The effect of mutation on NS1 structure stability was predicted using FoldX 5.0 (Schymkowitz et al. 2005 (link)). Three types of mutations lists were created from the sequence dataset:
To predict effect of mutations on protein stability, each mutation list was passed to FoldX. It first repairs the reference protein structure, and then calculates energy difference between wild-type and mutated structures (ΔΔG). Calculation was performed in triplicates and mean ΔΔG score was used to classify mutations. Mutations are classified into three categories: stabilizing (ΔΔG < −0.46 kcal), destabilizing (ΔΔG > −0.46 kcal), and neutral (−0.46 kcal/mol < ΔΔG ≤ + 0.46 kcal/mol). For detailed analysis, destabilizing and stabilizing groups were further divided into three subgroups based on ΔΔG scores. Highly stabilizing: ΔΔG < −1.84 kcal/mol, stabilizing: −1.84 kcal/mol ≤ ΔΔG < −0.92 kcal/mol, slightly stabilizing: −0.92 kcal/mol ≤ ΔΔG < −0.46 kcal/mol, slightly destabilizing: + 0.46 kcal/mol < ΔΔG ≤ + 0.92 kcal/mol, Destabilizing: + 0.92 kcal/mol < ΔΔG ≤ + 1.84 kcal/mol, highly destabilizing: ΔΔG > + 1.84 kcal/mol.
In case of virtual saturation mutagenesis, all stabilizing and neutral mutations were labeled as 1. On the other hand, all destabilizing mutations for a residue position were labeled as 0. Total aggregate score for each position was calculated and termed as Mutational capacity per residue positions (MC/RP). A Python script was used for calculation and plotting.
To predict effect of mutations on protein stability, each mutation list was passed to FoldX. It first repairs the reference protein structure, and then calculates energy difference between wild-type and mutated structures (ΔΔG). Calculation was performed in triplicates and mean ΔΔG score was used to classify mutations. Mutations are classified into three categories: stabilizing (ΔΔG < −0.46 kcal), destabilizing (ΔΔG > −0.46 kcal), and neutral (−0.46 kcal/mol < ΔΔG ≤ + 0.46 kcal/mol). For detailed analysis, destabilizing and stabilizing groups were further divided into three subgroups based on ΔΔG scores. Highly stabilizing: ΔΔG < −1.84 kcal/mol, stabilizing: −1.84 kcal/mol ≤ ΔΔG < −0.92 kcal/mol, slightly stabilizing: −0.92 kcal/mol ≤ ΔΔG < −0.46 kcal/mol, slightly destabilizing: + 0.46 kcal/mol < ΔΔG ≤ + 0.92 kcal/mol, Destabilizing: + 0.92 kcal/mol < ΔΔG ≤ + 1.84 kcal/mol, highly destabilizing: ΔΔG > + 1.84 kcal/mol.
In case of virtual saturation mutagenesis, all stabilizing and neutral mutations were labeled as 1. On the other hand, all destabilizing mutations for a residue position were labeled as 0. Total aggregate score for each position was calculated and termed as Mutational capacity per residue positions (MC/RP). A Python script was used for calculation and plotting.
