W458 Nucleic Acids Research, 2007, Vol. 35, Web Server issue Figure 3. Alignment of proteins containing the SKL motif found in the ‘peroxisomal matrix’ Gene Ontology data set. The shared position of the motif near the protein termini improves our overall conﬁdence in the true functionality of the motif. statistics (such as absolute charge, net charge and charge balance) for a user-deﬁned window size around each occurrence. The original SLiMDisc results can be re-ranked and/or ﬁltered on any of the statistics produced by the server, including novel scoring schemes created by the user. The CompariMotif server uses motif regular expres- sions to match putative motifs returned from the SLiMDisc analysis against libraries of known SLiMs. Libraries available include motif deﬁnitions drawn from the ELM database (2,version, 28 June 2005) and the MiniMotif database (11). The server allows ﬂexibility in motif–motif comparison, permitting incomplete matches (‘x’ of the ‘n’ positions in the motif must match), overlapping matches (neither motif is entirely contained within the other) and parent matches (one motif is longer and entirely contains the other motif). Ambiguity is also considered in the comparisons. Output is a sortable table of all pairs of matching motifs, along with their degree of similarity (based on information content) and their relationship to each other. Server details The SLiMDisc server is a Python-CGI server with a ﬁrst- in ﬁrst-out queuing system. The input is a data set of protein sequences which have been hypothesized to have a possible common motif due to a common attribute such as biological function, subcellular location or a common interaction. UNIPROT ﬂat ﬁle (12) and FASTA formats are fully supported. Most other major formats are also permitted. All the functionality of the standalone SLiMDisc method is available through the advanced user options in the web interface; however, these are set to the default and are therefore discretionary. The main advantages of the web-based SLiMDisc over the standalone version are the inclusion of interactive protein region masking, data visualizations, and post-processing ranking, ﬁltering and motif comparison options. Jobs take diﬀerent lengths of time depending on data set size and evolutionary relationship between proteins. Certain data sets will not be processed if they are estimated to be overly computationally expensive. In such cases, the user will be notiﬁed and will be advised to use the local version of the method. Post-processing and visualization tools may be used by uploading ﬁles generated using a local version of the SLiMDisc application. EXAMPLE OF USAGE Peroxisomal targeting signal (PTS) All peroxisomal proteins are synthesized in the cytoplasm before being transported to the peroxisome. The majority of peroxisomal proteins contain a PTS that binds a peroxin, Pex5, which mediates transport across the peroxisomal membrane to the peroxisomal matrix. We created a data set of 19 proteins by taking all Gene Product Associations for the cell component ‘peroxisomal matrix’ from the Gene Ontology database (13) and analysed it for any over-represented convergently evolving motifs. The top motif returned from the search was the motif SKL, occurring in eight of the proteins (with normalized MST support of 6.9). This motif is the archetypal, functional, peroxisomal targeting signal to which the peroxins bind before facilitating transport (14). Ignoring the context of this motif, it does not stand out very strongly compared with several other overrepresented motifs, with slightly lower rankings. However, upon inspection of the visualizations for the data set, all occurrences of the motif can be seen to occur within ﬁve residues of either the C-terminus or the N-terminus (Figure 3). Visualization in this case allows the user to see the context of the motif greatly improving their conﬁdence in the motif. The shared position of the motif at the C- or N- termini of seven proteins, from which only two have any evidence of common ancestry, is compelling support for true functionality. The classic PTS motif is serine-lysine-leucine (14); however, each position allows the substitution of similar residues so the true consensus motif is [SAPTC] [KRH][LMFI]. In an attempt to improve upon the motif descriptor for the targeting signal returned from the data set, the interactive masking feature of the web server was used to restrict analysis to the 20 residues nearest the C- and N-termini of the proteins and the analysis re-run allowing ambiguous positions (with equivalence groupings: KR, DE, ILMV, AG, QN, FYW, ST). The top ﬁve ranked motifs returned all describe PTS variants of which the third ranked motif, S[KR][ILMV], is the best descriptor, seen in 12 of the proteins (with normalized MST support 10.8). The data set and result ﬁles including visualizations for both analyses are available at the SLiMDisc website: http://bioware.ucd.ie/ slimdisc/example/peroxisome.html.