sigcleave Wiki The master copies of EMBOSS documentation are available at http://emboss.open-bio.org/wiki/Appdocs on the EMBOSS Wiki. Please help by correcting and extending the Wiki pages. Function Reports on signal cleavage sites in a protein sequence Description sigcleave predicts the site of cleavage between a signal sequence and the mature exported protein using the method of von Heijne. It reads one or more protein sequences and writes a standard EMBOSS report with the position, length and score of each predicted signal sequence. Optionally, you may specify the sequence is prokaryotic and this will change the default scoring data file used. The predictive accuracy is estimated to be around 75-80% for both prokaryotic and eukaryotic proteins. Algorithm sigcleave uses the method of von Heijne as modified by von Heijne in his later book where treatment of positions -1 and -3 in the matrix is slightly altered (see references). The minimum scoring weight value (-minweight) for the predicted cleavage site is specified. The value of -minweight should be at least 3.5. At this level, the method should correctly identify 95% of signal peptides, and reject 95% of non-signal peptides. The cleavage site should be correctly predicted in 75-80% of cases. Usage Here is a sample session with sigcleave % sigcleave Reports on signal cleavage sites in a protein sequence Input protein sequence(s): tsw:ach2_drome Minimum weight [3.5]: Output report [ach2_drome.sig]: Go to the input files for this example Go to the output files for this example Command line arguments Reports on signal cleavage sites in a protein sequence Version: EMBOSS:6.4.0.0 Standard (Mandatory) qualifiers: [-sequence] seqall Protein sequence(s) filename and optional format, or reference (input USA) -minweight float [3.5] Minimum scoring weight value for the predicted cleavage site (Number from 0.000 to 100.000) [-outfile] report [*.sigcleave] Output report file name (default -rformat motif) Additional (Optional) qualifiers: -prokaryote boolean Specifies the sequence is prokaryotic and changes the default scoring data file name Advanced (Unprompted) qualifiers: (none) Associated qualifiers: "-sequence" associated qualifiers -sbegin1 integer Start of each sequence to be used -send1 integer End of each sequence to be used -sreverse1 boolean Reverse (if DNA) -sask1 boolean Ask for begin/end/reverse -snucleotide1 boolean Sequence is nucleotide -sprotein1 boolean Sequence is protein -slower1 boolean Make lower case -supper1 boolean Make upper case -sformat1 string Input sequence format -sdbname1 string Database name -sid1 string Entryname -ufo1 string UFO features -fformat1 string Features format -fopenfile1 string Features file name "-outfile" associated qualifiers -rformat2 string Report format -rname2 string Base file name -rextension2 string File name extension -rdirectory2 string Output directory -raccshow2 boolean Show accession number in the report -rdesshow2 boolean Show description in the report -rscoreshow2 boolean Show the score in the report -rstrandshow2 boolean Show the nucleotide strand in the report -rusashow2 boolean Show the full USA in the report -rmaxall2 integer Maximum total hits to report -rmaxseq2 integer Maximum hits to report for one sequence General qualifiers: -auto boolean Turn off prompts -stdout boolean Write first file to standard output -filter boolean Read first file from standard input, write first file to standard output -options boolean Prompt for standard and additional values -debug boolean Write debug output to program.dbg -verbose boolean Report some/full command line options -help boolean Report command line options and exit. More information on associated and general qualifiers can be found with -help -verbose -warning boolean Report warnings -error boolean Report errors -fatal boolean Report fatal errors -die boolean Report dying program messages -version boolean Report version number and exit Input file format sigcleave reads one or more protein sequences. The input is a standard EMBOSS sequence query (also known as a 'USA'). Major sequence database sources defined as standard in EMBOSS installations include srs:embl, srs:uniprot and ensembl Data can also be read from sequence output in any supported format written by an EMBOSS or third-party application. The input format can be specified by using the command-line qualifier -sformat xxx, where 'xxx' is replaced by the name of the required format. The available format names are: gff (gff3), gff2, embl (em), genbank (gb, refseq), ddbj, refseqp, pir (nbrf), swissprot (swiss, sw), dasgff and debug. See: http://emboss.sf.net/docs/themes/SequenceFormats.html for further information on sequence formats. Input files for usage example 'tsw:ach2_drome' is a sequence entry in the example protein database 'tsw' Database entry: tsw:ach2_drome ID ACH2_DROME Reviewed; 576 AA. AC P17644; Q0KI18; Q9VC73; DT 01-AUG-1990, integrated into UniProtKB/Swiss-Prot. DT 01-AUG-1990, sequence version 1. DT 15-JUN-2010, entry version 110. DE RecName: Full=Acetylcholine receptor subunit alpha-like 2; DE Flags: Precursor; GN Name=nAcRalpha-96Ab; Synonyms=Acr96Ab, AcrE, sad; ORFNames=CG6844; OS Drosophila melanogaster (Fruit fly). OC Eukaryota; Metazoa; Arthropoda; Hexapoda; Insecta; Pterygota; OC Neoptera; Endopterygota; Diptera; Brachycera; Muscomorpha; OC Ephydroidea; Drosophilidae; Drosophila; Sophophora. OX NCBI_TaxID=7227; RN [1] RP NUCLEOTIDE SEQUENCE [GENOMIC DNA], TISSUE SPECIFICITY, AND RP DEVELOPMENTAL STAGE. RC TISSUE=Head; RX MEDLINE=90353591; PubMed=2117557; DOI=10.1016/0014-5793(90)81170-S; RA Jonas P., Baumann A., Merz B., Gundelfinger E.D.; RT "Structure and developmental expression of the D alpha 2 gene encoding RT a novel nicotinic acetylcholine receptor protein of Drosophila RT melanogaster."; RL FEBS Lett. 269:264-268(1990). RN [2] RP NUCLEOTIDE SEQUENCE [MRNA]. RX MEDLINE=90360975; PubMed=1697262; RA Sawruk E., Schloss P., Betz H., Schmitt B.; RT "Heterogeneity of Drosophila nicotinic acetylcholine receptors: SAD, a RT novel developmentally regulated alpha-subunit."; RL EMBO J. 9:2671-2677(1990). RN [3] RP NUCLEOTIDE SEQUENCE [MRNA], TISSUE SPECIFICITY, AND DEVELOPMENTAL RP STAGE. RC TISSUE=Head; RX MEDLINE=90301489; PubMed=2114015; DOI=10.1093/nar/18.12.3640; RA Baumann A., Jonas P., Gundelfinger E.D.; RT "Sequence of D alpha 2, a novel alpha-like subunit of Drosophila RT nicotinic acetylcholine receptors."; RL Nucleic Acids Res. 18:3640-3640(1990). RN [4] RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA]. RC STRAIN=Berkeley; RX MEDLINE=20196006; PubMed=10731132; DOI=10.1126/science.287.5461.2185; RA Adams M.D., Celniker S.E., Holt R.A., Evans C.A., Gocayne J.D., RA Amanatides P.G., Scherer S.E., Li P.W., Hoskins R.A., Galle R.F., RA George R.A., Lewis S.E., Richards S., Ashburner M., Henderson S.N., RA Sutton G.G., Wortman J.R., Yandell M.D., Zhang Q., Chen L.X., RA Brandon R.C., Rogers Y.-H.C., Blazej R.G., Champe M., Pfeiffer B.D., RA Wan K.H., Doyle C., Baxter E.G., Helt G., Nelson C.R., Miklos G.L.G., RA Abril J.F., Agbayani A., An H.-J., Andrews-Pfannkoch C., Baldwin D., [Part of this file has been deleted for brevity] DR GO; GO:0005515; F:protein binding; IPI:IntAct. DR GO; GO:0004872; F:receptor activity; IEA:UniProtKB-KW. DR GO; GO:0006811; P:ion transport; IEA:UniProtKB-KW. DR InterPro; IPR006202; Neur_chan_lig_bd. DR InterPro; IPR006201; Neur_channel. DR InterPro; IPR006029; Neurotrans-gated_channel_TM. DR InterPro; IPR018000; Neurotransmitter_ion_chnl_CS. DR InterPro; IPR002394; Nicotinic_acetylcholine_rcpt_N. DR Gene3D; G3DSA:2.70.170.10; Neur_chan_lig_bd; 1. DR PANTHER; PTHR18945; Neur_channel; 1. DR Pfam; PF02931; Neur_chan_LBD; 1. DR Pfam; PF02932; Neur_chan_memb; 1. DR PRINTS; PR00254; NICOTINICR. DR PRINTS; PR00252; NRIONCHANNEL. DR SUPFAM; SSF90112; Neu_channel_TM; 1. DR SUPFAM; SSF63712; Neur_chan_LBD; 1. DR TIGRFAMs; TIGR00860; LIC; 1. DR PROSITE; PS00236; NEUROTR_ION_CHANNEL; 1. PE 1: Evidence at protein level; KW Cell junction; Cell membrane; Complete proteome; Disulfide bond; KW Glycoprotein; Ion transport; Ionic channel; Membrane; KW Postsynaptic cell membrane; Receptor; Signal; Synapse; Transmembrane; KW Transport. FT SIGNAL 1 21 Probable. FT CHAIN 22 576 Acetylcholine receptor subunit alpha-like FT 2. FT /FTId=PRO_0000000300. FT TOPO_DOM 22 261 Extracellular (Potential). FT TRANSMEM 262 285 Helical; (Potential). FT TRANSMEM 293 311 Helical; (Potential). FT TRANSMEM 327 346 Helical; (Potential). FT TOPO_DOM 347 526 Cytoplasmic (Potential). FT TRANSMEM 527 545 Helical; (Potential). FT CARBOHYD 65 65 N-linked (GlcNAc...) (Potential). FT CARBOHYD 254 254 N-linked (GlcNAc...) (Potential). FT CARBOHYD 570 570 N-linked (GlcNAc...) (Potential). FT DISULFID 169 183 By similarity. FT DISULFID 243 244 Associated with receptor activation (By FT similarity). SQ SEQUENCE 576 AA; 65506 MW; 97D6A46CADC3F42F CRC64; MAPGCCTTRP RPIALLAHIW RHCKPLCLLL VLLLLCETVQ ANPDAKRLYD DLLSNYNRLI RPVSNNTDTV LVKLGLRLSQ LIDLNLKDQI LTTNVWLEHE WQDHKFKWDP SEYGGVTELY VPSEHIWLPD IVLYNNADGE YVVTTMTKAI LHYTGKVVWT PPAIFKSSCE IDVRYFPFDQ QTCFMKFGSW TYDGDQIDLK HISQKNDKDN KVEIGIDLRE YYPSVEWDIL GVPAERHEKY YPCCAEPYPD IFFNITLRRK TLFYTVNLII PCVGISYLSV LVFYLPADSG EKIALCISIL LSQTMFFLLI SEIIPSTSLA LPLLGKYLLF TMLLVGLSVV ITIIILNIHY RKPSTHKMRP WIRSFFIKRL PKLLLMRVPK DLLRDLAANK INYGLKFSKT KFGQALMDEM QMNSGGSSPD SLRRMQGRVG AGGCNGMHVT TATNRFSGLV GALGGGLSTL SGYNGLPSVL SGLDDSLSDV AARKKYPFEL EKAIHNVMFI QHHMQRQDEF NAEDQDWGFV AMVMDRLFLW LFMIASLVGT FVILGEAPSL YDDTKAIDVQ LSDVAKQIYN LTEKKN // Output file format The output is a standard EMBOSS report file. The results can be output in one of several styles by using the command-line qualifier -rformat xxx, where 'xxx' is replaced by the name of the required format. The available format names are: embl, genbank, gff, pir, swiss, dasgff, debug, listfile, dbmotif, diffseq, draw, restrict, excel, feattable, motif, nametable, regions, seqtable, simple, srs, table, tagseq. See: http://emboss.sf.net/docs/themes/ReportFormats.html for further information on report formats. By default the output is in 'motif' format. Output files for usage example File: ach2_drome.sig ######################################## # Program: sigcleave # Rundate: Fri 15 Jul 2011 12:00:00 # Commandline: sigcleave # -sequence tsw:ach2_drome # Report_format: motif # Report_file: ach2_drome.sig ######################################## #======================================= # # Sequence: ACH2_DROME from: 1 to: 576 # HitCount: 9 # # Reporting scores over 3.50 # #======================================= (1) Score 13.739 length 13 at residues 29->41 Sequence: LLVLLLLCETVQA | | 29 41 mature_peptide: NPDAKRLYDDLLSNYNRLIRPVSNNTDTVLVKLGLRLSQLIDLNLKDQIL (2) Score 12.135 length 13 at residues 26->38 Sequence: LCLLLVLLLLCET | | 26 38 mature_peptide: VQANPDAKRLYDDLLSNYNRLIRPVSNNTDTVLVKLGLRLSQLIDLNLKD (3) Score 10.465 length 13 at residues 28->40 Sequence: LLLVLLLLCETVQ | | 28 40 mature_peptide: ANPDAKRLYDDLLSNYNRLIRPVSNNTDTVLVKLGLRLSQLIDLNLKDQI (4) Score 7.360 length 13 at residues 528->540 Sequence: FLWLFMIASLVGT | | 528 540 mature_peptide: FVILGEAPSLYDDTKAIDVQLSDVAKQIYNLTEKKN (5) Score 6.981 length 13 at residues 330->342 Sequence: FTMLLVGLSVVIT | | 330 342 mature_peptide: IIILNIHYRKPSTHKMRPWIRSFFIKRLPKLLLMRVPKDLLRDLAANKIN (6) Score 5.057 length 13 at residues 24->36 Sequence: KPLCLLLVLLLLC | | 24 36 mature_peptide: ETVQANPDAKRLYDDLLSNYNRLIRPVSNNTDTVLVKLGLRLSQLIDLNL (7) Score 4.026 length 13 at residues 31->43 Sequence: VLLLLCETVQANP | | 31 43 mature_peptide: DAKRLYDDLLSNYNRLIRPVSNNTDTVLVKLGLRLSQLIDLNLKDQILTT (8) Score 3.751 length 13 at residues 527->539 Sequence: LFLWLFMIASLVG | | 527 539 mature_peptide: TFVILGEAPSLYDDTKAIDVQLSDVAKQIYNLTEKKN (9) Score 3.632 length 13 at residues 308->320 Sequence: LLISEIIPSTSLA | | 308 320 mature_peptide: LPLLGKYLLFTMLLVGLSVVITIIILNIHYRKPSTHKMRPWIRSFFIKRL #--------------------------------------- #--------------------------------------- #--------------------------------------- # Total_sequences: 1 # Total_length: 576 # Reported_sequences: 1 # Reported_hitcount: 9 #--------------------------------------- Data files EMBOSS data files are distributed with the application and stored in the standard EMBOSS data directory, which is defined by the EMBOSS environment variable EMBOSS_DATA. To see the available EMBOSS data files, run: % embossdata -showall To fetch one of the data files (for example 'Exxx.dat') into your current directory for you to inspect or modify, run: % embossdata -fetch -file Exxx.dat Users can provide their own data files in their own directories. Project specific files can be put in the current directory, or for tidier directory listings in a subdirectory called ".embossdata". Files for all EMBOSS runs can be put in the user's home directory, or again in a subdirectory called ".embossdata". The directories are searched in the following order: * . (your current directory) * .embossdata (under your current directory) * ~/ (your home directory) * ~/.embossdata Here is the default file for eukaryotic signals: # Amino acid counts for 161 Eukaryotic Signal Peptides, # from von Heijne (1986), Nucl. Acids. Res. 14:4683-4690 # # The cleavage site is between +1 and -1 # Sample: 161 aligned sequences # # R -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 +1 +2 Expect # - --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- ------ A 16 13 14 15 20 18 18 17 25 15 47 6 80 18 6 14.5 C 3 6 9 7 9 14 6 8 5 6 19 3 9 8 3 4.5 D 0 0 0 0 0 0 0 0 5 3 0 5 0 10 11 8.9 E 0 0 0 1 0 0 0 0 3 7 0 7 0 13 14 10.0 F 13 9 11 11 6 7 18 13 4 5 0 13 0 6 4 5.6 G 4 4 3 6 3 13 3 2 19 34 5 7 39 10 7 12.1 H 0 0 0 0 0 1 1 0 5 0 0 6 0 4 2 3.4 I 15 15 8 6 11 5 4 8 5 1 10 5 0 8 7 7.4 K 0 0 0 1 0 0 1 0 0 4 0 2 0 11 9 11.3 L 71 68 72 79 78 45 64 49 10 23 8 20 1 8 4 12.1 M 0 3 7 4 1 6 2 2 0 0 0 1 0 1 2 2.7 N 0 1 0 1 1 0 0 0 3 3 0 10 0 4 7 7.1 P 2 0 2 0 0 4 1 8 20 14 0 1 3 0 22 7.4 Q 0 0 0 1 0 6 1 0 10 8 0 18 3 19 10 6.3 R 2 0 0 0 0 1 0 0 7 4 0 15 0 12 9 7.6 S 9 3 8 6 13 10 15 16 26 11 23 17 20 15 10 11.4 T 2 10 5 4 5 13 7 7 12 6 17 8 6 3 10 9.7 V 20 25 15 18 13 15 11 27 0 12 32 3 0 8 17 11.1 W 4 3 3 1 1 2 6 3 1 3 0 9 0 2 0 1.8 Y 0 1 4 0 0 1 3 1 1 2 0 5 0 1 7 5.6 Notes Signal peptides mediate translocation across the endoplasmic reticulum (ER) membrane in eukaryotes. In prokaryotes signal peptides mediate translocation across the inner and outer membranes. sigcleave may predict any number of cleavage sites in a protein sequence but not all of these will be biologically relevant; the prediction algorithm is not perfect. There is no cutoff to eliminate sites because it is down to human expertise to decide what is relevant or not. Although the end of a protein sequence is usually easy to predict from a nucleotide sequence, the same cannot be said for the start which depends on such things as promoters, transcriptional control and splicing. This is why all predicted cleavage sites are reported. It is often useful to specify to use just the starting region of the input sequence using the in-built qualifier -send. For example, adding -send 50 to the command-line will check only the first 50 residues. References 1. von Heijne, G. "A new method for predicting signal sequence cleavage sites" Nucleic Acids Res.: 14:4683 (1986) 2. von Heijne, G. "Sequence Analysis in Molecular Biology: Treasure Trove or Trivial Pursuit" (Acad. Press, (1987), 113-117) Warnings The program will warn you if a nucleic acid sequence is given or if the data file is not mathematically accurate. Diagnostic Error Messages Exit status It exits with status 0 unless an error is reported. Known bugs None. See also Program name Description antigenic Finds antigenic sites in proteins epestfind Finds PEST motifs as potential proteolytic cleavage sites fuzzpro Search for patterns in protein sequences fuzztran Search for patterns in protein sequences (translated) patmatdb Searches protein sequences with a sequence motif patmatmotifs Scan a protein sequence with motifs from the PROSITE database preg Regular expression search of protein sequence(s) pscan Scans protein sequence(s) with fingerprints from the PRINTS database tmap Predict and plot transmembrane segments in protein sequences Author(s) Alan Bleasby European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK Please report all bugs to the EMBOSS bug team (emboss-bug (c) emboss.open-bio.org) not to the original author. Original program "SIGCLEAVE" (EGCG 1989) by Peter Rice European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK Please report all bugs to the EMBOSS bug team (emboss-bug (c) emboss.open-bio.org) not to the original author. History Completed 10th March 1999 Target users This program is intended to be used by everyone and everything, from naive users to embedded scripts. Comments None