W522 Nucleic Acids Research, 2012, Vol. 40, Web Server issue identifying the Ag-binding site of Abs, which can then be used as starting points for experiments, may help improve vaccine and Ab design and may serve for large scale analysis of Abs. DESCRIPTION OF WEB SERVER Input The input for the Paratome web server is either an amino acid sequence or a 3D structure (or PDB id) of an Ab. 3D structures must be in PDB ﬁle format (http://www. wwpdb.org/docs.html, 23 May 2012, date last accessed). Analysis of multiple Abs is available by uploading a com- pressed ﬁle containing a collection of either sequences or structures. Each submission allows the analysis of up to 100 MB of sequences or structures. Processing time is typically 5–15 s per query Ab. Output The ﬁrst analysis done by the server determines whether the input includes an Ab or a fragment thereof. If the input is not identiﬁed as such, the results page includes a link to a text ﬁles in which this result is stated and ex- plained (e.g. no BLAST hits found, see Supplementary Data S1C). Otherwise, the results page links to two ﬁles—a text ﬁle and an HTML ﬁle. These ﬁles provide a list of the residues that make up each ABR and their location. The HTML ﬁle provides also visualization of the ABRs highlighted in the sequence of the query Ab. For sequences, ABRs location is indicated according to their sequence position within the query sequence (see Supplementary Data S1A and S1B). Figure 1 shows the HTML results ﬁle of running Paratome on the structure of anti-IL-15 (PDB id 2xqb). For 3D structures, the location of each residue within the predicted ABRs is indicated according to its residue number as it appears in the input PDB (Figure 1A). Disordered ABRs residues (i.e. residues missing from the ATOM ﬁeld) are marked with an asterisk (Figure 1A). Additionally, the structure of the query Ab with its predicted ABRs highlighted is accessible using the Jmol Java applet (Figure 1B). MATERIALS AND METHODS ABRs identiﬁcation Paratome is based on a large MSTA of Abs, which revealed regions of structural consensus where the pattern of structural positions that bind the Ag is highly similar among all known Abs. These consensus regions, we found, cover virtually all the residues that bind the Ag. The algorithm is based on the fact that for each Ab in the MSTA we have already identiﬁed the ABRs. Thus, given a query Ab, the algorithm performs three consecutive steps: (i) A BLAST (20) search against all the sequences in the MSTA. If the query is a PDB ﬁle, the sequence is extracted from the SEQRES ﬁeld (if exists). Otherwise, it is ex- tracted from the ATOM ﬁeld. (ii) Next, a pairwise align- ment is performed between the query Ab and the top BLAST hit: If the input is a sequence, each framework region (FR) of the top BLAST hit Ab is locally aligned to the query Ab using the Smith–Waterman local align- ment algorithm (21). For a detailed example of sequence- based FRs pairwise alignment, see Supplementary Data S2. If the input is an Ab 3D structure, the best BLAST hit Ab and the query Ab are structurally aligned using the combinatorial extension (CE) structural alignment algo- rithm (22). For a detailed example of Abs pairwise struc- tural alignment, see Supplementary Data S3. (iii) ABRs identiﬁcation—the ABRs of the query Ab are identiﬁed according to the boundaries of the FRs of the top BLAST hit Ab with which it was aligned in the previous step. Figure 2 summarizes the process of ABRs identiﬁcation. PERFORMANCE The performance of Paratome has been tested on all Ab–Ag complexes that were added to the PDB after the Figure 1. An example HTML results ﬁle of running Paratome on anti IL-15 (PDB id 2xqb). (A) The sequences of Ab chains with ABRs highlighted. The location of ABRs residues is indicated according to the numbering in the ATOM ﬁeld within the input PDB ﬁle. Note that for ABR L1, the location of ﬁve residues (S, N, L, K and R) is indicated with an asterisk as they do not appear in the ATOM ﬁeld within the PDB ﬁle. A link to the visualization of the analyzed complex is located below the list of identiﬁed ABRs. (B) The visualization of the analyzed 2qxb complex using the Jmol Java applet.