Effect of charge on protein preferred orientation at the air-water interface in cryo-electron microscopy

The air-water interface (AWI) tends to absorb proteins and frequently causes preferred orientation problems in cryo-electron microscopy (cryo-EM). Here, we examined cryo-EM data from protein samples frozen with different detergents and found that both anionic and cationic detergents promoted binding of proteins to the AWI. By contrast, nonionic and zwitterionic detergents tended to prevent proteins from attaching to the AWI. This ability was positively associated with the critical micelle concentration of the detergent. The protein orientation distributions with different anionic detergents were similar and resembled that obtained without detergent. By contrast, cationic detergents gave distinct orientation distributions. The AWI is negatively charged and the absorption of cationic detergents to the AWI alters its charge. Our results indicates that proteins absorb to charged interface and the negative charge of the AWI plays an important role in absorbing proteins in the conventional cryo-EM sample preparation. According to these findings, a new method was developed to modify the charge distribution of the AWI by adding a very low concentration of anionic detergent. Using this method, the protein particles exhibited a more evenly distributed orientations and still absorbed to the AWI enabling them embedding in a thin layer of ice, which will benefit the cryo-EM structural determination.