4.4. Tertiary Structure Prediction, Structure Validation and Quality Prediction

Three-dimensional models of the selected putative enzymes were generated using different approaches. For sequences with acceptable homology in the template of the programs, UCSF Chimera (https://www.rbvi.ucsf.edu/chimera/) and the automated protein homology modeling server SWISS-MODEL (http://swissmodel.expasy.org/) were used. For sequences with low homology with the structures in the database, multiple-threading alignments using the I-TASSER approach (zhanglab.ccmb.med.umich.edu/I-TASSER/) was used. I-TASSER is an automated bioinformatics tool for predicting protein structures from an amino acid sequence followed by iterative structural assembly simulations and atomic-level structure refinement.

The predicted structures were evaluated to ensure correctness of the model stereochemistry, as checked by a Ramachandran plot (http://mordred.bioc.cam.ac.uk/~rapper/rampage.php) (Lovell et al., 2003) and Verify 3D [52]. The Ramachandran plot scores of the predicted structures showed more than 90% of the amino acids were in favorable regions. ProSA-web Z-score plot (https://prosa.services.came.sbg.ac.at/prosa.php) [53] was used to check whether the Z-score of the input structures is within the range of typically found for the native proteins of a similar size. The Z-score values of all protein structures checked in this study were highlighted as a black dot, which indicates being in the range of native conformations. The final modeled structures were further energetically minimized and molecular dynamics simulation was performed with CABS-flex 2.0 (http://212.87.3.12/CABSflex2). The latter program is an efficient simulation engine that allows modeling of the large-scale conformational change related to protein flexibility [54]. The models were comprehensively analyzed using PyMol (http://pymol.org/) to check for the presence of a lid, and the existence and orientation of the catalytic triad. The depth of the putative intramolecular tunnels was calculated with DEPTH [55] taking residues from the oxyanion hole in each candidate as the cavity end point.