Name: Fabricating Graphene-Coated Cryo-EM Grids Using Grid-Sized MMA-Coated CVD Graphene Squares
Uploaded: 2023-11-10T12:00:00
Description: Watch this Scientific Journal Video about Fabricating Graphene-Coated Cryo-EM Grids Using Grid-Sized MMA-Coated CVD Graphene Squares at JoVE.com

fabricating graphene-coated cryo-em grids using grid-sized mma-coated cvd graphene squares

Coating CryoEM Grids with Monolayer Graphene to Improve Cryo-Sample Preparation

Single particle cryogenic electron microscopy (cryoEM) has evolved into a widely used method for visualizing biological macromolecules1. Fueled by advances in direct electron detection2,3,4, data acquisition5, and image processing algorithms6,7,8,9,10, cryoEM is now capable of producing near-atomic resolution 3D structures of a fast-growing number of macromolecules11. Moreover, by leveraging the single-molecule nature of the approach, users can determine multiple structures from a single sample12,13,14,15, highlighting the promise of using the data generated to understand heterogeneous structural ensembles16,17. Despite this progress, bottlenecks in cryo-specimen grid preparation persist.

For structural characterization by cryoEM, biological samples should be well-dispersed in aqueous solution and then must be flash-frozen through a process called vitrification18,19. The goal is to capture particles in a uniformly thin layer of vitrified ice suspended across regularly spaced holes that are typically cut into a layer of amorphous carbon. This patterned amorphous carbon foil is supported by a TEM grid bearing a mesh of copper or gold support bars. In standard workflows, grids are rendered hydrophilic using a glow-discharge plasma treatment prior to the application of sample. Excess liquid is blotted with filter paper, allowing the protein solution to form a thin liquid film across the holes that can be readily vitrified during plunge-freezing. Common challenges include particle localization to the air-water interface (AWI) and subsequent denaturation20,21,22 or adoption of preferred orientations23,24,25, particle adherence to the carbon foil rather than migrating into the holes, and clustering and aggregation of the particles within the holes26. Nonuniform ice thickness is another concern; thick ice can result in higher levels of background noise in the micrographs due to increased electron scattering, whereas extremely thin ice can exclude larger particles27.
To address these challenges, a variety of thin support films have been used to coat grid surfaces, allowing particles to rest on these supports and, ideally, avoid interactions with the air-water interface. Graphene supports have shown great promise, in part due to their high mechanical strength coupled with their minimal scattering cross-section, which reduces the background signal added by the support layer28. In addition to its minimal contribution to background noise, graphene also exhibits remarkable electrical and thermal conductivity29. Graphene and graphene oxide coated grids have been shown to yield higher particle density, more uniform particle distribution30, and reduced localization to the AWI22. In addition, graphene provides a support surface that can be further modified to: 1) tune the physiochemical properties of the grid surface through functionalization31,32,33; or 2) couple linking agents that facilitate affinity purification of proteins of interest34,35,36.
In this article, we have modified an existing procedure for coating cryoEM grids with a single uniform layer of graphene30. The modifications aim to minimize grid handling throughout the protocol, with the goal of increasing yield and reproducibility. Additionally, we discuss our approach to evaluate the efficacy of various UV/ozone treatments in rendering grids hydrophilic prior to plunging. This step in cryoEM sample preparation using graphene-coated grids is critical, and we have found our straightforward method to quantify the relative hydrophilicity of the resulting grids to be useful. Using this protocol, we demonstrate the utility of employing graphene-coated grids for structure determination by generating a high-resolution 3D reconstruction of catalytically inactive S. pyogenes Cas9 in complex with guide RNA and target DNA.

Author Spotlight: Enhancing CryoEM Sample Preparation Using Graphene Monolayer on Microscopy Grids 

Application of Monolayer Graphene to Cryo-Electron Microscopy Grids for High-resolution Structure Determination

A common challenge in cryo-electron microscopy sample preparation is particle localization at the air-water interface, causing the particles to adopt preferred orientations and denature. Instead, it's better to have the particles rest on the grid surface, which thin film supports, like graphene promote. Another challenge is the requirement for relatively high sample concentrations, which could also be addressed by graphene.Graphene-coated grids provide numerous advantages for cryoEM structure determination, but have historically been difficult to fabricate in-house reproducibly, as well as cost prohibitive to purchase commercially. This protocol allows for the robust deposition of monolayer graphene, thereby lowering the barrier to users employing these grids in their research. Graphene is unique because of its material properties.It is a monolayer of carbon and therefore exhibits minimal background noise. Other cryoEM grid supports such as a thin layer of amorphous carbon or graphene oxide flakes cannot be applied as a single monolayer and contribute more background signal. Now that we have established a robust protocol for the application of graphene to cryoEM grids, we would like to pursue the functionalization of graphene-coated grids with affinity handles for on-grid capture directly from cell lysates.This could alleviate another bottleneck in cryoEM specimen preparation.

Watch this Scientific Journal Video about Fabricating Graphene-Coated Cryo-EM Grids Using Grid-Sized MMA-Coated CVD Graphene Squares at JoVE.com

Fabricating Graphene-Coated Cryo-EM Grids Using Grid-Sized MMA-Coated CVD Graphene Squares  

To attach the methyl methacrylate or MMA coated CVD graphene to the grids, use the previously prepared grid-sized coated graphene squares kept in a beaker containing molecular grade water. Using negative action tweezers. Gently dip a grid vertically into the water with the carbon side facing a floating MMA graphene square.Once in contact with the square, carefully remove the grid from the water while ensuring the square fully adheres to the carbon side of the grid. Place the grids on a clean cover slip with the MMA graphene side facing up and allow the grids to air-dry for one to two minutes. Using flat tip tweezers, transfer the cover slip with the grids to a hot plate set at 130 degrees Celsius.Incubate the grids after covering them with the top of a glass Petri dish for 20 minutes. Next, for washing MMA with acetone, transfer the entire cover slip into a Petri dish filled with 15 milliliters of acetone and incubate for 30 minutes. Then, using a clean glass serological pipette, transfer the acetone to a waste container.With another clean glass serological pipette, add 15 milliliters of fresh acetone to the Petri dish and incubate for 30 minutes. After three acetone washes, remove the acetone. And using a clean glass serological pipette, replace it with 15 milliliters of isopropanol.Then using tweezers, carefully remove the grids for isopropanol to air-dry them on a clean cover slip. Next place the cover slip with graphene-coated grids on a hot plate set to 100 degrees Celsius for 10 minutes to evaporate the residual organics. Using reverse action tweezers, gently place the grids into a clean illumination area of a UV ozone cleaner with the graphene-coated side facing up.Slide the illumination area closed and turn the machine on. Set the time dial to the designated treatment time to start UV ozone treating the grids. Finally, proceed to plunge a cryo-electron microscopy sample on the treated grids.

Research

JoVE Journal

Biochemistry

In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to ...