Volume 7 Issue 3
Jun.  2021
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Article Contents
Liu Nan, Zheng Liming, Xu Jie, Wang Jia, Hu Cuixia, Lan Jun, Zhang Xing, Zhang Jincan, Xu Kui, Cheng Hang, Yang Zi, Gao Xin, Wang Xinquan, Peng Hailin, Chen Yanan, Wang Hong-Wei. Reduced graphene oxide membrane as supporting film for high-resolution cryo-EM[J]. Biophysics Reports, 2021, 7(3): 227-238. doi: 10.52601/bpr.2021.210007
Citation: Liu Nan, Zheng Liming, Xu Jie, Wang Jia, Hu Cuixia, Lan Jun, Zhang Xing, Zhang Jincan, Xu Kui, Cheng Hang, Yang Zi, Gao Xin, Wang Xinquan, Peng Hailin, Chen Yanan, Wang Hong-Wei. Reduced graphene oxide membrane as supporting film for high-resolution cryo-EM[J]. Biophysics Reports, 2021, 7(3): 227-238. doi: 10.52601/bpr.2021.210007

Reduced graphene oxide membrane as supporting film for high-resolution cryo-EM

doi: 10.52601/bpr.2021.210007
Funds:  We thank Dr. Ning Gao, Dr. Xueming Li for kindly providing ribosome and 20S proteasome samples. We are grateful to Dr. Jianlin Lei, Dr. Lingpeng Cheng, Dr. Tao Yang, Dr. Xiaomin Li, Dr. Fan Yang, Danyang Li, Xiaofeng Hu, Jie Wen, Yakun Wang, and Anbao Jia at the Cryo-EM and High-Performance Computation platforms of Tsinghua University Branch of the National Protein Science Facility, for the technical support in cryo-EM data collection and analysis. This work is financially supported by the Ministry of Science and Technology of China (2016YFA0501100) and National Natural Science Foundation of China (31825009) to H-W Wang, and the National Natural Science Foundation of China (21525310) and the National Basic Research Program of China (2014CB932500 and 2016YFA0200101) to H Peng.
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  • Although single-particle cryogenic electron microscopy (cryo-EM) has been applied extensively for elucidating many crucial biological mechanisms at the molecular level, this technique still faces critical challenges, the major one of which is to prepare the high-quality cryo-EM specimen. Aiming to achieve a more reproducible and efficient cryo-EM specimen preparation, novel supporting films including graphene-based two-dimensional materials have been explored in recent years. Here we report a robust and simple method to fabricate EM grids coated with single- or few-layer reduced graphene oxide (RGO) membrane in large batch for high-resolution cryo-EM structural determination. The RGO membrane has decreased interlayer space and enhanced electrical conductivity in comparison to regular graphene oxide (GO) membrane. Moreover, we found that the RGO supporting film exhibited nice particle-absorption ability, thus avoiding the air–water interface problem. More importantly, we found that the RGO supporting film is particularly useful in cryo-EM reconstruction of sub-100-kDa biomolecules at near-atomic resolution, as exemplified by the study of RBD-ACE2 complex and other small protein molecules. We envision that the RGO membranes can be used as a robust graphene-based supporting film in cryo-EM specimen preparation.
  • † Nan Liu, Liming Zheng, Jie Xu and Jia Wang contributed equally to this work
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