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 24 mm) within a clean Petri dish and cover during transport to the spin coater. 
	&#8203;NOTE: An 18 mm x 18 mm square should yield 25-36 graphene-coated grids.</li>
</ol>
2. Coating CVD graphene with MMA
<ol>
	<li>Set the spin coater settings to a 60 s high speed spin at 2,500 rpm. Carefully place the CVD graphene on the appropriately sized chuck. 
	&#8203;NOTE: Ideally, the CVD graphene will extend 1-2 mm over the edge of the selected chuck to prevent aspiration of MMA into the vacuum system.</li>
	<li>Press the Take/Absorb button to engage the vacuum pump and affix the CVD graphene to the chuck. Apply MMA to the center of CVD graphene square, close lid, and press Start/Stop button immediately. For an 18 mm x 18 mm square of CVD graphene, 40 &#181;L of MMA is sufficient.</li>
	<li>Once spinning has stopped, disengage the vacuum pump and carefully retrieve the MMA coated CVD graphene with tweezers. Invert the CVD graphene such that the MMA coated side is facing down and place it back on the glass coverslip.</li>
</ol>
3. Plasma etching of the graphene back-side
<ol>
	<li>Transfer CVD graphene on the coverslip into the glow discharger and glow discharge for 30 s at 25 mA using flat-tip tweezers.</li>
	<li>Return the CVD graphene on the coverslip to the Petri dish and cover during transport to the copper etching area. MMA coating will protect the topside graphene layer from plasma etching.</li>
</ol>
4. Cutting grid-sized MMA coated CVD graphene squares
<ol>
	<li>Use two pairs of tweezers to support the CVD graphene square. Take note of the orientation of the CVD graphene square, keep the MMA side up while attached to the tweezers (CRITICAL).</li>
	<li>Cut CVD graphene into approximately 3 mm x 3 mm squares. Use&#160;two sets of reverse-action forceps for this step&#160;to hold the CVD graphene square right-side-up and anchor it in position, and also to hold the edge of a 3 mm x 3 mm square after cutting it away from the rest of the square.</li>
</ol>
5. Dissolving copper substrate from MMA coated CVD graphene
<ol>
	<li>Carefully float each 3 mm x 3 mm square in the APS solution. Make contact with the surface of the APS solution prior to releasing each square. Tilt the beaker such that each square can be placed in the solution at a shallow angle; this ensures that the square does not sink.</li>
	<li>Cover beaker with aluminum foil and incubate overnight at 25 &#176;C.</li>
</ol>
6. Removing MMA/graphene films from APS
<ol>
	<li>Use a glass coverslip with dimensions 12 mm x 50 mm and extract the MMA/graphene squares from APS by gently plunging the coverslip vertically into the APS and then moving the coverslip laterally such that it abuts a floating MMA/graphene square.</li>
	<li>Carefully remove the coverslip and ensure that the square adheres completely to the side of the coverslip upon removal.</li>
	<li>Transfer MMA/graphene squares to a clean 50 mL beaker filled with molecular grade water for 20 min. Gently plunge the coverslip with an MMA/graphene square attached into the water vertically and ensure that the square dislodges from the coverslip upon interaction with the surface of the water. Repeat for all MMA/graphene squares.</li>
</ol>
7. Adhering graphene to grids
<ol>
	<li>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 and ensure that the square adheres completely to the carbon side of the grid upon removal. Minimize lateral movement of the grid when submerged in water to prevent damaging grid squares (CRITICAL).</li>
	<li>Place grids on a clean coverslip MMA/graphene side up and air dry for 1 to 2 min. Transfer coverslip graphene/MMA coated grids to a hot plate set to 130 &#176;C using flat-tip tweezers.</li>
	<li>Cover with the top of a glass Petri dish and incubate for 20 min. Remove from heat and cool to room temperature for 1 to 2 min. 
	&#8203;CAUTION: Exercise caution as the Petri dish top will be extremely hot.</li>
</ol>
8. Dissolving MMA with acetone
<ol>
	<li>Transfer entire coverslip into a Petri dish filled with 15 mL of acetone. Incubate for 30 min.</li>
	<li>Using a clean glass serological pipette, transfer acetone, in which the grids were incubated, to a waste container. Carefully add 15 mL of fresh acetone to the Petri dish using a clean glass serological pipette. Incubate for 30 min.</li>
	<li>Repeat these wash steps for a total of 3 acetone washes.</li>
</ol>
9. Removing residual acetone with isopropanol
<ol>
	<li>Following 3 acetone washes, remove acetone and replace with 15 mL of isopropanol using a clean glass serological pipette. Incubate for 20 min.</li>
	<li>Repeat 3x for a total of 4 isopropanol washes. Carefully remove grids from isopropanol and air dry on a clean coverslip using tweezers. 
	NOTE: Residual isopropanol can make it difficult to release grids from tweezers. Making contact between the grid and the clean coverslip prior to releasing the grid from the tweezers can alleviate this problem.</li>
	<li>Evaporate residual organics by transferring the coverslip with graphene-coated grids onto a hotplate set to 100 &#176;C for 10 min. Cool to room temperature and store under vacuum until use.</li>
</ol>
10. UV/ozone treatment of graphene-coated grids
<ol>
	<li>Using reverse-action tweezers, gently place grids into a clean illumination area of a UV/ozone cleaner with the graphene-coated side facing up.</li>
	<li>Slide the illumination area closed and turn the machine ON. Turn the time dial to the designated treatment time to begin UV/ozone treating the grids.. 
	NOTE: Treatment duration is a tunable parameter, with excess treatment having the potential to damage the grid. Han et al.30 recommends 10 min of UV/ozone treatment, and we have also found a 10 min treatment to suffice. See step 12 for guidance on tuning this treatment time.</li>
	<li>Proceed directly to plunging a cryoEM sample on the treated grids by following plunging steps as described in Koh et al.37. 
	NOTE: Atmospheric hydrocarbons can accumulate on the grid surface after UV/ozone treatment and increase the hydrophobicity of the graphene surface38,39. To avoid this, plunge the UV/ozone treated grids immediately. It may be advantageous to UV/ozone treat grids in batches, for example, UV/ozone treating six grids and immediately plunging those six grids, then UV/ozone treating a second batch of grids, followed by plunging the second batch.</li>
</ol>
11. Capturing a diffraction image
<ol>
	<li>Ensure that the microscope is well tuned with parallel illumination established and set final defocus to -0.2 &#181;m.</li>
	<li>Insert the fluorescent screen and the beam stop fully. Enter diffraction mode to clearly visualize diffraction spots.</li>
	<li>Acquire an image using a CCD camera and evaluate it using image analysis software. 
	NOTE: The most easily observed and identifiable diffraction spots generated by a graphene monolayer are 6 spots corresponding to a spatial frequency of 2.13 &#197;. Use a measurement tool to estimate the distance from the center of the diffracted beam to one of these diffraction spots in reciprocal units. Notably 2.13 &#197; <img alt="Equation 1" src="/files/ftp_upload/66023/66023eq01.jpg" style="margin: auto;" /> 0.47 &#197;-1.</li>
</ol>
12. Assessing grid hydrophilicity
<ol>
	<li>Prepare the imaging setup. Locate a phone stand, tabletop imaging surface, phone with camera, glass coverslip, and paraffin film. Images shown here were obtained using a phone stand and tabletop imaging surface that were 3D printed using the provided .stl files, resulting in more easily reproducible and quantifiable contact angle measurements (Supplementary Figure 1A).</li>
	<li>Lay a glass coverslip onto the flat surface, then cut a 1 cm by 1 cm square of paraffin film and place it onto the glass coverslip. Place phone on the phone stand, orienting the phone such that the camera is in-plane with the glass coverslip. Secure phone in this position using rubber bands (Supplementary Figure 1B). Take a sample photo to verify that the camera is aligned with the imaging surface.</li>
	<li>Directly after UV/ozone treatment of graphene-coated grids, place a single grid onto the square of paraffin film on the glass coverslip. Ensure the graphene side is facing upwards. Add a 2 &#181;L water droplet onto the center of the grid surface with a pipette and immediately take a photo. 
	NOTE: Repeat this step after: i) desired intervals of UV/ozone treatment to determine a sufficient length of treatment; or ii) desired time intervals post UV/ozone treatment to measure how long after treatment the grid surface maintains its hydrophilic character.</li>
	<li>Calculate contact angles from photos by importing them into ImageJ43 and using the contact angle plugin.</li>
</ol>
13. Single particle analysis of the dCas9 complex dataset
NOTE: All image processing described in this protocol was performed using cryoSPARC version 4.2.1.
<ol>
	<li>Preprocess movies using Patch Motion Correction and Patch CTF Estimation jobs. Perform particle picking using the Blob picker job, using a spherical blob ranging in diameter from 115 &#197; to 135 &#197;.</li>
	<li>Extract particles using the Extract from micrographs job, using normalized correlation coefficient (NCC) and power thresholds resulting in approximately 200-300 particles per micrograph. Note that appropriate thresholds and resulting particle count may vary, and users should inspect pick quality across a range of micrographs to identify suitable conditions.</li>
	<li>Perform multiclass initial reconstructions using the Ab-initio reconstruction job, requiring three classes. Two of the three classes will likely contain non-Cas9 particles, including surface contaminants. Select the class resembling dCas9 for further processing. 
	NOTE: Additional rounds of multiclass Ab-initio reconstruction or Heterogeneous refinement may be applied to further refine the particle stack.</li>
	<li>Perform 3D refinement using the Non-uniform Refinement job selecting default parameters. Estimate resolution of the reconstruction using the Validation (FSC), and ThreeDFSC jobs, employing the maps and mask from the final 3D refinement.</li>
</ol>

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

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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. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contact+angle+measurement+of+free-standing+square-millimeter+single-layer+graphene.">Contact angle measurement of free-standing square-millimeter single-layer graphene.</a> Nat Commun. 9 (1), 4185 (2018).</li></ol>

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><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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Fisher</td><td>13-806-2</td><td></td></tr><tr><td>Methyl-Methacrylate EL-6&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>EMS</td><td>Q2100CR1</td><td></td></tr><tr><td>Spin coater&amp;nbsp;</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&amp;nbsp;</td><td>Grainger</td><td>3LRD1</td><td></td></tr><tr><td>UV/Ozone cleaner&amp;nbsp;</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></tbody></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.

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

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

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.

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 ...

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Biochemistry

1.7K Views.  Massachusetts Institute of Technology. Een veel voorkomende uitdaging bij de voorbereiding van cryo-elektronenmicroscopiemonsters is de lokalisatie van deeltjes op het grensvlak tussen lucht en water, waardoor de deeltjes voorkeursoriëntaties aannemen en denatureren. In plaats daarvan is het beter om de deeltjes op het roosteroppervlak te laten rusten, wat dunne film ondersteunt, zoals grafeen bevorderen. Een andere uitdaging is de eis van relatief hoge monsterconcentraties, die ook door grafeen kunnen worden aangepakt.Met...

Video: Auteur in de schijnwerpers: Verbetering van de voorbereiding van CryoEM-monsters met behulp van grafeenmonolaag op microscopieroosters 

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/&#945;

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its precursor p100. p52 functions as a homodimer or heterodimer with another NF-&#954;B subunit, RelB. These dimers in turn regulate the expression of hundreds of genes involved in inflammation, cell survival, and cell cycle. IKK1/&#945; primarily remains associated with IKK2/&#946; and NEMO as a ternary complex. However, a small pool of it is also observed as a low molecular weight complex(es). It is unknown if the p100 processing activity is triggered by activation of IKK1/&#945; within the larger or the smaller complex pool. Constitutive activity of IKK1/&#945; has been detected in several cancers and inflammatory diseases. To understand the mechanism of activation of IKK1/&#945;, and enable its use as a drug target, we expressed recombinant IKK1/&#945; in different host systems, such as E. coli, insect, and mammalian cells. We succeeded in expressing soluble IKK1/&#945; in baculovirus infected insect cells, obtaining mg quantities of highly pure protein, crystallizing it in the presence of inhibitors, and determining its X-ray crystal structure. Here, we describe the detailed steps to produce the recombinant protein, its crystallization, and its X-ray crystal structure determination.

I&#954;B Kinase 1/&#945; (IKK1/&#945; CHUK) is a Ser/Thr protein kinase that is involved in a myriad of cellular activities primarily through activation of NF-&#954;B transcription factors. Here, we describe the main steps necessary for the production and crystal structure determination of this protein.

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its ...

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures and information on more challenging systems. Sample preparation for cryo-EM is undergoing a similar revolution with new approaches being developed to supersede the traditional blotting systems. These include the use of piezo-electric dispensers, pin printing and direct spraying. As a result of these developments, the speed of grid preparation is going from seconds to milliseconds, providing new opportunities, especially in the field of time-resolved cryo-EM where proteins and substrates can be rapidly mixed before plunge freezing, trapping short lived intermediate states. Here we describe, in detail, a standard protocol for making grids on our in-house time-resolved EM device both for standard fast grid preparation and also for time-resolved experiments. The protocol requires a minimum of about 50 &#181;L sample at concentrations of &#8805; 2 mg/mL for the preparation of 4 grids. The delay between sample application and freezing can be as low as 10 ms. One limitation is increased ice thickness at faster speeds and compared to the blotting method. We hope this protocol will aid others in designing their own grid making devices and those interested in designing time-resolved experiments.

Here, we provide a detailed protocol for the use of a rapid grid making device for both fast grid-making and for rapid mixing and freezing to conduct time-resolved experiments.

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures ...

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the&#160;Saccharomyces cerevisiae.

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...

Single Particle Cryo-Electron Microscopy: From Sample to Structure

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The overall process involves i) vitrifying the specimen in a thin film supported on a cryoEM grid; ii) screening the specimen to assess particle distribution and ice quality; iii) if the grid is suitable, collecting a single particle dataset for analysis; and iv) image processing to yield an EM density map. In this protocol, an overview for each of these steps is provided, with a focus on the variables which a user can modify during the workflow and the troubleshooting of common issues. With remote microscope operation becoming standard in many facilities, variations on imaging protocols to assist users in efficient operation and imaging when physical access to the microscope is limited will be described.

Structure determination of macromolecular complexes using cryoEM has become routine for certain classes of proteins and complexes. Here, this pipeline is summarized (sample preparation, screening, data acquisition and processing) and readers are directed towards further detailed resources and variables that may be altered in the case of more challenging specimens.

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The ...

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules present in the cellular context and preserved in a near-native frozen-hydrated state. However, there are challenges associated with culturing and/or adhering cells onto TEM grids in a manner that is suitable for tomography while retaining the cells in their physiological state. Here, a detailed step-by-step protocol is presented on the use of micropatterning to direct and promote eukaryotic cell growth on TEM grids. During micropatterning, cell growth is directed by depositing extra-cellular matrix (ECM) proteins within specified patterns and positions on the foil of the TEM grid while the other areas remain coated with an anti-fouling layer. Flexibility in the choice of surface coating and pattern design makes micropatterning broadly applicable for a wide range of cell types. Micropatterning is useful for studies of structures within individual cells as well as more complex experimental systems such as host-pathogen interactions or differentiated multi-cellular communities. Micropatterning may also be integrated into many downstream whole-cell cryo-ET workflows, including correlative light and electron microscopy (cryo-CLEM) and focused-ion beam milling (cryo-FIB).

The goal of this protocol is to direct cell adhesion and growth to targeted areas of grids for cryo-electron microscopy. This is achieved by applying an anti-fouling layer that is ablated in user-specified patterns followed by deposition of extra-cellular matrix proteins in the patterned areas prior to cell seeding.

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules ...

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are influenced by its structure. Hence, a suitable extraction method that isolates representative samples of glycogen is crucial to the study of this macromolecule. Compared to other extraction methods, a method that employs a sucrose density gradient centrifugation step can minimize molecular damage. Based on this method, a recent publication describes how the density of the sucrose solution used during centrifugation was varied (30%, 50%, 72.5%) to find the most suitable concentration to extract glycogen particles of a wide variety of sizes, limiting the loss of smaller particles. A 10 min boiling step was introduced to test its ability to denature glycogen degrading enzymes, thus preserving glycogen. The lowest sucrose concentration (30%) and the addition of the boiling step were shown to extract the most representative samples of glycogen.

An optimal sucrose concentration was determined for the extraction of liver glycogen using sucrose density gradient centrifugation. The addition of a 10 min boiling step to inhibit glycogen-degrading enzymes proved beneficial.

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are ...

Coating TEM Grids with a Graphene Monolayer

Fabrication of Monolayer Graphene-Coated Grids for Cryoelectron Microscopy

Cryogenic electron microscopy (cryoEM) has emerged as a powerful technique for probing the atomic structure of macromolecular complexes. Sample preparation for cryoEM requires preserving specimens in a thin layer of vitreous ice, typically suspended within the holes of a fenestrated support film. However, all commonly used sample preparation approaches for cryoEM studies expose the specimen to the air-water interface, introducing a strong hydrophobic effect on the specimen that often results in denaturation, aggregation, and complex dissociation. Further, preferred hydrophobic interactions between regions of the specimen and the air-water interface impact the orientations adopted by the macromolecules, resulting in 3D reconstructions with anisotropic directional resolution. 
Adsorption of cryoEM specimens to a monolayer of graphene has been shown to help mitigate interactions with the air-water interface while minimizing the introduction of background noise. Graphene supports also offer the benefit of substantially lowering the required concentration of proteins required for cryoEM imaging. Despite the advantages of these supports, graphene-coated grids are not widely used by the cryoEM community due to the prohibitive expense of commercial options and the challenges associated with large-scale in-house production. This paper describes an efficient method for preparing batches of cryoEM grids that have nearly full coverage of monolayer graphene.

Author Spotlight: Enhancing CryoEM Resolution Using Graphene-Coated Grids 

Here, we describe a protocol for applying a single monolayer of graphene to electron microscopy grids and how to prepare them for use in cryoEM structure determination.

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

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