RT Journal Article
SR Electronic
T1 Effect of charge on protein preferred orientation at the air–water interface in cryo-electron microscopy
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.05.14.444152
DO 10.1101/2021.05.14.444152
A1 Bufan Li
A1 Dongjie Zhu
A1 Huigang Shi
A1 Xinzheng Zhang
YR 2021
UL http://biorxiv.org/content/early/2021/05/16/2021.05.14.444152.abstract
AB 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.Competing Interest StatementThe authors have declared no competing interest.