The correct interpretation of the electron density observed in a protein's binding site can sometimes be challenging. Many cases have shown that a density at the binding site, initially interpreted as a bound ligand, was actually due to water molecules, buffer components, or precipitant molecules. Therefore, it is essential to validate the quality of the atomic model of the ligand-protein complex. This validation is crucial for all subsequent steps, especially in computational methods used in structure-based drug discovery projects, which depend heavily on the overall quality of the 3D structure. Global statistics, such as resolution, the R-factor, and model geometry discussed above, ensure that the structure is well-refined and meets the general quality criteria set by the PDB. However, even well-refined protein structures may contain problematic regions with poor electron density, resulting in poor local model quality. In the case of a bound ligand (small molecule), poor electron density may result from partial occupancy of the binding site as well as conformational flexibility of the ligand in the crystal. Therefore, it is important to inspect the bound ligand using so-called real-space (in contrast to reciprocal space) quality indicators. Below we discuss some of the details. 1- While building the ligand into the electron density, we need to ensure that the electron density covers all ligand atoms and that no unaccounted-for density remains within the binding site. Unaccounted-for density, e.g., when running crystallographic fragment screening, may indicate that the bound molecule is not what we expected to find there. 2- The fit of the model to the electron density can be assessed by examining the Real Space Correlation Coefficient (RSCC) and the Real Space R-Value (RSR). These values are calculated per residue and can be obtained from the PDB validation report. RSCC is generally higher for higher-resolution and lower-B-factor models – a perfect fit of the model to the electron density would yield an RSCC of 1.0. The lower the RSCC, the greater the uncertainty in the ligand model's position relative to the electron density. Generally, values between 0.8 and 1.0 are considered satisfactory. 3- On some occasions, model bias may also result in spurious electron density, which is mistakenly interpreted as evidence of ligand presence. If in doubt, we can verify the presence of the ligand by first removing it from the coordinate file, running a few cycles of refinement, and then calculating a so-called omit electron density map (a map with the ligand molecule omitted from the coordinate file). If the ligand is present in the binding site at a high occupancy, an omit map will clearly reveal its density. 4- In addition, we need to examine the interactions between the ligand and the amino acid side chains within the protein-binding site. For example, chemically unrealistic interactions or atom-atom distances that are too short reveal potential problems with the ligand's positioning. 5- We also need to ensure that the ligand model complies with the expected stereochemical parameters of the chemical structure. 6- The atomic displacement factors (B-factors or temperature factors) of the ligand should be compared to the average B-factors of the protein atoms within the binding site. Too-high ligand temperature factors should also alert us to potential problems.
