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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 caring one electron using its HEME prosthetic group. There are many Cytochrome C homologues in 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 standard deviation of 3.3%. Thus, using this default means of collecting homologues, the vast majority of the residues appeared to be invariant, and was assigned the highest conservation level (Fig. B, maroon). Additionally, in some of the residues the data was considered as insufficient (Fig. B, 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 SwissProt, 124 were selected as homologues to be aligned and analyzed. Sequence identity to the query ranged from 22% to 91% with an average of 58% and standard deviation 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 insufficient (Fig. A). The MSA in ConSurf-HSSP for Cytochrome C shows similar sequence identity values.
The Evolutionary Trace Viewer analysis (Fig. C) and SiteFiNDER|3D prediction (Fig. D) resemble ConSurf-DB and show important (conserved) regions surrounding the HEME binding site. However, while ConSurf-DB marks the entire region with the highest conservation level (Fig. A, maroon), both the Evolutionary Trace Viewer and SiteFiNDER|3D divide it into different categories. Whether the division into different levels of importance is justified remains to be examined in wet-lab experiments.
In a HotPatch default analysis for predicting a "Generic Functional Site" two patches were found (Fig. E, red and green). Out of the total of 6 residues in these patches ConSurf-DB marks 5 as part of the most highly conserved region while the sixth is marked in the second highly conserved region.
Cytochrome C protein

Click here for ConSurf-DB full analysis of Cytochrome C protein (5CYT chain R).