Experiment-guided AlphaFold3 resolves measurement-consistent protein ensembles

AlphaFold3 predicts highly accurate protein structures from sequence but tends to collapse to a single dominant conformation, even when the underlying structure is inherently heterogeneous. Moreover, its predictions are oblivious to experimental conditions that can alter local sequence conformation. In this work, we show that AlphaFold3 can be guided to match data obtained by nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography and cryogenic electron microscopy (cryo-EM) experiments and combinations thereof. Our approach can also incorporate data that explicitly report on dynamics, such as site-resolved order parameters. We demonstrate that this methodology generates compact structural ensembles whose ensemble-averaged observables agree with experiment, with fewer distance restraint violations than traditionally resolved NMR structures and with unmodeled alternate conformations uncovered in electron density. This methodology paves the way for experimentally aware predictive models that generate structural ensembles consistent with the measurements, potentially over multiple modalities, and that can be further refined toward thermodynamically grounded ensembles by incorporating energetics.