Abstract text (incl. figure legends and references)

Introduction: As cryogenic electron microscopy (cryoEM) gains traction in the structural biology community as a method of choice for determining atomic structures of biological complexes, it has been increasingly recognized that many complexes that behave well under conventional negative-stain electron microscopy tend to have preferential orientation, aggregate or simply mysteriously "disappear" on cryoEM grids, but the reasons for such misbehavior are not well understood, limiting systematic approaches to solving the problem.

Objective: This study aims to develop a theoretical formulation that explains the phenomenon of particles preferential orientation and aggregation during cryoEM sample preparation.

Materials & methods: We first develop a mathematical model to calculate the surface energy of particles that migrate to the air-water interface (AWI). Then we employed GroEL as a model protein to confirm our formulations using cryogenic electron tomography (cryoET) technique.

Results: By conducting cryogenic electron tomography (cryoET) with the widely-tested sample, GroEL, we demonstrate that, in standard buffer solution, nearly all particles migrate to the AWI. Gradual reduction of the surface tension by introducing surfactants decreased the percentage of particles exposed to the surface. By conducting single-particle cryoEM, we confirm that the surfactants do not damage the biological complex, thus suggesting that they might offer a practical, simple and general solution to the problem for high-resolution cryoEM. Application of this solution to a real-world AWI adsorption problem with a more challenging membrane protein, namely, ClC-1 channel, has led to its first atomic structure using cryoEM.

Conclusion: Our formulation predicts that all particles migrate to the air-water interface (AWI) to lower the total potential surface energy —rationalizing the use of surfactant, which is a direct solution to lowering the surface tension of the aqueous solution.

Figure 1. Side-view schematics of a hole of cryoEM grid, highlighting particle behavior and distribution, in relation to AWI.

Figure 2. GroEL particle distribution in vitrified samples with different concentrations of FFC8.

Figure 3. Near-atomic cryoEM reconstructions of GroEL, demonstrating no structural damage introduced by the surfactants at the indicated concentrations—the proposed solution to the AWI problem.

Figure 4. Example of the AWI adsorption problem in cryoEM and application to real-world AWI adsorption problem.