Theoretical framework and experimental solution for the air-water interface adsorption problem in cryoEM
image: A A representative micrograph of single-particle cryoEM of purified human ClC-1 protein after applying FFC8 (at CMC, 3 mmol/L). As can be compared with Fig. 1, adding surfactant effectively solved the protein aggregation and deformation problem. B Two orthogonal shaded surface views of the resultant cryoEM map at an overall resolution of 3.6 Å, enabling de novo atomic modeling of the membrane domain (blue) of the complex (Wang et al. 2019). The intracellular domain is shown in yellow, and the detergent belt is in semi-transparent gray
Credit: Joon S. Kang, Xueting Zhou, Yun-Tao Liu, Kaituo Wang, Z. Hong Zhou
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. However, the reasons for such misbehavior are not well understood, which limits systematic approaches to solving the problem. Here, the authors have developed a theoretical formulation that explains these observations. Their 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 reduce the surface tension of the aqueous solution. By performing cryogenic electron tomography (cryoET) on the widely-tested sample, GroEL, they demonstrate that, in a standard buffer solution, nearly all particles migrate to the AWI. Gradually reducing the surface tension by introducing surfactants decreased the percentage of particles exposed to the surface. By conducting single-particle cryoEM, they confirm that suitable surfactants do not damage the biological complex, thus suggesting that they might provide a practical, simple, and general solution to the problem for high-resolution cryoEM. Applying this solution to a real-world AWI adsorption problem involving a more challenging membrane protein, namely, the ClC-1 channel, has resulted in its first near-atomic structure determination using cryoEM.
The work entitled “Theoretical framework and experimental solution for the air-water interface adsorption problem in cryoEM”was published on Biophysics Reports (published in Aug. 2023).