The reasons for control docking/ redocking more than 2 Å might due to:

1. Not included all the important amino acids in the box.

2. The protein protonation at the pocket or ligand charge, but if you follow the standard protocol, it should be no problem.

3. You might need to check for ligand interactions. If there is a covalent bond, you might need to use the covalent bond protocol.

To get the RMSD below 2, you might need to reevaluate the box size/parameters.

Reasons for RMSD > 2 Å:

Inaccurate Initial Structure: If the initial structure used for docking is inaccurate or deviates significantly from the experimental structure, it can lead to high RMSD values.

Ligand Flexibility: If the ligand undergoes significant conformational changes upon binding that are not adequately considered in the docking calculations, it can contribute to higher RMSD. Insufficient Sampling: Inadequate conformational sampling during the docking simulations may result in the omission of relevant binding modes, leading to higher RMSD. Scoring Function Limitations: The scoring function used by Auto Dock may have limitations in accurately ranking different binding poses, affecting the predicted binding mode.

Ways to Improve RMSD (Reduce it below 2 Å):

Refinement of Initial Structure: Ensure that the initial structures of the receptor and ligand are accurate. This may involve using experimental structures or refining homology models. Ligand Conformational Search: Allow for ligand flexibility during docking by performing a more extensive conformational search. This can be achieved by increasing the number of torsional degrees of freedom. Increased Sampling: Use more exhaustive search parameters or perform multiple independent docking runs to increase the conformational sampling and improve the chances of finding the correct binding mode. Ensemble Docking: Consider using an ensemble of receptor structures if there is significant flexibility in the binding site. This can account for variations in the binding site conformation. Scoring Function Adjustment: Evaluate and, if necessary, adjust the scoring function parameters in Auto Dock to better reflect the experimental binding affinity. Post-Docking Refinement: Apply post-docking refinement techniques to further optimize the predicted binding poses. Molecular dynamics simulations or energy minimization can be used for refinement. Experimental Validation: Validate the predicted binding poses experimentally, such as through site-directed mutagenesis or binding assays, to confirm the accuracy of the predictions.