Abstract
Graphene oxide (GO) sheets have been used successfully as a supporting substrate film in several recent cryogenic electron-microscopy (cryo-EM) studies of challenging biological macromolecules. However, difficulties in preparing GO-covered holey carbon EM grids have limited their widespread use. Here, we report a simple and robust method for covering holey carbon EM grids with GO sheets and demonstrate that these grids can be used for high-resolution single particle cryo-EM. GO substrates adhere macromolecules, allowing cryo-EM grid preparation with lower specimen concentrations and provide partial protection from the air-water interface. Additionally, the signal of the GO lattice beneath the frozen-hydrated specimen can be discerned in many motion-corrected micrographs, providing a high-resolution fiducial for evaluating beam-induced motion correction.
🔬 Techniques
🔭 Microscopes
🧬 Organisms
💻 Software
🧪 Sample Preparation
🏭 Microscope Brands
🧪 Reagent Suppliers
📷 Detectors
💻 Software Details
💾 Data Repositories
🏛️ Research Organizations (ROR)
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📊 Figures
Figure 1.
EM grids can be evenly coated with GO sheets using surface assembly
A . Schematic of the apparatus used for surface assembly of thin GO films and subsequent deposition onto holey carbon EM grids. B . Low magnification image of a holey carbon (Quantifoil) grid covered ...
Figure 2.
High-resolution single-particle cryo-EM on GO grids
A . Micrograph of 20S proteasome particles over a GO support film. The GO film adds minimal background contrast. The specimen was applied at a concentration 0.05mg/mL, approximately ten times less tha...
Figure 3.
Evaluation of motion correction using GO peak intensities
Power spectra from the image in Figure 2A before ( A. ) and after ( B. ) motion correction. Power spectra were calculated by averaging the periodograms of overlapping 512u00d7512 pixel windows using a...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
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