Sequence clustering confounds AlphaFold2

Though typically associated with a single folded state, some globular proteins remodel their secondary and/or tertiary structures in response to cellular stimuli. AlphaFold2¹ (AF2) readily generates one dominant protein structure for these fold-switching (a.k.a. metamorphic) proteins², but it often fails to predict their alternative experimentally observed structures^3,4. Wayment-Steele, et al. steered AF2 to predict alternative structures of a few metamorphic proteins using a method they call AF-cluster⁵. However, their Paper lacks some essential controls needed to assess AF-cluster's reliability. We find that these controls show AF-cluster to be a poor predictor of metamorphic proteins. First, closer examination of the Paper's results reveals that random sequence sampling outperforms sequence clustering, challenging the claim that AF-cluster works by "deconvolving conflicting sets of couplings." Further, we observe that AF-cluster mistakes some single-folding KaiB homologs for fold switchers, a critical flaw bound to mislead users. Finally, proper error analysis reveals that AF-cluster predicts many correct structures with low confidence and some experimentally unobserved conformations with confidences similar to experimentally observed ones. For these reasons, we suggest using ColabFold⁶-based random sequence sampling⁷-augmented by other predictive approaches-as a more accurate and less computationally intense alternative to AF-cluster.