General and robust covalently linked graphene oxide affinity grids for high-resolution cryo-EM.

GO Synthesis. GO was synthesized via the modified route developed by Marcano et al. ( 20 ). Into the mixture of concentrated sulfuric acid (120 mL; Ward’s Science, 470302-872) and phosphoric acid (13 mL; Sigma-Aldrich, 345245), graphite flakes (1 g; Sigma-Aldrich, 332461) were added. Potassium permanganate (6 g; Sigma-Aldrich, 223468) was slowly added, after which the mixture was placed in a water bath at 45 °C and stirred overnight. The reaction mixture was then moved into an ice bath and deionized (DI) water (100 mL) was poured in, followed by the addition of 30% H 2 O 2 (1.5 mL; Sigma-Aldrich, 216763). The mixture was allowed to sit for 2 h and was then centrifuged at 5,000 rpm for 20 min. The solid material at the bottom was retrieved and washed extensively with DI water by centrifugation until the pH reached 5. Finally, the remaining viscous material was collected and stirred overnight to make a GO stock solution in water. After drying at 80 °C, XPS measurements were performed on GO flakes with a PerkinElmer PHI 5600 X-ray photoelectron spectrometer. GO Deposition onto EM Grids. To coat GO sheets onto EM grids, we revised the Langmuir–Blodgett assembly method as described by Cote et al. ( 22 ) and also reported in our previous work ( 21 ). The GO water stock solution was diluted with methanol/water (5:1, v:v) to a concentration of 0.1 mg/mL. Mild stirring for 30 min rather than sonication was used to avoid destruction of GO sheets, producing a GO working solution. An epoxy-coated stainless steel mesh (McMaster–Carr) stand was placed at the bottom of a glass Petri dish (60 mm diameter, 15 mm tall), and DI water was filled to the top. EM grids (Au Quantifoil, 300 mesh) were used as received and placed on the mesh with the carbon side facing up. Then a total volume of 230 µL of GO working solution was spread dropwise onto the water surface at different spots at a speed of 50 µL/min using a syringe. Once the water was drained, the GO-coated grids were dried at room temperature overnight for use. Coverage of GO was examined on an FEI Tecnai 20 transmission electron microscope (Thermo Fisher Scientific) with an acceleration voltage of 200 kV. Surface Modification of GO-Coated EM Grids. In a 1.5-mL centrifuge microtube, one GO-coated EM grid (GO grids) was submerged in 20 µL of dibenzocyclooctyne-PEG-amine (DBCO-PEG4-amine; Click Chemistry Tools, A103P) solution in dimethyl sulfoxide (DMSO) at a concentration of 10 mM and shaken at room temperature overnight. Following that, the DBCO functionalized grid was washed three times with DMSO and DI water respectively, and then submerged in 20 μL of azide-PEG-maleimide (molecular weight 600 Da; Nanocs, PG2-AZML-600 or molecular weight 5,000 Da; Nanocs, PG2-AZML-5k) solution in DI water at a concentration of 4 mM. Alternatively, hydroxyl-capped PEG was introduced at this step to reduce nonspecific binding. Azide-PEG-maleimide (molecular weight 5,000 Da; Nanocs, PG2-AZML-5k) was mixed with azide-PEG-hydroxyl (molecular weight 5,000 Da; Nanocs, PG2-AZOH-5k) in DI water at a ratio of 1:9 to form a solution with a total PEG concentration of 4 mM. The reaction was shaken at room for 6 h, and the grid was washed three times with DI water and ethanol, respectively. The as-made maleimide grid or maleimide/hydroxyl grid was then dried in ambient air for 30 min and stored at −20 °C for future use. Details of protein purification, assembly and vitrification using affinity grids, cryo-EM data acquisition, image processing, and tomography analysis are provided in SI Appendix, Materials and Methods . The washing process during grid freezing is illustrated in Movie S2 .