## 3 Results
### Stability of Protein-Ligand Complexes
#### 3.9.2 Gossypetin and WT-EGFR Complex  
The Gossypetin with WT-EGFR simulation of the system was neutralized by adding eight Na\({}^{+}\) ions (12.778 mM concentration) and being subjected to a 100 ns run of production with a 10 ps recording interval. The backbone RMSD fluctuation was to be within 1.2 to 3.0 A, and the average RMSD was found to be \(\sim\)2.6 A for all three replicas (Figure 9A). The complex RMSD fluctuation was found to be within 0.699 to 3.53 A, and the average RMSD was found to be \(\sim\)2.6 A. In all three replicates, the complex RMSD was gradually increased with slight fluctuation from 0 to 50 ns (\(\sim\)0.7 to \(\sim\)2.2 A) and again attained stability from 50 to 100 ns (Figure 9B). Similarly, the initial average RMSD of ligand w.r.t. ligand for all three replicas was 0.294 A, and the final was 0.313 A. The total average RMSD of ligand w.r.t. ligand for all three replicas was 0.3 A. However, replica 3 of Gossypetin showed slight fluctuation from 35 to 50 ns (0.3 to 0.7 A) and again attained stability from 50 ns at 0.7 A. Further, a decrease in RMSD was seen at 75 ns (0.7 to 0.3 A) and was found to be stable throughout 100 ns (Figure 9C). The RMSF analysis was performed to infer residue-wise fluctuations observed during the production run. The average RMSF value of residues was \(\sim\)1.4 A. The c-terminal loop region showed maximum fluctuations due to the flexibility of terminal residues' maximum fluctuation (\(\sim\)2.5 A), whereas the RMSF of active residues involved in the ligand interactions, viz., Ser25, Lys50, Cys80, Thr95, Gln96, Met98, Pro99, Leu149, and Thr159 was \(<\)1.3 A (Figure 10A). The radius of gyration (rGyr) trajectory over the 100 ns production run was also analyzed. The rGyr deviation of Gossypetin with EGFR was found within 3.7 to 3.8 A, which indicates the higher compactness of the Gossypetin-EGFR complex (Figure 10B). Similarly, in all three replicas, the protein rGyr was also found to be uniform (within \(\sim\)20 to 20.8 A) throughout the simulation period (Figure 10C).  
Figure 8: EGFR-Andrographin complex sustained intermolecular interactions and percentage interaction fraction throughout 100 ns production run.  
Figure 10: EGFR-Gossypetin complex 100 ns MD production run. (**A**) Protein RMSF plot. (**B**) Gyration plot depicting the extendedness of a ligand. (**C**) Gyration plot of protein during a 100 ns production run.  
Figure 9: Gossypetin with WT-EGFR 100 ns MD production run. (**A**) RMSD plot depicting the \"protein backbone\". (**B**) Plot depicting the docked complex stability “ligand–protein complex RMSD”. (**C**) Plot depicting the stability of “ligand w.r.t. ligand\".  
Further, Gossypetin-EGFR contact analysis concluded that Gln96 formed stable hydrogen-bonded interactions for around \(\sim\)93% and 21% of the duration with the OH group of Gossypetin in replicas 1 and 3 and around 53% and 27% of the water-bridge in replicas 2 and 3. Met98, Thr95, and Asp105 residues were found to be common interactive residues and to be involved in hydrogen bonds, hydrophobic bonds, and water-bridge interactions in all three replicates. Figure 9, Figure 10, and Figure 11 represent the RMSD, RMSE, rGyr, and contacts of the Gossypetin-EGFR complex.