preparing mma-coated cvd graphene for fabrication of graphene-coated cryoem grids

Belegg av CryoEM-gitter med monolagsgrafen for å forbedre forberedelsen av kryoprøver

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.

Forfatter i søkelyset: Forbedring av CryoEM-prøveforberedelse ved bruk av grafenmonolag på mikroskopigitter 

Anvendelse av monolags grafen til kryo-elektronmikroskopigitter for høyoppløselig strukturbestemmelse

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 MMA-Coated CVD Graphene for Fabrication of Graphene-Coated CryoEM Grids at JoVE.com

Forbereder MMA-belagt CVD-grafen for fabrikasjon av grafenbelagte kryoEM-nett

Begynn med å forberede en grafenetseløsning i et 50 milliliter beger. For å gjøre det, tilsett 4, 6 gram ammonium per sulfat, eller APS, til 20 ml molekylært vann i begeret. Dekk den med aluminiumsfolie og la APS oppløses helt.For å forberede CVD-grafenseksjonen for MMA-belegg, kutt forsiktig en firkantet del av grafen. Overfør seksjonen til en dekselslip i en ren petriskål, og dekk fatet for å transportere det til spinnkoderen. Sett deretter spinnkoderen til et 60 sekunders høyhastighetsspinn ved 2.500 rotasjoner per minutt.Overfør forsiktig CVD-grafenseksjonen til den passende størrelsen chuck. Trykk på take or absorbere-knappen for å koble inn vakuumpumpen og feste grafen på chucken. Påfør deretter MMA til midten av CVD-grafenfirkanten.Lukk lokket. Trekk vakuumet og trykk umiddelbart på start- eller stoppknappen. Etter å ha koblet fra vakuumpumpen, når spinningen har stoppet, må du forsiktig hente den MMA-belagte CVD-grafenen ved hjelp av pinsett.Inverter grafénet slik at den belagte siden vender ned, og legg den tilbake på glassdekselet. Deretter overfører du CVD-grafen på dekselet ved hjelp av flat spisspinsett og glir inn i glødeladeren og glødutladningen i 30 sekunder ved 25 milliampere. Sett grafénet tilbake på omslagsseddelen til petriskålen.Til slutt, dekk den til for å transportere den til kobberetseområdet.

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Research

JoVE Journal

Biochemistry

Preparing MMA-Coated CVD Graphene for Fabrication of Graphene-Coated CryoEM Grids

172 Views.  Massachusetts Institute of Technology. Begin by preparing a graphene etching solution in a 50 milliliter beaker. To do so, add 4.6 grams of ammonium per sulfate, or APS, to 20 milliliters of molecular grade water in the beaker. Cover it with aluminum foil and allow the APS to dissolve completely.To prepare the CVD graphene section for MMA coating, carefully cut a square section of the graphene. Transfer the section to a cover slip in a clean petri dish, and cover the dish for transporting it to the spin coder. Next, set the...