3.11.  Molecular dynamics simulation
Figures 9 and 10, respectively, depict molecular simulation analysis of SARS-CoV-2 spike receptor-binding domain (PDB ID: 6M0J) bound with withanolide A and SARS-CoV-2 papain-like protease (PDB ID: 6W9C) bound with withanolide B. Both MD simulations showed an acceptable stability profile at a temperature of 300 K. Root mean square deviation (RMSD) is one of the most important fundamental properties to establish protein stability and its conformation to experimental structure (Kuzmanic & Zagrovic, 2010; Laskowski et al., 1997). RMSD is a measure of the deviation of the 3D or tertiary structure of a protein and is applied in order to get an insight into the stability of the protein in a biological system during a MD simulation. SARS-CoV-2 spike receptor-binding domain-withanolide A complex displayed constant RMSDs (0.5–2.0 angstrom) of both protein side chains and Cα atoms from the initial structure (before equilibrium) throughout the 3 ns time scale (Figure 9.1). Similarly, SARS-CoV-2 papain-like protease-withanolide B complex also exhibited constant RMSDs (0.8–2.9 angstrom) of both protein side chains and Cα atoms from the initial structure throughout the 3 ns time scale (Figure 10.1). Figures 11.1–11.3 and 12.1–12.3, respectively depict MS dynamics analyses of SARS-CoV spike glycoprotein (PDB ID: 5WRG) with withanolide B and SARS-CoV-2 main protease (PDB ID: 6LU7) with withanolide A.
Figure 9.  Molecular simulation of SARS–CoV–2 spike receptor–binding domain bound (6M0J) with withanolide A using Playmolecule open server (Table 1). Figures 9.1–9.2, Tables 2–4 here corresponds to the tables of MD simulation statistics. RMSD values were obtained as a function of time obtained at 300 K. Values were calculated with the use of Cα atoms. Figures 9.3–9.4. Average RMSF values obtained as a function of amino acid sequence numbers at 300 K. Values were calculated with the use of Cα atoms.
Figure 10.  Molecular simulation of papain–like protease (6W9C–A chain) with withanolide B using Playmolecule open server. Figures 10.1–10.2, Tables 2–4 here corresponds to the tables of MD simulation statistics. RMSD values were obtained as a function of time obtained at 300 K. Values were calculated with the use of Cα atoms. Figures 10.3–10.4. Average RMSF values obtained as a function of amino acid sequence numbers at 300 K. Values were calculated with the use of Cα atoms.
Figure 11.1.  MD simulation of SARS–CoV spike glycoprotein (PDB ID: 5WRG) with withanolide B using LARMD online server. (A) Ligand–protein conformation, (B) RMSD of receptor and ligand (C) RMSD histogram of receptor (D) RMSD histogram of ligand (E) Radius of gyration—Rg value (F) Fraction of native contacts analysis of SARS-CoV–2 PL-pro (PDB ID: 6W9C) with withanolide B over a time frame of 4000 ps (4 ns) (G) RMSF value of each residue (H) B–factor value (changing from blue to red with increase in value) and (I) B–factor analysis of defined complex.
Figure 11.2.  PCA of SARS–CoV spike glycoprotein (PDB ID: 5WRG) with withanolide B (A) PCA results for Trajectory (B) Simple clustering in PC subspace(C) Table data showing residue–wise loadings for PC1, PC2 and PC3 and residue number at each position (D) Clustering dendogram based on PC1, PC2 and PC3 (E) Dynamical residue cross–correlation map; the correlated residues are in blue, anti–correlated residues are in red; the pairwise residues with higher correlated coefficient (>0.8) and with higher anti–correlated coefficient (≤0.4) are linked with light pink and light blue (Int_mod) (F) Residue–wise loadings for PC1, PC2 and PC3 (G) Table showing pairwise cross–correlation coefficients; higher correlated coefficient value is >0.8 and higher anti–correlated coefficient value is ≤0.4.
Figure 11.3.  Energy, hydrogen bond analysis and decomposition analysis of SARS–CoV spike glycoprotein (PDB ID: 5WRG) with withanolide B (A) MM/PB(GB)SA result consists of electrostatic energy (ELE), van der Waals contribution (VDW), total gas phase energy (GAS), non–polar and polar contributions to solvation (PBSOL/GBSOL) (B,C) Statistics of hydrogen bonds (D) energy decompose of protein–ligand complex (Kcal/mol) (E) Graphical representation of decompose result (F) Showing the heatmap of decompose.
Figure 12.1.  MD Simulation of SARS-CoV-2 main protease (PDB ID: 6LU7) with withanolide A using LARMD online server. (A) Ligand-protein conformation (B) RMSD of receptor and ligand (C) RMSD histogram of receptor (D) RMSD histogram of ligand (E) Radius of gyration- Rg value (F) Fraction of native contacts analysis of SARS-CoV-2 PL-pro (PDB ID: 6W9C) with withanolide A, over a time frame of 4000ps (4 ns) (G) RMSF value of each residue (H) B-factor value (changing from blue to red with increase in value) and (I) B-factor analysis of defined complex.
Figure 12.2.  PCA of SARS-CoV-2 main protease (PDB ID: 6LU7) with withanolide A (A) PCA results for trajectory (B) Simple clustering in PC subspace(C) Table data showing residue-wise loadings for PC1, PC2 and PC3 and residue number at each position (D) Clustering dendogram based on PC1, PC2 and PC3 (E) Dynamical residue cross-correlation map; the correlated residues are in blue, anti-correlated residues are in red; the pairwise residues with higher correlated coefficient (>0.8) and with higher anti-correlated coefficient (≤0.4) are linked with light pink and light blue (Int_mod) (F) Residue-wise loadings for PC1, PC2 and PC3 (G) Table showing pairwise cross-correlation coefficients; higher correlated coefficient value is >0.8 and higher anti-correlated coefficient value is ≤0.4.
Figure 12.3.  Energy, hydrogen bond analysis and decomposition analysis of SARS-CoV spike glycoprotein (PDB ID: 5WRG) with withanolide B (A) MM/PB(GB)SA result consists of electrostatic energy (ELE), van der Waals contribution (VDW), total gas phase energy (GAS), non-polar and polar contributions to solvation (PBSOL/GBSOL) (B,C) Statistics of hydrogen bonds (D) Energy decompose of protein–ligand complex (Kcal/mol) (E) Graphical representation of decompose result (F) Showing the heatmap of decompose.
Vibrations around the equilibrium are not random, but depend on the local structure flexibility. In order to calculate the average fluctuation of all residues during simulations, the root mean square fluctuation (RMSF) of the Cα atoms of both target proteins were plotted from the primary structure of both proteins as a function of residue number (Kuzmanic & Zagrovic, 2010). The obtained patterns of RMSFs for both the proteins and ligands have been presented in Figures 11.1–11.3 and 12.1–12.3, respectively.