Table 5.

Important validation decisions

Decision	Available tools	Validation parameter
Does the electron density support the ligand model?	Inspect electron density using Twilight, PyMOL, Coot or EDS	RSCC < 0.8 suggest significant disagreement with electron density
Is the ligand in a region affected by crystal packing?	Inspect symmetry of model using PyMOL or Coot	Clashes of symmetry mates with ligand binding region suggest possible perturbations of conformation
Are the B-factors of the ligand significantly higher than the overall average?	Inspect B-factors of the ligand using PDB record or Twilight	B-factor >200 Å2 corresponds to ~1.6 Å displacement
Is the resolution of the structure sufficiently high to support the model of the ligand?	Inspect reflection count and resolution in PDB record	Resolutions >2.5 Å likely have a lower data-to-parameter ratio
Is the Kd of the ligand binding high in the crystallization conditions employed?	Further biophysical or biochemical studies required	Ligand occupancy is likely less than 100 %

Questions that any user of protein–ligand models should ask in order to use the ligand model with confidence