Hands-on: Ligand validation

When drawing functional conclusions about a small molecule ligand’s interactions with a protein, it is paramount to ensure the quality and reliability of that specific ligand’s model within the PDB entry. The validation report provides critical information to assess this.

In this exercise, we will examine the validation data for NADPH (ligand ID: NDP) in two distinct PDB entries: 7C83 and 5B1Y. We’ll focus on two key aspects: ligand geometry and fit to electron density.

Part 1: Ligand Geometry Assessment

The table below presents the bond length and bond angle outlier data for the NADPH molecule from PDB entry 7C83 (top) and 5B1Y (bottom).

• Z-score: The Z-score for a bond length or angle quantifies how many standard deviations the observed value deviates from its expected ideal value. A bond length or angle with an absolute Z-score (∣Z∣>2) is flagged as an outlier, warranting closer inspection.
• RMSZ: The Root-Mean-Square Z-score (RMSZ) provides an overall measure of the deviations for all bond lengths or angles within the ligand, summarising the geometric quality.

By interactively exploring these visualisation options, you can quickly identify and focus on areas of the structure that require closer inspection due to validation flags.

The images below, extracted from the respective validation reports, provide a visual representation of these geometric outliers. In these diagrams, all atoms and bonds of the ligand are shown, colour-coded to indicate outliers: green signifies no outliers, while pink highlights problematic bonds or atoms.

Question 1. Using the information provided above, which of these NADPH molecules has the most reliable geometry?

Part 2: Fit to electron density

Beyond geometry, a crucial aspect of ligand quality is how well it fits the experimental electron density data. The following images from the validation reports illustrate the fit of these ligands for 7c83 (top) and 5b1y (bottom). The green and purple meshes represent the Fo-Fc difference density map

• Green density indicates areas where there is positive electron density not accounted for by the current model, suggesting missing atoms or atoms placed too far from the true density peak.
• Purple (or red) density indicates areas where the model has atoms but there is a lack of corresponding electron density, suggesting incorrectly modelled atoms or atoms placed where they don’t belong

Question 2. Based on these images, the NADPH from which entry has been modelled using more reliable electron density data?