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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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

Abstract

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 °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 °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 °C as an example. The experiment made use of a vitrification apparatus modified using an additional centrifuge tube, and samples were incubated at 55 °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.

Introduction

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....

Protocol

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 °C. The overall experimental setup is shown in Figure 1. The protocol uses 55 °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

  1. Make a 1 cm hole in a 50 mL centrifuge tube on its closed end.
  2. Place the tube in the vitrification apparatus chamber at the ult....

Results

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.......

Discussion

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.......

Disclosures

The authors declare no competing financial interests.

Acknowledgements

The authors thank Dr. Hervé 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.

....

Materials

NameCompanyCatalog NumberComments
Falcon tubeFalcon352070size: 50 mL
Filter paperTed Pella47000-100Ø55/20mm, Grade 595
HI1210Leicawater bath
K100XElectron Microscopy Sciencesglow discharge
Quantifoil, 1.2/1.3 200Mesh Cu gridTed Pella658-200-CU-100
Titan Krios G3Thermo Fisher Scientific1063996low dose imaging
Vitrobot Mark IVThermo Fisher Scientific1086439
Vitrobot TweezerTed Pella47000-500

References

  1. Yip, K. M., Fischer, N., Paknia, E., Chari, A., Stark, H. Atomic-resolution protein structure determination by cryo-EM. Nature. 587, 157-161 (2020).
  2. Nakane, T., et al. Single-particle cryo-EM at atomic resolution.

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