▎ 摘　　要

Cryo-electron microscopy (cryo-EM) visualizes the atomic structure of macromolecules that are embedded in vitrified thin ice at their close-to-native state. However, the homogeneity of ice thickness, a key factor to ensure high image quality, is poorly controlled during specimen preparation and has become one of the main challenges for high-resolution cryo-EM. Here we found that the uniformity of thin ice relies on the surface flatness of the supporting film, and developed a method to use ultraflat graphene (UFG) as the support for cryo-EM specimen preparation to achieve better control of vitreous ice thickness. We show that the uniform thin ice on UFG improves the image quality of vitrified specimens. Using such a method we successfully determined the three-dimensional structures of hemoglobin (64 kDa), alpha-fetoprotein (67 kDa) with no symmetry, and streptavidin (52 kDa) at a resolution of 3.5 angstrom, 2.6 angstrom and 2.2 angstrom, respectively. Furthermore, our results demonstrate the potential of UFG for the fields of cryo-electron tomography and structure-based drug discovery.