The fast development of single-particle cryogenic electron microscopy (cryo-EM) has made it more feasible to obtain the 3D structure of well-behaved macromolecules with a molecular weight higher than 300 kDa at ~3 Å resolution. However, it remains a challenge to obtain the high-resolution structures of molecules smaller than 200 kDa using single-particle cryo-EM. In this work, we apply the Cs-corrector-VPP-coupled cryo-EM to study the 52 kDa streptavidin (SA) protein supported on a thin layer of graphene and embedded in vitreous ice. We are able to solve both the apo-SA and biotin-bound SA structures at near-atomic resolution using single-particle cryo-EM. We demonstrate that the method has the potential to determine the structures of molecules as small as 39 kDa.

The fast development of high-resolution electron microscopy (EM) demands a backgroundnoise-free substrate to support the specimens, where atomically thin graphene membranes can serve as an ideal candidate. Yet the preparation of robust and ultraclean graphene EM grids remains challenging. Here we present a polymer-and transfer-free direct-etching method for batch fabrication of robust ultraclean graphene grids through membrane tension modulation. Loading samples on such graphene grids enables the detection of single metal atoms and atomic-resolution imaging of the iron core of ferritin molecules at both room-and cryo-temperature. The same kind of hydrophilic graphene grid allows the formation of ultrathin vitrified ice layer embedded most protein particles at the graphene-water interface, which facilitates cryo-EM 3D reconstruction of archaea 20S proteasomes at a record high resolution of~2.36 Å. Our results demonstrate the significant improvements in image quality using the graphene grids and expand the scope of EM imaging.

The fast development of single particle cryo-EM has made it more feasible to obtain the 3D structure of well-behaved macromolecules with molecular weight higher than 300 kDa at ~3 Å resolution. It remains a challenge to obtain high resolution structure of molecules smaller than 100 kDa using single particle cryo-EM, mainly due to the low contrast of the molecules embedded in vitreous ice. In this work, we applied the Cs-corrector-VPP coupled cryo-EM to study 52 kDa streptavidin (SA) protein supported on a thin layer of graphene film and embedded in vitreous ice. We were able to solve both the apo-SA and biotin-bound SA at near-atomic resolution using single particle cryo-EM. We demonstrated that the method is capable to determine the structure of molecule as small as 39 kDa and potentially even smaller molecules. Furthermore, we found that using the graphene film to avoid the adsorption to the air-water interface is critical to maintain the protein's highresolution structural information.