The research groups of Peng Hailin, Wang Hongwei and Wei Xiaoding collaborated to develop ultra-flat graphene for high-resolution cryo-EM imaging

Single particle cryo-EM is one of the important means to reveal the fine structure and reaction mechanism of
biological macromolecules.
The preparation of a uniform thin ice layer can significantly improve the cryo-EM imaging quality during sample preparation, especially for biological macromolecules with small molecular weights (less than 100 kDa), because the thick ice layer will seriously affect the signal-to-noise ratio and cannot achieve high-resolution structural reconstruction
.
However, the thickness and uniformity of sample ice are still difficult to accurately control, which is one of
the main challenges of high-resolution cryo-EM imaging.

The research group of Peng Hailin of Peking University, the research group of Wang Hongwei of Tsinghua University and the group of Wei Xiaoding of Peking University revealed the relationship between the surface roughness of the cryo-EM sample support film and the homogeneity of the ice layer, and developed an ultra-flat graphene electron microscope carrier network, which solved the problem
of preparing uniform thin ice.
Experiments show that ultra-flat graphene support film is helpful for the preparation of thinner and more uniform glassy ice with low background contrast, which is conducive to cryo-EM imaging
with high signal-to-noise ratio and high resolution.
Using ultra-flat graphene support membranes, they performed structural elucidation on three smaller molecular weight proteins, streptavidinin protein (52 kDa), hemoglobin (64 kDa), and alpha-fetoprotein (67 kDa), resulting in high-resolution reconstruction
of 2.
2 ?, 3.
5 ?, and 2.
6 ?, respectively.
They also proved that the ultra-flat graphene carrier has good application prospects
in cryo-electron tomography reconstruction (cryo-ET) and other fields.

In this work, the research team 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, with the integrity of single-layer graphene suspension film as high as 98% and the average surface roughness as low as 0.
7 nm
.
The mechanical characterization results of atomic force microscopy nanoindentation technology 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 the rough undulating graphene support film
.

They used ultra-flat graphene carriers for cryo-EM imaging and found that uniform thin ice significantly improved image quality
.
On the one hand, the prestress of ultra-flat suspended graphene makes the ice layer less prone to warpage under electron beam irradiation, which effectively inhibits the drift of sample particles during imaging
.
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 ultra-flat graphene support film is about 20 nm, which is relatively uniform, which can effectively encapsulate biological macromolecular particles without introducing excessive background noise
.
Moreover, 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, the ultra-flat graphene support film ensures the collection of high-quality data and improves imaging efficiency
.

Fig.
1 The flatness of the suspended graphene support film affects the uniformity of ice thickness and imaging quality

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

Fig.
3 Ultra-flat graphene significantly improved the imaging quality of cryo-EM and successfully resolved the high-resolution three-dimensional structure of small molecular weight proteins

The research work, titled "Uniform thin ice on ultraflat graphene for high-resolution cryo-EM" was published in Nature Methods on December 15
, 2022 。 Professor Peng Hailin from the School of Chemistry and Molecular Engineering of Peking University, Professor Wang Hongwei and Dr.
Liu Nan from the School of Life Sciences of Tsinghua University, and Professor Wei Xiaoding from the School of Engineering of Peking University are co-corresponding authors of the paper, Liming Zheng, a PhD graduate of the School of Chemistry and Molecular Engineering of Peking University, Dr.
Nan Liu of the School of Life Sciences, Tsinghua University, Xiaoyin Gao, a 2020 doctoral student of the School of Chemistry and Molecular Engineering, Peking University, and Wenqing Zhu, a doctoral graduate of the School of Engineering of Peking University, are co-first authors
of the paper 。 This work has been supported
by the National Natural Science Foundation of China, the National Major Scientific Research Program, Beijing National Research Center for Molecular Sciences, Beijing Frontier Research Center for Biostructure, Tsinghua-Peking University Joint Center for Life Sciences, China Postdoctoral Science Foundation, Tencent Foundation, etc.

Link to the paper: