Help | ClustalW
- INTRODUCTION Multiple alignments of protein sequences are important tools in studying sequences. The basic information they provide is identification of conserved sequence regions. This is very useful in designing experiments to test and modify the function of specific proteins, in predicting the function and structure of proteins, and in identifying new members of protein families. Sequences can be aligned across their entire length (global alignment) or only in certain regions (local alignment). This is true for pairwise and multiple alignments. Global alignments need to use gaps (representing insertions/deletions) while local alignments can avoid them, aligning regions between gaps. ClustalW is a fully automatic program for global multiple alignment of DNA and protein sequences. The alignment is progressive and considers the sequence redundancy. Trees can also be calculated from multiple alignments. The program has some adjustable parameters with reasonable defaults. ClustalW Nucleotide Tutorial ClustalW Protein Tutorial For additional help on ClustalW also see: ClustalW FAQ ClustalW Improving Sensitivity YOUR SEQUENCES Please make sure that your sequences have different names as the first 30 characters of the name are significant. If clustalw finds two or more sequences with the same name it will fail! View example. Click here for more information on sequence errors. ClustalW currently supports 7 multiple sequence formats. These are: NBRF/PIR EMBL / UniProt/SwissProt Pearson (Fasta) GDE ALN/ClustalW GCG/MSF RSF More on Sequence Formats Please remove any white space space or empty lines from the beginning of your input. N.B. Bootstrapping is possible, you can paste your .aln files from your results back into the clustalw submission form. YOUR EMAIL You must type your email address in this text box if you are running a job via email. It is not necessary to fill in the box if you are running your search interactively. ALIGNMENT TITLE You may type any text you want to help you identify your search results. RESULTS This option lets you choose between email and interactive runs. The email run requires you to type an email address in the email text box, and your results will be delivered when they are ready to your email address, thus avoiding waiting for your results as with an interactive run. For example: joe@somewhere.domain.country. The default is interactive. ALIGNMENT You may choose to run a full alignment or using a stringent algorithm for generating the tree guide or a fast algorithm. CPU MODE The multiple CPU option run a special version of clustalw using several linux pc nodes in a parallel fashion to increase the speed of the job without compromising the quality of the results. This option is to be chosen when the user has a large number of sequences (50+ but less than 500) to align. However, care should be taken not to overestimate the quantification of the results. A very large alignment is difficult to read and handle by other software. OUTPUT Here you decide which output format you want your multiple sequence alignment in. The options are ALN, GCG, PHYLIP, PIR and GDE. The output results are stored in a ".aln" file which contains your results in ALN format. You can configure your browser to automatically load the results files from clustalw into an suitable application of your choice. Here is a small list of URL's for obtaining such applications for Win95, Macs and UNIX boxes: belvu - UNIX Multiple sequence alignment viewer - http://www.cgr.ki.se/cgr/groups/sonnhammer/Belvu.html njplot - UNIX, Mac & PC - Tree viewer - http://pbil.univ-lyon1.fr/software/njplot.html GeneDoc - GCG MSF file viewer - (NB - Use GCG format in the OUTPUT option) - http://www.psc.edu/biomed/genedoc/ TreeView - Tree viewer for Macs and PC (running Windows) http://taxonomy.zoology.gla.ac.uk/rod/treeview.html ClustalX - Graphical Interface X-Windows/Mac/Win9x based version of ClustalW ftp://ftp.ebi.ac.uk/pub/software/dos/clustalx/ ftp://ftp.ebi.ac.uk/pub/software/mac/clustalx/ ftp://ftp.ebi.ac.uk/pub/software/unix/clustalx/ ClustalW - Command line version ftp://ftp.ebi.ac.uk/pub/software/dos/clustalw/ ftp://ftp.ebi.ac.uk/pub/software/mac/clustalw/ ftp://ftp.ebi.ac.uk/pub/software/unix/clustalw/ CINEMA - A Colour INteractive Editor for Multiple Alignments http://www.bioinf.man.ac.uk/dbbrowser/CINEMA2.1/ EMMA- An EMBOSS interface to clustalw. http://www.emboss.org/ To configure your browser you must tell it how to handle the following non-registered MIME types: application/x-tree njplot %s (or whatever application you choose to handle the MIME type) application/x-align belvu %s (or whatever application you choose to handle the MIME type) JalView: A new experimental option has been added to the results page which involved using a Java Applet called JalView. This is a fully featured MSA editor which allows you not only to edit the alignment but also, to exchange the alignment formats. Please note that JalView is under development. For documentation please click on the JalView Hyperlink or visit the official website at http://www.jalview.org/. OUTORDER Decide which order the sequences should be printed in the alignment. COLOUR You can change this option in the output results, if you click the button, the alignment will be show in colour. NOTE: This option only works when you have chosen ALN or GCG as the output format. The colouring of residues takes place according to the following physiochemical criteria:
  AVFPMILW | RED | Small (small+ hydrophobic (incl.aromatic -Y))
  DE | BLUE | Acidic
  RHK | MAGENTA | Basic
  STYHCNGQ | GREEN | Hydroxyl + Amine + Basic - Q
  Others | Gray |
  The default is not to colour the alignments. CONSENSUS SYMBOLS: An alignment will display by default the following symbols denoting the degree of conservation observed in each column: "*" means that the residues or nucleotides in that column are identical in all sequences in the alignment. ":" means that conserved substitutions have been observed, according to the COLOUR table above. "." means that semi-conserved substitutions are observed. Example:
      CLUSTAL W (1.82) multiple sequence alignment

      FOSB_MOUSE      MFQAFPGDYDSGSRCSSSPSAESQYLSSVDSFGSPPTAAASQECAGLGEMPGSFVPTVTA 60
      FOSB_HUMAN      MFQAFPGDYDSGSRCSSSPSAESQYLSSVDSFGSPPTAAASQECAGLGEMPGSFVPTVTA 60
                      ************************************************************

      FOSB_MOUSE      ITTSQDLQWLVQPTLISSMAQSQGQPLASQPPAVDPYDMPGTSYSTPGLSAYSTGGASGS 120
      FOSB_HUMAN      ITTSQDLQWLVQPTLISSMAQSQGQPLASQPPVVDPYDMPGTSYSTPGMSGYSSGGASGS 120
                      ********************************.***************:*.**:******
More on sequence/alignment formats FAST PAIRWISE ALIGNMENT OPTIONS: KTUP This option allows you to choose which 'word-length' to use when calculating fast pairwise alignments.(note: make sure you have chosen 'fast' in the ALIGNMENT. WINDOW Use this option to set the window length when calculating fast pairwise alignments.(Note: make sure you have chosen 'fast' in the ALIGNMENT. SCORE This option allows you to decide which score to take into account when calculating a fast pairwise alignment. (Note: make sure you have chosen 'fast' in the ALIGNMENT. TOPDIAG Select here how many top diagonals should be integrated when calculating a fast pairwise alignment.(Note: make sure you have chosen 'fast' in the ALIGNMENT. PAIRGAP Select here to set the gap penalty when generating fast pairwise alignments. ### MULTIPLE SEQUENCE ALIGNMENT OPTIONS: MATRIX This option allows you to choose which matrix series to use when generating the multiple sequence alignment. The program goes through the chosen matrix series, spanning the full range of amino acid distances. BLOSUM (Henikoff). These matrices appear to be the best available for carrying out data base similarity searches. The matrix used is Blosum30. PAM (Dayhoff). These have been extremely widely used since the late '70s. We use the PAM 350 matrix. GONNET. These matrices were derived using almost the same procedure as the Dayhoff one (above) but are much more up to date and are based on a far larger data set. They appear to be more sensitive than the Dayhoff series. We use the GONNET 250 matrix. We also supply an identity matrix which gives a score of 10 to two identical amino acids and a score of zero otherwise. Default values are: DNA: DNA Identity matrix. Protein: Gonnet 250. More on Matrices GAPOPEN You can set here the penalty for opening a gap. The default value is 10. ENDGAP You can set here the penalty for closing a gap. GAPEXT You can set here the penalty for extending a gap. The default value is 0.05. GAPDIST You can set here the gap separation penalty. The default value is 8. More about gaps. EXAMPLE e.g. A multiple sequence alignment was done with ClustalW. 5 sequences were input in the fasta format (Download):

      >FOSB_MOUSE Protein fosB
           MFQAFPGDYD SGSRCSSSPS AESQYLSSVD SFGSPPTAAA SQECAGLGEM PGSFVPTVTA
           ITTSQDLQWL VQPTLISSMA QSQGQPLASQ PPAVDPYDMP GTSYSTPGLS AYSTGGASGS
           GGPSTSTTTS GPVSARPARA RPRRPREETL TPEEEEKRRV RRERNKLAAA KCRNRRRELT
           DRLQAETDQL EEEKAELESE IAELQKEKER LEFVLVAHKP GCKIPYEEGP GPGPLAEVRD
           LPGSTSAKED GFGWLLPPPP PPPLPFQSSR DAPPNLTASL FTHSEVQVLG DPFPVVSPSY
           TSSFVLTCPE VSAFAGAQRT SGSEQPSDPL NSPSLLAL

      >FOSB_HUMAN Protein fosB
           MFQAFPGDYD SGSRCSSSPS AESQYLSSVD SFGSPPTAAA SQECAGLGEM PGSFVPTVTA
           ITTSQDLQWL VQPTLISSMA QSQGQPLASQ PPVVDPYDMP GTSYSTPGMS GYSSGGASGS
           GGPSTSGTTS GPGPARPARA RPRRPREETL TPEEEEKRRV RRERNKLAAA KCRNRRRELT
           DRLQAETDQL EEEKAELESE IAELQKEKER LEFVLVAHKP GCKIPYEEGP GPGPLAEVRD
           LPGSAPAKED GFSWLLPPPP PPPLPFQTSQ DAPPNLTASL FTHSEVQVLG DPFPVVNPSY
           TSSFVLTCPE VSAFAGAQRT SGSDQPSDPL NSPSLLAL

      >FOS_CHICK Proto-oncogene protein c-fos
           MMYQGFAGEY EAPSSRCSSA SPAGDSLTYY PSPADSFSSM GSPVNSQDFC TDLAVSSANF
           VPTVTAISTS PDLQWLVQPT LISSVAPSQN RGHPYGVPAP APPAAYSRPA VLKAPGGRGQ
           SIGRRGKVEQ LSPEEEEKRR IRRERNKMAA AKCRNRRREL TDTLQAETDQ LEEEKSALQA
           EIANLLKEKE KLEFILAAHR PACKMPEELR FSEELAAATA LDLGAPSPAA AEEAFALPLM
           TEAPPAVPPK EPSGSGLELK AEPFDELLFS AGPREASRSV PDMDLPGASS FYASDWEPLG
           AGSGGELEPL CTPVVTCTPC PSTYTSTFVF TYPEADAFPS CAAAHRKGSS SNEPSSDSLS
           SPTLLAL

      >FOS_RAT Proto-oncogene protein c-fos
           MMFSGFNADY EASSSRCSSA SPAGDSLSYY HSPADSFSSM GSPVNTQDFC ADLSVSSANF
           IPTVTAISTS PDLQWLVQPT LVSSVAPSQT RAPHPYGLPT PSTGAYARAG VVKTMSGGRA
           QSIGRRGKVE QLSPEEEEKR RIRRERNKMA AAKCRNRRRE LTDTLQAETD QLEDEKSALQ
           TEIANLLKEK EKLEFILAAH RPACKIPNDL GFPEEMSVTS LDLTGGLPEA TTPESEEAFT
           LPLLNDPEPK PSLEPVKNIS NMELKAEPFD DFLFPASSRP SGSETARSVP DVDLSGSFYA
           ADWEPLHSSS LGMGPMVTEL EPLCTPVVTC TPSCTTYTSS FVFTYPEADS FPSCAAAHRK
           GSSSNEPSSD SLSSPTLLAL

      >FOS_MOUSE Proto-oncogene protein c-fos
           MMFSGFNADY EASSSRCSSA SPAGDSLSYY HSPADSFSSM GSPVNTQDFC ADLSVSSANF
           IPTVTAISTS PDLQWLVQPT LVSSVAPSQT RAPHPYGLPT QSAGAYARAG MVKTVSGGRA
           QSIGRRGKVE QLSPEEEEKR RIRRERNKMA AAKCRNRRRE LTDTLQAETD QLEDEKSALQ
           TEIANLLKEK EKLEFILAAH RPACKIPDDL GFPEEMSVAS LDLTGGLPEA STPESEEAFT
           LPLLNDPEPK PSLEPVKSIS NVELKAEPFD DFLFPASSRP SGSETSRSVP DVDLSGSFYA
           ADWEPLHSNS LGMGPMVTEL EPLCTPVVTC TPGCTTYTSS FVFTYPEADS FPSCAAAHRK
           GSSSNEPSSD SLSSPTLLAL
  Output was in the format: The sequences are aligned with each other, with the query sequence at the top and subsequent sequences below. Gaps are represented by the "-" symbol. The running total number of amino acids or nucleotides are shown on the right. The degree of similarity is illustrated underneath the alignments with a series of consensus symbols.
      FOS_RAT         MMFSGFNADYEASSSRCSSASPAGDSLSYYHSPADSFSSMGSPVNTQDFCADLSVSSANF 60
      FOS_MOUSE       MMFSGFNADYEASSSRCSSASPAGDSLSYYHSPADSFSSMGSPVNTQDFCADLSVSSANF 60
      FOS_CHICK       MMYQGFAGEYEAPSSRCSSASPAGDSLTYYPSPADSFSSMGSPVNSQDFCTDLAVSSANF 60
      FOSB_MOUSE      -MFQAFPGDYDS-GSRCSS-SPSAESQ--YLSSVDSFGSPPTAAASQE-CAGLGEMPGSF 54
      FOSB_HUMAN      -MFQAFPGDYDS-GSRCSS-SPSAESQ--YLSSVDSFGSPPTAAASQE-CAGLGEMPGSF 54
                       *:..* .:*:: .***** **:.:*   * *..***.*  :.. :*: *:.*.  ...*

      FOS_RAT         IPTVTAISTSPDLQWLVQPTLVSSVAPSQ-------TRAPHPYGLPTPS-TGAYARAGVV 112
      FOS_MOUSE       IPTVTAISTSPDLQWLVQPTLVSSVAPSQ-------TRAPHPYGLPTQS-AGAYARAGMV 112
      FOS_CHICK       VPTVTAISTSPDLQWLVQPTLISSVAPSQ-------NRG-HPYGVPAPAPPAAYSRPAVL 112
      FOSB_MOUSE      VPTVTAITTSQDLQWLVQPTLISSMAQSQGQPLASQPPAVDPYDMPGTS----YSTPGLS 110
      FOSB_HUMAN      VPTVTAITTSQDLQWLVQPTLISSMAQSQGQPLASQPPVVDPYDMPGTS----YSTPGMS 110
                      :******:** **********:**:* **... ::.    .**.:*  :    *: ..:

      FOS_RAT         KTMSGGRAQSIG--------------------RRGKVEQLSPEEEEKRRIRRERNKMAAA 152
      FOS_MOUSE       KTVSGGRAQSIG--------------------RRGKVEQLSPEEEEKRRIRRERNKMAAA 152
      FOS_CHICK       KAP-GGRGQSIG--------------------RRGKVEQLSPEEEEKRRIRRERNKMAAA 151
      FOSB_MOUSE      AYSTGGASGSGGPSTSTTTSGPVSARPARARPRRPREETLTPEEEEKRRVRRERNKLAAA 170
      FOSB_HUMAN      GYSSGGASGSGGPSTSGTTSGPGPARPARARPRRPREETLTPEEEEKRRVRRERNKLAAA 170
                         :** . * *.::: :::.. .: .: : .** : * *:********:******:***

      FOS_RAT         KCRNRRRELTDTLQAETDQLEDEKSALQTEIANLLKEKEKLEFILAAHRPACKIPNDLGF 212
      FOS_MOUSE       KCRNRRRELTDTLQAETDQLEDEKSALQTEIANLLKEKEKLEFILAAHRPACKIPDDLGF 212
      FOS_CHICK       KCRNRRRELTDTLQAETDQLEEEKSALQAEIANLLKEKEKLEFILAAHRPACKMPEELRF 211
      FOSB_MOUSE      KCRNRRRELTDRLQAETDQLEEEKAELESEIAELQKEKERLEFVLVAHKPGCKIPYEEG- 229
      FOSB_HUMAN      KCRNRRRELTDRLQAETDQLEEEKAELESEIAELQKEKERLEFVLVAHKPGCKIPYEEG- 229
                      *********** *********:**: *::***:* ****:***:*.**:*.**:* :

      FOS_RAT         PEEMSVTS-LDLTGGLPEATTPESEEAFTLPLLNDPEPK-PSLEPVKNISNMELKAEPFD 270
      FOS_MOUSE       PEEMSVAS-LDLTGGLPEASTPESEEAFTLPLLNDPEPK-PSLEPVKSISNVELKAEPFD 270
      FOS_CHICK       SEELAAATALDLG----APSPAAAEEAFALPLMTEAPPAVPPKEPSG--SGLELKAEPFD 265
      FOSB_MOUSE      PGPGPLAEVRDLPG-----STSAKEDGFGWLLPPPPPPP-----------------LPFQ 267
      FOSB_HUMAN      PGPGPLAEVRDLPG-----SAPAKEDGFSWLLPPPPPPP-----------------LPFQ 267
                      .   . :   ** .     :..  *:.*   *   . *                   **:

      FOS_RAT         DFLFPASSRPSGSETARSVPDVDLSG--SFYAADWEPLHSSSLGMGPMVTELEPLCTPVV 328
      FOS_MOUSE       DFLFPASSRPSGSETSRSVPDVDLSG--SFYAADWEPLHSNSLGMGPMVTELEPLCTPVV 328
      FOS_CHICK       ELLFSAGPR----EASRSVPDMDLPGASSFYASDWEPLGAGSGG------ELEPLCTPVV 315
      FOSB_MOUSE      --------------SSRDAP-PNLTA--SLFTHS----------------EVQVLGDPFP 294
      FOSB_HUMAN      --------------TSQDAP-PNLTA--SLFTHS----------------EVQVLGDPFP 294
                                    :::..*  :*..  *::: .                *:: *  *.

      FOS_RAT         TCTPSCTTYTSSFVFTYPEADSFPSCAAAHRKGSSSNEPSSDSLSSPTLLAL 380
      FOS_MOUSE       TCTPGCTTYTSSFVFTYPEADSFPSCAAAHRKGSSSNEPSSDSLSSPTLLAL 380
      FOS_CHICK       TCTPCPSTYTSTFVFTYPEADAFPSCAAAHRKGSSSNEPSSDSLSSPTLLAL 367
      FOSB_MOUSE      VVSP---SYTSSFVLTCPEVSAF---AGAQR--TSGSEQPSDPLNSPSLLAL 338
      FOSB_HUMAN      VVNP---SYTSSFVLTCPEVSAF---AGAQR--TSGSDQPSDPLNSPSLLAL 338
                      . .*   :***:**:* **..:*   *.*:*  :*..: .**.*.**:****

EDITING AN ALIGNMENT You can edit the alignment using jalview. Click on the button below to view the above alignment.

PHYLOGENETIC TREE Phylogram is a branching diagram (tree) assumed to be an estimate of a phylogeny, branch lengths are proportional to the amount of inferred evolutionary change. A Cladogram is a branching diagram (tree) assumed to be an estimate of a phylogeny where the branches are of equal length, thus cladograms show common ancestry, but do not indicate the amount of evolutionary "time" separating taxa. Tree distances can be shown, just click on the diagram to get a menu of options. The ".dnd" file is a file that describes the phylogenetic tree. These are now in controlled with new buttons in the output file as well as a pop up menu, that is available by right-clicking on the applet. The buttons on the page include "Show as Phylogram Tree", "Show as Cladogram Tree" and "Show Distances". IMPORTANT! Please note applets are not printed out with html pages, You will need to: Use the "Print Screen" button in the top right corner of your keyboard. Open an imaging application like paint or photoshop. Go "file>new" from the menu or "control+N" from the keyboard to create a new image. Go "edit>paste from the menu or "control+V" from the keyboard to paste your screen capture. The use the crop function to trim the image (e.g. "image>crop"). Then save or print the image. example: Right-click in the area below for options!

IMPORTANT: To use this option you will need to input a sequence alignment. Please make sure this alignment is in PIR or PHYLIP format. ALN and GCG MSF files are not supported so you will have to convert your MSF files to PIR format with, for example, GCG's ToPir:
    topir pileup.msf{*} -outf=pileup.pir
Please refer to the GCG documentation to find out how to use this program correctly. You may then use this file as input (cut and paste or upload) to this service. The method used is the NJ (Neighbour Joining) method of Saitou and Nei. First you calculate distances (percent divergence) between all pairs of sequence from a multiple alignment; second you apply the NJ method to the distance matrix. This option allows you to choose the following output formats for the tree: Neighbour Phylip Distance In order to view these trees you must have a program capable of displaying the data. Please refer to this pages section on OUTPUT for more information. Kimura Correction of distances This options allows you to set on distances correction (correction for multiple substitutions). This is because, as sequences diverge, more than one substitution will happen at many sites. However, you only see one difference when you look at the present day sequences. Therefore, this option has the effect of stretching branch lengths in trees (especially long branches). The corrections used here (for DNA or proteins) are both due to Motoo Kimura. Ignore Gaps in alignment With this option, any alignment positions where ANY of the sequences have a gap will be ignored. This means that 'like' will be compared to 'like' in all distances. It also, automatically throws away the most ambiguous parts of the alignment, which are concentrated around gaps (usually). The disadvantage is that you may throw away much of the data if there are many gaps. UPLOAD A FILE You may upload a file from your computer which containing a valid set of sequences in any format (GCG, FASTA, EMBL, GenBank, PIR, NBRF, Phylip or UniProt/Swiss-Prot) using this option. Please note that this option only works with Netscape Browsers or Internet Explorer version 5 or later. Some word processors may yield unpredictable results as hidden/control characters may be present in the files. It is best to save files with the Unix format option to avoid hidden windows characters. Some examples of common sequence formats may be seen here. SCORES TABLE Scores Table is a new view to ClustalW output. Users can sort the scores by Alignment Score, Sequence Number, Sequence Name and Sequence Length. REFERENCES Higgins D., Thompson J., Gibson T. Thompson J. D., Higgins D. G., Gibson T. J.(1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting,position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680. ClustalW WWW Service at the European Bioinformatics Institute http://www.ebi.ac.uk/clustalw Rodrigo Lopez, Services Program Andrew Lloyd The Clustal W WW server at the EBI embnet.news volume 4.2 1997 http://www.ebi.ac.uk/embnet.news/vol4_3/clustalw1.html The program in use in this service can be obtained freely from: http://www.sgi.com/chembio/resources/clustalw/parallel_clustalw.html Parry-Smith, D.J., Payne, A.WR, Michie, A.D. and Attwood, T. K. (1997) CINEMA - A novel Colour INteractive Editor for Multiple Alignments. Gene, 211(2), GC45-56. http://www.bioinf.man.ac.uk/dbbrowser/CINEMA2.1/ Attwood, T. K., Payne, A. W.R., Michie, A.D. and Parry-Smith, D.J. (1997) A Colour INteractive Editor for Multiple Alignments - CINEMA, EMBnet.news, 3 (3). Jalview - a java multiple alignment editor http://www.jalview.org pfaat (Protein Family Alignment Annotation Tool) http://pfaat.sourceforge.net/ OTHER SERVICES: This services is also available as an application from the EBI's srs server: http://srs.ebi.ac.uk/ N.B. DbClustal can be launched from Wu-Blast2 and NCBI-Blast2 you can choose some of the sequences from your score list and align them with your query sequence. You may also then paste your .aln files back into ClustalW (bootstrapping) in order to colour your alignments, view the phylogenetic trees or launch jalview.