_/_/_/_/ _/_/_/_/_/ _/_/_/ _/ _/ _/ _/ _/ _/ _/ _/_/ _/_/ _/ _/ _/ _/ _/ _/ _/ _/ _/_/_/_/ _/_/_/_/ _/_/_/_/_/ _/ _/ _/ _/ _/ _/ _/ _/ _/ _/ _/ _/ _/ _/ _/ _/ _/ _/ _/ _/ This document currently includes a more detailed description of the fields used in the Pfam database. The format of Pfam entries has become stricter and we now enforce some ordering of the fields. Pfam entries are composed of four sections shown in the figure below. __________________________________ | | | Header Section | | | |________________________________| | | | Reference Section | | | |________________________________| | | | Comment Section | | | |________________________________| | | | Alignment Section | | | |________________________________| Header Section: --------------- The header section mainly contains compulsory fields. These include Pfam specific information such as accession numbers and identifiers, as well as a short description of the family. The only non-compulsory field in the header section is the PI field. All the fields in this section are described below. AC Accession number: PFxxxxx or PBxxxxxx The Pfam-A accession numbers PFxxxxx are the stable identifier for each Pfam families. The Pfam-B accession PBxxxxxx numbers are not stable between releases of Pfam. PFxxxxx for Pfam entries PBxxxxxx for Pfam-B entries ID Identification: 15 characters or less This field is designed to be a meaningful identifier for the family. Capitalisation of the first letter will be preferred. Underscores are used in place of space, and hyphens are only used to mean hyphens. DE Definition: 80 characters or less This must be a one line description of the Pfam family. AU Author: Author of the entry. The format for this record is shown below, this is a comma separated list on a single line. AU Bloggs JJ, Bloggs JE AL Alignment method of seed: The method used to align the seed members. This field has a restricted vocabulary. Currently the approved AL lines are shown below. It is important to note that this field only gives a guide to the method used for alignment construction. You may find for example that Clustalw does not give an identical alignment to that found in Pfam even if the AL line shows Clustalw as the method. AL Clustalv AL Clustalw AL Clustalw_mask_xxxx AL Clustalw_manual AL Domainer AL HMM_built_from_alignment AL HMM_simulated_annealing AL Manual AL Prosite_pattern AL Prodom AL Structure_superposition AL Domainer AL pftools AL Unknown AL T_Coffee AL MAFFT AL Alignment kindly provided by SMART AL converted_from_SMART Any method can have _manual appended to it, to indicate minor changes. e.g AL Clustalw_manual Manual alignments are those from any method which have been altered by hand. BM HMM building command lines. See the HMMER 2 user's manual for full instructions on building HMMs. Also see URL: http://hmmer.wustl.edu/ An example of the BM lines from a single entry BM hmmbuild -F HMM_ls SEED BM hmmcalibrate --seed 0 HMM_ls All models are calibrated using a seed of zero to allow exact replication of HMM construction. SE Source of seed: The source suggesting seed members belong to a family. GA Gathering threshold: Search threshold to build the full alignment. GA lines are the thresholds in bits used in the hmmsearch command line. An example GA line is shown below: GA 25.00 15.00; 12.00 5.00 The order of the thresholds is ls mode sequence, ls mode domain, fs mode sequence, fs mode domain. The corresponding hmmsearch command line for the ls mode HMM would be: hmmsearch -T 25 --domT 15 HMM DB The -T option specifies the whole sequence score in bits, and the --domT option specifies the per-domain threshold in bits. NC Noise cutoff: This field refers to the bit scores of the highest scoring match not in the full alignment. An example NC line is shown below NC 19.50 18.10; 11.10 4.60 As with the GA line, this field contains two sets of two numbers - the first set refer to the ls mode HMM and the second set to the fs mode HMM. The first number in each set refers to the highest whole sequence score in bits of a match not in the full alignment, and the second number specifies the highest per-domain score in bits of a match not in the full alignment. These two scores may not refer to the same sequence. TC Trusted cutoff: This field refers to the bit scores of the lowest scoring match in the full alignment. An example TC line is shown below TC 23.00 16.10; 17.30 6.10 As with the GA line, this field contains two sets of two numbers - the first set refer to the ls mode HMM and the second set to the fs mode HMM. The first number in each set refers to the lowest whole sequence score in bits of a match in the full alignment, and the second number specifies the lowest per-domain score in bits of a match in the full alignment. These two scores may not refer to the same sequence. TP Type field: Single word The type field is a compulsory field describing the type of family. At present it can be one of: TP Family TP Domain TP Repeat TP Motif PI Previous IDs: Semi-colon list The most recent names are stored on the left. This field is non-compulsory. Reference Section: ------------------ The reference section mainly contains cross-links to other databases, and literature references. All the fields in this section are described below. DC Database Comment: Comment for database reference. DR Database Reference: Reference to external database. All DR lines end in a semicolon. Pfam carries links to a variety of databases, this information is found in DR lines. The format is DR Database; Primary-id; For SCOP links a third field is added indicating the level of placement in the SCOP hierarchy. Examples of each database link are shown below. For PDB links the second field contains the PDB identifier and chain identifier if present. The third and fourth fields contain the start and end points within the PDB entry. DR EXPERT; jeisen@leland.stanford.edu; DR MIM; 236200; DR PFAMB; PB000001; DR PRINTS; PR00012; DR PROSITE; PDOC00017; DR PROSITE_PROFILE; PS50225; DR SCOP; 7rxn; sf; DR SCOP; 1pii; fa; DR PDB; 2nad A; 123; 332; DR SMART; CBS; DR URL; http://www.gcrdb.uthscsa.edu/; DR LOAD; ku; DR HOMSTRAD; gdh; Links to PDBSUM at are also derived from the SCOP DR lines. RC Reference Comment: Comment for literature reference. RN Reference Number: Digit in square brackets Reference numbers are used to precede literature references, which have multiple line entries RN [1] RM Reference Medline: Eight digit number An example RM line is shown below RM 91006031 The number can be found as the UI number in pubmed http://www.ncbi.nlm.nih.gov/PubMed/ RT Reference Title: Title of paper. RA Reference Author: All RA lines use the following format RA Bateman A, Eddy SR, Mesyanzhinov VV; RL Reference Location: The reference line is in the format below. RL Journal abbreviation year;volume:page-page. RL Virus Genes 1997;14:163-165. RL J Mol Biol 1994;242:309-320. Journal abbreviations can be checked at http://expasy.hcuge.ch/cgi-bin/jourlist?jourlist.txt. Journal abbreviation have no full stops, and page numbers are not abbreviated. Comment Section: ---------------- The comment section contains functional information about the Pfam family. The only field in the comment section is the CC field. CC Comment: Comment lines provide annotation and other information. Annotation in CC lines does not have a strict format. Links to Pfam families can be provided with the following syntax: Pfam:PFxxxxx. Links to SWISS-PROT and SP-TrEMBL sequences can be provided with the following syntax: Swiss:Accession. Links to the enzyme classification database (EC) can be provided with the following syntax: EC:X.X.X.X Alignment Section: ------------------ AM buildmethod Indicates the order that ls and fs matches are aligned to the model to give the full alignment. globalfirst - ls matches are aligned first followed by fs matches that do not overlap. byscore - matches are aligned in order of evalue score - best first. localfirst - fs matches are aligned first followed by ls matches that do not overlap. NE Pfam accession; Pfam family may be nested within this family. Family aand this family are allowed to overlap. NL Sequence/start-stop; Indicates the location of the nested domain within the full alignment. Tied to a sequence SQ Sequence: Number of sequences, start of alignment. // End of alignment The alignment is in Stockholm format. This includes mark-ups of four types: #=GF #=GC #=GS #=GR Recommended placements: #=GF Above the alignment #=GC Below the alignment #=GS Above the alignment or just below the corresponding sequence #=GR Just below the corresponding sequence The alignment formats have the following size limits: : max 4096 characters. : max 50 characters. max 50 characters. These details can also be found on the web, see URL: http://www.cgr.ki.se/cgr/groups/sonnhammer/Stockholm.html Structural mark ups are now provided by Pfam. For each sequence of known structure we provide #=GR lines with feuturenames SS and SA for secondary structure and surface accessibility respectively. For the whole family we provide consensus #=GC lines with feature names SS_cons and SA_cons. Pfam marks up active site residues in the multiple sequence alignments. Active site residues are derived from the Swiss-Prot feature table. Active sites which are not annotated in the Swiss-Prot feature table as being probable, potential or by similarity are given the feature name AS. Pfam predicts a residue to be an active site residue if it aligns in a Pfam alignment with a Swiss-Prot annotated active site, and is of the same amino acid type. Active sites which are predicted by Pfam are given the feature name pAS. Active site residues which are annotated in Swiss-Prot as being probable, potential or by similarity are marked as predicted active sites, as long as they do no overlap with a Pfam predicted active site. Active sites which are annotated as probable, potential or by similarity by Swiss-Prot are given the feature name sAS. In all cases, active sites are marked with an asterix. Below is an example: #=GR SERA_ECOLI/12-325 AS ...........*.............. #=GR SERA_ECO57/12-325 pAS ...........*.............. #=GR YN14_YEAST/124-327 sAS ........*................. The consensus sequence for both the SEED and full alignment are provided by Pfam, denoted by #=GC seq_cons at the beginning of the line. In all cases a threshold of 60% is used (i.e 60% or above, of the amino acids in this column belong to this class of residue). Below is the key to the alignment mark up. The program used was orginally from the Consensus program by Nigel Brown of the EMBL. class key residues A A A C C C D D D E E E F F F G G G H H H I I I K K K L L L M M M N N N P P P Q Q Q R R R S S S T T T V V V W W W Y Y Y alcohol o S,T aliphatic l I,L,V any . A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y aromatic a F,H,W,Y charged c D,E,H,K,R hydrophobic h A,C,F,G,H,I,K,L,M,R,T,V,W,Y negative - D,E polar p C,D,E,H,K,N,Q,R,S,T positive + H,K,R small s A,C,D,G,N,P,S,T,V tiny u A,G,S turnlike t A,C,D,E,G,H,K,N,Q,R,S,T Flat-files: ----------- Historically, the Pfam library of HMMs has been searched against the UniProtKB database. As of release 22.0, we have started providing Pfam domain annotations for sequences from metagenomics projects and sequences from the GenPept database. The flat-files called Pfam-A.full and Pfam-A.seed are based on the UniProtKB database. Additional files for the GenPept and metagenomics sequences are outlined below. NCBI GenPept sequences We download a fasta file of the GenPept protein sequences from the NCBI ftp site. The name and version number of this file is in the form 'rel158.fsa.aa' and can be found on the Pfam ftp site. All GenPept sequences which score above the curated threshold for each Pfam-A family are included the in the full alignment for that family. The alignments for the GenPept data can be found in the file 'Pfam-A.full.ncbi'. Note that for this dataset we resolve overlaps between clan members, but not those that occur between families that are not in a clan. However, since we curate Pfam-A domain thresholds in a conservative manner to ensure high specificity (at the expense of some sensitivity), we expect the number of domain overlaps for the GenPept data to be low. Furthermore, approximately three-quarters of the sequences in GenPept are identical to a UniProtKB entry which are guaranteed to be non-overlapping. The file 'genpeptpfam' contains a graphical representation of the Pfam domain structure for GenPept sequences. Metagenomics The file 'metaseq' contains a fasta file of metagenomics sequences that we have collected from various sources. All metagenomics sequences which score above the curated threshold for each Pfam-A family are included the in the full alignment for that family. The alignments for the metagenomics data can be found in the file 'Pfam-A.full.metagenomics'. As with the GenPept data, we have not resolved any overlaps, but, unlike the situation with the GenPept data, we have not "competed" the overlapping sequence hits for families within clans, which means that there will inevitably be some overlapping hits between families that belong to the same clan. The file 'metagenomicspfam' contains a graphical representation of the Pfam domain structure for the metagenomics sequences contained in 'metaseq'. Active site alignments As of release 22.0 we have added an option in the program pfam_scan.pl to predict active sites. To use this feature, the user must download a set of Pfam alignments. These alignments are contained within the tarball AS.tgz.