Published online 29 October 2008 Nucleic Acids Research, 2009, Vol. 37, Database issue D323–D327 doi:10.1093/nar/gkn822 The ConSurf-DB: pre-calculated evolutionary conservation profiles of protein structures Ofir Goldenberg, Elana Erez, Guy Nimrod and Nir Ben-Tal* Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Israel Received September 15, 2008; Revised October 12, 2008; Accepted October 13, 2008 ABSTRACT ConSurf-DB is a repository for evolutionary conser- vation analysis of the proteins of known structures in the Protein Data Bank (PDB). Sequence homo- logues of each of the PDB entries were collected and aligned using standard methods. The evolution- ary conservation of each amino acid position in the alignment was calculated using the Rate4Site algo- rithm, implemented in the ConSurf web server. The algorithm takes into account the phylogenetic relations between the aligned proteins and the sto- chastic nature of the evolutionary process explicitly. Rate4Site assigns a conservation level for each position in the multiple sequence alignment using an empirical Bayesian inference. Visual inspection of the conservation patterns on the 3D structure often enables the identification of key residues that comprise the functionally important regions of the protein. The repository is updated with the latest PDB entries on a monthly basis and will be rebuilt annually. ConSurf-DB is available online at http://consurfdb.tau.ac.il/ INTRODUCTION The study of a protein raises many questions: What the protein function is? Does it have more than one function? How does the protein perform its functions? Is it acting alone? Where/when is the protein active? Where are the functional regions of the protein and what their nature is? Each of these questions can be further reﬁned into addi- tional, more speciﬁc, questions. Advances in sequencing technologies produce ever larger databases containing protein sequences from a large collection of species. Within these databases one can ﬁnd many protein families that can be analyzed in search for functional regions. Generally speaking, protein function is mediated through clusters of evolutionarily conserved amino acids that are located in close vicinity to each other. These clusters may be involved in enzymatic activity, ligand binding, protein–protein interactions, or in the folding and stabilization of the protein’s architecture (1). Typically, the detection of these clusters is useful for initial investigation of a protein by characterizing their properties. In addition, correlating the conservation pat- tern with other data is often insightful. The ConSurf-DB leverages the protein databases in order to aid in the detec- tion of such clusters. We introduced the original ConSurf, available as an online server (2) at http://consurf.tau.ac.il/, back in 2001 (3). ConSurf was developed for the identiﬁcation of func- tional regions in proteins based on the conservation of amino acids, taking into account the phylogenetic rela- tions between the proteins. In 2005 we introduced the ConSurf-HSSP (4) database which was a pre-calculated repository of ConSurf results based on multiple sequence alignments (MSAs) extracted from the HSSP database (5). The MSAs in HSSP do not include the gaps in the query sequence, i.e. positions in the aligned sequences which do not have corresponding positions in the query sequence are removed from the alignment. Consequently, the phylogenetic reconstruction of the protein family is prone to errors. The ConSurf-DB, presented here, replaces ConSurf-HSSP as our repository of pre-calculated ConSurf results. The MSAs in the ConSurf-DB include all sequence data needed for the phylogenetic reconstruc- tion, it also uses a more advanced Rate4Site (6) algorithm utilizing Bayesian inference rather than the Maximum Likelihood estimate that was used in ConSurf-HSSP. The conservation results of ConSurf-DB are presented in much more standard and cross platform formats. Other tools for predicting functional sites based on evo- lutionary conservation include the Evolutionary Trace Viewer (7) and SiteFiNDER|3D (8). Like ConSurf-DB, they take advantage of the evolutionary relationship between homologues to detect regions that are likely to be of functional importance. Other tools take a diﬀerent approach: The HotPatch (9) tool predicts functionally important regions by performing a statistical analysis and comparing the protein’s surface against the surfaces of a large set of proteins (not necessarily homologous to that protein) whose functional sites are known. For a brief *To whom correspondence should be addressed. Tel: +972-3-640-6709; Fax: +972-3-640-6834; Email: nirb@tauex.tau.ac.il 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.