期刊
NATURE COMMUNICATIONS
卷 12, 期 1, 页码 -出版社
NATURE PORTFOLIO
DOI: 10.1038/s41467-021-27596-8
关键词
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资金
- Ministry of Science and Technology of China [2017YFA0504700]
- National Natural Science Foundation of China [31830020]
- Chinese Academy of Sciences [XDB37040102]
- National Natural Science Foundation of China for Distinguished Young Scholars [31925026]
- Beijing Municipal Science and Technology Commission [Z181100004218002]
- SinoSwiss Scientific and Technological Cooperation Project by the Ministry of Science and Technology of China [2015DFG32140]
The amphiphilic low-molecular-weight protein hydrophobin (HFBI) can self-assemble into a crystalline monolayer film, which is used as a support film in cryo-EM to protect specimens and overcome the preferred orientation problem. By adjusting the buffer pH, the distribution of particle orientations can be regulated. This support film has been successfully applied to determine high-resolution structures of various specimens in cryo-EM.
The amphiphilic low-molecular-weight protein hydrophobin (HFBI) can self-assemble into a crystalline monolayer film. Here, the authors present a cryo-EM support film using such 2D crystals of hydrophobin HFBI and show that it protects specimen from the air-water interface and can help to overcome the preferred orientation problem of particles in cryo-EM. Cryo-electron microscopy (cryo-EM) has become a powerful tool to resolve high-resolution structures of biomacromolecules in solution. However, air-water interface induced preferred orientations, dissociation or denaturation of biomacromolecules during cryo-vitrification remains a limiting factor for many specimens. To solve this bottleneck, we developed a cryo-EM support film using 2D crystals of hydrophobin HFBI. The hydrophilic side of the HFBI film adsorbs protein particles via electrostatic interactions and sequesters them from the air-water interface, allowing the formation of sufficiently thin ice for high-quality data collection. The particle orientation distribution can be regulated by adjusting the buffer pH. Using this support, we determined the cryo-EM structures of catalase (2.29 angstrom) and influenza haemagglutinin trimer (2.56 angstrom), which exhibited strong preferred orientations using a conventional cryo-vitrification protocol. We further show that the HFBI film is suitable to obtain high-resolution structures of small proteins, including aldolase (150 kDa, 3.28 angstrom) and haemoglobin (64 kDa, 3.6 angstrom). Our work suggests that HFBI films may have broad future applications in increasing the success rate and efficiency of cryo-EM.
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