectures are
assigned. DomainFinder 3 is a novel algorithm that uses heaviest-weighted
clique finding to optimize the selection of matches into a multi-domain
architecture, and has significantly improved the overall quality
of the assignments (paper submitted).
Over the last 2 years, the underlying technology and software powering the Gene3D resource have been re-implemented, allowing faster generation of releases, easier updates of imported annotations and quicker, more sophisticated querying. These and other improvements are described in the following sections.
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IMPROVED DOMAIN ASSIGNMENT ACCURACY
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A total of 10 013 non-redundant domain representatives were
selected for all 2386 superfamilies in CATH v.3.3.0, and profiles
built by using each sequence to seed a SAM Target-2K (
9) iterative
search (strategy 10). A mask was added to the resulting sequence
alignment, so that only columns that align with the original
seed sequence are used to build a profile HMM with HMMER 3’s
‘hmmbuild’. The Gene3D v9 sequence database was
generated by merging UniProt, RefSeq and Ensembl 55 (
11), creating
a set of 9.5 million distinct protein sequences; ‘hmmsearch’
was then used to find matches with
E-value <0.001.
Once all significant matches have been gathered, DomainFinder is used to resolve them into a set of confident domain assignments. DomainFinder has been significantly redeveloped (version 3; paper submitted). This version significantly reduces the number of false negatives (missed domains) by using a more sophisticated representative match selection protocol based around searching graphs of overlapping matches with the exact-weighted clique finding algorithm of Cliquer (12). This translated into a 15% increase in the number of domains confidently identified by Gene3D in large-scale sequence databases.
For Gene3D v9.0.0, a match overlap of 30 or less residues was permitted, while if a sub-graph of matches contained more than 5900 nodes, then the old algorithm was used to reduce computational time (<0.005% of all sequences affected). In total, 9 050 048 domains were identified in 5 186 382 million sequences, a mean of 1.7 domains per annotated sequence, and an overall sequence coverage of 55%. By using the benchmark set generated for testing DomainFinder, we estimated the minimum and maximum bounds for the rate of false-positive predictions for Gene3D (for details see Supplementary Data) to be between 0.2% and 0.6% of all domains. However, many of these ‘false positives’ may signify complex relationships between two superfamilies or errors in the CATH classification due to incomplete information.
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A NEW WEBSITE FOR LARGE AND SMALL QUERIES
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The Gene3D website supports a wide range of query terms, from
protein and gene identifiers to superfamily codes, species names
and function codes. It returns either a detailed description
of the protein of interest or a summary view of the selected
set of proteins. Previously, the size of a returned set of proteins
was restricted to 5000, meaning that only subsets of the biggest
superfamilies could be retrieved. The new site is now capable
of retrieving annotations, species assignments and architectures
for the huge P-loop hydrolase superfamily (34 050 300), containing
over 530 000 domains in 440 000 sequences—although a few
seconds can be required to render some of the results tabs in
the browser.
Both the single protein and protein collection (i.e. a family or genome) views present a similar set of integrated information grouped into five categories: (i) taxonomic and genome data; (ii) sequence features and domain architectures; (iii) molecular and biochemical function; (iv) interactions and pathways; and (v) sequence database cross-references. The querying system then allows the user to select identifiers—for instance, the superfamily a protein domain belongs to, or proteins in the human genome with a specific GO term—and retrieve that subset or superset for further investigation. A query chain can be built up and followed by means of the ‘breadcrumb trail’ on the front summary tab in the results views. Imported annotations and descriptions, such as Pfam families (13), are also linked to the source database.
New genome assemblies and displays
The Ensembl Metazoa genome assemblies have been added to the Gene3D sequence database (currently Ensembl 55). All transcripts are included and individually scanned. Users may query using Ensembl gene, transcript and protein identifiers; mappings to UniProt and RefSeq identifiers are also shown. Gene3D annotates two sets of sequenced genomes: eukaryotic transcripts from Ensembl metazoan (49 species) and UniProt-based assemblies for archaea, bacteria and eukaryotes (1085 species) from Integr8 (14). In addition, a new dynamic web application for querying and visualizing genome coverage for specific superfamilies and genomes is available at: http://gene3d.biochem.ucl.ac.uk:8090/GenomeCoverageGraphs (Figure 1). As can be seen, coverage for different genomes can vary significantly, although this could reflect biases in gene annotation in addition to variation in domain content. Phylogenetic profiles for Ensembl 55 metazoa and Integr8 prokaryotes for the CATH-Gene3D superfamilies are available for download from the Gene3D FTP site (ftp://ftp.biochem.ucl.ac.uk/pub/gene3d_data/CURRENT_RELEASE/).
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Figure 1. The Gene3D genome coverage visualizer. Genome coverage—the number of genes with a match to a CATH superfamily—can be viewed using the new visualization web tool (http://gene3d.biochem.ucl.ac.uk:8090/GenomeCoverageGraphs/). Coverage for a specific superfamily can be retrieved using the top query bar. In the left-hand column, species can be removed from the graph or their species names manually edited. New species can be added via tables found by selecting the ‘Database Summaries’, ‘Ensembl Genome Data’ and ‘Integr8 Genome Data’ tab headers.
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A new aspect to the genome annotation is the inclusion of non-synonymous
single-nucleotide polymorphisms (SNPs) and other sequence variants
from Ensembl Variation (
11). These are included in the protein
feature display, allowing the correlation of altered activity
or phenotype with other annotations, such as the location of
binding sites or domains.
Sequence features and domain architectures
The Gene3D website uses a graphics package kindly provided by Pfam to draw sequence feature annotations (Figure 2), including domains, active sites, SNP locations, transmembrane regions, signal peptides and others. This has now been localized to the Gene3D server, greatly improving rendering speeds and site stability. For the single protein view (see above) a detailed description of the protein is provided (the ‘Features’ tab), with one ‘track’ per annotation type. A second visualization view (‘Sequence Image’) amalgamates all the annotations into a single ‘track’. The ‘Sequence Image’ tab is also available in the collection view (e.g. for a superfamily), along with a tab displaying the set of distinct multi-domain architectures found (‘Multi-Domain Architectures’). A useful facility of Gene3D is the inclusion of CATH domains based on the Protein Data Bank (PDB). We now use the SIFTS PDB-to-UniProt mapping, which has increased the number of domains we are able to map to Gene3D (15).
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Figure 2. Sequence feature annotation for human proto-oncogene vav. Displayed is the feature annotation returned for UniProt identifier VAV_HUMAN. Laying out each type and source of annotation against each other allows the user to easily cross-reference functional information. Clicking on domains brings up available functional descriptions and a link to the original resource. Detailed tables of assignments can be found in separate tabs. To aid users each family is given a unique colour that is consistent across the website; for CATH-Gene3D domains the colour-picking algorithm makes common superfamilies brighter, and ensures superfamilies within the same fold have similar hues.
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Molecular function, interactions, pathways and orthologues
The parsing and import of UniProt records has been improved,
leading to more descriptive terms being imported along with
more associated information. We have also added EggNOG orthologue
assignments (
16). EggNog orthologue families are generated by
large-scale clustering of protein sequences using an extension
of the COGS protocol and applying a rule-based annotation system.
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NEW DAS AND OTHER WEB SERVICES
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The current set of DAS sources provided by Gene3D and CATH can
be viewed at:
http://cathdb.info:9000. Recently added is the
‘gene3d_ensembl’ track, which can be queried with
Ensembl protein identifiers to retrieve domain annotations and
the results viewed in a DAS client.
Gene3D also underpins the FuncNet pipeline for analysis of protein function (paper submitted). This resource integrates various orthogonal methods that predict functional associations between proteins. Gene3D provides the Gene Expression Comparison, homology inherited Protein–Protein Interactions and Co-occurrence of Domains Analysis services, which can be queried directly via SOAP services or together through the FuncNet front-end service. Please refer to the FuncNet website for more information (http://funcnet.eu/).
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RELEASE SCHEDULE AND VERSIONING
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As of Gene3D_v9.0.0 release numbering system has been stabilized
to use a three-part identifier—‘Gene3D_v#.#.#’.
The first part signifies a major release, and is incremented
if there is a potential change to the domain assignments for
a given protein. This would be the case if there is a new CATH
release or if there is a change to the domain-identification
protocol. The second number is incremented if new sequences
are added to the database, such as a new set of Ensembl genomes;
while the third is incremented if any imported annotation is
updated or added. As such, there is no predefined schedule for
releases, but instead is done ‘as needed’. However,
major releases are expected two to three times a year, while
sequences will be updated more often.
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DISCUSSION
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The primary purpose behind the changes made to Gene3D is to
help support the investigations of the biological and medical
research communities. To this extent, Gene3D actively collaborates
within several teams and networks, providing data and tools,
including the FuncNet function prediction web services as well
as a member of the InterPro consortium of protein family databases
and the IMPACT network, and provides targets for structural
determination through the NIH PSI2 project. We also provide
individualized domain predictions and linked data sets on request.
Possible examples are lists of domains that contain annotated
active sites, or are adjacent to transmembrane regions. Through
these collaborations we hope to extend the web platform to provide
data in formats that can be used by the various research communities.
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SUPPLEMENTARY DATA
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Supplementary Data are available at NAR Online.
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FUNDING
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The European Commission’s BioSapiens and EMBRACE Networks
of Excellence funded under the Framework Program 6 (grant numbers
LSHG-CT-2003-503265 to C.Y., LSHG-CT-2004-512092 to A.C.); National
Institutes of Health’s PSI2 initiative (grant number DE-AC02-065CH11357);
ENFIN (grant number LSHG-CT-2005-518254 to J.L.). Funding for
open access charge: European Commission's EMBRACE NoE; LSHG-CT-2004-512092.
Conflict of interest statement. None declared.
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ACKNOWLEDGEMENTS
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We would like to thank the CATH team for their help; SIMAP for
providing significant computational services and annotations;
Prof. David Jones for providing Disopred2 predictions; InterPro
for reviews of the data quality; Alison Cuff for advice on distant
homologies between superfamilies; and the advice and support
of collaborators in the BioSapiens, EMBRACE, ENFIN and IMPACT
networks
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Footnotes
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The authors wish it to be known that, in their opinion, the
first two authors should be regarded as joint First Authors.
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ABSTRACT
INTRODUCTION