Name: Preparation of High-Temperature Sample Grids for Cryo-EM (Video) | JoVE Journal
Uploaded: 2021-07-26T15:00:00.000+00:00
Duration: 5 min 5 s
Description: Learn to prepare high-temperature sample grids for cryo-EM using a vitrification apparatus and plunge freezing. This protocol enables study of temperature-dependent protein structures up to 70°C.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

The authors thank Dr. Herv&#233; Remigy of Thermo Fisher Scientific for useful advice. The cryo-EM experiments were performed at the Academia Sinica Cryo-EM Facility (ASCEM). ASCEM is supported by Academia Sinica (Grant No. AS-CFII-108-110) and Taiwan Protein Project (Grant No. AS-KPQ-109-TPP2). The authors also thank Ms. Hui-Ju Huang for the assistance with the sample preparation.

NOTE: This protocol aims to use a modified commercial vitrification apparatus to prepare the cryo-electron microscopy (cryo-EM) samples at specific temperatures, especially higher than 37 &#176;C. The overall experimental setup is shown in Figure 1. The protocol uses 55 &#176;C as an example. For the specific conditions at other temperatures, please refer to Supplementary Table 2 in reference12.
1. Preparation of the vitrification apparatus
<ol>
	<li>Make a 1 cm hole in a 50 mL centrifuge tube on its closed end.</li>
	<li>Place the tube in the vitrification apparatus chamber at the ultrasonic water outlet, as shown in Figure 2. 
	NOTE: The purpose is to minimize water condensation by guiding water vapor to the heat exchanger through the tube before reaching the entire chamber.</li>
	<li>Set up the vitrification apparatus temperature to the specified temperature (e.g., 55 &#176;C, as shown in Figure 3), and allow the vitrification apparatus chamber to reach to 55 &#176;C and 100% relative humidity. Let it stand for at least half an hour to stabilize the conditions before starting the experiment.</li>
</ol>
2. Warming up the sample and the tools
<ol>
	<li>Place the water bath on a hotplate and set the hotplate to the desired temperature (here 55 &#176;C). Check with a thermometer to ensure that the water reaches 55 &#176;C.</li>
	<li>Incubate the sample in the water bath and preheat the pipette tip on the edge of the hotplate for 2 min or longer before the blotting experiment. 
	&#8203;NOTE: The highest temperature setting for the vitrification apparatus chamber is 60 &#176;C. To prepare higher temperature grids for cyro-EM experiment (e.g., 70 &#176;C), the sample is incubated in the water bath at 80 &#176;C, and the average between the sample temperature and the vitrification apparatus temperature is estimated to be the actual temperature of the sample on the grid (70 &#176;C in this case). See the Discussion section for further details and limitations on this estimation.</li>
</ol>
3. Preparation for the blotting experiment
<ol>
	<li>Glow discharge a holey carbon-supported grid at 25 mA for 30 s, or alternative to the values depending on the device used.</li>
	<li>Incubate the tweezers with the grid in the vitrification apparatus for 2 min or longer.</li>
	<li>Fill the ethane container with ethane according to standard procedures. Do not let the ethane overflow. 
	NOTE: This step takes about 10 min and should be followed immediately with the blotting experiment to avoid freezing.</li>
	<li>Place the vitrification filter paper into the vitrification apparatus chamber no sooner than 5 min before the blotting experiment. 
	NOTE: Placing the filter paper in the chamber too soon will cause it to become too wet.</li>
</ol>
4. Blotting experiment
NOTE: When holding the grid, ensure that the grid is stable and there is a minimal contact area with the tweezers (Figure 4). This is done to maintain the best cooling efficiency of ethane and to avoid non-vitreous ice.
<ol>
	<li>Use a pipette tip to apply 7-9 &#956;L of the sample to the grid. Then wait for 1-2 s, blot for 1-1.5 s, and quickly plunge the sample to liquid ethane.</li>
	<li>Transfer the grid from liquid ethane to the cryo box, which is stored in liquid nitrogen. 
	&#8203;NOTE: This step must be performed very carefully because the tweezers is still hot, so the entire cooling tank is full of vapor at this time.</li>
</ol>
5. Quality check for the grids
<ol>
	<li>Clip the grids and upload them to a cryo-EM instrument.</li>
	<li>Use the display screen of the cryo-EM instrument and the low dose function of the software to screen the ice condition on the grid and the distribution of the sample on the grid. 
	&#8203;NOTE: Often, the grids are very dry, or the ice layer is too thick. The success rate of the grids prepared at high temperatures is substantially lower than that at room temperature or 4 &#176;C. Representative results of good grids and non-satisfactory grids are shown in the next section.</li>
	<li>If the quality of the resulting grid is not good, repeat the process of grid preparation with varied conditions (such as waiting time, blotting time, etc.). If the quality of the resulting grid is good, repeat the same steps to make grids at a different temperature.</li>
</ol>
6. Data collection
<ol>
	<li>Transfer the good quality grids to a high resolution cryo-EM instrument.</li>
	<li>Perform data collection and data analyses according to established procedures. 
	NOTE: As shown in our previous publication, the resolution of the structure is not affected by the high temperature12.</li>
</ol>

<ol><li>Yip, K. M., Fischer, N., Paknia, E., Chari, A., Stark, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Atomic-resolution+protein+structure+determination+by+cryo-EM%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Atomic-resolution protein structure determination by cryo-EM.</a> Nature. 587, 157-161 (2020).</li>
<li>Nakane, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-particle+cryo-EM+at+atomic+resolution%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Single-particle cryo-EM at atomic resolution.</a> Nature. 587, 152-156 (2020).</li>
<li>Chen, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+Cryo-EM+to+uncover+structural+bases+of+pH+effect+and+cofactor+bi-specificity+of+ketol-acid+reductoisomerase%5BTitle%5D)+AND+%22Journal+of+the+American+Chemical+Society%22%5BJournal%5D)">Use of Cryo-EM to uncover structural bases of pH effect and cofactor bi-specificity of ketol-acid reductoisomerase.</a> Journal of the American Chemical Society. 141, 6136-6140 (2019).</li>
<li>Cabra, V., Samsó, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Do%27s+and+don%27ts+of+cryo-electron+microscopy%3A+A+primer+on+sample+preparation+and+high+quality+data+collection+for+macromolecular+3D+reconstruction%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Do's and don'ts of cryo-electron microscopy: A primer on sample preparation and high quality data collection for macromolecular 3D reconstruction.</a> Journal of Visualized Experiments. (95), e52311(2015).</li>
<li>Klebl, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Need+for+speed%3A+Examining+protein+behavior+during+CryoEM+grid+preparation+at+different+timescales%5BTitle%5D)+AND+%22Structure%22%5BJournal%5D)">Need for speed: Examining protein behavior during CryoEM grid preparation at different timescales.</a> Structure. 28 (11), 1238-1248 (2020).</li>
<li>Passmore, L. A., Russo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Specimen+preparation+for+high+resolution+cryo-EM%5BTitle%5D)+AND+%22J.+Methods+in+Enzymology%22%5BJournal%5D)">Specimen preparation for high resolution cryo-EM.</a> J. Methods in Enzymology. 579, 51-86 (2016).</li>
<li>Laughlin, T. G., Bayne, A. N., Trempe, J. -F., Savage, D. F., Davies, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+of+the+complex+I-like+molecule+NDH+of+oxygenic+photosynthesis%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Structure of the complex I-like molecule NDH of oxygenic photosynthesis.</a> Nature. 566, 411-414 (2019).</li>
<li>Gao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structures+and+operating+principles+of+the+replisome%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Structures and operating principles of the replisome.</a> Science. 363, (2019).</li>
<li>Zhao, Y., Chen, S., Swensen, A. C., Qian, W. -J., Gouaux, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Architecture+and+subunit+arrangement+of+native+AMPA+receptors+elucidated+by+cryo-EM%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Architecture and subunit arrangement of native AMPA receptors elucidated by cryo-EM.</a> Science. 364, 355-362 (2019).</li>
<li>Chen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+dynamics+of+ribosome+subunit+association+studied+by+mixing-spraying+time-resolved+cryogenic+electron+microscopy%5BTitle%5D)+AND+%22Structure%22%5BJournal%5D)">Structural dynamics of ribosome subunit association studied by mixing-spraying time-resolved cryogenic electron microscopy.</a> Structure. 23, 1097-1105 (2015).</li>
<li>Singh, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+basis+of+temperature+sensation+by+the+TRP+channel+TRPV3%5BTitle%5D)+AND+%22Nature+Structure+and+Molecular+Biology%22%5BJournal%5D)">Structural basis of temperature sensation by the TRP channel TRPV3.</a> Nature Structure and Molecular Biology. 26, 994-998 (2019).</li>
<li>Chen, C. Y., Chang, Y. C., Lin, B. L., Huang, C. H., Tsai, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Temperature-resolved+cryo-EM+uncovers+structural+bases+of+temperature-dependent+enzyme+functions%5BTitle%5D)+AND+%22Journal+of+the+American+Chemical+Society%22%5BJournal%5D)">Temperature-resolved cryo-EM uncovers structural bases of temperature-dependent enzyme functions.</a> Journal of the American Chemical Society. 141, 19983-19987 (2019).</li>
</ol>

The authors declare no competing financial interests.

In step 1 of the protocol, make sure that the centrifuge tube has been installed well and does not fall when the experiment is in progress. Due to the accumulation of a large number of water droplets in the chamber, which could change the adsorption capacity of the filter paper, it is recommended that the overall time of the experiment should not exceed 30 min after the vitrification apparatus chamber reached the equilibration temperature. If the operation time exceeds 30 min, the operator needs to replace the filter pap...

The cryo-EM technology for solving the structures of protein complexes has continued to improve, particularly in the direction of obtaining high-resolution structures1,2. In the meantime, the landscape of its application has also been expanded by varying the sample conditions such as pH or ligands prior to the vitrification process3, which involves the preparation of sample grids followed by plunge freezing4,5. Another important condition is the temperature. Although cryo-EM experiments, like X-ray crystallography, are performed at low temperatures, the structure solved by cryo-EM reflects the structure at the solution state prior to vitrification. Until recently, the majority of single particle analysis (SPA) cryo-EM studies use samples that are kept on ice (i.e., at 4 &#176;C) prior to vitrification6, though a number of studies use samples at around room temperature7,8,9,10 or as high as 42 &#176;C11. In a recent report, we performed temperature-dependent studies of the enzyme ketol-acid reductoisomerase (KARI) from the thermophilic archaeon Sulfolobus solfataricus (Sso) at six different temperatures from 4 &#176;C to 70&#176;C12. Our studies suggest that it is important to prepare sample grids at functionally relevant temperatures and that cryo-EM is the only structural method that is practically feasible for solving the structure of the same protein complex at multiple temperatures.
The major difficulty for vitrification at high temperatures is to minimize vapor condensation and achieve thin ice. Here we report the detailed protocol used for preparing sample grids at high temperatures in our previous study of the Sso-KARI12. We assume that the readers or viewers are already experienced in the overall sample preparation and data processing procedures for cryo-EM experiments and emphasize the aspects relevant to high temperature.

<table><tbody><tr><td>Falcon tube</td><td>Falcon</td><td>352070</td><td>size: 50 mL</td></tr><tr><td>Filter paper</td><td>Ted Pella</td><td>47000-100</td><td>&amp;Oslash;55/20mm, Grade 595</td></tr><tr><td>HI1210</td><td>Leica</td><td></td><td>water bath</td></tr><tr><td>K100X</td><td>Electron Microscopy Sciences</td><td></td><td>glow discharge</td></tr><tr><td>Quantifoil, 1.2/1.3 200Mesh Cu grid</td><td>Ted Pella</td><td>658-200-CU-100</td><td></td></tr><tr><td>Titan Krios G3</td><td>Thermo Fisher Scientific</td><td>1063996</td><td>low dose imaging</td></tr><tr><td>Vitrobot Mark IV</td><td>Thermo Fisher Scientific</td><td>1086439</td><td></td></tr><tr><td>Vitrobot Tweezer</td><td>Ted Pella</td><td>47000-500</td><td></td></tr></tbody></table>

preparation of high-temperature sample grids for cryo-em

Preparation of High-Temperature Sample Grids for Cryo-EM

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, mostly at 4 &#176;C and occasionally at room temperature. Recently, we discovered that the protein structure solved at low temperature may not be functionally relevant, particularly for proteins from thermophilic archaea. A procedure was developed to prepare protein samples at higher temperatures (up to 70 &#176;C) for cryo-EM analysis. We showed that the structures from samples prepared at higher temperatures are functionally relevant and temperature dependent. Here we describe a detailed protocol for preparing sample grids at high temperature, using 55 &#176;C as an example. The experiment made use of a vitrification apparatus modified using an additional centrifuge tube, and samples were incubated at 55 &#176;C. The detailed procedures were fine-tuned to minimize vapor condensation and obtain a thin layer of ice on the grid. Examples of successful and unsuccessful experiments are provided.

The low magnification overview is shown in Figure 5A,B. Panel A is an example of a successful grid. There is an ice gradient from top left (thicker) to bottom right (thinner or empty). Such a grid makes it easier to find an appropriate thickness of the ice layer in the middle area suitable for data collection, such as the blue and green boxes. The grid B is too dry. The squares in the grid have bright contrast, which means that the ice layer is too thin or there is no ice la...

This paper provides a detailed protocol for preparing sample grids at temperatures as high as 70 &#176;C, prior to plunge freezing for cryo-EM experiments.

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, ...

preparation-of-high-temperature-sample-grids-for-cryo-em

Research

JoVE Journal

Biochemistry

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

Miniaturized Sample Preparation for Transmission Electron Microscopy

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.

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The signal-to-noise ratio of data is one of the most important considerations in performing X-ray diffraction measurements from microcrystals. The VMXm beamline provides a low-noise environment and microbeam for such experiments. Here, we describe sample preparation methods for mounting and cooling microcrystals for VMXm and other microfocus macromolecular crystallography beamlines.

A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline

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.

Single Particle Cryo-Electron Microscopy: From Sample to Structure

This manuscript outlines the blot-and-plunge method to manually freeze biological specimens for single-particle cryogenic electron microscopy.

Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy

Here, we present a pipeline for 3D-correlative focused ion beam milling on guiding the preparation of cellular samples for cryo-electron tomography. The 3D position of fluorescently tagged proteins of interest is first determined by cryo-fluorescence microscopy, and then targeted for milling. The protocol is suitable for mammalian, yeast, and bacterial cells.

Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography

This method provides an accessible and flexible protocol for the preparation of electron microscopy (EM) grids for in situ cellular cryotomography and correlative light and electron microscopy (CLEM).

Sample Preparation for In Situ Cryotomography of Mammalian Cells

A step-by-step protocol for fabricating streptavidin affinity grids is provided for use in structural studies of challenging macromolecular samples by cryo-electron microscopy.

Streptavidin-Affinity Grid Fabrication for Cryo-Electron Microscopy Sample Preparation

This protocol presents a practical method using Methanocaldococcus jannaschii (MjsHSP16.5) as an example to address uneven particle distribution through sample preparation optimization, providing a reference for researchers to efficiently elucidate macromolecular structures using cryogenic electron microscopy (cryo-EM).

Optimizing Sample Preparation for Cryogenic Electron Microscopy

Learn to prepare high-temperature sample grids for cryo-EM using a vitrification apparatus and plunge freezing. This protocol enables study of temperature-dependent protein structures up to 70°C.

Preparation of High-Temperature Sample Grids for Cryo-EM

Explore More Videos