How are predicted protein structures validated?

Protein structure validation assesses quality at multiple levels: 1) Stereochemistry - Ramachandran plot analysis (most residues in favored regions), bond lengths and angles, rotamer conformations. Tools: MolProbity, PROCHECK, WHATCHECK. 2) Packing - check for atomic clashes, cavity analysis, and proper hydrophobic burial. 3) Energy-based - molecular mechanics scoring, compatibility with expected energy profiles. 4) Sequence-structure compatibility - 3D-1D profile methods (Verify3D), ProSA checks against statistical potentials. 5) For experimental structures - R-factor, resolution (X-ray), restraint violations (NMR). 6) For predicted structures - AlphaFold pLDDT scores, predicted aligned error (PAE), template modeling scores (GDT-TS, TM-score). Poor scores indicate potential errors requiring attention.