Cryo-EM is an important tool for determining the structures of electron-beam-sensitive biomolecules by embedding them in vitreous ice. Nowadays, the preparation of uniform thin ice has become increasingly critical to achieving high-resolution structural reconstruction, especially for small biomolecules with molecular weights less than 100 kDa. These have a relatively weak contrast during cryo-EM imaging and require uniform thin ice to minimize the background noise.
To obtain better control of vitreous ice thickness during cryo-EM specimen preparation, the teams led by Hailin Peng at the College of Chemistry and Molecular Engineering, Peking University, in collaboration with Hong-Wei Wang at the School of Life Sciences, Tsinghua University, and Xiaoding Wei at the College of Engineering, Peking University, developed ultraflat graphene (UFG) as the supporting film. They demonstrated that UFG could produce uniform thin ice of vitrified specimens, promoting image quality and data-processing efficiency. Using this method, the three-dimensional (3D) structures of hemoglobin (64 kDa), alpha-fetoprotein (67 kDa), and streptavidin (52 kDa) were determined at resolutions of 3.5 Å, 2.6 Å and 2.2 Å, respectively. This work was published online by Nature Methods on December 15, 2022 (https://www.nature.com/articles/s41592-022-01693-y), titled "Uniform thin ice on ultraflat graphene for high-resolution cryo-EM."
Hailin Peng and collaborators, reported for the first time that the uniformity of thin ice of vitrified specimens relies on the surface flatness of the supporting film. Based on the epitaxial growth of UFG on a Cu(111)/sapphire wafer developed by Hailin Peng and Zhongfan Liu's group, they achieved batch preparation of UFG EM grids using a face-to-face clean transfer method. The suspended UFG membrane featured high statistical intactness of ~98%, and its average surface roughness was reduced to 0.7 nm. In addition, the UFG membrane exhibited excellent mechanical properties, with its mechanical strength and Young's modulus comparable to those of mechanically exfoliated graphene. In the preparation procedure, the shear force generated by blotting the solution severely deformed the supporting film, resulting in variations in the ice thickness. However, the resistance to the deformation of UFG derives from a pre-tension of ~0.2 N/m, which is crucial to achieving uniform thin ice during cryo-EM specimen preparation. Therefore, pre-tensioned UFG is expected to be robust to the shear force and remain atomically flat throughout the blotting process.
When applied to cryo-EM imaging, UFG enabled a uniform and flat ice layer, with no wrinkles overlapping the target particle signals in tilted images (−60° and 60°). The ice thickness was approximately 20 nm, slightly larger than the biomolecular size. The particles were mainly distributed at the same defocus plane, that is, on the UFG surface. Moreover, the beam-induced particle motion was significantly reduced because of the pre-tension in UFG. These results collectively demonstrate that UFG improves the image quality and data-processing efficiency of vitrified specimens. Based on these advantages, the 3D structures of hemoglobin (64 kDa), alpha-fetoprotein (67 kDa) with no symmetry, and streptavidin (52 kDa) were determined at resolutions of 3.5 Å, 2.6 Å and 2.2 Å, respectively.
Fig. 1 Illustration of the correlation between graphene roughness andthe uniformity of ice thickness.
Fig. 2 Scalable preparation and characterization of suspended UFG membranes.
Fig. 3 Cryo-EM characterization of the UFG and rough graphene.
Liming Zheng, a Ph.D. alumnus at Peking University; Nan Liu, a postdoctoral researcher at Tsinghua University; Xiaoyin Gao, a Ph.D. candidate at Peking University; and Wenqing Zhu, a Ph.D. alumnus at Peking University, are the joint first authors of this paper. Prof. Hailin Peng from Peking University, Prof. Hong-Wei Wang, Dr. Nan Liu from Tsinghua University, and Prof. Xiaoding Wei from Peking University are the corresponding authors. This research was jointly supported by the National Natural Science Foundation of China, Beijing National Laboratory for Molecular Sciences, Ministry of Science and Technology of China, Beijing Municipal Science & Technology Commission, China Postdoctoral Science Foundation, and the XPLORER PRIZE.
Original link for the paper: https://www.nature.com/articles/s41592-022-01693-y
