Protein stability in solution—whether dispersions remain soluble or undergo aggregation, phase separation, or crystallization—is critical in biotechnology and pharmaceutical development. Predicting such behavior requires knowledge of protein pair interaction potentials (PIP), which quantify intermolecular interaction energies as a function of interparticle centroid-to-centroid distance. Despite the importance of PIP, no direct experimental method for measuring this parameter has been reported. Here, we show that cryo-electron tomography (cryo-ET) can directly measure PIP from the three-dimensional distributions of protein assemblies in vitrified solution. Unlike small-angle X-ray scattering (SAXS), this approach does not rely on model fitting or assumptions about potential shape. We applied the cryo-ET method to proteins of varying sizes and structures, including hemoglobin, ovalbumin, and serum albumins. The resulting interaction potentials were validated by two orthogonal techniques: structure factors from cryo-ET matched SAXS data, and Kirkwood-Buff integrals from cryo-ET agreed with second virial coefficients from analytical ultracentrifugation. We also compare the measured PIP with predictions from the theoretical model of the Derjaguin-Landau-Verwey-Overbeek framework and analyze its applicability for describing protein interactions. To conclude, this work demonstrates that cryo-ET provides direct, assumption-free measurements of protein interaction potentials, offering a versatile tool for studying protein interactions in solution.
Poliukhina et al. (Sun,) studied this question.