Name: preparation of high-temperature sample grids for cryo-em
Uploaded: 2021-07-26T15:00:00
Description: Watch this Scientific Journal Video about Preparation of High-Temperature Sample Grids for Cryo-EM at JoVE.com

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>Yip, K. M., Fischer, N., Paknia, E., Chari, A., Stark, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic-resolution+protein+structure+determination+by+cryo-EM.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-particle+cryo-EM+at+atomic+resolution.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Cryo-EM+to+uncover+structural+bases+of+pH+effect+and+cofactor+bi-specificity+of+ketol-acid+reductoisomerase.">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., Sams&#243;, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do's+and+don'ts+of+cryo-electron+microscopy:+A+primer+on+sample+preparation+and+high+quality+data+collection+for+macromolecular+3D+reconstruction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Need+for+speed:+Examining+protein+behavior+during+CryoEM+grid+preparation+at+different+timescales.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specimen+preparation+for+high+resolution+cryo-EM.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+complex+I-like+molecule+NDH+of+oxygenic+photosynthesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structures+and+operating+principles+of+the+replisome.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+and+subunit+arrangement+of+native+AMPA+receptors+elucidated+by+cryo-EM.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+dynamics+of+ribosome+subunit+association+studied+by+mixing-spraying+time-resolved+cryogenic+electron+microscopy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+of+temperature+sensation+by+the+TRP+channel+TRPV3.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-resolved+cryo-EM+uncovers+structural+bases+of+temperature-dependent+enzyme+functions.">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>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>&#216;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

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...

Watch this Scientific Journal Video about Preparation of High-Temperature Sample Grids for Cryo-EM at JoVE.com

Preparation of High-Temperature Sample Grids for Cryo-EM

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, ...

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