pepstats 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 Calculates statistics of protein properties Description pepstats reads one or more protein sequences and writes an output file with various statistics on the protein properties. This includes: * Molecular weight * Number of residues * Average residue weight * Charge * Isoelectric point * For each type of amino acid: number, molar percent, DayhoffStat * For each physico-chemical class of amino acid: number, molar percent * Probability of protein expression in E. coli inclusion bodies * Molar extinction coefficient (A280) * Extinction coefficient at 1 mg/ml (A280) Usage Here is a sample session with pepstats % pepstats Calculates statistics of protein properties Input protein sequence(s): tsw:laci_ecoli Pepstats program output file [laci_ecoli.pepstats]: Go to the input files for this example Go to the output files for this example Command line arguments Calculates statistics of protein properties Version: EMBOSS:6.4.0.0 Standard (Mandatory) qualifiers: [-sequence] seqall Protein sequence(s) filename and optional format, or reference (input USA) [-outfile] outfile [*.pepstats] Pepstats program output file Additional (Optional) qualifiers: (none) Advanced (Unprompted) qualifiers: -aadata datafile [Eamino.dat] Amino acid properties -mwdata datafile [Emolwt.dat] Molecular weight data for amino acids -[no]termini boolean [Y] Include charge at N and C terminus -mono boolean [N] Use monoisotopic weights 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 -odirectory2 string Output directory 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 pepstats 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:laci_ecoli' is a sequence entry in the example protein database 'tsw' Database entry: tsw:laci_ecoli ID LACI_ECOLI Reviewed; 360 AA. AC P03023; O09196; P71309; Q2MC79; Q47338; DT 21-JUL-1986, integrated into UniProtKB/Swiss-Prot. DT 19-JUL-2003, sequence version 3. DT 15-JUN-2010, entry version 117. DE RecName: Full=Lactose operon repressor; GN Name=lacI; OrderedLocusNames=b0345, JW0336; OS Escherichia coli (strain K12). OC Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacteriales; OC Enterobacteriaceae; Escherichia. OX NCBI_TaxID=83333; RN [1] RP NUCLEOTIDE SEQUENCE [GENOMIC DNA]. RX MEDLINE=78246991; PubMed=355891; DOI=10.1038/274765a0; RA Farabaugh P.J.; RT "Sequence of the lacI gene."; RL Nature 274:765-769(1978). RN [2] RP NUCLEOTIDE SEQUENCE [GENOMIC DNA]. RA Chen J., Matthews K.K.S.M.; RL Submitted (MAY-1991) to the EMBL/GenBank/DDBJ databases. RN [3] RP NUCLEOTIDE SEQUENCE [GENOMIC DNA]. RA Marsh S.; RL Submitted (JAN-1997) to the EMBL/GenBank/DDBJ databases. RN [4] RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA]. RC STRAIN=K12 / MG1655 / ATCC 47076; RA Chung E., Allen E., Araujo R., Aparicio A.M., Davis K., Duncan M., RA Federspiel N., Hyman R., Kalman S., Komp C., Kurdi O., Lew H., Lin D., RA Namath A., Oefner P., Roberts D., Schramm S., Davis R.W.; RT "Sequence of minutes 4-25 of Escherichia coli."; RL Submitted (JAN-1997) to the EMBL/GenBank/DDBJ databases. RN [5] RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA]. RC STRAIN=K12 / MG1655 / ATCC 47076; RX MEDLINE=97426617; PubMed=9278503; DOI=10.1126/science.277.5331.1453; RA Blattner F.R., Plunkett G. III, Bloch C.A., Perna N.T., Burland V., RA Riley M., Collado-Vides J., Glasner J.D., Rode C.K., Mayhew G.F., RA Gregor J., Davis N.W., Kirkpatrick H.A., Goeden M.A., Rose D.J., RA Mau B., Shao Y.; RT "The complete genome sequence of Escherichia coli K-12."; RL Science 277:1453-1474(1997). RN [6] RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA]. RC STRAIN=K12 / W3110 / ATCC 27325 / DSM 5911; RX PubMed=16738553; DOI=10.1038/msb4100049; RA Hayashi K., Morooka N., Yamamoto Y., Fujita K., Isono K., Choi S., RA Ohtsubo E., Baba T., Wanner B.L., Mori H., Horiuchi T.; RT "Highly accurate genome sequences of Escherichia coli K-12 strains [Part of this file has been deleted for brevity] FT CHAIN 1 360 Lactose operon repressor. FT /FTId=PRO_0000107963. FT DOMAIN 1 58 HTH lacI-type. FT DNA_BIND 6 25 H-T-H motif. FT VARIANT 282 282 Y -> D (in T41 mutant). FT MUTAGEN 17 17 Y->H: Broadening of specificity. FT MUTAGEN 22 22 R->N: Recognizes an operator variant. FT CONFLICT 286 286 L -> S (in Ref. 1, 4 and 7). FT HELIX 6 11 FT TURN 12 14 FT HELIX 17 24 FT HELIX 33 45 FT HELIX 51 56 FT STRAND 63 69 FT HELIX 74 89 FT STRAND 93 98 FT STRAND 101 103 FT HELIX 104 115 FT TURN 116 118 FT STRAND 122 126 FT HELIX 130 139 FT TURN 140 142 FT STRAND 145 150 FT STRAND 154 156 FT STRAND 158 161 FT HELIX 163 177 FT STRAND 181 186 FT HELIX 192 207 FT STRAND 213 217 FT HELIX 222 234 FT STRAND 240 246 FT HELIX 247 259 FT TURN 265 267 FT STRAND 268 271 FT HELIX 277 281 FT STRAND 282 284 FT STRAND 287 290 FT HELIX 293 308 FT STRAND 314 319 FT STRAND 322 324 FT STRAND 334 338 FT HELIX 343 353 FT HELIX 354 356 SQ SEQUENCE 360 AA; 38590 MW; 347A8DEE92D736CB CRC64; MKPVTLYDVA EYAGVSYQTV SRVVNQASHV SAKTREKVEA AMAELNYIPN RVAQQLAGKQ SLLIGVATSS LALHAPSQIV AAIKSRADQL GASVVVSMVE RSGVEACKAA VHNLLAQRVS GLIINYPLDD QDAIAVEAAC TNVPALFLDV SDQTPINSII FSHEDGTRLG VEHLVALGHQ QIALLAGPLS SVSARLRLAG WHKYLTRNQI QPIAEREGDW SAMSGFQQTM QMLNEGIVPT AMLVANDQMA LGAMRAITES GLRVGADISV VGYDDTEDSS CYIPPLTTIK QDFRLLGQTS VDRLLQLSQG QAVKGNQLLP VSLVKRKTTL APNTQTASPR ALADSLMQLA RQVSRLESGQ // Output file format Output files for usage example File: laci_ecoli.pepstats PEPSTATS of LACI_ECOLI from 1 to 360 Molecular weight = 38590.16 Residues = 360 Average Residue Weight = 107.195 Charge = 1.5 Isoelectric Point = 6.8820 A280 Molar Extinction Coefficients = 22920 (reduced) 23045 (cystine bridges) A280 Extinction Coefficients 1mg/ml = 0.594 (reduced) 0.597 (cystine bridges) Improbability of expression in inclusion bodies = 0.660 Residue Number Mole% DayhoffStat A = Ala 44 12.222 1.421 B = Asx 0 0.000 0.000 C = Cys 3 0.833 0.287 D = Asp 17 4.722 0.859 E = Glu 15 4.167 0.694 F = Phe 4 1.111 0.309 G = Gly 22 6.111 0.728 H = His 7 1.944 0.972 I = Ile 18 5.000 1.111 J = --- 0 0.000 0.000 K = Lys 11 3.056 0.463 L = Leu 41 11.389 1.539 M = Met 10 2.778 1.634 N = Asn 12 3.333 0.775 O = --- 0 0.000 0.000 P = Pro 14 3.889 0.748 Q = Gln 28 7.778 1.994 R = Arg 19 5.278 1.077 S = Ser 32 8.889 1.270 T = Thr 19 5.278 0.865 U = --- 0 0.000 0.000 V = Val 34 9.444 1.431 W = Trp 2 0.556 0.427 X = Xaa 0 0.000 0.000 Y = Tyr 8 2.222 0.654 Z = Glx 0 0.000 0.000 Property Residues Number Mole% Tiny (A+C+G+S+T) 120 33.333 Small (A+B+C+D+G+N+P+S+T+V) 197 54.722 Aliphatic (A+I+L+V) 137 38.056 Aromatic (F+H+W+Y) 21 5.833 Non-polar (A+C+F+G+I+L+M+P+V+W+Y) 200 55.556 Polar (D+E+H+K+N+Q+R+S+T+Z) 160 44.444 Charged (B+D+E+H+K+R+Z) 69 19.167 Basic (H+K+R) 37 10.278 Acidic (B+D+E+Z) 32 8.889 Data files The Dayhoff statistic is read from the EMBOSS data file 'Edayhoff.freq'. You can inspect and modify this file by copying it into your current directory with the command: 'embossdata -fetch'. Absorption coefficients use values read from the EMBOSS data file 'Eamino.dat'. Values in this file assume cysteines are reduced. If cysteines are in disulphide bridges the value should be adjusted as documented at the top of the file, and a local copy used to override the default values. Molecular weights are read from a local data file Emolwt.dat. 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 Notes DayhoffStat is the amino acid's molar percentage divided by the Dayhoff statistic. The Dayhoff statistic is read from the EMBOSS data file Edayhoff.freq and is the amino acid's relative occurence per 1000 aa normalised to 100. The probability of expression in inclusion bodies is sometimes referred to as a type of solubility measure. If, however, a recombinant protein is expressed in Escherichia coli, it can be expressed as soluble in the cytosol or insoluble in inclusion bodies. If the Harrison model predicts a given protein to be probably expressed in includion bodies, this doesn't mean that it is not possible to get it soluble in the cytosol. One example: Thermatoga maritima cell divison protein FtsA with a C-terminal His-Tag has a 58% Harrison probability of being expressed in inclusion bodies. However, there was plenty of soluble protein in the E. coli cytosol (F. van den Ent and J. Lowe, EMBO J. 19, 5300-5307 2000). If the protein is expressed in inclusion bodies or not is not only dependent on the sequence, but also on many other factors, such as E. coli strain, incubation temperature, type of expression vector, strength of promoter and medium. References 1. Roger G. Harrison "Expression of soluble heterologous proteins via fusion with NusA protein" in inNovations 11, June 2000, p 4 - 7. Warnings None. Diagnostic Error Messages None. Exit status It always exits with a status of 0. Known bugs None. See also Program name Description backtranambig Back-translate a protein sequence to ambiguous nucleotide sequence backtranseq Back-translate a protein sequence to a nucleotide sequence charge Draw a protein charge plot compseq Calculate the composition of unique words in sequences emowse Search protein sequences by digest fragment molecular weight freak Generate residue/base frequency table or plot hmoment Calculate and plot hydrophobic moment for protein sequence(s) iep Calculate the isoelectric point of proteins mwcontam Find weights common to multiple molecular weights files mwfilter Filter noisy data from molecular weights file octanol Draw a White-Wimley protein hydropathy plot oddcomp Identify proteins with specified sequence word composition pepdigest Reports on protein proteolytic enzyme or reagent cleavage sites pepinfo Plot amino acid properties of a protein sequence in parallel pepwindow Draw a hydropathy plot for a protein sequence pepwindowall Draw Kyte-Doolittle hydropathy plot for a protein alignment wordcount Count and extract unique words in molecular sequence(s) 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. History Written (1999) - Alan Bleasby Target users This program is intended to be used by everyone and everything, from naive users to embedded scripts. Comments None