of the process for all the polypeptide chains in the PDB within less than 5 days. Rate4Site output includes a Newick formatted phylogenetic tree of the homologues and a list of conservation scores for each of the amino acids positions in the original sequence. The last step includes parsing of the Rate4Site scores and formatting them to create a range of output data. The scores are normalized and classiﬁed into nine conser- vation levels, as explained in the Introduction section above. These levels are subsequently used for visualization (e.g. Figure 1), using RasMol (18) coloring script and FirstGlance in Jmol. The conﬁdence interval, which is assigned to each amino acid position, represents the reli- ability of the conservation score of that position. For example, a conservation score for a position that consists mostly of gaps will have a large conﬁdence interval, i.e. low reliability. Low reliability positions are marked yellow in the 3D visualization (2). All data including intermediate calculations are saved in each chain’s directory and a user-friendly HTML page is created to allow viewing the results using a web browser. ConSurf-DB IN NUMBERS The build statistics for the ﬁrst full version of the ConSurf-DB database are presented in Table 1. The initial version of ConSurf-DB was built based on a PDB Nucleic Acids Research, 2009, Vol. 37, Database issue D325 containing 48 091 entries, using PSI-BLAST v2.2.14 on UniProtKB/SwissProt v54.6 and Clean_UniProt contain- ing 4 225 158 sequences. A total of 117 384 chains were found, 30 918 of which were unique amino acid polypep- tides, conforming to our length and modiﬁcation percen- tile requirements. At peak level, the build process was using 70–150 CPUs, ranging from Pentium III to Xeon. The total CPU time for building the entire ConSurf-DB database was 14 000 CPU hours with an average CPU time of 30 min per chain. EXAMPLE AND COMPARISON The cytochrome c protein (PDB ID: 5cyt) can be found in many species including plants, animals and unicellular organisms. It comprises a single polypeptide chain and is absorbed on the inner membrane of the mitochondrion. It participates in the electron transport chain by carrying one electron using its HEME prosthetic group. There are many cytochrome c homologues in UniProtKB/SwissProt and many of them are highly similar to each other. Therefore, the MSA that was constructed using the default parameters of the ConSurf server includes 50 homologues of over 75% sequence identity to the query with an average of 83% and SD of 3.3%. Thus, using this default means of collecting homologues, the vast majority of the residues appeared to be invariant, Figure 1. Cytochrome c. (A) The conservation coloring proﬁle from the ConSurf-DB repository, mapped onto a space-ﬁlling representation of the protein. The conservation coloring scale is shown below. The HEME group, in stick representation, is colored green. (B) The same view as calculated by the ConSurf server using default parameters. Table 1. Build statistics for the ﬁrst full version of ConSurf-DB dated February 2008 PDB chains PDB entries processed Total chains found Filtered Chains containing nucleic acids Chains of less than 30 residues Chains containing more than 15% modiﬁcations Total chains meeting our requirements Total distinct chains meeting our requirements 48 091 117 384 8237 5594 281 103 272 30 918 MSA sizes Chains with less than 5 homologues (insuﬃcient) MSAs Created Chains with 5-10 homologues Chains with 11-20 homologues Chains with 21-50 homologues Chains with 51-100 homologues Chains with 101-200 homologues Chains with 201-300 homologues 1348 29 570 859 1059 2332 7297 14 945 3078