A cryo-electron microscopy support film formed by 2D crystals of hydrophobin HFBI

Hongcheng Fan, Bo Wang, Yan Zhang, Yun Zhu, Bo Song, Haijin Xu, Yujia Zhai, Mingqiang Qiao () and Fei Sun ()
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Hongcheng Fan: Chinese Academy of Sciences
Bo Wang: Nankai University
Yan Zhang: Chinese Academy of Sciences
Yun Zhu: Chinese Academy of Sciences
Bo Song: Nankai University
Haijin Xu: Nankai University
Yujia Zhai: Chinese Academy of Sciences
Mingqiang Qiao: Nankai University
Fei Sun: Chinese Academy of Sciences

Nature Communications, 2021, vol. 12, issue 1, 1-13

Abstract: Abstract 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 Å) and influenza haemagglutinin trimer (2.56 Å), 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 Å) and haemoglobin (64 kDa, 3.6 Å). Our work suggests that HFBI films may have broad future applications in increasing the success rate and efficiency of cryo-EM.

Date: 2021
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DOI: 10.1038/s41467-021-27596-8

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