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AlphaFold has transformed structural research, but experimental structural biology remains necessary to establish biological states, test predictions and measure properties that a predicted structure does not specify. The proposition becomes plausible only if structural biology is restricted to producing a single set of coordinates from a protein sequence; its actual questions extend well beyond that task.

The achievement should not be minimized. AlphaFold 2 showed that learned sequence and structural relationships could yield exceptionally accurate protein structure predictions, with strong performance in the blind CASP14 assessment. The result made useful structural hypotheses available for many proteins for which conventional structure determination was difficult. Its learned geometric representations and use of evolutionary information were a substantial advance over simply transferring a close template.^[ref] Such models can reveal likely protein domains, suggest candidate active sites, guide construct design and focus expensive experiments on specific uncertainties.

By the examination date, the scope had already expanded. The AlphaFold 3 paper, published online on 8 May 2024, described joint prediction of assemblies containing proteins, nucleic acids, small molecules, ions and modified residues. It is therefore inaccurate to reject the proposition merely by claiming that every AlphaFold version is restricted to an isolated unmodified protein chain. Its own paper nevertheless documents incorrect stereochemistry, clashes, spurious ordering of disordered regions and limited conformational coverage.^[ref]

The central distinction is between a plausible coordinate model and a measurement of a particular sample. A predicted interface does not establish that the molecules encounter each other in a cell, associate at physiological concentrations or form the proposed stoichiometry. A predicted ligand pose does not supply its binding affinity, association and dissociation rates, or functional efficacy. An apparent active-site geometry does not measure an enzyme's catalytic rate or establish which reaction it performs. Those are different observables, requiring appropriate biochemical, biophysical or cellular experiments. Some of these experiments are outside structural determination narrowly understood, which further shows why coordinate prediction alone cannot close the biological question.

Many proteins also act through a conformational ensemble. Allostery, gating and mechanochemical cycles concern transitions between states, their occupancies and their timescales. One accurate fold is not a measurement of that ensemble. Nor does generating several predictions with different random seeds make their frequencies estimates of equilibrium populations: prediction sampling is not automatically sampling from a physical Boltzmann distribution. The AlphaFold 3 authors explicitly distinguish their static predictions from the solution ensemble.^[ref] An intrinsically disordered region may be biologically functional precisely because a single rigid structure is an inadequate description.

Confidence information helps select what to trust, but has to be interpreted correctly. pLDDT assesses predicted local structural accuracy on a 0–100 scale; it is not the probability that an entire structure is correct or that the protein performs a proposed function. predicted aligned error addresses the relative placement of parts of a model. Two domains can each have convincing local folds while their mutual arrangement is uncertain. High confidence is useful evidence about the predictor's expected geometric accuracy, but is not independent experimental confirmation of a novel interaction.^[ref]

The different experimental methods answer complementary questions. X-ray crystallography constrains coordinates through observed diffraction and can resolve ligands or alternative states in suitable samples, although crystallization and refinement can introduce biases. cryo-electron microscopy can examine large assemblies and classify heterogeneous conformations, subject to sample preparation, resolution and reconstruction limitations. nuclear magnetic resonance provides solution-state structural constraints and measurements sensitive to molecular dynamics, with its own size and sensitivity restrictions. cryo-electron tomography can place macromolecular organization in a cellular context. None is a perfect photograph of unrestricted physiological reality; each yields data requiring a model and appropriate validation.

The practical relationship is increasingly reciprocal. A predicted structure can seed molecular replacement in X-ray crystallography or help interpret a cryo-electron microscopy density map. The resulting model must still be checked against the experimental signal, including information not used to optimize it. Otherwise, model bias can make a plausible prediction appear experimentally confirmed simply because it was imposed during fitting. Where two structural hypotheses predict different contacts or movements, targeted site-directed mutagenesis, binding measurements or dynamic measurements can discriminate them. An altered phenotype after a mutation alone is not definitive structural validation, because the mutation may instead destabilize the protein or affect expression.

Experiments also underpin evaluation of the predictor itself. Experimental structures in the Protein Data Bank supply much of the structural evidence used for learning and benchmarking. Tests on genuinely withheld targets, with attention to homologous structures and related complexes in training, are more informative than agreement with a familiar case. Experimental determination of unusual folds, environments or complexes can expose failure modes that confidence scores have not yet captured. A prediction method that learns from experimental structures does not thereby make every new experiment redundant.

The justified conclusion is a change in the allocation of effort. Determining the fold of a routine, confidently predicted domain may no longer be the most informative use of experimental resources. Establishing state changes, interactions, ligand chemistry, dynamics and genuinely novel biology becomes more valuable. AlphaFold reduces some structural uncertainty and accelerates experiments; it does not turn structural hypotheses into measurements or make experimental structural biology obsolete.