D326 Nucleic Acids Research, 2009, Vol. 37, Database issue and was assigned the highest conservation level (Figure 1B, maroon). Additionally, in some of the residues the data was considered as insuﬃcient (Figure 1B, yellow). Overall, the results were unsatisfactory. The approach that was used to create the ConSurf-DB managed to deal much better with cytochrome c and its ample homologues. From over 180 similar sequences that were found in UniProtKB/SwissProt, 123 were selected by the ConSurf-DB ﬁltering process as homologues to be aligned and analyzed. Sequence identity to the query ranged from 22% to 91% with an average of 58% and SD of 17.6%. Thus, the evolutionary proﬁle obtained makes much more sense in view of the protein function: Highly conserved residues delineate the HEME binding site and no position in the MSA was classiﬁed as insuﬃ- cient (Figure 1A). The MSA in ConSurf-HSSP for cyto- chrome c shows similar sequence identity values. Comparison with results from the ‘Evolutionary Trace Viewer’, ‘SiteFiNDER|3D’ and ‘HotPatch’ servers can be found in Figure 3 of the supporting materials. CONCLUSIONS ConSurf-DB is a new addition to the ConSurf set of online tools for creating evolutionary conservation proﬁles of proteins. In most cases it gives better results than the ordinary ConSurf running with default parameters due to the more advance homologues selection process. Moreover, since all the data is pre-calculated there is no waiting time. This makes ConSurf-DB a preferred tool for initial investigation of proteins. The evolutionary proﬁle of the protein can be correlated with results obtained using other computational tools and experimental data to gain functional insight. The conservation proﬁles can also be linked to other online web servers. It is important to note that the quality of the results of any evolutionary algorithm depends on the amount of homologous proteins and their diversity over the phyloge- netic tree. For that reason the ConSurf-DB repository will be rebuilt annually to incorporate new homologues, which were sequenced during the year. The automatic procedure that was used here represents a quasi-optimum with regards to the search for homo- logous proteins and their alignment. However, very often manual intervention can be used to improve this process further, especially when conducted by an expert on a speciﬁc protein. Thus, users may still prefer to use the original ConSurf server that allows inputting custom MSAs and phylogenetic trees, as well as changing key parameters. Generally speaking, functional regions are highly con- served. However, it is noteworthy that there are exceptions to this rule. One particularly interesting case is the recog- nition region in antibodies and MHC molecules, which are hyper-variable (19). The ConSurf-DB can be used to recognize these regions as well, if the user knows what to look for. It is also important to notice that in some cases we had to abort the ConSurf analysis of chains not conforming to our basic thresholds of length, modiﬁcations percentile and the number of homologues found. One of the key reasons for that was an insuﬃcient number of homo- logues. It is anticipate that as the various genome and meta-genome projects are moving forward and sequences accumulate, we will be able to cover the entire PDB. Until then, complementary tools, such as THEMATICS (20) and HotPatch, may be used to ﬁnd functional regions without the need to look for homologous proteins. We are hopeful that ConSurf-DB will be a valuable tool for researchers and anticipate that it will assist in the dis- covery of protein function. To this end, we are constantly working on adding ConSurf-DB results to online protein databases. The PDBsum (21) database will present direct links to ConSurf-DB and the Proteopedia Project (22) will integrate the ConSurf-DB data, allowing users to browse conservation scores without leaving the site. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS We thank Eric Martz for his constant feedback on the various ConSurf tools. This feedback was reﬂected in the design of ConSurf-DB. FUNDING This work was supported by the German Federal Ministry of Education and Research (BMBF) within the framework of German-Israeli Project Cooperation (DIP) (grant number K5.1). Funding for open access charge: The German Federal Ministry of Education and Research (BMBF) within the framework of German- Israeli Project Cooperation (DIP) (grant number K5.1). Conﬂict of interest statement. None declared. REFERENCES 1. 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