Wang Hongwei and his collaborators jointly developed a new functional graphene for improving the advantage orientation of cryo-EM

Single-particle cryo-EM three-dimensional reconstruction technology is one of
the mainstream methods for resolving the high-resolution structure of biological macromolecules.
However, high-quality cryo-EM sample preparation still faces many challenges, such as gas-liquid interface, dominant orientation, and background noise, which greatly limit the efficiency of
structure elucidation.
In response to these problems, Wang Hongwei's research group, Rao Liao's research group and Peking University Peng Hailin's group jointly developed a new functional graphene electron microscope carrier network, which can help solve the problems of
sample particle advantage orientation and gas-liquid interface.

In this study, they synthesized a variety of diazo salt molecules with groups with different charge properties (such as amino and sulfonate), and used these diazo salt molecules to functionalize the CVD-grown graphene membrane to obtain graphene support films
with different charge properties.
Using paraffin wax as the transfer medium, they cleanly transferred the graphene support film to the electron microscope carrier for cryo-EM sample preparation
.

After cryo-electron tomography reconstruction and characterization, this functional graphene support film ensures the effective adsorption of target biological macromolecules and avoids the potential risks
caused by the gas-liquid interface.
In addition, because the graphene surface modified groups have different charge properties, they provide different interaction methods with the target biological macromolecules and achieve the purpose of
rich orientation distribution.
Single-particle cryo-EM data analysis showed that graphene with negative electrical modification tended to bind to the positive region on the surface of biological macromolecule particles, while graphene with positive electric modification combined with negative electric region of biological macromolecular particles to achieve the orientation distribution
of regulating biological macromolecules.
LtrB RNP, a type II intron of Lactococcus lactis, has serious dominant orientation problems on conventional support membranes, making it difficult to obtain high-resolution reconstruction
.
In this study, this functional graphene support film with different electrical modifications can regulate the orientation distribution of LtrB RNP, successfully solve the problem of dominant orientation, and finally obtain a three-dimensional reconstruction result
with a resolution of 3.
2 ?.
Moreover, in the process of three-dimensional reconstruction, compared with the ordinary graphene support film without modification, the effective utilization rate of particles of LtrB RNP on the functional graphene film is also significantly increased
.
These data show that this functional graphene support film provides a more friendly interface and helps to protect the three-dimensional structure
of biological macromolecules.

Fig.
20S proteasome and ribosome graphene with different electrical modifications (NFG: positive electric modification; SFG: Negative Electrical Modification).

The research work, published online in Nature Communications on November 7, 2022, is titled "Functionalized graphene grids with various charges for single-particle cryo-EM
.
" 。 Professor Hongwei Wang, School of Life Sciences/Center for Advanced Innovation in Structural Biology, Nan Liu, Postdoctoral Fellow, School of Life Sciences, Professor Liao Rao, School of Pharmacy, Tsinghua University, and Professor Hailin Peng, School of Chemistry and Molecular Engineering, Peking University, are co-corresponding authors, and Ye Lu, postdoctoral fellow Liu Nan, 2019 doctoral student of School of Life Sciences, Tsinghua University, Yongbo Liu, 2019 doctoral student of School of Pharmacy, and Liming Zheng, a doctoral graduate of School of Chemistry and Molecular Engineering, Peking University, are co-first authors
of this paper.
Dr.
Wang Jia, Yang Junhao, Jia Xia, Zi Qinru and other members of the laboratory provided important assistance
to this work.
This work is supported
by the National Natural Science Foundation of China, the National Key Research and Development Program of China, the Beijing Frontier Research Center for Biostructures, the Tsinghua-Peking University Joint Center for Life Sciences, the Xplorer Award, and the China Postdoctoral Science Foundation.

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