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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.