preparing grid-sized mma-coated cvd graphene squares for attaching to cryoem grids

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 Preparing Grid-Sized MMA-Coated CVD Graphene Squares for Attaching to CryoEM Grids at JoVE.com

Preparing Grid-Sized MMA-Coated CVD Graphene Squares for Attaching to CryoEM Grids  

Begin by cutting the methylmethacrylate or MMA coated graphene squares into grid sized pieces. To do so, use two sets of reverse action forceps to support the CVD graphene square, while ensuring the MMA side faces up while attaching it to the forceps. To cut the graphene into small squares, use the two sets of reverse action forceps to hold the graphene right side up, anchoring it in position, as well as to hold the edge of the small square after cutting it away from the rest of the big square.Tilt the beaker containing ammonium per sulfate, or APS, to place the squares in it at a shallow angle without sinking. Then pick a square and make it contact the surface of the APS solution before releasing it. After covering the beaker with aluminum foil, incubate it overnight at 25 degrees Celsius.Next, to remove the MMA graphene films from the APS solution, first, plunge a glass cover slip vertically into the solution and then move it laterally such that it touches a floating square. Then carefully remove the cover slip while ensuring that the film adheres completely to the side of the cover slip upon removal. Plunge the cover slip vertically into molecular grade water in a 50 milliliter beaker to dislodge the attached film into the water.In a similar manner, transfer all the films to the beaker. Let the films remain in the beaker for 20 minutes before they are ready to be adhered to cryo EM grids.

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Research

JoVE Journal

Biochemistry

70 次观看.  Massachusetts Institute of Technology. 首先将甲基丙烯酸甲酯或MMA涂层石墨烯方块切割成网格大小的块。为此，使用两组反向作用镊子来支撑 CVD 石墨烯方块，同时确保 MMA 面朝上，同时将其连接到镊子上。要将石墨烯切割成小方块，请使用两组反向作用镊子将石墨烯右侧朝上，将其固定到位，并在将其与大方块的其余部分切开后保持小方块的边缘。倾斜含有硫酸铵或 APS 的烧杯，将正方形以浅角度放入其中?...