AlphaFold 3 Prediction Accuracy and Reliability across RNA Structures and Protein–RNA Interfaces

AlphaFold 3 (AF3) models RNA and protein–RNA complexes, but when its predictions and confidence metrics can be trusted is unclear. We evaluated AF3 on more than 1,300 protein–RNA complexes and about 270 RNA-only systems, and compared post-cutoff RNAs with pre-cutoff candidates selected by sequence and fold similarity. On structurally novel RNAs, AF3 largely preserves the protein fold, while RNA-fold and interface accuracy drop sharply with little change in predicted RNA confidence score. Input ablations show that protein alignments and templates from RNA-bound proteins govern complex assembly, whereas RNA alignments matter most for single-chain and long RNAs. The most frequent failed interface retains protein-side contacts but places an often-misfolded RNA in the wrong pose. RNA interface confidence only partly distinguishes this error from correct placements. Against CLIP-seq, SELEX and RNAcompete motifs, AF3 usually places the protein against the experimentally observed motif-bearing RNA segment, even where the interface fails, but often not in the native pose. In protein–RNA complexes released in 2026, a score combining AF3 confidence with RNA familiarity and template use ranked RNA chains with accurate folds and correctly placed interfaces better than AF3’s ranking score.