Peng Hailin's research group and collaborators published an article in Nature Methodology to report the new ultra-flat graphene electron microscope carrier

On December 15, 2022, Professor Peng Hailin's research group from the School of Chemistry and Molecular Engineering of Peking University, Professor Wang Hongwei's group from the School of Life Sciences of Tsinghua University, and Professor Wei Xiaoding's group from the School of Engineering of Peking University jointly published the title "Ultra-flat graphene/uniform thin ice support film for high-resolution cryo-EM imaging" in Nature Methods The research paper "Uniform thin ice on ultraflat graphene forhigh-resolution cryo-EM" reports a novel ultra-flat graphene electron microscope carrier network, which solves the problem
of preparing uniform thin ice in high-resolution cryo-EM characterization.
This work shows that the ultra-flat graphene/uniform thin ice support film can significantly improve the imaging quality and efficiency of cryo-EM, and realize the high-resolution three-dimensional reconstruction
of a variety of small proteins (molecular weight less than 70 kDa).

Figure 1.
The flatness of the support film of suspended graphene cryo-EM affects the uniformity of ice thickness and imaging quality

Single-particle cryo-EM is one of the important means to reveal the fine structure and reaction mechanism of
biological macromolecules.
During cryo-EM imaging, biological macromolecules are encapsulated in a thin layer of glassy ice that, under electron beam irradiation, remains in its original structure and eigenic state
.
Experiments show that the preparation of uniform thin ice layer is one of the key factors to achieve high-resolution cryo-EM imaging, especially for proteins with small molecular weight (less than 100 kDa), because too thick ice will seriously affect the signal-to-noise ratio and cannot achieve high-resolution structural reconstruction
.
However, ice thickness and homogeneity during sample preparation are still difficult to precisely control and have become one of
the main challenges for high-resolution cryoimaging.

Figure 2.
Design, preparation and characterization of mechanical properties of ultra-flat graphene carriers

In response to this key problem, Peng Hailin's group, Wang Hongwei's group and Wei Xiaoding's group cooperated to reveal the relationship between the surface roughness of the cryo-EM sample support film and the uniformity of the ice layer, and developed an ultra-flat graphene electron microscope carrier network, which solved the problem
of preparing uniform thin ice.
Studies have shown that ultra-flat graphene support film can form thinner and more uniform glassy ice with low background contrast, which is conducive to high signal-to-noise ratio and high-resolution cryo-EM imaging
.
Based on the ultra-flat graphene/uniform thin ice support membrane, they performed structural elucidation of three small molecular weight streptavidin, 52 kDa, hemoglobin (64 kDa) and alpha α-fetoprotein (67 kDa), and obtained high-resolution reconstruction
of 2.
2?, 3.
5?, and 2.
6?, respectively.
Furthermore, they also demonstrated that the ultra-flat graphene carrier has good application prospects
in cryo-electron tomography (cryo-ET) and other fields.

Peng Hailin's research group and the research group of Academician Liu Zhongfan of the School of Chemistry and Molecular Engineering of Peking University have cooperated in the early stage to develop an epitaxial growth method and batch preparation technology
for ultra-flat graphene single crystals on copper (111)/sapphire wafers.
On this basis, Peng Hailin's research group successfully realized the batch preparation of ultra-flat suspended graphene electron microscope carrier network based on ultra-flat graphene single wafer and "face-to-face" ultra-clean glue-free transfer method, and the integrity of single-layer graphene suspension film was as high as 98%, and the average surface roughness was as low as 0.
7nm
.
The mechanical characterization results of atomic force microscopy (AFM) nanoindentation show that the ultra-flat graphene support film has a Young's modulus and breaking strength close to the theoretical value of graphene, which is much higher than that of rough undulating and wrinkled graphene
.
Further analysis showed that the ultra-flat graphene support film had a prestress of about 0.
2N/m, and prestress was a key factor in the preparation of
uniform thin ice.
During freeze preparation, after the sample solution is added dropwise, excess liquid on the support film needs to be sucked away with filter paper to prepare a thin liquid film
.
This process generates a large shear force (on the order of kPa), which causes the sample liquid film to undulate, which in turn causes the ice to undulate
.
The presence of prestress in ultra-flat graphene allows it to counteract the effects of shear forces and remain flat
even under shear forces of 100 kPa.

Figure 3.
Ultra-flat graphene significantly improved the imaging quality of cryo-EM and successfully resolved the high-resolution 3D reconstruction of streptavidin (52kDa).

The researchers used ultra-flat graphene carriers for cryo-EM imaging and found that the formation of uniform thin ice significantly improved imaging quality
.
On the one hand, the prestress makes the ice layer less prone to warpage under electron beam irradiation, which effectively inhibits the drift of the sample during characterization
.
On the other hand, at different tilt angles (-60°~60°), there is no fold structure on the surface of graphene, which avoids the mask of the sample signal by folds during high-angle imaging
.
After measurement, the thickness of the ice layer prepared by the ultra-flat graphene support film is about 20nm, and the thickness is relatively uniform, which can effectively encapsulate biological macromolecular particles without introducing excessive background noise
.
Most of the biomacromolecular particles are adsorbed on the graphene surface at almost the same height, reducing the difference
in local underfocus values under the same field of view during imaging.
Compared with rough and undulating graphene, ultra-flat graphene ensures the collection of high-quality data and improves imaging efficiency
.
Based on the above advantages, the authors used the novel ultra-flat graphene carrier to analyze the structure of three small proteins streptavidin (streptavidin, 52kDa), hemoglobin (hemoglobin, 64kDa) and α-fetoprotein (alpha-fetoprotein, 67kDa), and obtained high-resolution structural reconstruction
of 2.
2?, 3.
5?, and 2.
6?, respectively.

Hailin Peng, Hongwei Wang, Dr.
Nan Liu and Xiaoding Wei of the School of Life Sciences, Tsinghua University are co-corresponding authors of the paper, Liming Zheng and Nan Liu, Ph.
D.
graduates of the School of Chemistry and Molecular Engineering of Peking University, Xiaoyin Gao, doctoral students of the School of Chemistry and Molecular Engineering, Peking University, and Wenqing Zhu, a doctoral graduate of the School of Engineering, Peking University, are co-first authors
of the paper 。 This work was supported by the National Natural Science Foundation of China, the National Major Scientific Research Program, the Beijing National Research Center for Molecular Sciences, the Beijing Frontier Research Center for Biostructures, the Tsinghua-Peking University Joint Center for Life Sciences, the China Postdoctoral Science Foundation, and the Tencent Foundation, and was supported
by the instrument platform of the Laboratory of Molecular Materials and Nanoprocessing (MMNL) of the School of Chemistry and Molecular Engineering of Peking University.