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Biophysics Reports 2021, 7(3): 227-238 https://doi.org/10.52601/bpr.2021.210007
Method | Open Access | Issue | Published: 07 July 2021

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

Show Author's Information Nan Liu1,2,  Liming Zheng3,  Jie Xu1,2,  Jia Wang1,  Cuixia Hu1,  Jun Lan1,  Xing Zhang1,  Jincan Zhang3,  Kui Xu1,2,  Hang Cheng1,4,  Zi Yang1,  Xin Gao3,  Xinquan Wang1,2,4,7,  Hailin Peng3,5( ),  Yanan Chen1,6( ),  Hong-Wei Wang1,2,4,7( )
Ministry of Education Key Laboratory of Protein Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China
Tsinghua-Peking Joint Center for Life Sciences, Tsinghua University, Beijing 100084, China
Center for Nanochemistry, Beijing Science and Engineering Center for Nanocarbons, Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China
Joint Graduate Program of Peking-Tsinghua-NIBS, School of Life Sciences, Tsinghua University, Beijing 100084, China
Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China
School of Materials Science and Engineering, Tianjin University, Tianjin 300072, China
Beijing Frontier Research Center for Biological Structures, Tsinghua University, Beijing 100084, China
Keywords:
3D reconstruction, Reduced graphene oxide, Cryo-EM, Air–water interface
Cite this article:
Liu N, Zheng L, Xu J, et al. Reduced graphene oxide membrane as supporting film for high-resolution cryo-EM. Biophysics Reports, 2021, 7(3): 227-238. https://doi.org/10.52601/bpr.2021.210007
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Abstract Full text Electronic supplementary material About this article

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.


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Abstract
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Electronic supplementary material
About this article

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

Show Author's information Nan Liu1,2, Liming Zheng3, Jie Xu1,2, Jia Wang1, Cuixia Hu1, Jun Lan1, Xing Zhang1, Jincan Zhang3, Kui Xu1,2, Hang Cheng1,4, Zi Yang1, Xin Gao3, Xinquan Wang1,2,4,7, Hailin Peng3,5( ), Yanan Chen1,6( ), Hong-Wei Wang1,2,4,7( )
Ministry of Education Key Laboratory of Protein Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China
Tsinghua-Peking Joint Center for Life Sciences, Tsinghua University, Beijing 100084, China
Center for Nanochemistry, Beijing Science and Engineering Center for Nanocarbons, Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China
Joint Graduate Program of Peking-Tsinghua-NIBS, School of Life Sciences, Tsinghua University, Beijing 100084, China
Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China
School of Materials Science and Engineering, Tianjin University, Tianjin 300072, China
Beijing Frontier Research Center for Biological Structures, Tsinghua University, Beijing 100084, China

Abstract

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.

Keywords: 3D reconstruction, Reduced graphene oxide, Cryo-EM, Air–water interface

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Publication history

Received: 17 March 2021
Accepted: 13 April 2021
Published: 07 July 2021
Issue date: June 2021

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© The Author(s) 2021

Acknowledgements

Acknowledgements

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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