Pfam 3.1: 1313 multiple alignments and profile HMMs match the majority of proteins.

Pfam is a collection of multiple alignments and profile hidden Markov models of protein domain families. Release 3.1 is a major update of the Pfam database and contains 1313 families which are available on the World Wide Web in Europe at http://www.sanger.ac.uk/Software/Pfam/ and http://www.cgr.ki.se/Pfam/, and in the US at http://pfam.wustl.edu/. Over 54% of proteins in SWISS-PROT-35 and SP-TrEMBL-5 match a Pfam family. The primary changes of Pfam since release 2.1 are that we now use the more advanced version 2 of the HMMER software, which is more sensitive and provides expectation values for matches, and that it now includes proteins from both SP-TrEMBL and SWISS-PROT.     

The Pfam Protein Families Database

Pfam is a large collection of protein multiple sequence alignments and profile hidden Markov models. Pfam is available on the World Wide Web in the UK at http://www.sanger.ac.uk/Software/Pfam/, in Sweden at http://www.cgb.ki.se/Pfam/, in France at http://pfam.jouy.inra.fr/ and in the US at http://pfam.wustl.edu/. The latest version (6.6) of Pfam contains 3071 families, which match 69% of proteins in SWISS-PROT 39 and TrEMBL 14. Structural data, where available, have been utilised to ensure that Pfam families correspond with structural domains, and to improve domain-based annotation. Predictions of non-domain regions are now also included. In addition to secondary structure, Pfam multiple sequence alignments now contain active site residue mark-up. New search tools, including taxonomy search and domain query, greatly add to the functionality and usability of the Pfam resource.     

The Pfam protein families database

Pfam is a large collection of protein multiple sequence alignments and profile hidden Markov models. Pfam is available on the WWW in the UK at http://www.sanger.ac.uk/Software/Pfam/, in Sweden at http://www.cgr.ki.se/Pfam/ and in the US at http://pfam.wustl.edu/. The latest version (4.3) of Pfam contains 1815 families. These Pfam families match 63% of proteins in SWISS-PROT 37 and TrEMBL 9. For complete genomes Pfam currently matches up to half of the proteins. Genomic DNA can be directly searched against the Pfam library using the Wise2 package.     

iPfam: visualization of protein-protein interactions in PDB at domain and amino acid resolutions

SUMMARY: There are many resources that contain information about binary interactions between proteins. However, protein interactions are defined by only a subset of residues in any protein. We have implemented a web resource that allows the investigation of protein interactions in the Protein Data Bank structures at the level of Pfam domains and amino acid residues. This detailed knowledge relies on the fact that there are a large number of multidomain proteins and protein complexes being deposited in the structure databases. The resource called iPfam is hosted within the Pfam UK website. Most resources focus on the interactions between proteins; iPfam includes these as well as interactions between domains in a single protein. AVAILABILITY: iPfam is available on the Web for browsing at http://www.sanger.ac.uk/Software/Pfam/iPfam/; the source-data for iPfam is freely available in relational tables via the ftp site ftp://ftp.sanger.ac.uk/pub/databases/Pfam/database_files/.     

InterPro as a new tool for whole genome analysis. A comparative analysis of Mycobacterium tuberculosis, Bacillus subtilis and Escherichia coli as a case study

InterPro was developed as a new integrated documentation resource for protein families, domains and functional sites to rationalize the complementary efforts of the PROSITE, PRINTS, Pfam and ProDom database projects and has applications in computational functional classification of newly determined sequences lacking biochemical characterization and in comparative genome analysis. InterPro contains over 3500 entries, with more than 1000000 hits in SWISS-PROT and TrEMBL. The database is accessible for text- and sequence-based searches at http://www.ebi.ac.uk/interpro/. InterPro was used for whole proteome analysis of the pathogenic microorganism, Mycobacterium tuberculosis, and comparison with the predicted protein coding sequences of the complete genomes of Bacillus subtilis and Escherichia coli. 64.8% of the M. tuberculosis proteins in the proteome matched InterPro entries, and these could be classified according to function. The comparison with B. subtilis and E. coli provided information on the most common protein families and domains, and the most highly represented families in each organism. InterPro thus provides a useful tool for global views of whole proteomes and their compositions.     

Application of InterPro for the functional classification of the proteins of fish origin in SWISS-PROT and TrEMBL

InterPro (http://www.ebi.ac.uk/interpro/) is an integrated documentation resource for protein families, domains and sites, developed initially as a means of rationalizing the complementary efforts of the PROSITE, PRINTS, Pfam and ProDom database projects. It is a useful resource that aids the functional classification of proteins. Almost 90% of the actinopterygii protein sequences from SWISS-PROT and TrEMBL can be classified using InterPro. Over 30% of the actinopterygii protein sequences currently in SWISS-PROT and TrEMBL are of mitochondrial origin, the majority of which belong to the cytochrome b/b6 family. InterPro also gives insights into the domain composition of the classified proteins and has applications in the functional classification of newly determined sequences lacking biochemical characterization, and in comparative genome analysis. A comparison of the actinopterygii protein sequences against the sequences of other eukaryotes confirms the high representation of eukaryotic protein kinase in the organisms studied. The comparisons also show that, based on InterPro families, the trans-species evolution of MHC class I and II molecules in mammals and teleost fish can be recognized.     

InterPro as a new tool for complete genome analysis: An example of comparative analysis

InterPro, an integrated documentation resource for protein families, protein domains, and functional sites, was developed to amalgamate the individual efforts of the PROSITE, PRINTS, Pfam, and ProDom databases. InterPro can be used for the computational functional classification of newly determined amino acid sequences that lack biochemical characterization and for comparative genome analysis. InterPro contains over 3500 entries for more than 1 000 000 hits in SWISS-PROT and TrEMBL. The database is accessible for text-and sequence-based searches at http://www.ebi.ac.uk/interpro/. InterPro was used for the complete analysis of the proteome of the pathogenic microorganism Mycobacterium tuberculosis and the comparison with the predicted protein-coding sequences of the complete genomes of Bacillus subtilis and Escherichia coli. It was found that 64.8% of proteins in the proteome of M. tuberculosis matched InterPro entries and can be classified by their functions. The comparison with B. subtilis and E. coli provided information on the most common protein families and domains and on the most highly represented protein families in each organism. Thus, InterPro is a useful tool for general comparison of complete proteomes and their compositions.     

Most partial domains in proteins are alignment and annotation artifacts

BackgroundProtein domains are commonly used to assess the functional roles and evolutionary relationships of proteins and protein families. Here, we use the Pfam protein family database to examine a set of candidate partial domains. Pfam protein domains are often thought of as evolutionarily indivisible, structurally compact, units from which larger functional proteins are assembled; however, almost 4% of Pfam27 PfamA domains are shorter than 50% of their family model length, suggesting that more than half of the domain is missing at those locations. To better understand the structural nature of partial domains in proteins, we examined 30,961 partial domain regions from 136 domain families contained in a representative subset of PfamA domains (RefProtDom2 or RPD2).ResultsWe characterized three types of apparent partial domains: split domains, bounded partials, and unbounded partials. We find that bounded partial domains are over-represented in eukaryotes and in lower quality protein predictions, suggesting that they often result from inaccurate genome assemblies or gene models. We also find that a large percentage of unbounded partial domains produce long alignments, which suggests that their annotation as a partial is an alignment artifact; yet some can be found as partials in other sequence contexts.ConclusionsPartial domains are largely the result of alignment and annotation artifacts and should be viewed with caution. The presence of partial domain annotations in proteins should raise the concern that the prediction of the protein’s gene may be incomplete. In general, protein domains can be considered the structural building blocks of proteins.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0656-7) contains supplementary material, which is available to authorized users.     

NIFAS: visual analysis of domain evolution in proteins

MOTIVATION: Multi-domain proteins have evolved by insertions or deletions of distinct protein domains. Tracing the history of a certain domain combination can be important for functional annotation of multi-domain proteins, and for understanding the function of individual domains. In order to analyze the evolutionary history of the domains in modular proteins it is desirable to inspect a phylogenetic tree based on sequence divergence with the modular architecture of the sequences superimposed on the tree. RESULT: A Java applet, NIFAS, that integrates graphical domain schematics for each sequence in an evolutionary tree was developed. NIFAS retrieves domain information from the Pfam database and uses CLUSTAL W to calculate a tree for a given Pfam domain. The tree can be displayed with symbolic bootstrap values, and to allow the user to focus on a part of the tree, the layout can be altered by swapping nodes, changing the outgroup, and showing/collapsing subtrees. NIFAS is integrated with the Pfam database and is accessible over the internet (http://www.cgr.ki.se/Pfam). As an example, we use NIFAS to analyze the evolution of domains in Protein Kinases C.     

Identification and distribution of protein families in 120 completed genomes using Gene3D

Using a new protocol, PFscape, we undertake a systematic identification of protein families and domain architectures in 120 complete genomes. PFscape clusters sequences into protein families using a Markov clustering algorithm (Enright et al., Nucleic Acids Res 2002;30:1575-1584) followed by complete linkage clustering according to sequence identity. Within each protein family, domains are recognized using a library of hidden Markov models comprising CATH structural and Pfam functional domains. Domain architectures are then determined using DomainFinder (Pearl et al., Protein Sci 2002;11:233-244) and the protein family and domain architecture data are amalgamated in the Gene3D database (Buchan et al., Genome Res 2002;12:503-514). Using Gene3D, we have investigated protein sequence space, the extent of structural annotation, and the distribution of different domain architectures in completed genomes from all kingdoms of life. As with earlier studies by other researchers, the distribution of domain families shows power-law behavior such that the largest 2,000 domain families can be mapped to approximately 70% of nonsingleton genome sequences; the remaining sequences are assigned to much smaller families. While approximately 50% of domain annotations within a genome are assigned to 219 universal domain families, a much smaller proportion (< 10%) of protein sequences are assigned to universal protein families. This supports the mosaic theory of evolution whereby domain duplication followed by domain shuffling gives rise to novel domain architectures that can expand the protein functional repertoire of an organism. Functional data (e.g. COG/KEGG/GO) integrated within Gene3D result in a comprehensive resource that is currently being used in structure genomics initiatives and can be accessed via http://www.biochem.ucl.ac.uk/bsm/cath/Gene3D/.     

CDD: a curated Entrez database of conserved domain alignments

The Conserved Domain Database (CDD) is now indexed as a separate database within the Entrez system and linked to other Entrez databases such as MEDLINE(R). This allows users to search for domain types by name, for example, or to view the domain architecture of any protein in Entrez's sequence database. CDD can be accessed on the WorldWideWeb at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=cdd. Users may also employ the CD-Search service to identify conserved domains in new sequences, at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi. CD-Search results, and pre-computed links from Entrez's protein database, are calculated using the RPS-BLAST algorithm and Position Specific Score Matrices (PSSMs) derived from CDD alignments. CD-Searches are also run by default for protein-protein queries submitted to BLAST(R) at http://www.ncbi.nlm.nih.gov/BLAST. CDD mirrors the publicly available domain alignment collections SMART and PFAM, and now also contains alignment models curated at NCBI. Structure information is used to identify the core substructure likely to be present in all family members, and to produce sequence alignments consistent with structure conservation. This alignment model allows NCBI curators to annotate 'columns' corresponding to functional sites conserved among family members.     

SUPFAM—a database of potential protein superfamily relationships derived by comparing sequence-based and structure-based families: implications for structural genomics and function annotation in genomes

Members of a superfamily of proteins could result from divergent evolution of homologues with insignificant similarity in the amino acid sequences. A superfamily relationship is detected commonly after the three-dimensional structures of the proteins are determined using X-ray analysis or NMR. The SUPFAM database described here relates two homologous protein families in a multiple sequence alignment database of either known or unknown structure. The present release (1.1), which is the first version of the SUPFAM database, has been derived by analysing Pfam, which is one of the commonly used databases of multiple sequence alignments of homologous proteins. The first step in establishing SUPFAM is to relate Pfam families with the families in PALI, which is an alignment database of homologous proteins of known structure that is derived largely from SCOP. The second step involves relating Pfam families which could not be associated reliably with a protein superfamily of known structure. The profile matching procedure, IMPALA, has been used in these steps. The first step resulted in identification of 1280 Pfam families (out of 2697, i.e. 47%) which are related, either by close homologous connection to a SCOP family or by distant relationship to a SCOP family, potentially forming new superfamily connections. Using the profiles of 1417 Pfam families with apparently no structural information, an all-against-all comparison involving a sequence-profile match using IMPALA resulted in clustering of 67 homologous protein families of Pfam into 28 potential new superfamilies. Expansion of groups of related proteins of yet unknown structural information, as proposed in SUPFAM, should help in identifying ‘priority proteins’ for structure determination in structural genomics initiatives to expand the coverage of structural information in the protein sequence space. For example, we could assign 858 distinct Pfam domains in 2203 of the gene products in the genome of Mycobacterium tubercolosis. Fifty-one of these Pfam families of unknown structure could be clustered into 17 potentially new superfamilies forming good targets for structural genomics. SUPFAM database can be accessed at http://pauling.mbu.iisc.ernet.in/~supfam.     

Integration of related sequences with protein three-dimensional structural families in an updated version of PALI database

The database of Phylogeny and ALIgnment of homologous protein structures (PALI) contains three-dimensional (3-D) structure-dependent sequence alignments as well as structure-based phylogenetic trees of protein domains in various families. The latest updated version (Release 2.1) comprises of 844 families of homologous proteins involving 3863 protein domain structures with each of these families having at least two members. Each member in a family has been structurally aligned with every other member in the same family using two proteins at a time. In addition, an alignment of multiple structures has also been performed using all the members in a family. Every family with at least three members is associated with two dendrograms, one based on a structural dissimilarity metric and the other based on similarity of topologically equivalenced residues for every pairwise alignment. Apart from these multi-member families, there are 817 single member families in the updated version of PALI. A new feature in the current release of PALI is the integration, with 3-D structural families, of sequences of homologues from the sequence databases. Alignments between homologous proteins of known 3-D structure and those without an experimentally derived structure are also provided for every family in the enhanced version of PALI. The database with several web interfaced utilities can be accessed at: http://pauling.mbu.iisc.ernet.in/~pali.     

pDAWG: An Integrated Database for Plant Cell Wall Genes

We have recently developed a database, pDAWG, focused on information related to plant cell walls. Currently, pDAWG contains seven complete plant genomes, 12 complete algal genomes, along with computed information for individual proteins encoded in these genomes of the following types: (a) carbohydrate active enzyme (CAZy) family information when applicable; (b) phylogenetic trees of cell wall-related CAZy family proteins; (c) protein structure models if available; (d) physical and predicted interactions among proteins; (e) subcellular localization; (f) Pfam domain information; and (g) homology-based functional prediction. A querying system with a graphical interface allows a user to quickly compose information of different sorts about individual genes/proteins and to display the composite information in an intuitive manner, facilitating comparative analyses and knowledge discovery about cell wall genes. pDAWG can be accessed at http://csbl1.bmb.uga.edu/pDAWG/.     

The InterPro Database, 2003 brings increased coverage and new features

InterPro, an integrated documentation resource of protein families, domains and functional sites, was created in 1999 as a means of amalgamating the major protein signature databases into one comprehensive resource. PROSITE, Pfam, PRINTS, ProDom, SMART and TIGRFAMs have been manually integrated and curated and are available in InterPro for text- and sequence-based searching. The results are provided in a single format that rationalises the results that would be obtained by searching the member databases individually. The latest release of InterPro contains 5629 entries describing 4280 families, 1239 domains, 95 repeats and 15 post-translational modifications. Currently, the combined signatures in InterPro cover more than 74% of all proteins in SWISS-PROT and TrEMBL, an increase of nearly 15% since the inception of InterPro. New features of the database include improved searching capabilities and enhanced graphical user interfaces for visualisation of the data. The database is available via a webserver (http://www.ebi.ac.uk/interpro) and anonymous FTP (ftp://ftp.ebi.ac.uk/pub/databases/interpro).     

The homeodomain resource: a prototype database for a large protein family

The Homeodomain Resource is an annotated collection of non-redundant protein sequences, three-dimensional structures and genomic information for the homeodomain protein family. Release 2.0 contains 765 full-length homeodomain-containing sequences, 29 experimentally derived structures and 116 homeobox loci implicated in human genetic disorders. Entries are fully hyperlinked to facilitate easy retrieval of the original records from source databases. A simple search engine with a graphical user interface is provided to query the component databases and assemble customized data sets. A new feature for this release is the addition of more automated methods for database searching, maintenance and implementation of efficient data management. The Homeodomain Resource is freely available through the WWW at http://genome.nhgri.nih.gov/homeodomain     

CluSTr: a database of clusters of SWISS-PROT+TrEMBL proteins

The CluSTr (Clusters of SWISS-PROT and TrEMBL proteins) database offers an automatic classification of SWISS-PROT and TrEMBL proteins into groups of related proteins. The clustering is based on analysis of all pairwise comparisons between protein sequences. Analysis has been carried out for different levels of protein similarity, yielding a hierarchical organisation of clusters. The database provides links to InterPro, which integrates information on protein families, domains and functional sites from PROSITE, PRINTS, Pfam and ProDom. Links to the InterPro graphical interface allow users to see at a glance whether proteins from the cluster share particular functional sites. CluSTr also provides cross-references to HSSP and PDB. The database is available for querying and browsing at http://www.ebi.ac.uk/clustr.     

Pfam: A comprehensive database of protein domain families based on seed alignments

Databases of multiple sequence alignments are a valuable aid to protein sequence classification and analysis. One of the main challenges when constructing such a database is to simultaneously satisfy the conflicting demands of completeness on the one hand and quality of alignment and domain definitions on the other. The latter properties are best dealt with by manual approaches, whereas completeness in practice is only amenable to automatic methods. Herein we present a database based on hidden Markov model profiles (HMMs), which combines high quality and completeness. Our database, Pfam, consists of parts A and B. Pfam-A is curated and contains well-characterized protein domain families with high quality alignments, which are maintained by using manually checked seed alignments and HMMs to find and align all members. Pfam-B contains sequence families that were generated automatically by applying the Domainer algorithm to cluster and align the remaining protein sequences after removal of Pfam-A domains. By using Pfam, a large number of previously unannotated proteins from the Caenorhabditis elegans genome project were classified. We have also identified many novel family memberships in known proteins, including new kazal, Fibronectin type III, and response regulator receiver domains. Pfam-A families have permanent accession numbers and form a library of HMMs available for searching and automatic annotation of new protein sequences. Proteins: 28:405–420, 1997. © 1997 Wiley-Liss, Inc.     

WWW access to the SYSTERS protein sequence cluster set

SUMMARY: We present a Web server where the SYSTERS cluster set of the non-redundant protein database consisting of sequences from SWISS-PROT and PIR is being made available for querying and browsing. The cluster set can be searched with a new sequence using the SSMAL search tool. Additionally, a multiple alignment is generated for each cluster and annotated with domain information from the Pfam protein family database. AVAILABILITY: The server address is http://www.dkfz-heidelberg.de/tbi/services/cluster/ systersform