Pymol molecular graphic system

All structural figures were produced using PyMOL (PyMOL Molecular Graphic System, Delano Scientific) with the following structures: unliganded CTLA-4 (Protein Databank (PDB) accession no. 3OSK)^{51 (link)}, CTLA-4 complexed with CD80 (PDB accession no. 1I8L)^{52 (link)} and CTLA-4 complexed with CD86 (PDB accession no. 1I85)^{53 (link)}.

Using the solved structure of gap junction protein alpha 8 as template (Protein Data Bank accession No.6MHY_A), the model structure of homomeric wild and the mutant GJA8 were constructed by Swiss Model Server (https://swissmodel.expasy.org) and shown with the PyMOL Molecular Graphic system (Delano Scientific). Meanwhile, we used online programs to predict the possible functional impact of the amino acid change, including BayesDel addAF, BayesDel no AF, DANN, DEOGEN2, EIGEN, FATHMM, LRT, MVP, MetaLR, MetaSVM, MutPred, MutationTaster, PROVEAN, PrimateAI, REVEL, SIFT, etc. (http://varsome.com). In addition, multiple sequence alignment from different species was performed by CLUSTALW (https://www.genome.jp/tools-bin/clustalw).

To define the coordinates of the active site to launch the docking assay, a structural alignment between the candidate TB506 and the CYP450 prototype (PDB code: 2HPD) (Schuler and Werck-Reichhart, 2003 (link)) was performed by the PyMOL Molecular graphic system (DeLano scientific: San Carlos, CA, United States). Molecular dynamics (MD) calculations were performed by NAMD software using the apo form of the enzyme containing the heme-group (Phillips et al., 2005 (link)). In previous MD calculations, an energy minimization was carried out using standard conditions (10,000 cycles, 1 femtosecond per cycle and a dielectric constant of 80). The MD calculations gave 220 derivative structures from the candidate TB506. Putative substrates were constructed and topologically optimized by ACD/ChemSketch software v1.7 2007 (ACD/Labs: Toronto, ON, Canada). Molecular docking was performed by AutoDockTools 4 (Morris et al., 2009 (link)) providing the coordinates corresponding to the active site of the molecule.