EFI - Enzyme Similarity Tool

This web resource is supported by a Research Resource from the National Institute of General Medical Sciences (R24GM141196-01).

The tools are available without charge or license to both academic and commercial users.

Please cite your use of the EFI tools:

Rémi Zallot, Nils Oberg, and John A. Gerlt, The EFI Web Resource for Genomic Enzymology Tools: Leveraging Protein, Genome, and Metagenome Databases to Discover Novel Enzymes and Metabolic Pathways. Biochemistry 2019 58 (41), 4169-4182. https://doi.org/10.1021/acs.biochem.9b00735

Nils Oberg, Rémi Zallot, and John A. Gerlt, EFI-EST, EFI-GNT, and EFI-CGFP: Enzyme Function Initiative (EFI) Web Resource for Genomic Enzymology Tools. J Mol Biol 2023. https://doi.org/10.1016/j.jmb.2023.168018

The EFI Tools were upgraded on 10/26/26. While the updated site is functionally identical to the old site, jobs run prior to 10/24/26 will only be available through the results link in the finalization email (not the Previous Jobs tab). User accounts created on the old site will no longer be available, and users are encouraged to create new accounts.

If you encounter issues, please report it to us via the Feedback page and our team will address your specific problem.

A sequence similarity network (SSN) allows for visualization of relationships among protein sequences. In SSNs, the most related proteins are grouped together in clusters. The Enzyme Similarity Tool (EFI-EST) makes it possible to easily generate SSNs. Cytoscape is used to explore SSNs.

InterProScan sequence search can be used to find matches within the InterPro database for a given sequence.

Information on Pfam families and clans and InterPro family sizes is available on the Family Information page.

Previous Jobs

Create a user account or login to see job history.

Generate a SSN for a single protein and its closest homologues in the UniProt, UniRef90, or UniRef50 database.

The input sequence is used as the query for a search of the UniProt, UniRef90, or UniRef50 database using BLAST. For the UniRef90 and UniRef50 databases, the sequence of the cluster ID (representative sequence) is used for the BLAST.

The database is selected using the BLAST Retrieval Options.

An all-by-all BLAST is performed to obtain the similarities between sequence pairs to calculate edge values to generate the SSN.

Query Sequence:

Input a single protein sequence only. The default maximum number of retrieved sequences is 1,000. A FASTA header line is permitted at the top of the input.

BLAST Retrieval Options

UniProt BLAST query e-value:

Negative log of e-value for retrieving similar sequences (≥ 1; default: 5)

Maximum number of sequences retrieved:

(≤ 10,000, default 1,000)

Sequence database:

Fragment Option

UniProt designates a Sequence Status for each member: Complete if the encoding DNA sequence has both start and stop codons; Fragment if the start and/or stop codon is missing. Approximately 10% of the entries in UniProt are fragments.

Exclude Fragments:

Check to exclude UniProt-defined fragments in the results. [default: off]

For the UniRef90 and UniRef50 databases, clusters are excluded if the cluster ID ("representative sequence") is a fragment.

UniProt IDs in UniRef90 and UniRef50 clusters with complete cluster IDs are removed from the clusters if they are fragments.

Filter by Taxonomy

From preselected conditions, the user can select "Bacteria, Archaea, Fungi", "Eukaryota, no Fungi", "Fungi", "Viruses", "Bacteria", "Eukaryota", or "Archaea" to restrict the retrieved sequences to these taxonomy groups, i.e., the taxonomy filter is applied after the specified number of sequences is retrieved.

"Bacteria, Archaea, Fungi", "Bacteria", "Archaea", and "Fungi" select organisms that may provide genome context (gene clusters/operons) useful for inferring functions.

The retrieved sequences also can be restricted to taxonomy categories within the Superkingdom, Kingdom, Phylum, Class, Order, Family, Genus, and Species ranks. Multiple conditions are combined to be a union of each other.

The sequences from the UniRef90 and UniRef50 databases are the UniRef90 and UniRef50 clusters for which the cluster ID ("representative sequence") matches the specified taxonomy categories. The UniProt members in these clusters that do not match the specified taxonomy categories are removed from the cluster.

The taxonomy database used for filtering is maintained by the UniProt group is based on the NCBI taxonomy database https://www.ncbi.nlm.nih.gov/taxonomy, and is supplemented with data specific to the UniProt Knowledgebase (UniProtKB).

SSN Edge Calculation Option

SSN Edge BLAST E-value: Negative log of e-value for all-by-all BLAST (≥1; default 5)

Input an alternate e-value for BLAST to calculate similarities/edge alignment scores similarities. The default parameter (5) is useful for most sequences. However, a larger e-value/smaller negative log should be used for short sequences or when low pairwise identities may be useful for separating functionally distinct SSN clusters.

Protein Family Addition Options

Pfam and/or InterPro Families and/or Pfam clans:

The input format is a single family or comma/space separated list of families. Families should be specified as PFxxxxx (five digits), IPRxxxxxx (six digits) or CLxxxx (four digits) for Pfam clans.

(Type at least 3 characters)

Fraction: Reduce the number of sequences used to a fraction of the full family size (≥ 1; default: 1)

Selects every Nth sequence in the family; the sequences are assumed to be added randomly to UniProtKB, so the selected sequences are assumed to be a representative sampling of the family. This allows reduction of the size of the SSN. Sequences in the family with SwissProt annotations will always be included; this may result in the size of the resulting data set being slightly larger than the fraction specified.

UniRef90 clusters contain UniProt IDs that share ≥90% sequence identity and have 80% overlap with the longest sequence in the cluster ("seed sequence"); as a result, the UniProt IDs in the cluster usually are functionally homogeneous, i.e., orthologues. UniRef50 clusters contain UniProt IDs that share ≥50% sequence identity and have 80% overlap with the seed sequence; as a result, the UniProt IDs in the cluster often are functionally heterogeneous, e.g., paralogues.

The sequences from the UniRef90 and UniRef90 databases are the UniRef90 and UniRef50 clusters for which the cluster ID ("representative sequence") matches the specified families. The UniProt members in these UniRef90 and Uni/Ref50 clusters that do not match the specified families are removed from the cluster.

Job name:

Database Version:

E-mail:

You will be notified by e-mail when your submission has been processed.

Generate a SSN for a protein family.

The members of the input Pfam families, InterPro families, and/or Pfam clans are selected from the UniProt, UniRef90, or UniRef50 database.

Pfam and/or InterPro Families and/or Pfam clans:

Sequence database:

The input format is a single family or comma/space separated list of families. Families should be specified as PFxxxxx (five digits), IPRxxxxxx (six digits) or CLxxxx (four digits) for Pfam clans.

(Type at least 3 characters)

Fragment Option

Exclude Fragments:

Check to exclude UniProt-defined fragments in the results. [default: off]

For the UniRef90 and UniRef50 databases, clusters are excluded if the cluster ID ("representative sequence") is a fragment.

UniProt IDs in UniRef90 and UniRef50 clusters with complete cluster IDs are removed from the clusters if they are fragments.

Filter by Taxonomy

"Bacteria, Archaea, Fungi", "Bacteria", "Archaea", and "Fungi" select organisms that may provide genome context (gene clusters/operons) useful for inferring functions.

Protein Family Size Options

Fraction: Reduce the number of sequences used to a fraction of the full family size (≥ 1; default: 1)

Family Domain Boundary Option

Pfam and InterPro databases define domain boundaries for members of their families.

Domain:

Sequences trimmed to the domain boundaries defined by the input family will be used for the calculations.

Region:

N-Terminal

Domain [default]

C-Terminal

If enabled, only the sequence corresponding to the defined domain for the family(ies) will be used for the computation. If the N-terminal option is selected, the sequence that is N-terminal to the specified domain will be used. If the C-terminal option is selected, the sequence that is C-terminal to the specified domain will be used.

SSN Edge Calculation Option

SSN Edge BLAST E-value: Negative log of e-value for all-by-all BLAST (≥1; default 5)

Job name:

Database Version:

E-mail:

You will be notified by e-mail when your submission has been processed.

Generate a SSN from FASTA-formatted UniProt sequences.

An all-by-all BLAST is performed to obtain the similarities between sequence pairs to calculate edge values to generate the SSN.

Input multiple sequences in FASTA format into the Sequences text input or upload a FASTA-formatted sequence file.

The sequences in the FASTA file are used to calculate edge values.

The ID in the header that immediately follows the ">" is used to retrieve node attribute information. Acceptable IDs include UniProt IDs, PDB IDs, and NCBI GenBank IDs that have equivalent entries in the UniProt database. ?

If the header for a sequence does not contain an acceptable ID for retrieving node attribute information, the SSN provides node attributes for only the sequence, sequence length, and the header as the Description.

If the user identifies the input sequences as UniRef50 or UniRef90, the node attributes will include the UniRef Cluster Size and UniRef Cluster IDs node attributes. The other node attributes will be lists of the values for UniRef cluster IDs in the node.

Use UniProt IDs
Use UniRef50 or UniRef90 Cluster IDs

Input FASTA-formatted sequences with UniProt accession IDs in the header or upload a file.

Sequences:

FASTA File:

Select a FASTA file containing UniProt IDs and sequences to upload

Input FASTA-formatted sequences with UniRef50 or UniRef90 accession IDs in the header or upload a file.

Sequences:

FASTA File:

Select a FASTA file containing UniRef IDs and sequences to upload

Input accession IDs are:

Filter by Taxonomy

"Bacteria, Archaea, Fungi", "Bacteria", "Archaea", and "Fungi" select organisms that may provide genome context (gene clusters/operons) useful for inferring functions.

Protein Family Addition Options

Pfam and/or InterPro Families and/or Pfam clans:

The input format is a single family or comma/space separated list of families. Families should be specified as PFxxxxx (five digits), IPRxxxxxx (six digits) or CLxxxx (four digits) for Pfam clans.

(Type at least 3 characters)

Fraction: Reduce the number of sequences used to a fraction of the full family size (≥ 1; default: 1)

SSN Edge Calculation Option

SSN Edge BLAST E-value: Negative log of e-value for all-by-all BLAST (≥1; default 5)

Job name:

Database Version:

E-mail:

You will be notified by e-mail when your submission has been processed.

Generate a SSN from a list of UniProt, UniRef, NCBI, or Genbank IDs.

An all-by-all BLAST is performed to obtain the similarities between sequence pairs to calculate edge values to generate the SSN.

Use UniProt IDs
Use UniRef50 or UniRef90 Cluster IDs

Input a list of UniProt, NCBI, or Genbank (protein) accession IDs, or upload a text file.

Sequences:

Accession IDs File:

Select a text file containing UniProt IDs to upload

Input a list of UniRef50 or UniRef90 cluster accession IDs, or upload a text file.

Sequences:

Accession IDs File:

Select a text file containing UniRef IDs to upload

Input accession IDs are:

Fragment Option

Exclude Fragments:

Check to exclude UniProt-defined fragments in the results. [default: off]

For the UniRef90 and UniRef50 databases, clusters are excluded if the cluster ID ("representative sequence") is a fragment.

UniProt IDs in UniRef90 and UniRef50 clusters with complete cluster IDs are removed from the clusters if they are fragments.

Filter by Taxonomy

"Bacteria, Archaea, Fungi", "Bacteria", "Archaea", and "Fungi" select organisms that may provide genome context (gene clusters/operons) useful for inferring functions.

Filter by Family

The input list of UniRef90 or UniRef50 cluster IDs should (must!) be filtered with the same list of Pfam families, InterPro families, and/or Pfam clans used to generate the IDs, if:

The input list of UniRef90 or UniRef50 IDs is obtained from 1) the Color SSN or Cluster Analysis utility for a Families option (Option B) EFI-EST SSN, 2) the Families option of the Taxonomy Tool, or 3) the Accession IDs option of the Taxonomy Tool.

Input a list of Pfam families, InterPro families, and/or Pfam clans to restrict the UniProt and/or UniRef IDs in the SSN to these families.

Family(s):

The input format is a single family or comma/space separated list of families. Families should be specified as PFxxxxx (five digits), IPRxxxxxx (six digits) or CLxxxx (four digits) for Pfam clans.

For input lists of UniRef90 and UniRef50 clusters, the cluster ID (representative sequence) is used to identify those that match the list of families and are included in the SSN. The UniProt members in these clusters that do not match the input families are removed from the cluster and are not included in the SSN node attributes.

Protein Family Addition Options

Pfam and/or InterPro Families and/or Pfam clans:

The input format is a single family or comma/space separated list of families. Families should be specified as PFxxxxx (five digits), IPRxxxxxx (six digits) or CLxxxx (four digits) for Pfam clans.

(Type at least 3 characters)

Fraction: Reduce the number of sequences used to a fraction of the full family size (≥ 1; default: 1)

Family Domain Boundary Option

Pfam and InterPro databases define domain boundaries for members of their families.

Domain:

Sequences trimmed to the domain boundaries defined by the input family will be used for the calculations.

Family: Use domain boundaries from the specified family (enter only one family).

Region:

N-Terminal

Domain [default]

C-Terminal

SSN Edge Calculation Option

SSN Edge BLAST E-value: Negative log of e-value for all-by-all BLAST (≥1; default 5)

Job name:

Database Version:

E-mail:

You will be notified by e-mail when your submission has been processed.

EFI - Enzyme Similarity Tool

Previous Jobs

BLAST Retrieval Options

Fragment Option

Filter by Taxonomy

SSN Edge Calculation Option

Protein Family Addition Options

Fragment Option

Filter by Taxonomy

Protein Family Size Options

Family Domain Boundary Option

SSN Edge Calculation Option

Filter by Taxonomy

Protein Family Addition Options

SSN Edge Calculation Option

Fragment Option

Filter by Taxonomy

Filter by Family

Protein Family Addition Options

Family Domain Boundary Option

SSN Edge Calculation Option

Sequence Filter

WebLogos

Consensus Residues

HMMs

Length Histograms