Flowchart of the method.

<p>A reference structural alignment of the target and the template is generated by the MALIGN3D command in MODELLER <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002645#pone.0002645-Sanchez2" target="_blank">[11]</a> (step 1). The initial target-template sequence alignment is realized by T_COFFEE (step 2). The regions of interest (ROI), defined as misaligned secondary structure elements together with their adjacent loops, are identified by comparison of the initial target-template sequence alignment with the reference structural alignment. A set of alignments to evaluate is generated using an exhaustive ungapped search in the ROI (step 3). Hundred models for each alignment are built using MODELLER (step 4). For each model, an energy minimization is done in vacuum using CHARMM (step 5). The energy for the minimized models is calculated (step 6). The secondary structure is assigned with DSSP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002645#pone.0002645-Qiu1" target="_blank">[24]</a> and the predictor's scores are calculated (step 7). After all alignments are processed, a statistical analysis using the statistical package R (<a href="http://www.R-project.org" target="_blank">http://www.R-project.org</a>) is further performed on the predictor to associate a degree of confidence to the prediction (step 8) and the best alignment is determined (step 9). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002645#s2" target="_blank">Materials and Methods</a> for details.</p

Influence of energy minimization for six different predictors CDIE/All (A), RDIE/All (B), GBMV2/All (C), ProsaII/All (D), ANOLEA/All (E) and ANOLEA/SSE (F) applied to the test case 1flp helix1.

<p>The six corresponding profiles and their standard deviation are shown for minimized and non minimized models, with plain and dotted lines, respectively.</p

Optimization of the ANOLEA/SSE Local predictor specificity.

<p>The highest specificity is attained for an inter-residue distance cut-off of all pairs of heavy atoms of 4 Å as deduced from the scan by step of 0.5 Å from 2 to 6 Å for all the test cases. ANOLEA/SSE Local predictor scores for 1flp helix1 test case versus alignment offset to the structural alignment and for different inter-residue distance of all pairs of heavy atoms (from 2 Å (smallest sphere) to 6 Å (biggest sphere)).</p

Mean backbone (bb) RMSD per residue for 100 models of 1flp h1 testcase for Δseq = 0.

<p>Secondary structure elements limits are indicated by horizontal bars on the upper x axis. Regions of high variability correspond to loops.</p

For each test case, the SSE in the ROI is colored in red on a protein ribbon representation and is defined by a red rectangle in the alignments.

<p>The different alignments are represented and the corresponding score using the ANOLEA/SSE Local predictor is plotted. In T0141 test case, the “new strands” label in the alignment picture represents the position of a beta finger present in the structure of T0141 but absent in the 1aro_L template.</p

ANOLEA-based predictors' values distribution for the three different residues selections.

<p>A hundred models of 1flp helix1 test case are superimposed. Only parts of the protein involved in each selection are represented and the corresponding predictor values distribution are shown. The SSE in the ROI is represented in red. A, Predictor ANOLEA/All. B, Predictor ANOLEA/ROI Local. C, Predictor ANOLEA/SSE Local. The black and gray colored histograms represent the predictors values distributions for the lowest predictor scores (structural alignment, ∶seq = 0) and for the second lowest one, respectively. The bin width is set equal to the quarter of the variance (10 for A, 5 for B and C).</p

Impact of the distance between the evaluated alignment and the reference alignment (∶seq, X axis) on the three ANOLEA-based predictors (Y axis), for T0178 helix 7 case.

<p>The most favorable predictor score is indicated by a red circle and the structural alignment by a green circle. A, Predictor <i>ANOLEA/All</i>. B, Predictor <i>ANOLEA/ROI Local</i>. C, Predictor <i>ANOLEA/SSE Local</i>. Δ, Μεαν Χα RMSD in the region of interest of the models to crystal structure for each alignment.</p