octanol draws a hydropathy plot for an input protein sequence. This plots the free energy difference calculated for windows over the protein sequence, of the residues in water compared to two lipid environments: i. Octanol (equivalent to inside a lipid bilayer). ii. The interface of a synthetic lipid bilayer. Free energy differences are calculated for each position in a window of 19 residues by default, about the size of a membrane spanning alpha-helix. The energy values for each residue are summed to get two values for each window. By default, the value plotted is the free energy difference between the interface and octanol environments, which is the best indicator of the location of probable transmembrane regions. Command line options allow the display of the octanol and interface values, or hiding the difference values. The experimental free energy values for the water-interface and water-octanol transitions are read from a datafile (Ewhite-wimley.dat)
% octanol Draw a White-Wimley protein hydropathy plot Input protein sequence: tsw:opsd_human Graph type [x11]: ps Created octanol.ps
Standard (Mandatory) qualifiers: [-sequence] sequence Protein sequence filename and optional format, or reference (input USA) [-graph] xygraph [$EMBOSS_GRAPHICS value, or x11] Graph type (ps, hpgl, hp7470, hp7580, meta, cps, x11, tekt, tek, none, data, xterm, png, gif) Additional (Optional) qualifiers: -datafile datafile [Ewhite-wimley.dat] White-Wimley data file -width integer  Window size (Integer from 1 to 200) -octanolplot boolean [N] Display the octanol plot -interfaceplot boolean [N] Display the interface plot -[no]differenceplot boolean [Y] Display the difference plot Advanced (Unprompted) qualifiers: (none) Associated qualifiers: "-sequence" associated qualifiers -sbegin1 integer Start of the sequence to be used -send1 integer End of the 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 "-graph" associated qualifiers -gprompt2 boolean Graph prompting -gdesc2 string Graph description -gtitle2 string Graph title -gsubtitle2 string Graph subtitle -gxtitle2 string Graph x axis title -gytitle2 string Graph y axis title -goutfile2 string Output file for non interactive displays -gdirectory2 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. 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
|Standard (Mandatory) qualifiers||Allowed values||Default|
|Protein sequence filename and optional format, or reference (input USA)||Readable sequence||Required|
|Graph type||EMBOSS has a list of known devices, including ps, hpgl, hp7470, hp7580, meta, cps, x11, tekt, tek, none, data, xterm, png, gif||EMBOSS_GRAPHICS value, or x11|
|Additional (Optional) qualifiers||Allowed values||Default|
|-datafile||White-Wimley data file||Data file||Ewhite-wimley.dat|
|-width||Window size||Integer from 1 to 200||19|
|-octanolplot||Display the octanol plot||Boolean value Yes/No||No|
|-interfaceplot||Display the interface plot||Boolean value Yes/No||No|
|-[no]differenceplot||Display the difference plot||Boolean value Yes/No||Yes|
|Advanced (Unprompted) qualifiers||Allowed values||Default|
ID OPSD_HUMAN Reviewed; 348 AA. AC P08100; Q16414; Q2M249; DT 01-AUG-1988, integrated into UniProtKB/Swiss-Prot. DT 01-AUG-1988, sequence version 1. DT 20-MAR-2007, entry version 91. DE Rhodopsin (Opsin-2). GN Name=RHO; Synonyms=OPN2; OS Homo sapiens (Human). OC Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; OC Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini; OC Catarrhini; Hominidae; Homo. OX NCBI_TaxID=9606; RN  RP NUCLEOTIDE SEQUENCE [GENOMIC DNA]. RX MEDLINE=84272729; PubMed=6589631; RA Nathans J., Hogness D.S.; RT "Isolation and nucleotide sequence of the gene encoding human RT rhodopsin."; RL Proc. Natl. Acad. Sci. U.S.A. 81:4851-4855(1984). RN  RP NUCLEOTIDE SEQUENCE [GENOMIC DNA]. RA Suwa M., Sato T., Okouchi I., Arita M., Futami K., Matsumoto S., RA Tsutsumi S., Aburatani H., Asai K., Akiyama Y.; RT "Genome-wide discovery and analysis of human seven transmembrane helix RT receptor genes."; RL Submitted (JUL-2001) to the EMBL/GenBank/DDBJ databases. RN  RP NUCLEOTIDE SEQUENCE [LARGE SCALE MRNA]. RC TISSUE=Retina; RG The German cDNA consortium; RL Submitted (JUN-2003) to the EMBL/GenBank/DDBJ databases. RN  RP NUCLEOTIDE SEQUENCE [LARGE SCALE MRNA]. RX PubMed=15489334; DOI=10.1101/gr.2596504; RG The MGC Project Team; RT "The status, quality, and expansion of the NIH full-length cDNA RT project: the Mammalian Gene Collection (MGC)."; RL Genome Res. 14:2121-2127(2004). RN  RP NUCLEOTIDE SEQUENCE [GENOMIC DNA] OF 1-120. RX PubMed=8566799; DOI=10.1016/0378-1119(95)00688-5; RA Bennett J., Beller B., Sun D., Kariko K.; RT "Sequence analysis of the 5.34-kb 5' flanking region of the human RT rhodopsin-encoding gene."; RL Gene 167:317-320(1995). RN  RP REVIEW ON RP4 VARIANTS. RX MEDLINE=94004905; PubMed=8401533; RA Al-Maghtheh M., Gregory C., Inglehearn C., Hardcastle A., RA Bhattacharya S.; [Part of this file has been deleted for brevity] FT /FTId=VAR_004816. FT VARIANT 209 209 V -> M (effect not known). FT /FTId=VAR_004817. FT VARIANT 211 211 H -> P (in RP4). FT /FTId=VAR_004818. FT VARIANT 211 211 H -> R (in RP4). FT /FTId=VAR_004819. FT VARIANT 216 216 M -> K (in RP4). FT /FTId=VAR_004820. FT VARIANT 220 220 F -> C (in RP4). FT /FTId=VAR_004821. FT VARIANT 222 222 C -> R (in RP4). FT /FTId=VAR_004822. FT VARIANT 255 255 Missing (in RP4). FT /FTId=VAR_004823. FT VARIANT 264 264 Missing (in RP4). FT /FTId=VAR_004824. FT VARIANT 267 267 P -> L (in RP4). FT /FTId=VAR_004825. FT VARIANT 267 267 P -> R (in RP4). FT /FTId=VAR_004826. FT VARIANT 292 292 A -> E (in CSNBAD1). FT /FTId=VAR_004827. FT VARIANT 296 296 K -> E (in RP4). FT /FTId=VAR_004828. FT VARIANT 297 297 S -> R (in RP4). FT /FTId=VAR_004829. FT VARIANT 342 342 T -> M (in RP4). FT /FTId=VAR_004830. FT VARIANT 345 345 V -> L (in RP4). FT /FTId=VAR_004831. FT VARIANT 345 345 V -> M (in RP4). FT /FTId=VAR_004832. FT VARIANT 347 347 P -> A (in RP4). FT /FTId=VAR_004833. FT VARIANT 347 347 P -> L (in RP4; common variant). FT /FTId=VAR_004834. FT VARIANT 347 347 P -> Q (in RP4). FT /FTId=VAR_004835. FT VARIANT 347 347 P -> R (in RP4). FT /FTId=VAR_004836. FT VARIANT 347 347 P -> S (in RP4). FT /FTId=VAR_004837. SQ SEQUENCE 348 AA; 38893 MW; 6F4F6FCBA34265B2 CRC64; MNGTEGPNFY VPFSNATGVV RSPFEYPQYY LAEPWQFSML AAYMFLLIVL GFPINFLTLY VTVQHKKLRT PLNYILLNLA VADLFMVLGG FTSTLYTSLH GYFVFGPTGC NLEGFFATLG GEIALWSLVV LAIERYVVVC KPMSNFRFGE NHAIMGVAFT WVMALACAAP PLAGWSRYIP EGLQCSCGID YYTLKPEVNN ESFVIYMFVV HFTIPMIIIF FCYGQLVFTV KEAAAQQQES ATTQKAEKEV TRMVIIMVIA FLICWVPYAS VAFYIFTHQG SNFGPIFMTI PAFFAKSAAI YNPVIYIMMN KQFRNCMLTT ICCGKNPLGD DEASATVSKT ETSQVAPA //
The line on the default plot is the difference between the interface and octanol free energy calculations. Command line options allow the display of the interface and octanol values, or hiding the difference values.
In the example, the human opsin protein has 7 transmembrane regions: 37-61, 74-98, 114-133, 153-176, 203-230, 253-276 and 285-309. Each is about 20 residues in length, which is also the gap between tick marks on the sequence axis. All have energetic preferences for being in the lipid (octanol) enviroment - shown as being above the zero line - or have at least no clear preference.
Running octanol with all three plots:
% octanol -interface -octanol Input sequence: tsw:opsd_human Graph type [x11]:gives a graph with the water-interface and water-octanol plots.
For those regions where the diference plot is close to zero, both the other two plots are above the line, showing a preference for either the octanol or the interface membrane environments rather than water.
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:
Protein sequences that form transmembrane regions are assumed to have a thermodynamic preference for a hydrophobic environment (inside the membrane lipid bilayer), rather than an aqueous environment in water. The free energy change for each amino acid residue between a lipid and a water environment can be measured experimentally, and the values for peptides can be shown to be additive (White and Wimley 1999).
For each amino acid residue in the protein, the free energy difference of the residue in lipid and water environments is measured in two ways. The first is the free energy difference between the protein in water and the protein associated with the interface (glycerol group) of a POPC (palmitoyloleoylphosphocholine) bilayer. The second is the free energy difference of the protein in water and the protein in octanol, equivalent to the environment inside a lipid bilayer.
Residues which can be buried inside a lipid bilayer must be in a region of the peptide where most residues show a free energy difference in favour of being in an octanol environment or at least being in the lipid/water interface region. White and Wimley (1999) showed that a sliding window of either free energy difference will indicate the location of probable transmembrane regions, but that the best indicator is the difference between the two values, which is the free energy difference between the interface and octanol environments.
|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|
|checktrans||Reports STOP codons and ORF statistics of a protein|
|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|
|iep||Calculate the isoelectric point of proteins|
|mwcontam||Find weights common to multiple molecular weights files|
|mwfilter||Filter noisy data from molecular weights file|
|pepinfo||Plot amino acid properties of a protein sequence in parallel|
|pepstats||Calculates statistics of protein properties|
|pepwindow||Draw a Kyte-Doolittle hydropathy plot for a protein sequence|
|pepwindowall||Draw Kyte-Doolittle hydropathy plot for a protein alignment|
|wordcount||Count and extract unique words in DNA sequence(s)|