Specimens were prepared and imaged at the CryoEM Facility in MIT.nano on microscopes acquired thanks to the Arnold and Mabel Beckman Foundation. Contact angle imaging devices were printed at the MIT Metropolis Maker Space. We thank the laboratories of Nieng Yan and Yimo Han, and staff at MIT.nano for their support throughout the adoption of this method. In particular, we extend our thanks to Drs. Guanhui Gao and Sarah Sterling for their insightful discussions and feedback. This work was supported by NIH grants R01-GM144542, 5T32-GM007287, and NSF-CAREER grant 2046778. Research in the Davis lab is supported by the Alfred P. Sloan Foundation, the James H. Ferry Fund, the MIT J-Clinic, and the Whitehead Family.

1.&#160;Preparation of CVD graphene
<ol>
	<li>Prepare graphene etching solution as described below.
	<ol>
		<li>Dissolve 4.6 g ammonium persulfate (APS) in 20 mL of molecular grade water in a 50 mL beaker for a 1 M solution and cover with aluminum foil. Allow APS to completely dissolve while proceeding to step 1.2.</li>
	</ol>
	</li>
	<li>Prepare a section of CVD graphene for methyl methacrylate (MMA) coating. Carefully cut a square section of CVD graphene. Transfer the square to a coverslip (50 mm x 2...

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

<ol>
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(181), (2022).</li><li>Schweizer, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+cleaning+of+graphene+using+in+situ+electron+microscopy.">Mechanical cleaning of graphene using in situ electron microscopy.</a> Nat Commun. 11 (1), 1743 (2020).</li><li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+airborne+contaminants+on+the+wettability+of+supported+graphene+and+graphite.">Effect of airborne contaminants on the wettability of supported graphene and graphite.</a> Nat Mater. 12 (10), 925-931 (2013).</li><li>Jinek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+programmable+dual-RNA-guided+DNA+endonuclease+in+adaptive+bacterial+immunity.">A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.</a> Science. 337 (6096), 816-821 (2012).</li><li>Zhu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-EM+structures+reveal+coordinated+domain+motions+that+govern+DNA+cleavage+by+Cas9.">Cryo-EM structures reveal coordinated domain motions that govern DNA cleavage by Cas9.</a> Nat Struct Mol Biol. 26 (8), 679-685 (2019).</li><li>Croll, T. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ISOLDE:+a+physically+realistic+environment+for+model+building+into+low-resolution+electron-density+maps.">ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps.</a> Acta Crystallogr D Struct Biol. 74, 519-530 (2018).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9 (7), 671-675 (2012).</li><li>Prydatko, A. V., Belyaeva, L. A., Jiang, L., Lima, L. M. C., Schneider, G. 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The authors have no conflicts to disclose.

CryoEM sample preparation involves a host of technical challenges, with most workflows requiring researchers to manually manipulate fragile grids with extreme care to avoid damaging them. Additionally, the amenability of any sample to vitrification is unpredictable; particles often interact with the air-water-interface or with the solid support foil overlaying the grids, which can lead to particles adopting preferred orientations or failing to enter the imaging holes unless very high protein concentrations are applied<su...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>250 mL beaker (3x)</td>
			<td>Fisher</td>
			<td>02-555-25B</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL beaker (2x)</td>
			<td>Corning</td>
			<td>1000-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher</td>
			<td>A949-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Fisher</td>
			<td>15-078-292</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Fisher</td>
			<td>(I17874</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips 50 mm x 24 mm</td>
			<td>Mattek</td>
			<td>PCS-1.5-5024</td>
			<td></td>
		</tr>
		<tr>
			<td>CVD graphene</td>
			<td>Graphene Supermarket</td>
			<td>CVD-Cu-2x2</td>
			<td></td>
		</tr>
		<tr>
			<td>easiGlow discharger</td>
			<td>Ted-Pella</td>
			<td>91000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Millipore-Sigma</td>
			<td>1.11727</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat-tip tweezers&#160;</td>
			<td>Fisher</td>
			<td>50-239-60</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass cutter</td>
			<td>Grainger</td>
			<td>21UE26</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass petri plate and cover&#160;</td>
			<td>VWR</td>
			<td>75845-544</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass serological pipette</td>
			<td>Fisher</td>
			<td>13-676-34D</td>
			<td></td>
		</tr>
		<tr>
			<td>Grid Storage Case</td>
			<td>EMS</td>
			<td>71146-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Fisher</td>
			<td>07-770-108</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma</td>
			<td>W292907</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipe</td>
			<td>Fisher</td>
			<td>06-666</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab scissors&#160;</td>
			<td>Fisher</td>
			<td>13-806-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl-Methacrylate EL-6&#160;</td>
			<td>Kayaku</td>
			<td>MMA M310006 0500L1GL</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular grade water</td>
			<td>Corning</td>
			<td>46-000-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Negative action tweezers (2x)</td>
			<td>Fisher</td>
			<td>50-242-78</td>
			<td></td>
		</tr>
		<tr>
			<td>P20 pipette</td>
			<td>Rainin</td>
			<td>17014392</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 pipette</td>
			<td>Rainin</td>
			<td>17008652&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Fisher</td>
			<td>13-374-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>Rainin</td>
			<td>30389291</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantifoil grids with holey carbon&#160;</td>
			<td>EMS</td>
			<td>Q2100CR1</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater&#160;</td>
			<td>SetCas</td>
			<td>KW-4A</td>
			<td>with chuck SCA-19-23</td>
		</tr>
		<tr>
			<td>Straightedge</td>
			<td>ULINE</td>
			<td>H-6560</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermometer&#160;</td>
			<td>Grainger</td>
			<td>3LRD1</td>
			<td></td>
		</tr>
		<tr>
			<td>UV/Ozone cleaner&#160;</td>
			<td>BioForce</td>
			<td>SKU: PC440</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum desiccator</td>
			<td>Thomas Scientific</td>
			<td>1159X11</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman paper</td>
			<td>VWR</td>
			<td>28297-216</td>
			<td></td>
		</tr>
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</table>

application of monolayer graphene to cryo-electron microscopy grids for high-resolution structure determination

In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to remove excess liquid and rapidly frozen in a roughly 20-100 nm thick layer of vitreous ice, suspended across roughly 1 &#181;m wide foil holes. The resulting sample is imaged using cryogenic transmission electron microscopy, and after image processing using suitable software, near-atomic resolution structures can be determined. Despite cryoEM&#39;s widespread adoption, sample preparation remains a severe bottleneck in cryoEM workflows, with users often encountering challenges related to samples behaving poorly in the suspended vitreous ice. Recently, methods have been developed to modify cryoEM grids with a single continuous layer of graphene, which acts as a support surface that often increases particle density in the imaged area and can reduce interactions between particles and the air-water interface. Here, we provide detailed protocols for the application of graphene to cryoEM grids and for rapidly assessing the relative hydrophilicity of the resulting grids. Additionally, we describe an EM-based method to confirm the presence of graphene by visualizing its characteristic diffraction pattern. Finally, we demonstrate the utility of these graphene supports by rapidly reconstructing a 2.7 &#197; resolution density map of a Cas9 complex using a pure sample at a relatively low concentration.

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.

Successful fabrication of graphene-coated cryoEM grids using the equipment (Figure 1) and protocol (Figure 2) outlined here will result in a monolayer of graphene covering the foil holes that can be confirmed by its characteristic diffraction pattern. To promote protein adsorption to the graphene surface, UV/ozone treatment can be used to render the surface hydrophilic by installing oxygen-containing functional groups. However, hydrocarbon contaminants in the ai...

Watch this Scientific Journal Video about Enhancing CryoEM Sample Preparation Using Graphene Monolayer on Microscopy Grids at JoVE.com

The application of support layers to cryogenic electron microscopy (cryoEM) grids can increase particle density, limit interactions with the air-water interface, reduce beam-induced motion, and improve the distribution of particle orientations. This paper describes a robust protocol for coating cryoEM grids with a monolayer of graphene for improved cryo-sample preparation.

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

application-monolayer-graphene-to-cryo-electron-microscopy-grids-for

Research

JoVE Journal

Biochemistry

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

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 spin coder to a 60 second high speed spin at 2, 500 rotations per minute.Carefully transfer the CVD graphene section to the appropriately-sized chuck. Press the take or absorb button to engage the vacuum pump and affix the graphene to the chuck. Then apply MMA to the center of the CVD graphene square.Close the lid. Pull the vacuum and immediately press the start or stop button. After disengaging the vacuum pump, once the spinning has stopped, using tweezers, carefully retrieve the MMA-coated CVD graphene.Invert the graphene such that the coated side is facing down, and place it back on the glass cover slip. Next, using flat tip tweezers, transfer the CVD graphene on the cover slip into the glow discharger and glow discharge for 30 seconds at 25 milliampere. Return the graphene on the cover slip to the petri dish.Finally, cover it for transporting it to the copper etching area.

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.

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.

1.1K Views.  Massachusetts Institute of Technology.  The application of support layers to cryogenic electron microscopy (cryoEM) grids can increase particle density, limit interactions with the air-water interface, reduce beam-induced motion, and improve the distribution of particle orientations. This paper describes a robust protocol for coating cryoEM grids with a monolayer of graphene for improved cryo-sample preparation.

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

概要