What is a conserved domain?

Domains can be thought of as distinct functional and/or structural units of a protein. These two classifications coincide rather often, as a matter of fact, and what is found as an independently folding unit of a polypeptide chain also carries specific function. Domains are often identified as recurring (sequence or structure) units, which may exist in various contexts. The image below illustrates four "domains" identified as structural units in the MMDB-entry 1IGR, chain A, as segments colored in magenta, blue, brown, and green.

In molecular evolution such domains may have been utilized as building blocks, and may have been recombined in different arrangements to modulate protein function. We define conserved domains as recurring units in molecular evolution, the extents of which can be determined by sequence and structure analysis.

Conserved domains contain conserved sequence patterns or motifs, which allow for their detection in polypeptide sequences. The distinction between domains and motifs is not sharp, however, especially in the case of short repetitive units. Functional motifs are also present outside the scope of structurally conserved domains. The CD database is not meant to systematically collect such motifs.

For this query sequence, a good correspondence exists between structural units (3D domains), identified by purely geometric criteria, and units asserted to be evolutionary conserved (domain families). The region annotated as "FU" (furin-repeat like) overlaps with a domain-split that was suggested by the MMDB domain parser. Click anywhere on the image to open the complete, interactive 3D record for this protein structure (1IGR) in Cn3D, a free helper application available for Windows, Macintosh, and Unix platforms. Note: Cn3D must be present on your computer, however, in order for the link to work. Cn3D installation takes only a couple of minutes and a tutorial describes the program's features and functions. Open the 1IGR structure summary record in the Molecular Modeling Database (MMDB) to access more information about the protein, its conserved domains, and ligands (small molecules). On that page, follow the "show annotation" link to open an interactive graphic in which you can click on a conserved domain or ligand of interest to view its complete information in the Conserved Domain Database or PubChem, respectively. Click on the grey bar representing the overall protein, or on a colored bar representing an individual 3D domain of interest, to retrieve similar 3D structures, as identified by the Vector Alignment Search Tool (VAST). View the CD-Search help document for more details about the program that was used to identify the conserved domains in the protein chain. The concise display of the conserved domains is shown here and includes specific hits, superfamilies, and multi-domains. (Open the current, interactive CD-Search results for this protein to view alignments of its sequence to a conserved domain's consensus sequence, and/or to access a full display of all domain models found.)

Multiple sequence alignments provide basis for conserved domain models

The two types of domains shown in the 1IGR illustration above -- 3D domains and conserved domains (or "domain families") -- often coincide with each other. However, because they represent two distinct types of data -- 3D structures and protein sequences, respectively -- they reside in two distinct databases: the Entrez Structure (Molecular Modeling Database, MMDB) and the Conserved Domain Database (CDD). The former includes the spatial (X,Y,Z) coordinates of each atom in a structure (where 3D domains are identified algorithmically), while the latter shows the span and composition of a conserved protein sequence region.

Specifically, conserved domain models are based on multiple sequence alignments of related proteins spanning a variety of organisms to reveal sequence regions containing the same, or similar, patterns of amino acids. The illustration below provides an example, showing the multiple sequence alignment for the Furin-like domain, which is present in the Type 1 Insulin-like Growth Factor Receptor (1IGR) protein. Click anywhere on the image to open the complete, interactive CDD record for that domain model, cd00064. A separate section of this help document provides additional information about multiple sequence alignment display options.

In the CDD database, protein sequences from three-dimensional structures are included in domain models whenever possible, as one goal of the NCBI conserved domain curation effort is to make multiple sequence alignments agree with what we can infer from three-dimensional structure and three-dimensional structure superposition, in order to understand sequence/structure/function relationships. The sequence-based domain models and corresponding 3D structures are also cross-referenced to each other through Entrez "Links" between CDD and structure records.

This illustration shows the multiple sequence alignment for the Furin-like domain, which is present in the Type 1 Insulin-like Growth Factor Receptor (1IGR) protein. Click anywhere on the illustration above to open the live CDD domain summary record for CD00064: FU, Furin-Like Repeats and view the complete multiple sequence alignment for the Furin-like domain model. Separate sections of this CDD help document provide additional details about the source databases from which domain models are collected, the conserved domain assembly process, including the generation of a position-specific scoring matrix (PSSM) for each domain model, and multiple sequence alignment display options.

Source Databases:  Where does CDD content come from?

Conserved Domains can be described by local multiple sequence alignments (illustration) spanning a variety of organisms to reveal sequence regions that contain the same, or similar, patterns of amino acids. Computational biologists from all over the world have compiled collections of such alignments representing conserved domains. CDD includes domains curated at NCBI as well as data imported from the external sources listed below, and data sources are indicated by their accession number prefixes.

The source databases differ in their scope of coverage and the method by which they develop their models. Therefore, each source database may have its own model for a given conserved domain, in addition to some domain models found only in that database. To provide a non-redundant view of the data, CDD clusters similar domain models from various sources into superfamilies. The data sources include:

NCBI-Curated Domains

NCBI-curated domains use 3D-structure information to explicitly to define domain boundaries, aligned blocks, and amend alignment details. More details about the unique features of NCBI-curated domains are below.

The goal of the curation project is to provide CDD users with insights into how patterns of residue conservation and divergence in a family relate to functional properties, and to provide useful links to more detailed information that may help to understand those sequence/structure/function relationships. The presence of conserved features help to affirm family membership in search results with borderline significance, for example. NCBI CDD Curators provide feature annotation and associated evidence in a computer friendly way, so that the scientific community can build software tools for the automation of tasks like annotation transfer, for example.

NCBIfams

NCBIfams is a collection of protein family hidden Markov models (HMMs) for improving bacterial genome annotation. A paper by Haft et al. (2018) provides additional information about NCBIfams, which is part of NCBI's Reference Sequence (RefSeq) project.

External Data Sources

In addition, CDD imports data from five other major sources, below. The version number (as available) of each source database that is imported into CDD is provided in the CDD News page.

Abbreviation	Database Name	Description
SMART	Simple Modular Architecture Research Tool	SMART is a web tool for the identification and annotation of protein domains, and provides a platform for the comparative study of complex domain architectures in genes and proteins. SMART is maintained by Chris Ponting, Peer Bork and colleagues, mainly at the EMBL Heidelberg. CDD contains a large fraction of the SMART collection.
Pfam	Protein families	Pfam is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains and families. Pfam is maintained by Alex Bateman and colleagues, mainly at the Wellcome Trust Sanger Institute. CDD contains a large fraction of the Pfam collection.
COGs	Clusters of Orthologous Groups of proteins	COGs is an NCBI-curated protein classification resource. Sequence alignments corresponding to COGs are created automatically from constituent sequences and have not been validated manually when imported into CDD.
TIGRFAMs	The Institute for Genomic Research's database of protein families	TIGRFAMs, a research project of the J. Craig Venter Institute, is a collection of manually curated protein families from The Institute for Genomic Research and consists of hidden Markov models (HMMs), multiple sequence alignments, Gene Ontology (GO) terminology, cross-references to related models in TIGRFAMs and other databases, and pointers to literature.
PRK	PRotein K(c)lusters	Protein Clusters is an NCBI collection of related protein sequences (clusters) consisting of Reference Sequence proteins encoded by complete prokaryotic and chloroplast plasmids and genomes. It includes both curated and non-curated (automatically generated) clusters.

CDD also contains data from additional research projects, such as KOGs (a eukaryotic counterpart to COGs) and the Library of Ancient Domains (LOAD), contributed by I. Aravind, E. Koonin, and colleagues. The KOGs data set is accessible as a separate CD-Search database/Batch CD-Search database, and the LOAD data set is available on the FTP site, but neither of those data sets is directly searchable by text term in Entrez CDD.

The content of imported domain models is determined by the providers of the source database, with slight modifications made at NCBI to link a domain model's member sequences to corresponding, complete protein sequence and 3D structure records in Entrez databases, when possible. The method by which imported domain models are integrated into the CDD database is described in the CD assembly process section of this help document.

Accession Prefixes indicate data sources:

Source databases are evident from CD accessions:

Accession starts with:	Source Database
cd	Curated at NCBI
sd	Domain models specifically built to annotate structural motifs; this is a subset of the NCBI-curated domain models.
NF	NCBIfams
pfam	Pfam
smart	SMART
COG	COGs
KOG	KOGs (available as a separate search set via CD-Search (RPS-BLAST); not searchable by text term in Entrez)
PRK	PRotein K(c)lusters (Entrez database)
CHL	Chloroplast and organelle proteins; subset of the PRK database.
MTH	Mitochondrial proteins; subset of the PRK database.
PHA	Phage proteins; subset of the PRK database.
PLN	Plant-specific (non-chloroplast) proteins; subset of the PRK database.
PTZ	Protozoan proteins; subset of the PRK database.
TIGR	TIGRFAMs
LOAD_	Library of Ancient Domains (LOAD) data set. (available as a separate data set via FTP; not searchable by text term in Entrez)

Accessions that start with "cl" are for superfamily cluster records, which can contain domain models from one or more source databases.

When searching CDD, it is possible to limit search results to domains from any given source database by using the Database Search Field.

CD Assembly Process:  How have CDs been assembled?

NCBI-curated domain models are assembled using the methods briefly described in the source databases section of this document. More details about the NCBI curation process are provided by Marchler-Bauer, et al. (2007). An example of a multiple sequence alignment on which a model is based is shown in an illustration of the Furin-like domain.

Domain models from external data sources are assembled by various methods, ranging from automated processing to manual curation, depending on the individual source database. Upon import into CDD, protein sequence alignments (illustration) from each of the source databases are processed in an automated way to provide links from each aligned sequence to the corresponding, complete record in the Entrez Protein database. Occasionally, sequences that cannot be identified in Entrez's databases are omitted or substituted for closely related matches. Whenever possible, sequences in PFAM, SMART, and COGs alignments are substituted for closely related sequences (passing a stringent sequence similarity threshold) that have direct links to three-dimensional structures in the Moleclular Modeling Database (MMDB).

A representative sequence is chosen for each domain model, preferably with a structure-link, for technical reasons. The representative sequence is generally shown as the first member of the multiple sequence alignment for a domain model. By default, this representative is the 3D structure shown when CD alignments are visualized with Cn3D.

A consensus sequence is computed from the imported alignments. Alignment columns have to be represented in at least 50% of all aligned sequences (weighted by diversity) to determine the extent of the consensus. The most frequently occurring residue in each column (after weighting to account for redundancy) is reported.

A position-specific scoring matrix (PSSM) is calculated for the extent of the consensus sequence. The PSSM profiles the various amino acids that were present in a given position of the multiple sequence alignment for a domain model and how frequently each one was observed. The consensus sequence does not contribute to the residue frequency statistics. Each PSSM receives a unique identifier (PSSM ID).

A PSSM ID is the unique identifier for a domain model's position-specific scoring matrix (PSSM). If a domain model's PSSM changes in any way as a result of updates to its multiple sequence alignment, it receives a new PSSM ID. This happens because a conserved domain model can evolve over time. For example, as new sequence data become available, the curators of a source database might add sequences to a multiple sequence alignment or update the sequences already present. As a result of such changes to the domain model, the PSSM and its ID can change. (Additional notes: Each superfamily record in the Conserved Domain Database also has a PSSM ID, which refers to the specific set of conserved domain PSSM IDs that comprise the superfamily, rather than to an actual position-specific scoring matrix for the overall superfamily. Obsolete PSSMs (e.g., 667) cannot be retrieved through the Entrez CDD search interface because they are no longer indexed. However, they can be retrieved from the archival copy of the database by using the "Direct Fetch via UID" option on the CDD Search Methods page.)

Search databases compiled of these PSSMs are available through the CD-Search service (see help document) and on the NCBI FTP site as collections of pre-computed RPS-BLAST databases that can be used for locally installed versions of that program.

What is unique about NCBI-curated domains?

As noted in the section on CDD data sources, NCBI-curated domains use 3D-structure information to explicitly to define domain boundaries, aligned blocks, and amend alignment details. The goal of the NCBI conserved domain curation project is to provide database users with insights into how patterns of residue conservation and divergence in a family relate to functional properties, and to provide useful links to more detailed information that may help to understand those sequence/structure/function relationships. To do this, CDD Curators include the following types of information in order to supplement and enrich the traditional multiple sequence alignments that form the foundation of domain models: 3-dimensional structures and conserved core motifs:   NCBI Conserved Domain Curators have re-evaluated and modified multiple sequence alignments imported from outside sources, and made them agree with what we can infer from three-dimensional structure and three-dimensional structure superposition. Curated alignments contain aligned blocks spanning all rows (with no gaps allowed inside blocks) and unaligned regions between blocks. The blocks are meant to represent conserved structural core motifs of the corresponding domain family. The 3D structures can be viewed interactively with the Cn3D structure viewing program. More information about viewing structures is provided in the section of this document on CD summary pages, and the illustration at the right provides an example of a protein structure that has been annotated by NCBI curators to highight the Cl- binding residues. (Click on the illustration to open the current, interactive record for the Voltage-Gated Chloride Channel domain model, cd00400, in the Conserved Domain Database (CDD). From there, you can open an interactive version of the 3D structure, with conserved feature annotations, in the free Cn3D structure viewing program.) Conserved features/sites:   In addition to working on the alignment model (illustration), NCBI curators also record, when possible, the location and nature of features conserved in the domain family. Typically these would describe catalytic residues, binding sites, or motifs commonly referred to in the literature.

Features are added if they seem applicable to the family described in the CD's scope and if there is evidence linking the feature to a set of addresses on the alignment. Such evidence is recorded and available for inspection; it may be free-text comments, citations linked to PubMed, or "structure evidence" - exemplifying the existence of a site by highlighting an actual molecular complex, for example. Both features and evidence can be visualized on CD summary pages (in the conserved features/sites summary box, and as hash marks (#) in the multiple sequence alignment displays), and with the Cn3D structure viewing program. An example is shown in the illustration at the right. (Click on the illustration to open the current, interactive record for the Voltage-Gated Chloride Channel domain model, cd00400, in the Conserved Domain Database (CDD). Note that the live web page may look different from the illustration shown here, because the Conserved Domain Database continues to evolve with the addition of new data; however, the concepts shown in the illustration remain stable.) In addition, the CD-Search tool can be used to identify conserved features in a query protein sequence, designated by small triangles (illustrated example) in the search results graphical summary, when such features can be mapped from the conserved domain annotations to the query sequence. Phylogenetic organization:   Based on evidence from sequence comparison, NCBI Conserved Domain Curators attempt to organize related domain models into phylogenetic family hierarchies (illustrated example). The CDTree program used by NCBI curators can be downloaded in order to view NCBI-curated domains interactively and in greater detail. Links to electronic literature resources:   NCBI curated domains also provide links to citations in PubMed and NCBI Bookshelf that discuss the domain. These references are selected by curators and, whenever possible, include articles that provide evidence for the biological function of the domain and/or discuss the evolution and classification of a domain family. NCBI-curated domains can be recognized in CDD search results by their "cd" accession number prefix. It is also possible to limit CDD search results to domain models from any given source database by using the Database Search Field.

What is a domain family hierarchy?

A domain family hierarchy is a set of related domains that share a common ancestor, a common set of conserved residues, and a common general function, but differ from each other in their specific phylogeny, specific functions, and additional spans of conserved residues. Domain hierarchies are present in NCBI-curated domains in order to provide insights into how patterns of residue conservation and divergence in a family relate to functional properties.

Some domain families have only a single node, while others have a hierarchy that is two or more levels deep, sometimes with numerous nodes ("subfamilies")at each level. Such hierarchies have generic "parent" models and more specific "children". The parent node contains a span of conserved residues that is also present in each of the children. Each of the child nodes can have additional conserved residues that extend beyond that span and help to further characterize the members of the child node.

NCBI CDD Curators attempt to split "children" nodes where they see evidence for ancient gene duplications resulting in orthologous groups, often occurring together with functional divergence. The CDTree program used by NCBI curators can be downloaded in order to view NCBI-curated domains interactively and in greater detail, with or without a query sequence embedded.

An illustrated example of a subfamily hierarchy is provided below.

Click anywhere on the image to open the current, interactive record for the Voltage-Gated Chloride Channel domain model, cd00400, in the Conserved Domain Database (CDD). Note that the live web page may look different from the illustration shown here, because the Conserved Domain Database continues to evolve with the addition of new data; however, the concepts shown in the illustration remain stable.

What is a superfamily?

A superfamily cluster is a set of conserved domain models that generate overlapping annotation on the same protein sequences. These models are assumed to represent evolutionarily related domains and may be redundant with each other. A superfamily accession number begins with the prefix "cl" for "cluster". (Some superfamilies contain only a single conserved domain model (singleton), and these are not indexed in Entrez. Only superfamilies that contain two or more conserved domain models are indexed in Entrez and will therefore appear in search results.)

Clustering methodology:

Superfamily members are clustered through an automated process that involves the following steps:

1. Identify domain models that have overlapping hits on sequences in the Entrez Protein database from at least five different identical protein groups (IPGs).

   Technical note: In the data processing pipeline at NCBI, protein sequence records that contain an identical sequence, regardless of TaxID, are placed in an identical protein group (IPG), and each group is given a stable unique identification number (referred to as IPG ID or UID).

2. Store the overlapping domain models as pairwise associations, and use those pairwise associations to populate a similarity matrix.


3. Refine the similarity matrix by comparing it against a "blacklist" (to remove unacceptable pairwise associations), and against a "whitelist" (to add pairwise associations that known to be valid but are not yet listed):


• The "blacklist" is used to separate domain models that should never be paired. The blacklist overrides all other aspects of the clustering algorithm.
  For example:
  • cd00538 (PA domain hierarchy) is blacklisted against pfam00082 (a serine protease model, subtilase family)
  • cd00538 (PA domain hierarchy) is blacklisted against cd08022 (an M28-family metalloprotease)
  These pairings above are forbidden because the PA (protease-associated) domain is often inserted in a protease domain, yet the proteases that contain the insert are distinct from each other. For example, if a PA domain is in a serine protease and also in a metalloprotease, the two types of protease would be clustered together by the algorithm in the absence of a black list. However, the metalloprotease and serine protease are actually distinct, and simply represent convergent evolution to similar function.


• The "whitelist" is used to add pairwise associations of domain models that are known to be related, but that might not have been listed in the initial, unrefined similarity matrix.
  For example:
  • NCBI-curated domain models that are organized hierarchically are part of the white list.
  • Conserved domain models from external databases can also be grouped together, if those domains are known to be related but were not grouped automatically by the clustering algorithm. An example of such a whitelisted pair is shown in cluster cl23875: MvaI_BcnI Superfamily, which includes pfam15515 (MvaI/BcnI restriction endonuclease family) and pfam09562 (LlaMI restriction endonuclease), as of 30 January 2018. Those two conserved domain models for restriction endonucleases (REs) have relatively few hits in common. RE superfamilies are very sequence-diverse, meaning that models for specific subfamilies can differ quite a bit in terms of overall length and conserved residue patterns/signatures. Nevertheless, the models are whitelisted together because they are known to be related.


4. Take the refined similarity matrix and feed it to the Python "fastcluster" package (https://pypi.python.org/pypi/fastcluster), to create clusters using the "complete linkage" algorithm.


5. Implement a post-processing step to compare the Python-generated clusters against the whitelist, in case Python did not put two domain models in the same cluster but should have.

NOTE: Multi-domain models that were computationally detected are not included in Superfamily clusters. These models are likely to contain multiple single domains and might falsely join superfamily clusters.

Rationale:
Superfamilies provide a method for organizing data within CDD in a non-redundant way. CDD contains conserved domains from a number of different source databases, each of which may have its own model for a given conserved domain. The models might share many similiarities in their reported residue conservation patterns, but differ in the specific protein sequences used in the multiple alignment, their footprint length [domain boundaries], and biological annotations. Because of the similarities, RPS-BLAST might find that multiple domain models align to the same general region of a query protein, but have different footprints and E-value scores relative to the query protein. If the footprints of two or more domain models overlap on the query, those models are clustered into the same superfamily, then the superfamily continues to be extended using the methodology described above.

Example:
One example of a superfamily is Cluster ID cl02915, which contains various domain models for the voltage-gated chloride channel. Superfamily members include the NCBI-curated domain cd00400 and all members of that family hierarchy plus domain models from external resources.

Selection of Superfamily Representative:
A superfamily can contain one to many domain models. As of spring 2008, approximately 70% of the ~9,000 superfamilies contain a single model and the rest contain multiple models. Single model superfamilies often represent proteins specific to certain organisms or taxonomic lineages (for example, viruses). The numbers of superfamilies containing single or multiple domain models will continue to evolve as new domains are imported and new NCBI-curated hierarchies are added.

In superfamilies contatining multiple domain models, one of the models is selected as the source of the superfamily name and description. The representative is one of the following, listed in priority order:

• the parent node of an NCBI-curated domain family hierarchy, if one is present in the superfamily cluster. In the few cases where a superfamily contains more than one NCBI-curated domain, the parent of the hierarchy with the largest number of sequence hits is chosen as the superfamily representative.
• the Pfam domain model that hits the largest number of Entrez protein sequences in an RPS-BLAST search
• the SMART, COG, PRK, or CHL model that hits the largest number of Entrez protein sequences in an RPS-BLAST search
• the sole member of a superfamily

Superfamily can change over time:
The composition of a cluster can change over time due to a variety of factors, such as:

1. availability of new domain models as the Conserved Domain Database continues to grow
2. changes to previously existing models
3. new and/or updated sequence records in the Entrez Protein database
4. refinements to the automated clustering procedures

A superfamily cluster accession number will remain the same if at least 50 percent of its member models (conserved domain accessions) have not changed relative to the previous version of the cluster.

If more than 50 percent of the conserved domain accessions from a previous version of a cluster are no longer present in the new build of that cluster, or if the cluster size more than doubles with a new build, then the superfamily cluster accession is retired and replaced by a new accession(s). If two previous clusters merge into a single new cluster, the superfamily cluster accession number of the larger component cluster is used for the new grouping.

A superfamily also has a PSSM ID, which refers to the specific set of PSSM IDs for the domain models that comprise the superfamily, rather than to an actual position-specific scoring matrix for the overall superfamily. The superfamily PSSM ID will change if there is any change to the set of member PSSM IDs relative to the previous version of the cluster (e.g., if a member conserved domain gets a new PSSM ID due to changes in its multiple sequence alignment, of if a new conserved domain model is added to the superfamily as the result of a CDD database update).

The CD summary page for a Superfamily record does not include a multiple sequence alignment display; rather, it provides the name and description of the superfamily and lists the domain models that belong to it. The multiple sequence alignment for any member domain model can be viewed by clicking on it to open its CD summary page.

Superfamilies that contain a single domain model ("singletons")

The concept of superfamilies was applied to CDD in order to cluster related conserved domain models together and provide a non-redundant view of the available domain models. After the superfamily clustering algorithm is applied to the domain models in CDD, all resulting clusters are referred to as superfamilies, regardless of how many domain models they contain. The non-redundant view of CDD therefore includes superfamilies with a single domain model ("singletons") as well as superfamilies containing two or more domain models.

In the user interface, however, superfamilies that contain only one model are not displayed in search results, or listed as links from the domain model, because they look very similar to the model itself.

In contrast, superfamilies that contain two or more models ("multi-model superfamilies") are displayed in search results, and are also accessible as links from their member domain models. The number of multi-model superfamilies is provided in the Database Statistics box on the "Conserved Domains and Protein Classification News" page, and they can be retrieved by clicking on that statistic.

Search Tips: How to find conserved domains

| Protein query sequence | Text term search in Entrez CDD | Protein → Conserved Domains links | Domain architecture |

Protein Query Sequence (CD-Search):

Most users will explore conserved domains starting from CD-Search results for a protein of interest.

The query can be a protein sequence in FASTA format or the GI or Accession of a protein sequence that exists in the Entrez Protein database.

The search results will show the conserved domains found in the protein. The colored bars that depict the domain footprints (shown in both the concise display and full display of CD-Search results) are active hotlinks that open the corresponding CD summary pages with your query sequence embedded in the multiple sequence alignment of proteins used to create the domain model.

The second half of this help document provides details on how to use the CD-Search service, including input required and output shown.

Text Term Search in Entrez CDD:

| allowable search terms | search methods:  basic and advanced | search fields | quotes | wild card * |
| search results | document summary page | "display", "show, "sort by", "send to" menus) |

Allowable search terms

Conserved domains can be searched by text term in the Entrez CDD database. The Entrez query interface allows searching for keywords, publication dates, and taxonomic span, accesssion numbers, and more. The search field summary table in this document shows the variety of terms that can be used to query the database and provides sample searches. It is also possible to use quotes to force multiple terms to be searched as a phrase, and to use an asterisk (*) as a wild card to search for a word stem.

For example, search the Entrez CDD database for strings like "Kinase" or "pfam023*" or "Tetratrico*" to see how it works: for

A number of techniques can be used to search the database, offering varying degrees of control over your query. The search methods summary table provides examples of basic and advanced searches. In basic searches, you can just enter one or more search terms without specifying search fields, Boolean operators, or other search criteria. These searches are quick and easy but can result in some extraneous hits. Advanced search methods, on the other hand, allow you to exercise greater control over your search, for example, by specifying which search field to use for each query term, limiting search results to a particular type of record or source database, or refining your search in other ways. A separate section of this help document describes the CDD search results.

(The PubMed help document and Entrez help document provide additional, general information about using the Entrez search system.)

Search Methods

A variety of techniques can be used to search the Entrez CDD database, offering varying degrees of control over your query. In some cases, they offer alternative ways of executing the same search (as is true for sample searches #4, #5, and #6 below), with each method offering different benefits. The search methods include:

• Basic search (& search details)
• Limits
• Advanced search (Search builder, Show index list, History)
• Complex Boolean query
• Batch query

Method Description Example Basic Search Just enter search terms without specifying search fields, other limits, or Boolean operators. The "Search Details" box in the right margin of the search results page shows exactly how Entrez parsed and handled your query. If desired, you can edit the query in that box and press the "Search" button to run the modified query. The "See more..." link a the bottom of the "Search Details" box opens a more detailed display: The Query Translation box shows the search strategy used to run the search To edit the search in the Query Translation box, add or delete terms and then click Search. Click URL to display the current search as a URL to bookmark for future use. Searches created using History numbers can not be saved using the URL feature. You may also save your search using My NCBI. The Result number link retrieves the documents found and displays them in a search results page. Translations details how each term was translated using Entrez's search rules and syntax for the database. User Query shows the search terms as you entered them in the search box and any syntax errors with the query. Search #1: mismatch repair eukaryotes will retrieve biosystems with those terms anywhere in the record. Some of the records might include aligned sequences from organisms other than eukaryotes because we did not limit that search term to the Organism search field. Because of this, we might also retrieve conserved domain records they happen to contain the term "eukaryotes" in a comment or some other field of the record. Similarly, the term "mismatch repair" can appear anywhere in the record. The terms entered in a basic search may or may not be adjacent to each other in the retrieved records, depending on how Entrez parsed the query (as shown in the Search Details for a given search). To force terms to be searched as a phrase, use quotes. To refine your search in other ways, use the Limits option or the Advanced Search methods described below. Limits The Limits page allows you to restrict your search in various ways. At a minimum, the Limits page displays the list of available search fields. You can do a separate search for each term or phrase in your query, as shown in sample Search #2 and #3 to the right, and select the desired search field for each one. (If desired, you can then combine the searches by using the Search Builder or History section of the Advanced Search page.) For some databases, the Limits page also provides other commonly used options, as check boxes and/or pull-down menus, for restricting your search results to records with specific characteristics. These check boxes and pull-down menus generally represent a commonly used subset of the choices that are available from the Advanced Search page and are placed on the Limits page for easy access. IMPORTANT NOTE: Once you have used a particular Limit, warning sign will appear near the top of your search results page that indicates which Limit(s) are currently in effect, for example: Note that the Limit will remain in effect for all subsequent searches in the current database unless you change or remove that limit. In the illustrated example above, any search you do will be limited to the Titles of records, until you remove the limit. Search #2: On the Entrez CDD search page, click on the Limits link, select the Text Word, enter the following query: "mismatch repair" and press "GO". That will retrieve only records which contain those terms in the conserved domain's description. The quotes surrounding the terms force them to be searched as a phrase. Search #3: Open the Limits page again and clear your previous search. Change the search field selection to Organism, enter the following query: eukaryotes and press "GO". That will retrieve conserved domain models containing only eukaryotic sequences in their multiple sequence alignments (i.e., eukaryota will be the root taxonomic node of the sequences in a domain model's alignment). If desired, you can then combine the searches on the Advanced Search page, either by using the Search Builder, as shown in sample Search #4, or by using the History section of that page, as shown in sample Search #5. Advanced Search The Advanced Search page allows you to exercise greater control over your search, for example, by enabling you to: Build a search one step at a time. Browse the index of any search field and add term(s) of interest from the index to the active query box at the top of the page. View your search History and combine or subtract searches from each other. As you build a query, either by using the Search Builder's pull-down menus, or by using the "Add" links in the "History" portion of the page to combine previous searches, the grey text box at the top of the page will display your current query. You can also manually edit the current query by clicking the "Edit" link beneath the grey text box. That will allow you to type terms/search numbers/etc. directly into the box, add parentheses for nesting if desired, change Boolean operators, etc. In addition, the following types of advanced searches can be entered in the query box of any Entrez search page (i.e., in the query box of the database's Home page, Limits page, or Advanced Search page): Complex Boolean query Batch query Search Builder The "Search Builder" section of the Advanced Search page allows you to build your query step by step, adding a new search term and selecting a new search field at each step. It also allows you to browse the index of any search field to view the available terms. To build a query: (1) Select the Search Field of interest using the pull-down menu. (2) Type a term(s) in the text box beside the search field menu. Or, use the "Show index list" link to see the index of the search field and select the desired term from the index. (tips on using the "Show Index List") (3) Select the Boolean operator (AND, NOT, OR) that should precede the term when it is added to the active query at the top of the page. Continue the above steps, as desired, to add more term/search field combinations to your query. As you use the Search Builder, the grey text box at the top of the page will show your current query. You can manually edit the current query by clicking the "Edit" link beneath the grey text box. That will allow you to type terms/search numbers/etc. directly into the box, add parentheses for nesting if desired, change Boolean operators, etc. Press the Search button to display the records retrieved by your search (i.e., it displays the search results page). Click on the "Add to history" link if you prefer to simply add the query to your search history and remain on the Advanced Search page, where you can continue building your query. Tips on using the "Show Index List" function on the Advanced Search page: The "Show Index List" function allows you to browse the index of any Search Field. If you select a search field and press the "Show Index" link without entering a term in the box, you will be taken to the top of the index. If you enter a term first, you will be taken to the part of the index that contains your term (or the closest alphabetical location, if your term is not present in the index). The number of records that contain the term will appear in parentheses. You can also browse the index to explore the variety of terms available (for example, select "All Fields", enter "Huntington", and click on the "Show Index" link to see additional spellings and/or related terms, such as Huntington disease, Huntington's, Huntington's disease). To select a range of terms from the index, use the Shift key while selecting the first and last term. Then use the AND, OR, or NOT buttons to add that group of terms to the active query. To select multiple terms that do not fall within a continuous range from the index, use the Control key while selecting the terms of interest. Then use the AND, OR, or NOT buttons to add that group of terms to the active query. Note: When multiple terms are selected from the index window, they are OR'ed together within parentheses and then appended to your query with whatever Boolean operator you have selected. Search #4: On the Entrez CDD search page, click on Advanced Search and build your search one step at a time: (a) Using the first pull-down menu in Search Builder, select the Text Word search field and enter the following query: "mismatch repair" and select "AND" as the Boolean operator. That term/search field selection will automatically be displayed in the grey text box at the top of the page, which shows your current query. (b) Using the second pull-down menu in Search Builder, select the Organism search field and enter the following query: eukaryotes and select "AND" as the Boolean operator. That newest term/search field selection will automatically be added to the grey text box at the top of the page. (c) Your query will now appear as: "mismatch repair"[Text Word] AND eukaryotes[Organism] Press the Search button if you want to display the records retrieved by your search (i.e., it displays the search results page). Or, click on the "Add to history" link if you prefer to just add the query to your search history and remain on the Advanced Search page, where you can continue building your query. Note that this search will produce the same results as sample searches #5 and #6. It is simply executed in a different way. That is, you remain on a single query page (Advanced search) and can browse the index of any search field as you build your query one step at a time. History The "History" section of the Advanced Search page displays the searches you have done in the current database. You can combine or subtract searches from each other by entering the search numbers and the AND, OR, or NOT Boolean operators in the query box, for example: #2 AND #3. If the query contains several search numbers and Boolean operators, the Boolean operators are processed from left to right unless parentheses are used for nesting. If parentheses are used, the portions of the query in parentheses will be processed first, then the remaining Boolean operators will be processed from left to right. Additional details about Search History: The Search History will be lost after 8 hours of inactivity. (To save a search indefinitely, click on the search # and select "Save in My NCBI.") Click "Clear History" to delete all searches from History. Entrez will move a search statement number to the top of the History if a new search is the same as a previous search. History search numbers may not be continuous because some numbers are assigned to intermediate processes, such as displaying a citation in another format. The maximum number of searches held in History is 100. Once the maximum number is reached, PubMed will remove the oldest search from the History to add the most current search. A separate Search History will be kept for each database, although the search statement numbers will be assigned sequentially for all databases. PubMed uses cookies to keep a history of your searches. For you to use this feature, your Web browser must be set to accept cookies. Database records that you have copied to the Clipboard are represented by the search number #0, which may be used in Boolean search statements. For example, to limit the records you have collected in the Clipboard to those from human, use the following search: #0 AND human[organism]. This does not change or replace the Clipboard contents. Search #5: Use the search numbers shown in the "History section" of the advanced search page to combine previous searches (for example, searches #2 and #3 shown above). To do that, you can either: Click on the "Edit" link beneath the grey text box and type in a search statement such as: #2 AND #3 Or, instead of typing the search statement, use the "Add" link beside any search number in the "History" section of the Advanced Search page to add that search number into the grey text box. That will retrieve records containing "mismatch repair" in the Text Word field and "eukaryotes" in the Organism field. Compare the retrieval from this search with that of the sample basic search above. (Note that your search numbers might be different from those shown here, if you did earlier searches in the Entrez system before trying these examples.) Complex Boolean Whether you are on the Basic search page (i.e., the database's home page), the Limits page, or the Advanced search page, you can: Enter a search in command language, specifying your exact combination of desired search terms, search fields, and Boolean operators, as shown in the examples to the right. The syntax is:     term[field] BOOLEAN term[field] BOOLEAN term[field] etc. Search Field names must be placed in square brackets [], and can be written as either the full name, for example, [Database], or as the corresponding search field abbreviation, for example, [db]  (additional examples). Boolean operators (AND, OR, NOT) must be written in UPPER CASE. Boolean operators are processed from left to right unless parentheses are used for nesting. If parentheses are used, the portions of the query in parentheses will be processed first, then the remaining Boolean operators will be processed from left to right. Boolean operators can also be used to combine or subtract searches from each other (i.e., to find the union, difference, or intersection of the data sets retrieved by various searches). To do this, use the History section of the Advanced Search page and simply enter the search numbers and desired Boolean operators in the query box. For example, to identify the records that were retrieved by Search #2 of your search history, and also by Search #3, you could enter the following query: #2 AND #3 To identify the records that were retrieved by Search #2 but not by Search #3, you could enter the following query: #2 NOT #3 Search #6: Simply enter all search terms and search fields as a single statement into the query box: "mismatch repair"[Text Word] AND eukaryotes[Organism] Note that this search will produce the same results as sample searches #4 and #5, but it takes only a single step when entered directly into the search box as a Boolean query. Search #7: ("chloride channel"[All] OR ClC[All]) AND (cdd[Database] OR pfam[Database]) This search will retrieve biosystem records that contain the phrase "chloride channel" or the abbreviation "ClC" in any field of the record, and that are from the NCBI-curated or PFAM source databases. Batch query If you have a list of conserved domain accession numbers or PSSM IDs, you can use Batch Entrez to retrieve them. Be sure to select the appropriate database from the pull-down menu at the top of the page, then "Browse" to find the file of UIDs you'd like to upload, and press "Retrieve." Batch Entrez will then display a report summarizing the data it found in your input file, such as: (1) number of lines that were present in the file (there should be one UID per line); (2) rejected lines (indicating how many invalid UIDs were detected); (3) removed duplicates (indicating how many duplicate UIDs were detected/removed); (4) passed to Entrez (indicating how many of the UIDs from your list are valid and will therefore be acted upon). The latter may include obsolete UIDs that have been superceded by newer UIDs. The obsolete UIDs, as well as any invalid UIDs that were present in your file, are explicitly reported at the top of the Batch Entrez report. If you have a list of protein sequences in which you'd like to identify conserved domains, you can use the Batch CD-Search service. See the Batch CD-Search help for additional information. (To search for conserved domains on a single protein sequence, the original CD-Search service continues to be available, along with the CD-Search help.)   Additional details about search methods and options are provided in the: (1) PubMed help document (including information about temporarily saving records from your search results to the Clipboard); (2) My NCBI help document (including information about Saving search strategies and indefinitely saving records from your search results into your My NCBI Collections); and (3) general Entrez help document.

Search Fields

By default, the Entrez system searches all fields of a record. If you want to narrow your query by searching for your term(s) in a specific search field, you can select the desired field by using the pull-down menus on either the Limits and Advanced search page, or you can type the search field directly in your query (surrounding field names with square brackets [], for example, [Organism] or [Orgn]).* The Show index link on the Advanced search page allows you to browse the index of each search field, where you can see the available terms, the number of records containing each term or phrase, as well as the syntax for entering values in search fields such as Modification Date or Publication Date.

The currently available fields include:

Field name Abbreviation* Description Sample Search All Fields [all] Searches the complete database record "chloride channel"[All] will retrieve the CDD records that contain the phrase "chloride channel" in any field of the record. The quotes surrounding the search terms ensure they are searched as a phrase.** Accession [accn] Searches only the accession number of the record, which is always an alphanumeric combination. The accession number prefix indicates the source database. The accession number applies to the complete conserved domain record. Note: An additional unique identifier, the PSSM ID, is assigned to the position specific scoring matrix that is derived from the conserved domain's multiple sequence alignment. Conserved domains can also be retrieved by entering their PSSM ID (without a search field specifier). cd00400[Accn] will retrieve the CDD record that contains the specified unique identifier in the accession number field. Alternative Accession [AltAccn] Native accession format from an external source database. For example, the PFAM database uses accessions with a format such as pf08617. When these are imported into CDD, the accessions are represented in a format such as pfam08617. Similarly, the SMART database uses a format such as sm00100, while records that have been imported into CDD have a format such as smart00100. This is primarily done to indicate that SMART and PFAM domain alignments may have been modified slightly by NCBI staff, for example by the substitution of a protein sequence that does not have 3D structure with a highly similar one that does (as explained in the help document section on the CD assembly process). pf08617[AltAccn] will retrieve the pfam08617 record from CDD. Database [db] Use this field to limit your search to a particular source database. cdd[db] will retrieve the NCBI curated domain models and superfamily records, which are also created at NCBI, from CDD. pfam[db] will retrieve the domain models that were imported from the PFAM database. Filter [filt] The "Filter" search field allows you to narrow your retrieval to records that have certain attributes, such as curated or uncurated, or records that have links to other Entrez databases of interest. Many attributes from the Filter field are provided in the "Links" menus that are present on an Entrez search results page, and in the "Links" box on an individual CD Summary page. A detailed explanation of each type of link is provided in the description of the "Links" box. cdd_gene[filt] will retrieve the CDD records that have associated data in the Entrez Gene database. On the CDD search results page, you can then open "Display" menu and select the Gene Links option to view the corresponding Entrez Gene records. Modification Date [mdat] Date of the most recent changes to the alignment model and/or descriptive information Number of Sites [ns] The number of conserved features, such as catalytic or binding sites, that have been annotated on a domain. Conserved features are available on NCBI-curated domains. As of April 2008, this ranges from zero to 21 sites. (To see the current range, select the "Number of Sites" search field on the "Search Builder" section of the "Advanced search" page, then use the "Show index" link to view the index of that search field and see available values.) 4[ns] will retrieve the NCBI curated domain models that contain four sites (i.e., four conserved features). Organism [Orgn] The root taxonomy node of a conserved domain. This is the highest node in the NCBI Taxonomy database that encompasses all organisms whose protein sequences are in the multiple sequence alignment for a domain model. eukaryotes[orgn] will retrieve conserved domains found in eukaryotes. PSSM Length [plen] Length of the PSSM or domain search model. This is the same as the length of the consensus sequence. Publication Date [pdat] the date on which a CD was published Structure Representative [strp] The number of structures that have protein sequences in the multiple sequence alignment for a domain model. As of January 2010, this ranges from zero to 70 protein sequences from structures. (To see the current range, select the "Structure Representative" search field on the "Search Builder" section of the "Advanced search" page, then use the "Show index" link to view the index of that search field and see available values.) NCBI-curated domain models tend to have more structure representatives because the curation process includes incorporation of protein sequences from resolved structures. However, domain models from external data sources may also contain structure representatives. As noted in the section on data processing, sequences in PFAM, SMART, and COGs alignments are substituted, whenever possible, for closely related sequences that have direct links to three-dimensional structures in the Moleclular Modeling Database (MMDB). 6[strp] will retrieve domain models that contain six protein sequences from 3D structures in their multiple sequence alignment. Subtitle [subtitle] The subtitle of a conserved domain, which may contain descriptive terms not present in the conserved domain's title. An example of a title:subtitle combination is present in pfam00654: "Voltage_CLC: Voltage gated chloride channel" A search of the subtitle field for "chloride channel" will retrieve that record and others. Note: Not all conserved domain models have subtitles, and search of the Text Word field may therefore retrieve more comprehensive results. "chloride channel"[subtitle] will retrieve the CDD records that have the phrase "chloride channel" as part of their subtitle. The quotes surrounding the search terms ensure they are searched as a phrase.** Text Word [word] The long description (text summary) of the conserved domain. The Description of Sites [sd] Brief descriptions of conserved features. Title [titl] The short name of a conserved domain, which concisely defines the domain. Example:  "Voltage gated ClC" is the short title of the NCBI-curated conserved domain model (cd00400) for the voltage gated chloride channel. Note: A search of this field will also retrieve superfamily clusters (cl* accessions) that contain one or more domain models with the search term in their short title, even if the title of the superfamily cluster itself does not contain the search string. voltage*[titl] will retrieve the CDD records that have the term "voltage" as part of their short name, such as cd00400: Voltage gated ClC and pfam00654: Voltage CLC, which represent NCBI-curated and externally imported domain models, respectively, for the voltage gated chloride channel. (The asterisk (*) is a wild card that can be used to search for a word stem.) UID [UID] Retrieves a conserved domain record by its PSSM ID. If you enter a string of digits as a query and do not specify a search field, the UID field will be searched by default. Note: As mentioned in the section on CD Assembly Process, when domain models evolve as new sequence data becomes available, their PSSMs can change, and in such cases, they received new PSSM IDs. Obsolete PSSMs (e.g., 667) cannot be retrieved through the Entrez CDD search interface, even with direct searches of the UID field, because they are no longer indexed. However, those obsolete PSSMs can be retrieved from the archival copy of the database by using the "Direct Fetch via UID" option on the CDD Search Methods page. 238233[UID] will retrieve the conserved domain record cd00400, whose PSSM ID is 79359. 79359 will also retrieve that same conserved domain record, because the UID field is searched by default for queries that are only a string of digits. * In a query, the field name may be typed as the full name or abbreviation, and may be in upper, lower, or mixed case. It must be surrounded by square brackets []. A space between the search term and the field specifier is optional. If desired, surround a phrase with quotes to force an adjacency search. For example, the sample queries below will work equally:       "chloride channel" [WORD]       "chloride channel"[WORD]       "chloride channel" [word]       "chloride channel"[Text Word] ** The quotes surrounding the search terms in the All Fields example ensure the terms are searched as a phrase. If quotes are not used and the terms are not automatically recognized as a phrase by the Entrez system, Entrez will insert a Boolean AND between the terms and they may or may not appear adjacent to each other in the retrieved records. More search tips are provided in the PubMed help document and Entrez help document. It is also possible to search for a word stem by using an asterisk (*) as a wild card; for example, chlori* will retrieve records with terms such as chloride, chlorin, chlorinate, chlorite. The Entrez Help document provides additional information about truncating search terms in this way.

Search Results

| document summary page | display settings: format, items per page, sort by | send to | filter your results | find related data |

Document Summary (DocSum) Page

After querying Entrez CDD by text term, the initial search results page (also referred to as the document summary, or "DocSum") provides a list of the conserved domain records that contain your search terms. The terms can appear in any field of the record, unless a search field was specified in the query.  (Note:  A separate part of this document describes the results of a search by protein query sequence using CD-Search.)

Click on the accession number or thumbnail image of any record on the DocSum page to view its conserved domain (CD) summary page.

If desired, you can narrow your search by restricting the query to a search field of interest or adding more terms with a Boolean AND.

Alternatively, you can broaden your search by adding more terms (e.g., synonyms) to your query with a Boolean OR, or by following links to Superfamily Members.

SAMPLE SEARCH RESULTS DISPLAY Click on the image above to open the live search results page in CDD. Note that the number of items retrieved may be different than shown here because the Conserved Domain Database continues to evolve with the addition of new data.

READ MORE ABOUT: Limits Advanced Search Search Builder Show Index History Display Settings Format Items per page Sort by "Send To" menu Subsets of Results Filter your results Find Related Data Related Structures Literature Domains Chemicals Other Links Search Details See My NCBI help for: Save Search Save Results View details for individual conserved domain

Display Settings:

The "Display settings" menu on acts upon all of the conserved domain records (default) in your search results, or on the subset you have selected with checkboxes. You can select items from multiple pages of the search results, if desired.

Format	Summary -- a summary of all of the structure records (default) retrieved by your search, or for those you have selected with checkboxes, in HTML format.The information shown for each record may include the following, as available: Short name, which concisely defines the conserved domain Thumbnail image indicating if the conserved domain includes a protein sequence from a 3D structure. If a 3D structure is included, the thumbnail will be a still graphic of the actual domain structure. If no 3D structure is available for the protein family from which the domain model was created, the thumbnail icon will show a schematic of a multiple sequence alignment. First 100 characters of the text summary, which provides a synopsis of biological function and salient features of the domain Accession number PSSMid A subset of links to additional information about the domain, including a "View in Cn3D" link that opens an interactive view of the domain's 3D structure in NCBI's free Cn3D structure viewing program and links to related data in other Entrez databases. (Note: The "Find Related Data" menu in the right margin of the search results page provides a complete list of links. That menu retrieves related data for all records (default) retrieved by your search, or for the subset of records you have selected with checkboxes.) Summary (text) -- a summary of the records retrieved by your search, in plain text format. By default, all records from your search result are listed. If you are interested only in specific records, select their checkboxes, select the desired display settings, and press "Apply" to view only those records. The information shown for each record is the same as in the "Summary" format described above, but does not include the subset of links to additional information. UI List -- a list of the unique identifiers (UI's) for all of the conserved domain records (default) retrieved by your search, or for those you have selected with checkboxes.
Items per page	By default, 20 documents are listed per page. If desired, decrease (to a minimum of 5) or increase (to a maximum of 200) the number of documents displayed per page then press the "Apply" button.
Sort by	Search results are displayed in order of decreasing relevance with respect to the query. Many search fields have a score or rank associated with them; for example, the Title and Organism fields have a high rank, while the Description of Sites field has a lower rank. The presence of a search term in any one or more of the fields is scored accordingly by the search system, and the total score given to a hit is used in determining its relevance to the query and therefore its placement on the search results page. Additional options are available to sort records by descending or ascending order of Accession, Database, Modification Date, Number of Sites, PSSM Length, Publication Date, and Structure Representatives. A few of these sort options will cause certain types of records to cluster at the top or bottom of the search results, depending on whether ascending (up) or descending (down) order is chosen. For example, if you sort by: Number of Sites - NCBI-curated domain models will appear at the top of search results (if "sort by number of sites (down)" order is selected) because conserved features, such as catalytic or binding sites, are annotated only on those domain models. PSSM Length - the superfamily records will appear at the bottom (if "sort by PSSM length (down)" is selected) because they do not have an actual Position Specific Scoring Matrix (PSSM). Rather, each member of a superfamily has a PSSM and corresponding PSSM ID. A superfamily's PSSM ID refers to the specific set of conserved domain PSSM IDs that comprise the superfamily, rather than to an actual position-specific scoring matrix for the overall superfamily. Structure Representatives - NCBI-curated domain models will tend to appear at the top of search results (if "sort by structure representatives (down)" order is selected) because the curation process includes incorporation of protein sequences from resolved structures. Domain models from external data sources may also contain structure representatives. As noted in the section on data processing, sequences in PFAM, SMART, and COGs alignments are substituted, whenever possible, for closely related sequences that have direct links to three-dimensional structures in the Moleclular Modeling Database (MMDB). Technical note: If you retrieve all records in the database by searching the Filter field for All[Filt], the records are simply displayed in descending order of UID (i.e., PSSM ID).

"Send To" menu options

The "Send To" menu options act upon all the hits retrieved by your search (default), or those you have selected by using their checkboxes.

File	Saves all the hits retrieved by your search into a plain text file, in either "Summary (text)" or "UI List" format.
Clipboard	Copies all the hits retrieved by your search (default), or those you have selected with check boxes, into a Clipboard, which temporarily stores up to 500 items (they will be lost after 8 hours of inactivity). Click on the "Clipboard: XX items" link in the upper right corner of the page to view the items in any format for up to 8 hours after your last activity in the database. The Clipboard will not add an item that is currently in the Clipboard; it will not create duplicate entries. You can remove items from the Clipboard, if desired. Entrez uses cookies to add your selections to the Clipboard. For you to use this feature, your Web browser must be set to accept cookies. Items in the Clipboard are represented by the search number #0, which may be used in Boolean search statements. For example, to limit the items you have collected in the Clipboard to those from human, use the following search: #0 AND human[organism]. This does not affect or replace the Clipboard contents. The Clipboard's "Send to" menu offers you the same "File" and "Collections" options as offered on the original search results page. The latter option saves all items (default), or the subset of items selected with check boxes, indefinitely in the My NCBI Collections section of your My NCBI account.
Collections	Saves all the hits retrieved by your search (default), or those you have selected by using their checkboxes, into the My NCBI Collections section of your My NCBI account.

Filter your results

The "Filter your results" area in the upper right corner of a search results page allows you to see all the records (default) retrieved by your search, or subsets of your search results that reflect commonly requested categories of records, and shows the corresponding number of records in each case.

The links for "NCBI-curated," "imported," "families" (individual conserved domain models), and "superfamilies" (clusters of evolutionarily related conserved domain models into which the individual conserved domain models fall) show the number of retrieved records that fall into each of those categories, and allow you to view those subsets of your search results, if desired.

Find related data:

The "Related information" box that appears in the right margin of the display for an individual record allows you to retrieve related data for that particular domain model. (For example, the "Related CDs/Superfamily Members" link for accession cd00400 will retrieve the other domain models in the Conserved Domain Database that appear to be evolutionarily related to or redundant with cd00400.)

A "Find Related Data" box (instead of an "Related information" box) will appear in the right margin of a CDD search results page if you retrieved two or more records. The "Find Related Data" box allows you to retrieve related data for all the models retrieved by your search (default), or for the domain models you have selected with checkboxes. (For example, the "Find Related Data" option for Related CDs/Superfamily Member Links will retrieve the other domain models in CDD that appear to be evolutionarily related to or redundant with the domains retrieved by your search, or with the domains you have selected with checkboxes.)

The links in either display can include the following, depending on the related data that are available for the domains you have retrieved:

• Related CDs
• Literature
• Sequence
• Structure
• BioSystems
• Other Links

A "Links" box also appears in the displays of individual conserved domain records. All links are described in the help document section on "CDD Record (CD summary page): What information is displayed for each domain model on its CD Summary page?" : "Links to related data in Entrez". The number and type of links that exist vary among CDD records, depending on the related data that are available for any given record.

Most links are accessible on both the search results page and on a CD summary page, although a few of the links are available in only one of those places (*), such as Representatives and Books links, which are available only on the CD summary page.

Entrez Protein links to Conserved Domains:

Another (indirect) way to search the Conserved Domain Database is to start in a database such as Protein or Structure and use the "Find Related Data" ad in the right margin of the search results page, or use the "Related Information" ad in the right margin of the display for an individual record, to traverse to conserved domains.

For example, all sequence records in the Entrez Protein database have been RPS-BLASTed against the Conserved Domain database. These pre-calculated search results are available as "Conserved Domains" links from protein sequence records, making protein functional information one click away from the sequence record.

Several different types of links are available, allowing you to choose: (a) the format in which you want to view the conserved domains (e.g., in graphical format as domain footprints aligned to the protein sequence, or as a list of records from the Conserved Domain Database, each of which includes a multiple sequence alignment of the proteins used to create the domain model), and (b) the level of redundancy in the list of conserved domain models (e.g., a concise list of the top scoring models or a full list of all models that have a statistically significant RPS-BLAST hit to the protein).

The number of conserved domain models retrieved, and the order in which they are sorted/presented, depends upon the view you select:

Click on the image to open the actual Entrez protein sequence record and follow the live links to conserved domains. You may need to scroll down the display to see the links.

CDD Search Results -- opens a graphical display (illustrated example) of conserved domain model footprints, ranked by their RPS-BLAST score and hit type. A model may appear more than once if it aligns to multiple regions of the query sequence. A concise display showing only the top-scoring hits is presented by default, and it can be changed to a full display of all hits if desired. more... Conserved Domains (Concise) -- opens a concise list of the conserved domain models that are the top-scoring RPS-BLAST hits to the protein query sequence. Each domain model is listed only once, even if a model had a hit to more than one region on the query sequence. more... Conserved Domains (Full) -- opens a full list of all the conserved domain models that have a statistically significant RPS-BLAST hit to the protein. Each domain model is listed only once, even if a model had a hit to more than one region on the query sequence. more... Domain Relatives -- opens a graphical display of similar domain architectures, as determined by the CDART tool, with links to the proteins that have each similar architecture. more...

More details about each link are provided in the table below:

Link Name	What you will get:
CDD Search Results	The protein sequence → "CDD Search Results" link will open a graphical display, in the CD-Search tool, that shows conserved domain footprints aligned to the protein sequence, based on the results of an RPS-BLAST search of the protein sequence against the PSSMs of all the domain models in the Conserved Domain Database (CDD). A concise display (depicted below and in a more detailed illustrated example), is shown by default and features only the top scoring models from various hit types. A toggle switch near the upper right corner of the display allows you to see the full display (illustrated example), if desired, which shows all domain models that meet or exceed the RPS-BLAST threshold for statistical significance, i.e., the E-value cutoff. (Open the protein sequence record GI 157830769, featured in the illustration below, and follow the "CDD Search Results" link to try it yourself.) In both the concise and full views, small triangles indicate the amino acids involved in conserved feaures/sites, such as catalytic and binding sites, when such annotations are available in a domain model. In these graphical views, the domain hits are ordered not only by E-value, but also by classification (specific hits, non-specific hits, superfamilies, multidomains) and model origin (with priority given to NCBI-curated domains if they meet or exceed a domain-specific threshhold score). If a domain model aligns to more than one region of the query sequence, it will be listed multiple times in the list of domain hits. This is true because the alignment coordinates and score of the domain model vary among different regions of the query sequence, and each hit is reported separately. (The list of domain hits is not shown in the illustration above but is visible beneath the graphical summary on an actual CD-Search results page.)
Conserved Domains (Concise)	The protein sequence → "Conserved Domains (Concise)" link will open a concise list, in the Entrez Conserved Domain Database, of the conserved domain models that were top-scoring hits to the protein query sequence. Each domain model will be listed only once, even if a model aligned to more than one region on the query sequence. This list of domains corresponds to those shown in the concise graphical display of CD-Search results. The same domains will appear in both views, but the order in which they are presented might vary between the displays because the graphical display sorts hits by RPS-BLAST score and hit type, and the list display does not. Also, if a superfamily on the "CDD Search Results" graphical display contains only one domain model, the "Conserved Domains (Concise)" link will retrieve the record for that sole domain model, rather than retrieving the superfamily record. For example, compare the concise set of top-scoring conserved domains on protein sequence GI 157830769, Cyclodextrin Glucanotransferase, in list vs. graphical format. Note that each link will open in a new window: Conserved Domains (Concise) -- list format CDD Search Results -- graphical display
Conserved Domains (Full)	The protein sequence → "Conserved Domains (Full)" link will open a full list, in the Entrez Conserved Domain Database, of the all the conserved domain models that have a statistically significant RPS-BLAST hit to the protein query sequence. Each domain model will be listed only once, even if a model aligned to more than one region on the query sequence. This list of domains corresponds to those shown in the full graphical display of CD-Search results. The same domains will appear in both views, but the order in which they are presented might vary between the displays because the graphical display sorts hits by RPS-BLAST score and hit type, and the list display does not. Also, if a superfamily on the "CDD Search Results" graphical display contains only one domain model, the "Conserved Domains (Full)" link will retrieve the record for that sole domain model, rather than retrieving the superfamily record. For example, compare the full set of top-scoring conserved domains on protein sequence GI 157830769, Cyclodextrin Glucanotransferase, in list vs. graphical format. Note that each link will open in a new window: Conserved Domains (Full) -- list format CDD Search Results -- graphical display, with "View Full Result" option activated
Domain Relatives	The protein sequence → "Domain Relatives" link opens a graphical display, in the Conserved Domain Architecture Retrieval (CDART) tool, of similar domain architectures, with links to the proteins that have each similar architecture. More information about CDART is provided below, in the "domain architecture" section of this document.

Domain architecture: CDART:

The Conserved Domain Architecture Retrieval Tool (CDART) program has been used to analyze the domain architecture of all sequence records in the Entrez Protein database, and to identify proteins with similar architecture. Those proteins are accessible by selecting "Domain Relatives" in the "Links" menu of a protein sequence record of interest (illustrated example).

Or, you can search CDART directly by entering a query protein sequence in FASTA format, or entering the GI or Accession number of a protein sequence that already exists in the Entrez Protein database. CDART will then retrieve proteins that contain one or more of the domains present in the query sequence.

More information about CDART is available in the overview, help document, and corresponding publication.

CDD Record (CD Summary page):   What information is displayed for each domain model?

As you are viewing a search results page, click on the thumbnail image or title for any conserved domain model to see it's summary page. The thumbnail will show a snapshot of the 3D structure if a domain model includes a protein sequence(s) from a resolved 3D structure. The thumbnail will depict a sequence alignment if a domain model does not include protein(s) from a resolved 3D structure.

A CD-summary page provides the following information for a domain model (example: cd00400: voltage-gated chloride channel):

1. text summary (synopsis of function)
2. links to the source database, literature references, and related data in Entrez, as available
3. bioassay targets and results, as available
4. statistics summarizing salient features of the domain model, such as number of protein sequence rows in the alignment, PSSM identifier, and more
5. structure viewing options to display the 3D structure(s), if available, of protein sequences used to curate the domain model.
6. conserved features (available for NCBI-Curated domains only)
7. sequence cluster phylogenetic tree for protein sequences used to curate the domain (available for NCBI-Curated domains only)
8. domain family hierarchy (available for NCBI-Curated domains only)
9. multiple sequence alignments of the proteins used to develop the domain model.

More details about each section of the CD-summary page are provided below.

Text Summary (synopsis of function):

• The text summary shown at the top of a CD summary page was written by curators at the source database and provides a synopsis of the domain's biological function. In NCBI curated domains, it also describes the taxonomic extent of the domain, whether it is a monomer or dimer, and any salient features. The text summary in a superfamily record is derived from the representative domain.

Links to related data in Entrez:

The "Links" box (illustrated at right) on an individual CD Summary page allows you to retrieve additional data associated with the domain model, including related models from the Conserved Domain Database, as well as literature, sequence, and other types of data from across the Entrez system. Most of the links are accessible from both the search results page (as "Related information" or "Find Related Data" ads in the right margin) and from the "Links" box on an individual CD Summary page. Links that are present on only one of the pages are noted with an asterisk (*), below. The number and type of links that exist vary among CDD records, depending on the related data that are available for any given record. The link types can include: Related CDs, Literature, Sequence, Structure, BioSystems, and Other Links. The "Source" link shown in the illustration opens either: (a) the root (parent) node of an NCBI-curated domain hierarchy, or (b) the corresponding domain record in the external source database, depending upon what type of domain model you are currently viewing. For example, if you are viewing a child or intermediate parent node of a hierarchy, the "Source" link will open the root node. If you are viewing the root node of the hierarchy, the "Source" link will open the corresponding domain model from an external database that was used as a seed for the curated model. If you are viewing the CDD display of a model that was imported from an external database, the "Source" link will open the corresponding domain record on the web site of the source database. In the latter case, note that CDD's display of the model might vary slightly from the source record due to the data processing procedures used by CDD.

Link Group	Link Name *	Description
Related CDs	Superfamily	This links to the record for the CDD superfamily to which this domain belongs.
	Superfamily Members	This retrieves all the other domain models that belong to the superfamily.
Literature	PubMed	PubMed citations annotated on the domain. All references have been identified by curators, either by NCBI staff for the NCBI-curated domains, or by the staff of the external databases represented in CDD. For NCBI-curated domains, the PubMed link leads to the citations that have been annotated on that particular node of a domain family hierarchy, not for all nodes in the tree. Whenever possible, the citations include articles that provide evidence for the biological function of a domain and/or discuss the evolution and classification of a domain family.
	Free in PMC	The subset of PubMed links that are available as free full text in PubMed Central.
	Books *	Full text information in the Entrez Books database that further clarifies or elucidates the domain's function, the protein's role in metabolic pathways, and other broad overview information, including diagrams and illustrations.
Sequence	Representatives *	The set of protein sequences that are present in the multiple sequence alignment for the domain model. Following the "Representatives" link will retrieve the complete sequence records from the Entrez Protein database. The number of records retrieved will be identical or similar to the number of Aligned Rows shown in the Statistics box of the CD Summary page.
	Specific Protein	The set of protein sequences found by RPS BLAST to contain the domain (with an E-value that is equal to or lower than a domain-specific Threshold E-value). These are called specific hits and represent a very high confidence that the query sequence belongs to the same protein family as the sequences use to create the domain model. The number of proteins you will retrieve by following this link is greater than retrieval from the "Representatives" link, but less than retrieval from "Related Protein" link.
	Related Protein (Protein †)	Superset of all protein sequences found by RPS BLAST to contain the domain (with an E-value equal to or better than the default cutoff of 0.01). Therefore, this superset includes two CD-Search hit types: specific hits and non-specific hits.
	Architectures (Architecture †)	Proteins found by CDART to contain one or more of the domains present in the proteins that are hit by domains found in the domain superfamily
	Gene *	Links from the RPS BLAST concise display hits to the protein sequences listed in Entrez Gene records. Details: Each protein listed in an Entrez Gene record has been RPS BLASTed against the domain models in CDD. Links are then created between specific regions of those protein sequences and top-scoring domain models which align to them. Top-scoring domain models are shown either as specific-hits, or as the superfamily to which the highest-ranking non-specific hit belongs.
	HomoloGene *	Links from the RPS BLAST concise display hits to the protein sequences listed in HomoloGene records. (The details provided for Gene links, above, also apply to HomoloGene links.)
Structure	Related Structure (Structure †)	Three-dimensional structures containing at least one protein molecule that has an RPS BLAST hit (specific hit or non-specific hit) to this domain model's PSSM (compare with "Family Structure").
BioSystems	BioSystems *	BioSystems containing proteins that have specific hits to the conserved domain. The proteins that have been associated with the BioSystem via the method descibed in data processing/create direct links/proteins. Note that the BioSystems link can appear in two different places on a search results page: The "BioSystems" link in the Find Related Data ad in the right margin of the search results page will retrieve the biosystems associated with all of the conserved domains retrieved by your search, or with those you have selected with checkboxes The "BioSystems" link (when present) that is listed beneath an individual conserved domain record on a search results page (or in the "All links for this record" ad in the right margin of the display for an individual record) will open the subset of biosystems containing proteins annotated with that specific domain model. NOTE: If a conserved domain did not get any specific hits to proteins in any BioSystem, it will not have a BioSystem link.
Other Links	Taxonomy	The highest node in the NCBI Taxonomy database that encompasses all organisms whose protein sequences are in the multiple sequence alignment for a domain model. The taxonomy link for a superfamily retrieves the highest taxonomic node for all of its constituent domain models.
	LinkOut *	External data repositories can link to records in Entrez databases, including the Conserved Domain Database, by using the LinkOut tool. Selecting this option will display brief information about each conserved domain record you have retrieved (such as its accession and short name), followed by the names of (and links to) any external data providers that have chosen to link to the conserved domain (e.g., see cd05582, which has a LinkOut to "Domain Mapping of Disease Mutations").
	Family Structure *	Three-dimensional structures containing at least one protein molecule that has an RPS BLAST specific hit to this domain model's PSSM (compare with "Related Structure").

* Most of the links from conserved domain records to related data are accessible from both the search results page (as "Find Related Data" or "Related information" menus) and from the "Links" box on an individual CD Summary page. Links that are present on only one of the pages are noted with an asterisk (*) in the table above, specifically:

The "representatives" and "books" links are found only on the CD summary page for a conserved domain; they are not present in the "Links" menus on a search results page.

Conversely, the "Gene," "HomoloGene," "LinkOut," "Family Structure," and "BioSystems" link currently appear only on the search results page.

† Some links have a short name and a long name. The search results page often uses short names in its "Links" menus due to space limitations, such as "Protein" "Architecture," and "Structure." Those same links are called "Related Protein," Architectures," and "Related Structure," respectively, on an individual CD Summary page.

BioAssay Targets and Results:

• A section entitled "BioAssay Targets and Results" appears on a conserved domain's summary page only if one or more members of the protein family have been used as targets in PubChem BioAssay records, and if at least one chemical was identified in the experiment(s) to be active against one of the targets defined by the domain family. As examples:
  • view the conserved domain summary page for:
  
  
  • cd09816: prostaglandin_endoperoxide_synthase (Animal prostaglandin endoperoxide synthase and related bacterial proteins)
  
  
  • cd05061: PTKc_InsR (Catalytic domain of the Protein Tyrosine Kinase, Insulin Receptor)


• The "BioAssay Targets and Results" section lists bioassays that have tested the activities of small molecules (e.g., chemicals) against protein sequences that have a specific hit to this domain model, and are therefore considered to be members of this protein family. Some of the information from the bioassays may be generalizable to other members of the protein family, depending on how narrowly a family is defined.



• Up to three representative bioassays are listed as examples in the "BioAssay Targets and Results" box. Click on "Explore more" at the bottom of the box to see a complete list of experiments in the PubChem BioAssay database that have tested small molecules against protein targets belonging to this protein family. From there, you can open the "BioAssays" or "Compounds" folder tabs and click on the counts in the columns such as "active," "inactive," "tested" to see the chemicals that have been screened and their activity potency in the respective bioassays.

Statistics:

What is CD-Search, and what information can it provide about a protein?

The CD-Search service is a web-based tool for the detection of conserved domains in protein sequences. It can therefore help to elucidate the protein's function.

The CD-Search service uses RPS-BLAST to compare a query protein sequence against conserved domain models that have been collected from a number of source databases, and presents results as a concise display (default), standard display, or full display.

If CD-Search finds a specific hit, there is a high confidence in the association between the protein query sequence and a conserved domain, resulting in a high confidence level for the inferred function of the protein query sequence. The other types of hits that can be found also shed light on the putative function of the query protein.

The CD-Search tool can also identify putative conserved features in a query protein sequence, when such features can be mapped from the conserved domain annotations to the query sequence. If conserved features are found, they designated by small triangles in the search results graphical summary, indicating the specific amino acids likely involved in functions such as catalysis or binding.

What is RPS-BLAST?

The CD-Search service uses RPS-BLAST, which stands for "Reverse Position-Specific BLAST". This is a variant of the popular PSI-BLAST program ("Position-Specific Iterated BLAST"). PSI-BLAST finds sequences significantly similar to the query in a database search and uses the resulting alignments to build a Position-Specific Score Matrix (PSSM) for the query. With this PSSM the database is scanned again to eventually pull in more significant hits, and further refine the scoring model.

RPS-BLAST uses the query sequence to search a database of pre-calculated PSSMs, and report significant hits in a single pass. The role of the PSSM has changed from "query" to "subject", hence the term "reverse" in RPS-BLAST.

RPS-BLAST is the search tool used in the CD-Search service. The CD-Search service provides a web-interface to the RPS-BLAST program, the CD search databases, and interactive alignment visualization including 3D structures. The search results can include several types of RPS-BLAST hits that represent various confidence levels (specific hits, non-specific hits) and domain model scope (superfamilies, multi-domains).

A standalone version of the RPS-BLAST program is available as part of the NCBI toolkit distribution. A separate section of this document describes the differences between the CD-Search web tool and the standalone RPS BLAST program.

What input is required to do a CD-Search?

| query sequence | options: database selection, expect value, Composition-corrected scoring, low complexity filter, force live search, rescue borderline hits, suppress weak overlapping hits, maximum number of hits, result mode | retrieve previous CD-Search result by RID |

Query Sequence:

You can submit a protein or nucleotide query sequence to CD-Search, either as a sequence identifier (i.e., as an accession or GI number that is valid in the NCBI Entrez system), or as FASTA-formatted or bare sequence data. Hitting the submit button will start CD-search with default settings for search sensitivity and display options.

Protein sequence queries:  There is no length limit on a protein query (CD-Search does not check the length of a protein query sequence). Searches with multiple protein query sequences can be submitted to the Batch CD-Search tool, which also accepts input as either protein sequence identifiers or raw sequence data. The Batch CD-Search Help document provides details about formatting the input, including inserting line breaks between the query proteins. If multiple protein query sequences are entered in this way on the regular CD-Search page, your query will be automatically redirected to the Batch CD-Search tool. If there are no line breaks between the query proteins, however, an error message will be displayed and no redirect will occur. (Note that Batch CD-search only works for protein sequences; it does not accept nucleotide sequences.)

Nucleotide sequence queries:  The maximum length of a nucleotide query is 200,000 base pairs. The search system translates all 6 reading frames and scans the RPS-BLAST databases with the protein products. If a nucleotide query is input as a sequence identifier (accession or GI number), it will be translated using the genetic code that corresponds to the source organism of the sequence. If a nucleotide query is input as a FASTA-formatted or bare sequence data, it will be translated using the standard genetic code. Each translation is essentially processed like a separate protein query. In other words, 1 nucleotide sequence query = 6 protein queries. CD-Search will combine the results into a single page, but will only display the translated reading frames that picked up a match in CDD.

Options:
The options below are only available when using the actual CD-Search form. Searches launched from the CDD home page or together with protein BLAST requests use default search parameters. (The CDD home page does allow you to select the database, however.)

• Database Selection:  currently, CD-Search is offered with the following search databases. Note that if you use the default "CDD" database, CD-Search automatically returns precalculated search results, unless you select the option to "force live search." If you select a database other than the default "CDD," the CD-Search program automatically uses the live search mode.



• CDD - this is a superset including NCBI-curated domains and data imported from Pfam, SMART, COG, PRK, and TIGRFAMs. It is the default database for searches.
• NCBI_Curated - NCBI-curated domains, which use 3D-structure information to explicitly to define domain boundaries, aligned blocks, and amend alignment details, and which aim to provide insights into how patterns of residue conservation and divergence in a family relate to functional properties.
• Pfam - a mirror of a recent Pfam-A database of curated seed alignments. Pfam version numbers do change with incremental updates. As with SMART, families describing very short motifs or peptides may be missing from the mirror. An HMM-based search engine is offered on the Pfam site.
• SMART - a mirror of a recent SMART set of domain alignments. Note that some SMART families may be missing from the mirror due to update delays or because they describe very short conserved peptides and/or motifs, which would be difficult to detect using the CD-Search service. You may want to try the HMM-based search service offered on the SMART site. Note also that some SMART domains are not mirrored in CD because they represent "superfamilies" encompassing several individual, but related, domains; the corresponding seed alignments may not be available from the source database in these cases. Note also that SMART version numbers do not change with incremental updates of the source database (and the mirrored CD-Search database).
• PRK - "PRK," short for Protein Clusters, is an NCBI collection of related protein sequences (clusters) consisting of Reference Sequence proteins encoded by complete prokaryotic and chloroplast plasmids and genomes. It includes both curated and non-curated (automatically generated) clusters.
• TIGRFAMs - a mirror of a recent TIGRFAMs set of domain alignments.
• COG - a mirror of the current COG database of orthologous protein families focusing on prokaryotes. Seed alignments have been generated by an automated process. An alternative search engine, "Cognitor", which runs protein-BLAST against a database of COG-assigned sequences, is offered on the COG site.
• KOG - a eukaryotic counterpart to the COG database. KOGs are not included in the CDD superset, but are searchable as a separate data set.

More information about each database is provided in the section on "Where does CDD content come from?" and the version number (as available) of each source database is provided in the CDD News page.

• Maximum number of hits: limits the size of the hit list produced by CD-Search. Typically, for average sized proteins, the number of expected domain-hits is small and the default setting of 500 should be more than sufficient.

• Expect Value (E-value): is a parameter that describes the number of hits one can "expect" to see by chance when searching a database of a particular size. The E-value setting can be modified to adjust the statistical significance threshold used for reporting matches against PSSMs in the database. False positive results should be very rare with the default setting of 0.01 (use a more conservative, i.e. lower, setting for more reliable results). Results with E-values in the range of 1 and above should be considered putative false positives. Additional information about E-value is available in the Glossary of the NCBI Handbook and in the BLAST help document.
  
  Note that the E-values you get (for any given protein query--conserved domain hit pair) on the CD-Search web service might differ from those you get when using standalone RPS-BLAST on your local PC. A separate section of this document describes the differences between the web service and standalone program and provides a tip on how you can generate the same results in standalone RPS-BLAST as those produced by the web service.

• Composition-corrected scoring, which is employed by RPS-BLAST version 2.2.28 (March 19, 2013) and up, abolishes the need to mask out compositionally biased regions in query sequences. This option is on by default.
  Note: In general, when composition-corrected scoring is on, the low complexity filter should be turned off. However, it is possible to have both options on at the same time (to filter false-positives that slip through the cracks of the composition-correction), or off at the same time (to find more distant relatives for compositionally biased queries), if desired.

• Low Complexity Filter: filters query sequences for compositionally biased regions. These regions are flagged as such and largely ignored during the search phase if filtering is turned ON (the default setting is OFF).
  Note: In general, when the low complexity filter is turned on, the composition-corrected scoring should be turned off. However, it is possible to have both options on at the same time (to filter false-positives that slip through the cracks of the composition-correction), or off at the same time (to find more distant relatives for compositionally biased queries), if desired.
  
  If the low-complexity filter is turned on and compositially biased regions are detected, they are shown in the CD-Search output as cyan regions in the bar graphic that represents the query sequence, as illustrated below. More information about the low complexity filter is also available in the BLAST help document.



• If the low-complexity filter was ON for the search, the compositionally biased regions were NOT USED in the search against the domain database and are shown as SOLID cyan blocks. (As an example, open the default CD-Search results for P14780, GI 269849668, with filtering turned ON.) However, those regions may still overlap with or be included in a domain footprint and the pair-wise alignment generated by RPS-BLAST.
  



• If the low-complexity filter was turned OFF for the search, the compositionally biased regions were USED in the search and are shown as blocks OUTLINED in cyan. (As an example, open the CD-Search results for P14780, GI 269849668, with filtering turned OFF.) Please keep in mind, however, that compositionally biased regions can cause inaccurate annotation of the query sequence.
  



• If the low complexity filter DID NOT DETECT any compositionally biased regions in the query sequence, then it is displayed as a plain grey bar (with no cyan regions), as shown in the illustrations of the sample concise display and full display of CD-Search results.

• Force Live Search - Use this option if your query is a GI or accession number of a protein sequence already in the Entrez Protein database and you prefer to see live rather than precalculated CD-Search results. Note that precalculated searches use default parameters (options), while live searches allow you to change those parameters, if desired.


• Normally, CD-search will display precalculated search results for queries that contain a GI or accession number of a sequence already in the Entrez Protein database. This is because CD-Searches are done as part of the automated processing of the Entrez Protein database, and the stored search results are readily available. If that is true for your query, the "BLAST search parameters" information shown at the bottom of a Full Display of search results will say: Data Source: Precalculated Data, and will show the parameters (options) that are used by default.


• A Live search is done automatically IF: (a) your query is a FASTA formatted sequence, and the FASTA defline does not include a GI or accession number of a sequence record in Entrez protein, or (b) your query includes a GI or accession number but you selected a search database other than the default CDD or you changed any other parameters (options) from their default settings. If a Live Search was done, the "BLAST search parameters" information shown at the bottom of a Full Display of search results will say: Data Source: Live blast search, RID = XXNNNXXNNN. The RID is a "request ID" and will enable you to retrieve the results of that particular search for the next 36 hours. The display will also show the search parameters that were applied.

• Rescue Borderline Hits: This option allows you to see hits that have an E-value above the RPS-BLAST reporting threshold (anywhere between 0.01 and 1.0), and that are consistent with known domain architectures. A rescued hit is displayed with a dashed border, and its e-value is displayed in red, as shown in the illustrated example below. Additional details about rescued hits are provided in:
  
  Derbyshire MK, Gonzales NR, Lu S, He J, Marchler GH, Wang Z, Marchler-Bauer A. Improving the consistency of domain annotation within the Conserved Domain Database. Database (Oxford) 2015 Mar 12; 2015. pii: bav012. doi: 10.1093/database/bav012. Print 2015. [PubMed PMID: 25767294] [Full Text at Oxford Journals] [Full Text in PubMedCentral]

• Suppress Weak Overlapping Hits: This option suppresses hits that have an e-value close to the RPS-BLAST reporting threshold (in between 0.01 and 0.001) but overlap with stronger hits. A suppressed hit is displayed with a strikethrough, as shown in the illustrated example below. Additional details about suppressed hits are provided in:
  
  Derbyshire MK, Gonzales NR, Lu S, He J, Marchler GH, Wang Z, Marchler-Bauer A. Improving the consistency of domain annotation within the Conserved Domain Database. Database (Oxford) 2015 Mar 12; 2015. pii: bav012. doi: 10.1093/database/bav012. Print 2015. [PubMed PMID: 25767294] [Full Text at Oxford Journals] [Full Text in PubMedCentral]

• Result Mode: allows you to select the level of detail displayed in the search results: Concise mode (illustrated example) shows only the best scoring domain model, as available for each region on the query sequence. Standard mode (illustrated example) shows the best scoring domain model from each source database, for each region on the query sequence. Full mode (illustrated example) shows all hits for each region on the query sequence. Once you are viewing the search results, you can use the "View Full Result/View Concise Result" button in the upper right corner of the search results to toggle between the two views.

Retrieve a previous CD-Search result by entering its Request ID (RID):
A Request ID (RID) is assigned to a CD-Search if it was done as a live search. In such a case, the "BLAST search parameters" information shown at the bottom of a Full Display of search results will say: Data Source: Live BLAST search, RID = XXNNNXXNNN. The RID enables you to retrieve the results of that particular search for the next 36 hours by entering that number in the "Retrieve previous CD-Search result" section of the CD-Search page. (RIDs are not assigned to CD-Searches that use precalculated results.)

What output is shown on the CD-Search results page?

The CD-Search results page provides the following display options and information for the conserved domains that align to your query sequence:

• three levels of detail in CD-Search results displays: concise results | standard results | full results
• types of RPS-BLAST hits: specific | non-specific | superfamily | multidomain
• display elements: protein classification | domain colors/shapes | jagged edges (partial matches) | double-headed arrows (structural motifs) | small triangles (conserved features/sites) | compositionally biased regions
• display controls: horizontal zoom | zoom to residue level | refine search | search for similar domain architectures
• tabular list of domain hits

Three Levels of Detail in CD-Search Results Displays:

A pull-down "View" menu in the upper right corner of a search results page allows you to to select the desired view: Concise Results, Standard Results, or Full Results. This enables you to control the level of detail shown in both the Graphical Summary (shown in the illustrations below) and Tabular List of Domain Hits (not shown in the illustrations below for brevity, but available on the actual, interactive CD-Search results page for the example featured in the illustrations).

CD-Search results can include up to four hit types that represent various confidence levels (specific hits, non-specific hits) and scope (superfamilies, multi-domains) of domain hits. In the search result displays, hits are ranked by E-value, although NCBI-curated models are ranked ahead of other hits to the region if their E-value exceeds a threshold of 1e-05.

If the protein query sequence contains compositially biased regions, those will be detected by the low-complexity filter and shown in the graphical output as cyan regions in the grey bar graphic that represents the query sequence (illustrated example). The filter can be turned ON or OFF (default) when you submit a query by using the options on the search form.

Concise Results:

The Concise display is the default output for CD-Search results and shows only the best scoring domain model, as available for each region on the query sequence, in each of three hit types: specific hits, the superfamily to which the highest-ranking hit belongs, and multi-domain models. In addition, small triangles (illustrated example) indicate the amino acids involved in conserved feaures/sites, such as catalytic and binding sites, when such annotations are available in a domain model.

If CD-Search finds both specific and non-specific hits for a region of a protein query sequence, only the highest ranking specific hit and its superfamily will be shown. If CD-Search finds only non-specific hits for a region of a protein query sequence, only the superfamily to which the hits belong will be shown, but not the non-specific hits themselves. The latter are provided only in the full display.

The top-scoring multi-domain model is shown in the concise display only if: (a) it meets or exceeds the specific hit threshold, OR (b) if it does not overlap with a specific hit or superfamily annotation and if ≥50% of the domain model's length aligns to the query protein sequence. If the top-scoring multi-domain model does not meet the 50% length threshold, it is shown on the concise display only if there is no specific hit or superfamily annotation on that query sequence region at all.

Standard Results:

The Standard result lists the best scoring domain model from each source database, as available for each region on the query sequence. In some cases, two NCBI-curated models might be shown for a given region of a protein, if the immediate parent of the highest ranking NCBI-curated conserved domain model is also in the search results. (A separate section of this document provides more information about domain family hierarchies.) The top-scoring multi-domain model from each source database is also shown.

Full Results:

The Full display shows all domain models, as available for each region on the query sequence, that meet or exceed the RPS-BLAST threshold for statistical significance (i.e., the E-value cutoff). The hit types can include specific hits, non-specific hits, the superfamily(ies) to which those hits belong, and multi-domain models. Hits are ranked by E-value, although NCBI-curated models are ranked ahead of other hits to the region if their E-value exceeds a threshold of 1e-05. In addition, small triangles indicate the amino acids involved in conserved feaures/sites, such as catalytic and binding sites, when such annotations are available in a domain model.

The bottom of the Full Display (not shown in the image below but viewable by clicking on that image to open the actual, interactive CD-Search results page) also provides a summary of BLAST search parameters, which includes information such as the database which you searched against, whether the low complexity filter was used, the expect value (E-value) threshold, the BLAST software version number, and whether RPS-BLAST did a live search or retrieved precalculated search results. If a live search was done, the BLAST Request ID (RID) is also shown in the "BLAST search parameters" section and allows you to retrieve the search results by RID anytime within 36 hours following the search, without having to re-execute it.

Types of RPS-BLAST hits:

CD-Search results can include hit types that represent various confidence levels (specific hits, non-specific hits) and domain model scope (superfamilies, multi-domains). They can be seen in both the Concise display and Full display, except for non-specific hits, which are shown only in the Full Display.

1. Specific hit is the top-ranking RPS-BLAST hit (compared to other hits in overlapping intervals) that meets or exceeds a domain-specific E-value threshold (details and illustration). It represents a very high confidence that the query sequence belongs to the same protein family as the sequences used to create the domain model, and therefore a high confidence level for the inferred function of the protein query sequence.



2. Non-specific hits meet or exceed the RPS-BLAST threshold for statistical significance (default E-value cutoff of 0.01, or an E-value selected by the user with advanced search options). (NOTE: Non-specific hits are shown only in the full display (illustration) of search results. In contrast, the concise display (illustration) shows only the superfamily to which the top-scoring non-specific hit for a given sequence region belongs.)



3. Superfamily is the domain cluster to which the specific and/or non-specific hits belong. This is a set of conserved domain models that generate overlapping annotation on the same protein sequences and are assumed to represent evolutionarily related domains. (See additional details, including information about clustering methodology, under "What is a superfamily?") In the Concise Display, if a region of the query sequence has only non-specific hits to domain models from a given superfamily, only the superfamily footprint will be displayed -- not the individual superfamily members to which the query sequence had non-specific hits. To see the latter, view the Full Display of search results. In that display, the width of the box that encloses superfamily members is determined by the alignment span of the highest scoring superfamily member.



4. Multi-domains are domain models that were computationally detected and are likely to contain multiple single domains. They are typically shown as grey-colored bars. (Examples are shown in the concise display and full display illustrations.)

Display Elements:

A number of display elements are used to graphically convey conserved domain annotations on the query sequence. Those elements are used in all three views of search results: Concise Results, Standard Results, or Full Results. The display elements, described below, include:

• protein classification
• domain colors/shapes
• jagged edges (partial matches)
• double-headed arrows (structural motifs)
• small triangles (conserved features/sites)
• compositionally biased regions

The Tabular List of Domain Hits, which appears beneath the graphical summary of search results, provides additional details about, and viewing options for, each conserved domain model that has been mapped to your query sequence. Each domain model's accession number links to the corresponding record in the Conserved Domain Database, where the model and its member sequences can be launched in free software such as the Cn3D structure viewing program, or the CDTree alignment viewer/editor.

Protein Classification:

• How is the protein classification determined?  
  
  A protein classification is shown in the CD-Search results, when possible, and provides a functional characterization of the conserved domain architecture found in the protein query (illustrated example). The protein classification section appears in CD-Search results only if we have a curated label for the protein family to which the query sequence belongs.
  
  A domain architecture is defined as the sequential order of conserved domains in a protein, and the architectures are computationally identified by the Conserved Domain Architecture Retrieval Tool (CDART).
  
  A tool called SPARCLE (Subfamily Protein Architecture Labeling Engine) is used to label the proteins that contain a given architecture.
  
  Each conserved domain architecture can be assigned a unique, functional name based on the composition of the architecture. The names are assigned either manually, through a curation process, or computationally, by an autoname algorithm or a namedbydomain algorithm.
  
  The domains used for architectures may include ancient superfamilies (like ATPase) or much more recently evolved protein subfamilies (like RAS).
  
  In the case of curated architectures, the functional characterization of each architecture is written by Conserved Domain Database Curators, based on a review of the publications associated with the proteins that contain the domain architecture.
  
  To given an example of proteins that have similar function but different domain architectures:
  
  
  • DNA gyrase B (NP_387887), an antibiotic target, has a conserved domain architecture that includes a histidine kinase-like ATPase domain, a transducer domain, a topoisomerase-primase domain, followed by a type II topoisomerase carboxy domain.
  
  
  • In contrast, enzymes of similar function, such as topoisomerase IV (Q45066), have a different domain architecture.
    
    Note: in each of the examples above, the default graphic that appears when you click on the architecture link depicts the full length protein model; click on the option to "View: Full Results" link in the upper right hand corner of the display to see the individual conserved domains that compose the full length protein model.
    
    Also, in each example, you can follow the "domain architecture ID xxxxxxx" link that appears in the "Protein Classification" section of the display to open the corresponding SPARCLE record. The SPARCLE record, in turn, lists the evidence that was used to name the architecture and contains links to other protein sequences that have the same architecture.
  
  
  There are several types of architectures:
  
  
  • superfamily architectures - domain architectures consisting solely of superfamilies.
  
  
  • subfamily architectures - domain architectures that mix superfamilies and subfamilies (i.e., conserved domain models that get a specific hit to the protein query sequence).
    
    Note: It is also possible for a domain architecture to consist of a single conserved domain footprint.
  
  
  Separate sections of this help document provide additional information about domain family hierarchies, and the hit types you see in CD-Search results, such as specific hits, non-specific hits, the superfamily(ies) to which those hits belong, and multi-domain models. Each superfamily is represented by a cartoon with a distinct color/shape combination, in order to distinguish domains from each other.
  
  The SPARCLE Help document provides additional information about that resource, including an overview, examples of how SPARCLE can be used to learn more about proteins, allowable input, a description of the search output and the contents of a SPARCLE record, and details about the data processing pipeline.

Domain colors:

• What is the significance of the domain colors/shapes?  
  
  Each superfamily is represented by a cartoon with a distinct color/shape combination, in order to distinguish domains from each other. (Because the number of superfamilies exceeds the number of different color/shape combinations, some of the superfamilies share the same display style. It should be rare, though, to encounter such pairs in the same domain architecture.)
  
  The color/shape combination remains stable for a given superfamily cluster ID, and is consistent across all NCBI tools that show conserved domain footprints on protein sequences (i.e., CD-Search, Batch CD-Search, and CDART).
  
  If the cluster ID for a superfamily changes (due to significant changes in the superfamily composition), the color/shape combination may also change.
  
  "Specific hits" are shown as bright colors, with full saturation. The "superfamily" to which a "specific hit" belongs is shown as a pastel color, with lower saturation.
  
  If there are no specific hits to a region of a protein query sequence, then the "Concise display" will show only the superfamily. Regardless whether there are specific hits or not, "Standard display" will show the superfamily plus highest ranking hit from each source database, and the "Full display" will show the all the domain models in each hit type (specific hits, non-specific hits, the superfamily(ies) to which those hits belong, and multi-domain models).

Jagged Edges:

• What do domain cartoons with jagged edges mean?  Occasionally domain-cartoons have jagged edges (illustrated example). This means that the alignment found by RPS-BLAST omitted more than 20% of the CD's extent at the n- or c-terminus (or both, as indicated by the cartoons). This feature may give hints towards truncated query sequences, false-positive hits, or unusual domain architectures involving long insertions. The exact percentage of the CD's extent used in the alignment is listed in detail in the pairwise alignment section.

Double-headed Arrows (structural motifs)

• What do the double-headed arrows mean in the Graphical Summary?  
  
  Double-headed arrows appear on a CD-Search results graphical summary only if the query protein contains structural motifs. Structural motifs are regions in proteins and protein domains that are too small to be modelled as individual evolutionarily conserved domains and too extensive to be characterized as conserved features/sites. They play a structural and/or functional role that CDD curators chose to document, as their presence contributes to functional annotation and/or protein classification. Structural motifs are particularly useful in annotating the locations of specific repeats. Examples are blades in beta-propeller structures ("closed solenoid proteins"), super-helical repeats such as Armadillo and HEAT ("open solenoid proteins"), various zinc fingers, various calcium-binding motifs, coiled coils, or transmembrane segments.
  
  The structural motifs cannot be modeled as evolutionarily conserved domains because the properties of the PSSMs as search models require a minimum length to be effective (exacerbated by the fact that many of the structural motif regions have compositional bias), and because the evolutionary history of most of these structural motifs is not clear enough to enable a representation of that history.

Small Triangles

• What do the small triangles mean in the Graphical Summary?  
  
  The small triangles beneath the query protein on a CD-Search results page indicate the residues that comprise conserved features/sites, such as binding or catalytic sites, as mapped from the conserved domain annotations to the query sequence. An illustrated example is below.
  
  The triangles appear if a region of the query protein sequence either:
  
  
  • gets a specific hit to an NCBI-curated domain domain model on which conserved features/sites have been annotated. In such a case, the conserved features/sites that have been annotated on the domain model will be mapped to the query sequence.
    
    OR
  
  
  
  • gets a non-specific hit to an NCBI-curated domain model that belongs to a superfamily whose representative has conserved/feature site annotations. In such a case, the conserved features/sites from the superfamily representative will be mapped to the query sequence. (Note that the non-specific hit will not appear on the concise display of the CD-Search results -- only the site annotations will appear there. View the full display to see both the triangles and the hit.)
  
  The triangles are shown in the same color as the domain on which they have been annotated.
  
  Click on the triangles to view details about the feature, including a multiple sequence alignment of your query sequence and the protein sequences used to curate the domain model, where hash marks (#) above the aligned sequences (illustration) show the location of the conserved feature residues. A thumbnail image, if present, provides an approximate view of the feature's location in three dimensions and options for interactive 3D structure viewing.
  
  Conserved features/sites, if present, are shown by default in the graphical display. If desired, they can be hidden by clicking on show options in the graphical summary header bar, then deactivating the show site features checkbox and pressing the update button.

Compositionally Biased Regions:

• On the CD-Search results page, what do the cyan regions mean in the bar graphic that represents the query sequence?
  These represent compositially biased regions detected in the query sequence by the low-complexity filter.


• If the low-complexity filter was ON for the search, the compositionally biased regions were NOT USED in the search against the domain database and are shown as SOLID cyan blocks. (As an example, open the default CD-Search results for P14780, GI 269849668, with filtering turned ON.) However, those regions may still overlap with or be included in a domain footprint and the pair-wise alignment generated by RPS-BLAST.
  


• If the low-complexity filter was turned OFF (default) for the search, the compositionally biased regions were USED in the search and are shown as blocks OUTLINED in cyan. (As an example, open the CD-Search results for P14780, GI 269849668, with filtering turned OFF.) Although compositionally biased regions can cause inaccurate annotation of the query sequence, their effect is ameliorated to a great extent by composition-corrected scoring, which is turned on by default.
  


• If the low complexity filter DID NOT DETECT any compositionally biased regions in the query sequence, then it is displayed as a plain grey bar (with no cyan regions), as shown in the illustrations of the sample concise display and full display of CD-Search results.

Display Controls:

The CD-Search results display can be customized with the following controls:

• horizontal zoom
• zoom to residue level
• refine search
• search for similar domain architectures

The Tabular List of Domain Hits, which appears beneath the graphical summary of search results, provides additional details about, and viewing options for, each conserved domain model that has been mapped to your query sequence. Each domain model's accession number links to the corresponding record in the Conserved Domain Database, where the model and its member sequences can be launched in free software such as the Cn3D structure viewing program, or the CDTree alignment viewer/editor.

Horizontal Zoom

• If a query sequence is very long and contains many domains (e.g., human titin isoform N2-B, gi 291045223), the details of the graphical summary might be difficult to read. In that case, you can click on show options in the graphical summary header bar, enter the desired magnification level in the horizontal zoom box, and press the update button to refresh the display.
  
  There is no specific maximum value that can be entered in the horizontal zoom box. Rather, the limit is determined by the pixel width of the graphic image displayed.
  
  
  • If the zoom value you enter is too large, the system will display the message: "invalid zoom factor". In that case, enter a smaller zoom value.
  
  
  • There might be other cases in which the zoom value is acceptable but it takes some time to generate the display. In such cases, you might get an option to stop script or continue. Choose the latter if you would like the process of generating the enlarged graphic display to continue.


• Zoom to Residue Level
  
  
  • This option displays the amino acids ("residues") in the query sequence. It also highlights the amino acids in the query that are mapped to conserved features/sites, which are denoted by small triangles in the graphical summary. As an example of the "zoom to residue level" view, see the human regulator of G-protein signaling 12 isoform 2.
  
  
  • When you activate the "zoom to residue level" setting, the "horizontal zoom" text box (which is visible when you press "show extra options") will still retain the zoom value that was used before the "zoom to residue level" option was activated. This makes it possible to easily toggle between the residue level view and the previous zoom level.
    
    Note: The "show extra options/horizontal zoom" text box will generally contain the default value of 1, unless you viewed a different magnification before zooming in to the residue level. The actual horizontal zoom level that is applied by the CD-Search program when the "zoom to residue level" option is checked varies based on length of sequence and is determined automatically by the program.
  
  
  • Note about display limits: If a protein sequence is very long, the individual amino acids might not be visible when the "zoom to residue level" option is checked. This is because images wider than approximately 35000 pixels cannot be displayed correctly in browsers. Therefore, the "zoom to residue level" option limits the display to 35000 pixels wide. If the query sequence is very long (as an example, see human titin isoform N2-B, 26,926 amino acids long), the program will still draw the residues, but they will be squeezed together and will not be easily readable as individual letters.
  
  

Refine Search

• The Refine Search button on a CD-Search results page allows you to modify your query to search against a different database and/or use advanced search options.
  

Search for Similar Domain Architectures

• The Search for Similar Domain Architectures button on a CD-Search results page retrieves proteins that contain one or more of the domains present in the query sequence, using the Conserved Domain Architecture Retrieval Tool, "CDART" (illustrated example).
  

Tabular List of Domain Hits

• Beneath the CD-Search results graphical summary is a Tabular List of Domain Hits. This table appears in all three views: Concise Display, Standard Display, and Full Display.
  
  When you mouse over any conserved domain footprint in the graphical summary, the corresponding CD accession number and description will be highlighted in the tabular list of domain hits.
  
  If a domain model aligns to more than one region of the query sequence, it will be listed multiple times in the tabular list of domain hits. This is true because the alignment coordinates and score of the domain model vary among different regions of the query sequence, and each hit is reported separately.
  
  Click on the [+] to the left of the CD accession to see a pairwise alignment of your query sequence and the consensus sequence for the domain model. (Residues that are identical between your query sequence and the consensus sequence are shown in red.) Click on the CD accession number to view the domain model's summary record in the Conserved Domain Database (CDD).
  
  If you'd like to see your query sequence embedded in the domain model's multiple sequence alignment, click on the domain footprint for any specific hit, non-specific hit, or multidomain of interest in the graphical portion of the concise or full CD-search results page. That will open the Entrez CDD record for the domain model, with your query sequence embedded in the model's multiple sequence alignment. In that view, you can change the color bits setting to increase or decrease the threshhold that determines which columns of the alignment are displayed in red. (Note: Superfamily records do not include a multiple sequence alignment display, so if you click on the footprint of any superfamily, you will see a CDD summary page that provides general information about the superfamily and lists the domain models that belong to it. Only the individual domain models will have multiple sequence alignments, and you must click on the footprints of those models in the graphical summary of a CD-Search results page (not on the superfamily's CDD summary page) in order to see your query sequence embedded in the alignment.)
  
  The tablular list of domain hits also provides a link from each domain model's accession number to the corresponding record in the Conserved Domain Database, where the model and its member sequences can be launched in free software such as the Cn3D structure viewing program, or the CDTree alignment viewer/editor.
  

What is a Specific Hit?

A specific hit is a high confidence association between a protein query sequence and a conserved domain, resulting in a high confidence level for the inferred function of the protein query sequence. It is one of four types of RPS-BLAST Hits. (See illustrations of CD-Search results concise display and full display for examples.)

In order to be considered a specific hit, an alignment of a domain model to a query protein sequence must meet two criteria:

1. The domain model must be either: (1) the top-ranked (best E-value) NCBI-Curated domain, or (2) the top-ranked domain model from an external source, if there is no NCBI-curated domain that meets all the criteria for a specific hit.
   
   If domain models from both the NCBI-curated data set and external sources meet a domain-specific threshold, the NCBI-Curated domain domain will be listed preferentially as the specific hit because it has been annotated with fine-grained evolutionary relationships, conserved sequence blocks, specific functions, and conserved features/sites based on careful review of sequence data, 3D structures, and literature. However, if no NCBI-curated domain meets the criteria for a specific hit, then the top-ranked domain model from an external source will be shown in the CD-Search results concise display if it meets all the criteria for a specific hit.



2. The E-value of the RPS-BLAST hit must be equal to or lower than a domain-specific threshold E-value.
   The domain-specific threshold is the weakest E-value obtained when each of the protein sequences used to curate a domain are RPS-BLAST'ed against that domain's Position-Specific Scoring Matrix (PSSM). In other words, the threshold is the weakest E-value among self-hits of a domain�s member protein sequences to the resulting domain model. The illustration below provides an example, showing the domain-specific threshold for cd03683, ClC-1-like chloride channel proteins. Domain-specific threshold scores are displayed (in the form of bit score) in the statistics box of a domain model's CD-summary page.

If a specific hit IS found on a protein sequence, then:

• There is a high confidence level that the query protein sequence is a member of the protein family represented by the domain model and has the specific function annotated on that domain.
• If the query sequence resides in the Entrez Protein database, the inferred function is annotated as "region" on the protein sequence record, showing the name of the high-scoring domain model and its base span. If the specific hit is to an NCBI-curated domain model that includes conserved features (residues involved in catalysis or binding), those are annotated on the protein sequence record as "sites." If the specific hit is to a domain model from an external source, and the model belongs to a superfamily whose representative is an NCBI-curated domain that has such annotations, then the conserved features/sites that have been annotated on the superfamily representative will be mapped to the query sequence.

If a specific hit IS NOT found on a query protein sequence, but the protein has an otherwise statistically significant hit (E-value cutoff of 0.01) to any domain model in CDD, the domain model is regarded as a non-specific hit. In that case:

• The general function of the domain superfamily can be inferred for the query protein sequence, but the specific function is less certain.
• If the query protein sequence resides in the Entrez Protein database, the name and general function of the domain superfamily is annotated in the protein sequence record (as a "region"). The name and function text is derived from the domain model which has been selected as the superfamily representative. Conserved features ("sites") are also annotated on the protein sequence record if the superfamily representative is an NCBI-curated domain that has such annotations.

* Domain-specific threshold scores are displayed in the statistics box of a domain model's CD summary page. In the actual calculation of domain-specific thresholds, bit scores are used rather than E-values. (A bit score is defined in the NCBI Handbook glossary and Field Guide glossary.) NOTE: The image above reflects the cd03683 domain alignment as of April 20, 2008. The scientific community's understanding of molecular data continues to evolve as research progresses, and as new as well as updated sequence data are regularly deposited into the databases. If a member sequence used in a domain alignment is later superceded by an updated version, the new sequence data and gi number will replace the old one during review/update cycles of curated domains. Some revisions to sequence data, such as upstream or downstream extensions, do not affect the domain model, but the gi number and amino acid span will change to reflect the updated sequence record.

How can I view multiple sequence alignments with my query sequence embedded?

When you click on the cartoon (colored bar representing a domain footprint) in the graphical display on the CD-search results page, an alignment view will be opened, which adds the query sequence to the multiple CD-alignment. It is possible to modify the number and type of sequences shown, as described in the help document section on CDD Record : multiple sequence alignment displays.

Alignment visualization including 3D-structures

If you display an alignment view that includes a query sequence, you can also view the same alignment in the Cn3D program by pressing the Structure View button. (Cn3D installation takes only a couple of minutes and a tutorial describes the program's features and functions. The program must be installed in order for the Structure View button to work.)

If a protein sequence from a 3D structure is included among the sequences used to curate a domain model, Cn3D will show the 3D structure as well. If the domain model includes sequences from more than one 3D structure, all of the structures will be displayed, superimposed upon each other, and their sequences will be displayed in the multiple sequence alignment.

Cn3D offers column-specific coloring by sequence conservation when invoked with multiple alignment views. This is a convenient feature to study sequence conservation within a CD-alignment and to find out how well the aligned query fits the existing patterns of conservation and variability.

How long do I have to wait for CD-Search results?

CD-search requests are submitted to the BLAST servers immediately. A typical search should take a few seconds only, depending on the size of the search database chosen, the length of the query sequence, and the load on the servers. Click here to test response time with a typical query.
CD-Search requests can also be sent to the BLAST Queuing system (this happens by default for searches launched in parallel with protein BLAST requests), use the optional button at the bottom of the CD-Search page. Requests sent to the query will take longer, but the results can be retrieved at a later time using the RID ("Request ID"), without having to re-calculate the search. A form at the bottom of the CD-Search page can be used to retrieve earlier search results by RID.

CD-Search run in parallel to protein BLAST:  When do search requests end up in the BLAST-Queue?

When CD-search is run as an integral part of protein-BLAST search requests, the jobs are put in the BLAST queue and may take a little longer to complete (depending on the system load and length of query sequence). Queued CD-search will try to retrieve the finished results every few seconds until they are available. You may also store the request-id (RID) and retrieve results later here.

Can I run RPS-BLAST locally?
How can I make my own search database for local searching?
How can I get NCBI's CDD search database for local searching?

Yes, you can run RPS-BLAST locally. A standalone version of RPS-BLAST is packaged in with the BLAST executables available on the NCBI FTP site, and is also available as part of the NCBI toolkit distribution (see ftp://ftp.ncbi.nih.gov/toolbox).

Separate directories on the FTP site provide documents that describe each of the BLAST applications, including documents for RPS-BLAST and a Formatrpsdb application that can be used to build search databases that are properly formatted for use with RPS-BLAST.

Pre-formatted search databases, which have already been processed by Formatrpsdb, are available on the CDD FTP site. A README file on the CDD FTP site also provides more details about customizing search databases.

What accounts for the differences in search results generated by the CD-Search web service and standalone RPS-BLAST?

There are several differences between the CD-Search web service and standalone RPS-BLAST, as distributed by NCBI and used with search databases as distributed by the CDD group.

The web server is optimized for the most common use of the CDD resource, which is to annotate protein sequences with clearly identified and well understood protein domains, and is also optimized for speed in order to accomodate a high volume of searches.

As part of the optimization, we use some different statistical parameters for the web service than for the standalone RPS-BLAST application. Specifically, we use a constant, assumed search "database size" setting on the web server for calculating E-values. This means that the actual size of the search database can change (we are adding new models every few weeks), but the E-value computed for any individual GI -- PSSM match will remain constant. This approach: (a) ensures that pre-calculated residues are not dependent on the actual size of the model collection (which is redundant and mostly grows by increasing that redundancy); (b) facilitates incremental updates of pre-computed sequence annotation with conserved domains; and (c) is used for the creation of protein-CDD links.

In contrast, standalone RPS-BLAST does not employ the constant, assumed database size parameter. So when you use a search set downloaded from the CDD FTP site, the database size might be different than the one used by the CD-Search web service, and the same hit of your query protein to a model will receive a different E-value in the standalone result. For example, if the size of the FTP'ed database is smaller than what the CD-Search web service assumes in its database size parameter, the same hit of your query protein to a model will receive a lower E-value in the standalone. Conversely, if the size of the FTP'ed database is larger than what the CD-Search web service assumes in its database size parameter, the same hit of your query protein to a conserved domain model will receive a higher E-value in the standalone.

If you want standalone RPS-BLAST to use the same database size parameter that is used for the web server (and thereby reproduce the same E-values with standalone RPS-BLAST that are generated by the web service), you can do that by creating an "alias" file on your local computer and placing it in the same directory as the standalone RPS-BLAST executable. The file can have a name such as "mycdd.pal" and can have contents such as the following (where lines starting with "#" are comments):

	    #
	    # RPSBLAST alias file
	    #
	    TITLE mycdd
	    #
	    DBLIST ./Cdd
	    #
	    STATS_TOTLEN    13521388
	    STATS_NSEQ      59695
	    

This will now let you search against the database named "Cdd" using the two search set size parameters as specified, e.g.:

	    ~$ rpsblast -i rpstest.tfa -d mycdd -F T -e 0.01 -m 9
	    # RPSBLAST 2.2.26 [Sep-21-2011]
	    # Query: gi|156356500|ref|XP_001623960.1| predicted protein [Nematostella vectensis]
	    # Database: mycdd
	    # Fields: Query id, Subject id, % identity, alignment length, mismatches,
	    gap openings, q. start, q. end, s. start, s. end, e-value, bit score
	    gi|156356500|ref|XP_001623960.1|        gnl|CDD|197660  31.91   47      29
	    2       432     475     4       50      7e-04 36.9
	    gi|156356500|ref|XP_001623960.1|        gnl|CDD|197660  31.48   54      31
	    3       493     545     6       54      8e-04 36.5
	    gi|156356500|ref|XP_001623960.1|        gnl|CDD|197660  33.33   42      27
	    1       312     352     2       43      0.003 35.3
	    gi|156356500|ref|XP_001623960.1|        gnl|CDD|119391  23.53   51      34
	    2       493     542     1       47      8e-04 36.4
	    gi|156356500|ref|XP_001623960.1|        gnl|CDD|119391  21.57   51      35
	    2       375     424     1       47      0.003 34.5
	    gi|156356500|ref|XP_001623960.1|        gnl|CDD|177721  24.47   94      56
	    3       463     541     18      111     0.005 38.6
	    

In addition to the different statistical parameters, the CD-Search web service does not filter out compositionally biased regions in the query sequence by default. It uses composition-corrected scoring to mitigate the effects of compositional bias. In contrast, standalone RPS-BLAST filters out compositionally biased segments and does not employ composition-corrected scoring. In the current RPS-BLAST version 2.13.0 (as of Oct. 2022), you can set parameters to replicate CD-Search settings by specifying "-comp_based_stats 1" and "-seg no" on the command line. If those options are not specified, standalone RPS-BLAST may retrieve somewhat different results. Finally, some advanced options in standalone RPS-BLAST are not available in the web service, such as the ability to use a single-hit/two-pass mode in order to detect more distant homologous relationships. Users who select such options in the standalone version may get different search results with the web service.

| What is batch CD-Search? | input | output | scripted data downloads | references |

WHAT IS BATCH CD-SEARCH?

INPUT

Protein sequences only | protein unique identifiers (UIDs) or protein sequences | maximum input
Adjust search options: search mode, database selection, expect value, composition-corrected scoring, low complexity filter, maximum hits, include retired sequences
Optional job title | e-mail notification when job is done | retrieve previous search result

OUTPUT

| job summary, statistics, & sample data table | download options:  data type (target data),  data mode | graphical display |

SCRIPTED DATA DOWNLOADS (WEB API)

| HTTP GET or POST requests | maximum input |search ID |
| submit:  base URL, parameters, examples | check status | retrieve |
| sample PERL script for HTTP POST operations:  input, PERL script, sample run, output |