W488 Nucleic Acids Research, 2009, Vol. 37, Web Server issue PDB ﬁle of each template. By default, this includes small organic molecules but it can also include ions, peptides, sugar compounds and nucleic acids (upon selection in the submission form). All the ligands in close contact (one atom at <4 A˚ from one atom of the template polypeptide) are taken into consideration. Contacts are classiﬁed according to three distance thresholds (4, 6 and 8 A˚ , respectively). In order to increase the speed of ligand detection, all ligand–protein distances have been precom- puted for the whole PDB database and stored in a local database called CBE for ‘Contact Between Entities’. Access to the database CBE is available at http:// abcis.cbs.cnrs.fr/AT2/cbe.html. The number of ligands detected is shown for the corresponding template on the result page. Upon clicking on this number, the ligands are listed to show their name, size, nature, the number of residues in contact and the highest similarity to a biolog- ical ligand listed in PROCOGNATE (28). The residues that are in contact with the selected ligand(s) are high- lighted on a copy of the SA. Highlighting the positions in contact can be used for the selection of the ligand, of the 3D template or of the best alignment. A box can be clicked to select the desired ligand. Comparative docking can be performed for a set of selected 3D-models and ligands. It may preferentially be done using constrained 3D models built using TITO or SCWRL, especially at low level of sequence identity (below 25%). 3D models built by MODELLER might be used for docking only when excellent SAs have been obtained. In all cases, 3D models and the template structure in complex with the desired ligand are superposed using MaxCluster (18). Then, the novel protein–ligand interactions (but those including metal ions) are computed using MedusaScore (23) and AUTODOCK (24). The best interaction score is displayed in the main result page. A link brings the user to a docking result page containing a score table and a Jmol applet for visualization of the protein–ligand complexes and the corresponding protein–protein super- position. Ranking by superposition score or interaction score can be done. A table of results is drawn for each type of 3D models (built by TITO, SCWRL or MODELLER). This may highlight trouble in model completion or the role of the strictly conserved residues in the interactions. In this way, comparative docking can provide a local evaluation of sequence conserva- tion (e.g. within active sites). This may distinguish homologous from analogous templates or suggests some functional variations (e.g. ligand speciﬁcity). Finally, a short and focused simulation can be performed using AUTODOCK 4.0 (24) to reﬁne the ligand positions. All the computed complexes can be downloaded. Longer sim- ulation might be necessary for complete exploration of the binding pocket and ligand conformational space. Thus, all the necessary input ﬁles can be also downloaded. Finally, the main menu allows the user to archive the desired SAs in various formats (such as PIR and FASTA). A set of 3D models (or templates) can be superposed using the program Matt (29) and then saved as a compressed tar ﬁle. The SAs saved in PIR format can be edited using ViTO (11) or used in MODELLER oﬄine. All the mentioned options should help recognizing the proper templates for comparative modeling, potentially some ligands, and tentatively combine them. The usefulness of the described pipeline is exempliﬁed below for several case studies. Case studies Example 1: Ssu72 from yeast. Ssu72 is a small protein previously shown to be conserved in eukaryotes. It was genetically linked with a serine/threonine kinase and was known to physically interact with transcription initiation and termination complexes (30). In parallel, we have pro- vided evidence that yeast Ssu72 is a phosphatase of unknown speciﬁcity by means of extensive sequence ana- lysis and molecular modeling. Ligand docking by similar- ity allowed the correct identiﬁcation of the active site in agreement with subsequent mutagenesis studies. We also successfully predicted its inhibition by ortho-vanadate, a generic inhibitor of the phosphatase/arsenic reductase superfamily (30). Identical predictions can now be per- formed using @TOME-2 within a few clicks and in a semi-automatic manner (see: http://abcis.cbs.cnrs.fr/ ATV2/EG/70890/atome.html). Clustering of the sequence conservation in the catalytic center is rapidly highlighted, while the prediction of the substrate speciﬁcity appears elusive as the remaining of the sequence seems highly divergent (overall sequence identity 15%). The role of best conserved motif (‘C-x2-N-x2-R-S’) can be readily deduced from comparative docking (see Figures 2 and 3). As shown previously (30), the resulting conclusions can be tested more easily and more accurately than usual biophysical evaluation of a global structure conservation. Indeed, we are still awaiting the resolution of its structure but this enzyme has been already shown to be a serine/ threonine phosphatase with an original substrate speciﬁ- city (31). Example 2: CggR from Bacillus subtilis. CggR belongs to the SorC family of bacterial transcriptional regulators, which control the expression of genes and operons involved in carbohydrate catabolism (32). Automatic fold recognition and molecular modeling of CggR can rapidly reveal the presence of a winged-helix DNA-bind- ing motif followed by a C-terminal domain presenting weak similarity (10% of global sequence identity) with glucosamine-6-phosphate deaminases from the NagB family. Before the 3D structure of the latter domain was completed, a predictive analysis of structure/function rela- tionship suggested that the C-terminal region of CggR could be involved in binding of a phosphorylated sugar. Indeed, in vitro, fructose-1,6-biphosphate was the only sugar compound capable of interfering with CggR coop- erative binding to DNA (32). The same conclusions can be drawn using @TOME-2, ﬁrst, to delimit the domain boundaries (see: http://abcis.cbs.cnrs.fr/ATV2/EG/87746/ atome.html), and then to build satisfactory 3D models. Comparative ligand docking highlights the role of some conserved residues in the recognition of one of the two phosphate groups of the actual ligand of CggR. Their importance for the function of this repressor has been conﬁrmed by means of mutagenesis and