{"doi":"10.1101/2023.07.28.550908","title":"Application of monolayer graphene to cryo-electron microscopy grids for high-resolution structure determination","abstract":"ABSTRACT In cryogenic electron microscopy (cryo-EM), purified macromolecules are typically applied to a grid bearing a holey carbon foil, blotted to remove excess liquid and rapidly frozen in a roughly 20-100 nm thick layer of vitreous ice that is suspended across roughly 1 μm-wide foil holes. The resulting sample is then imaged using cryogenic transmission electron microscopy and, after substantial image processing, near-atomic resolution structures can be determined. Despite cryo-EM’s widespread adoption, sample preparation remains a severe bottleneck in cryo-EM workflows, with users often encountering challenges related to samples behaving poorly in the suspended vitreous ice. Recently, methods have been developed to modify cryo-EM grids with a single continuous layer of graphene, which acts as a support surface that often increases particle density in the imaged area and can reduce interactions between particles and the air-water interface. Here, we provide detailed protocols for the application of graphene to cryo-EM grids, and for rapidly assessing the relative hydrophilicity of the resulting grids. Additionally, we describe an EM-based method to confirm the presence of graphene by visualizing its characteristic diffraction pattern. Finally, we demonstrate the utility of these graphene supports by rapidly reconstructing a 2.7 Å resolution density map of an exemplar Cas9 complex using a highly pure sample at a relatively low concentration. SUMMARY The application of support layers, such as graphene, to cryo-electron microscopy grids can increase the density of particles imaged, limit particle interactions with the air-water interface, reduce the extent of beam-induced motion, and, in some instances, improve the distribution of particle orientations. This paper describes a robust protocol for coating cryo-EM grids with a monolayer of graphene for improved cryo-sample preparation.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":392515,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":5,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9535,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":395411,"name":"Mira B. May","orcid":"0000-0002-4545-4001","position":1,"is_corresponding":false},{"id":555652,"name":"Joseph H. Davis","orcid":"0000-0002-8858-8907","position":2,"is_corresponding":false},{"id":510134,"name":"Andrew V. Grassetti","orcid":"0000-0002-8874-5992","position":0,"is_corresponding":true}],"reference_count":47,"raw_metadata":null,"created_at":"2026-07-19T01:18:56.730121Z","pmid":"37546934","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}