A novel cryo-electron microscopy support film based on 2D crystal of HFBI protein
A novel cryo-electron microscopy support film based on 2D crystal of HFBI protein
Abstract Cryo-electron microscopy (cryo-EM) has become the most powerful tool to resolve the high-resolution structures of biomacromolecules in solution. However, the air-water interface induced preferred orientation, dissociation or denaturation of biomacromolecules during cryo-vitrification is still a major limitation factor for many specimens. To solve this bottleneck, we developed a new type of cryo-EM support film using the 2D crystal of hydrophobin I (HFBI) protein. The HFBI-film utilizes its hydrophilic side to adsorb protein particles via electrostatic interactions and keep air-water interface away, allowing thin-enough ice and high-quality data collection. The particle orientation distribution can be optimized by changing the buffer pH. We, for the first time, solved the cryo-EM structure of catalase (2.28-?) that exhibited strong preferred orientation using conventional cryo-vitrification protocol. We further proved the HFBI-film is suitable to solve the high-resolution structures of small proteins including aldolase (150 kDa, 3.34-?) and hemoglobin (64 kDa, 3.6-?). Our work implied that the HFBI-film will have a wide application in the future to increase the successful rate and efficiency of cryo-EM.
Song Bo、Sun Fei、Wang Bo、Zhang Yan、Zhai Yujia、Zhu Yun、Qiao Mingqiang、Xu Haijin、Fan Hongcheng
The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, College of Life Sciences, Nankai UniversityNational Key Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences||The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, College of Life Sciences, Nankai University||University of Chinese Academy of Sciences||Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences||Physical Science Laboratory, Huairou National Comprehensive Science Center||Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory)The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, College of Life Sciences, Nankai UniversityNational Key Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences||University of Chinese Academy of SciencesNational Key Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences||University of Chinese Academy of SciencesNational Key Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences||University of Chinese Academy of SciencesThe Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, College of Life Sciences, Nankai University||School of Life Science, Shanxi universityThe Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, College of Life Sciences, Nankai UniversityNational Key Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences||University of Chinese Academy of Sciences
生物科学现状、生物科学发展生物科学研究方法、生物科学研究技术生物物理学
Song Bo,Sun Fei,Wang Bo,Zhang Yan,Zhai Yujia,Zhu Yun,Qiao Mingqiang,Xu Haijin,Fan Hongcheng.A novel cryo-electron microscopy support film based on 2D crystal of HFBI protein[EB/OL].(2025-03-28)[2025-05-24].https://www.biorxiv.org/content/10.1101/2021.11.09.467987.点此复制
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