![]() ![]() We synthesized two diazonium salts (Supplementary Fig. 3).įull size image Preparation and characterization of functionalized grapheneīased on the paraffin-aided transfer method, we further developed a procedure to functionalize the freshly-grown graphene before transferring onto EM grids to prevent potential contamination’s effect on the functionalization reaction (Fig. The graphene surface was fairly flat with the roughness of less than 1 nm under AFM (Fig. 2), suitable for high-quality cryo-EM specimen preparation. ![]() ![]() The sharp diffraction spots under TEM indicating the presence of single-crystal graphene, with significantly reduced contaminations compared with PMMA-assistant graphene transfer method, where 87% holes displayed no or no-more-than-five contamination spots (Fig. In such a setup, we normally can prepare tens of graphene-coated EM grids at once (Supplementary Fig. The paraffin was then removed by petroleum ether from the graphene surface. In our setup, we first synthesized single-layer graphene on copper foil by CVD method 16 and then used paraffin to support the graphene during the fabrication procedure (Fig. Moreover, the graphene-adsorption energy of paraffin is reported to be much smaller than PMMA 15, theoretically making it easier to be cleaned from the graphene surface. Paraffin is a chemically inert alkane in solid state with relatively lower melting temperature (50–60 ☌) compared to other commonly used transfer mediator such as PMMA, which is normally dissolved and removed by organic solvents. We found that paraffin is an ideal transfer mediator. Paraffin-aided transfer method enabled clean graphene grid fabricationĪs the clean transfer of graphene remains one of the major practical barriers for the application of graphene-based supporting films in cryo-EM, we aimed to develop a robust procedure to transfer graphene on EM grids without contamination. Finally, we successfully determine the cryo-EM structure of Ll.LtrB RNP at 3.2-Å resolution, the highest resolution of the endogenously purified group II intron RNP, whose structural reconstruction is hindered by severe preferential orientation problem on conventional EM grids. Applied onto 20S proteasome and ribosome complex, the modified graphene with different charge properties enables particle adsorption on the surface with favorite orientation distribution, thus solving the AWI and preferred orientation problems in cryo-EM specimen. In this work, to overcome the graphene functionalization and transfer challenges and solve the preferential orientation problem, we develop a method to produce robust graphene grids functionalized with different electrostatic charges, which provide various electrostatic interaction interface for target macromolecules to bind in and enrich orientations. Although the graphene functionalization has been used to specifically capture and anchor target particles to avoid the AWI 6, 12, 13, 14, the current protocols of the bioactive functionalization process of graphene, based on the electrophilic reaction or conjugation interaction with the π-π bonds, are sub-optimal due to contamination built-up on the graphene surface during the transfer process or storage. The fabrication of clean graphene grids also remains a practical challenge for cryo-EM specimen preparation. Graphene membrane of sole nature, however, might induce preferential orientation problem of particles adsorbed to its surface 11. Graphene 10, an atomically thin film comprised of sp 2-bonded carbon atoms, has a superior electrical and thermal conductivity, mechanical strength and negligible background noise. Many efforts have been also made to solve the preferential orientation as well as AWI problem by introducing supporting films 4, 5, 6, 7, 8, 9, among which graphene membrane is the most promising one. To alleviate the preferential orientation problem, tilting the cryo-specimen during data collection has been applied 3, which, however, may result in large beam-induced motion and inaccurate defocus estimation during data processing, thereby impairing the high-resolution reconstruction. However, high-quality cryo-specimen preparation still faces many challenges, exemplified by the preferential orientation and air-water interface (AWI) problems 2, reducing the success rate and efficiency of high-resolution structure determination by cryo-EM. Cryo-electron microscopy (cryo-EM) technique has been successfully applied to reveal the molecular basis of many essential macromolecules at atomic resolution 1. ![]()
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