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Bioquímica

用于单颗粒低温电子显微镜的生物标本的手动印迹和切入式冷冻

Published: February 07, 2022
doi:
10.3791/62765
, , ,
1Department of Chemistry and Biochemistry,University of California, San Diego

Summary

本手稿概述了手动冷冻生物标本以进行单颗粒低温电子显微镜的印迹和暴跌方法。

Abstract

通过单颗粒低温电子显微镜（cryoEM）用电子成像生物标本以进行高分辨率结构测定，需要一层含有目标生物分子的玻璃体冰薄层。尽管近年来有许多技术进步将单颗粒冷冻电镜推向了结构生物学的前沿，但对标本进行玻璃化以进行高分辨率成像的方法通常仍然是限速步骤。尽管最近的许多努力为克服样品玻璃化过程中经常遇到的障碍提供了手段，包括开发新型样品支架和创新的玻璃化仪器，但由于购买成本低且易于操作，传统的手动操作柱塞仍然是cryoEM社区的主要产品。在这里，我们提供了使用标准的断头台式手动印迹和插孔装置对生物标本进行玻璃化的详细方法，以便通过单颗粒冷冻EM进行高分辨率成像。此外，还描述了标准制备未能产生合适样品时经常遇到的问题和故障排除建议。

Introduction

单粒子低温电子显微镜（cryoEM）是一种强大的结构技术，可用于将动态生物标本的结构求解为近原子分辨率1，2，3，4。事实上，直接电子探测器技术的最新进展4，5，6，7，8，9，10，电子源的改进4，11，12，13，14和电磁透镜稳定性15，以及数据采集的持续发展16，17 和分析软件包18，19使研究人员能够常规地确定性能良好的标本的结构，分辨率为3 Å或更好4，11，13，14，20，21，22，23.尽管这些改进了成像和数据处理能力，但冷冻电镜网格准备仍然是成功进行高分辨率结构测定的最大障碍，并且经常成为EM工作流程中相当大的瓶颈24，25，26，27。

CryoEM依赖于对水溶液中的生物样品进行成像，这些溶液被冷冻以形成”玻璃状”冰的薄膜 – 这一过程称为玻璃化 – 保留了天然生化状态。用于冷冻电镜的生物样品的玻璃化可追溯到40多年前28，29，30，为该过程开发的许多技术和设备都依赖于最初详细的印迹和暴跌法31，32，33，34，35，其中将少量样品（例如，1-5μL）施加到专用的EM网格上，然后使用网格与转印纸的物理相互作用去除多余的溶液。该过程的时间通常由经验确定，因为冷冻样品的关键组成部分是玻璃体冰膜的厚度 – 如果冰太厚，则成像质量会由于电子束的散射增加而急剧下降，而太薄的冰会限制蛋白质取向和/或从网格箔孔的中心排除颗粒36.这种对单颗粒冷冻机电偶术完美冰厚的依赖导致了可以冷冻样品的各种技术和设备，包括机器人37，38，微流体42和超声波或喷涂设备27，39，40，41，42，43，44.近年来，一些最流行的样品制备设备依赖于使用机器人技术，使用印迹和暴跌技术自动冷冻样品45。虽然这些设备旨在可重复地为成像创建适当的冰厚度，但它们通常对于单个实验室来说仍然过于昂贵，并且通常以每小时的使用费率在cryoEM设施中找到。近年来，最初的手动印迹和插孔技术已重新投入使用3，47，48，49，50，51，52。事实上，手动操作的印迹和插孔设备可以实现高质量的冷冻EM网格，而成本只是机器人对应物的一小部分。此外，手动印迹还为用户提供了对印迹的更多控制，因为研究人员可以根据每个单独的样品和研究问题调整印迹类型（即网格的反转印，网格的前印转印等）和印迹时间。

在本文中，我们将详细介绍如何使用传统的手动转印和骤入式玻璃化装置以及定制设计的杜瓦瓶平台53来有效冷冻生物样品。提供了最佳实践，包括冷冻剂的制备、网格处理、样品应用和印迹，以及常见的陷阱和如何克服这些障碍的建议。讨论了如何提高网格制备之间的冰厚度再现性以及如何根据生物样品类型修改样品印迹的建议。鉴于本手稿中描述的手动柱塞的购买和操作成本低廉，全球的实验室可以以具有成本效益和可重复的方式为冷冻电镜制备生物标本。

Protocol

1. 准备手动插孔环境 注：预计运行时间：5-30 分钟 将手动柱塞放置在4°C的冷室中，加湿器可以放在同一位置，以保持房间接近100%相对湿度（RH）（图1A）。注意：请查阅机构的环境健康和安全指南，了解手动柱塞的安全位置和建议的操作。 在准备电网之前，打开冷室中的加湿器，以确保冷室的RH≥95%。注：低湿度下的网格制备可能?…

Representative Results

成功执行此处描述的印迹和暴跌方案将导致一层薄而均匀的玻璃体冰，该玻璃体冰层没有任何六角形冰，污染物和可以在电子显微镜下观察到的大梯度不可用的冰（图3）。转印纸与网格表面的接触不一致、过早取下印迹纸或在网格接触期间移动印迹纸都会降低玻璃体冰的质量，并导致整个EM网格上的冰厚度不一致（图4） <p class="jove_content" fo:keep-toget…

Discussion

通过单颗粒低温电子显微镜（cryoEM）对用于成像的生物标本进行玻璃化仍然是成功进行结构测定的关键步骤。该协议中描述的手动印迹和切入方法代表了一种经济高效，可靠且可靠的方法，用于在玻璃体冰薄膜中快速冷冻生物样品以进行冷冻EM成像。使用手稿中概述的方法，研究人员将能够组装和操作手动柱塞，制备适用于闪冻生物样品的冷冻剂，并手动印迹和插入含有生物标本的EM网格。虽然这…

Declarações

The authors have nothing to disclose.

Acknowledgements

我们感谢Herzik实验室成员对这份手稿和视频内容的批判性思考和反馈。M.A.H.Jr.由NIH R35 GM138206和Searle学者提供支持。H.P.M.N由分子生物物理学培训补助金（NIH T32 GM008326）提供支持。我们还要感谢斯克里普斯研究所的Bill Anderson，Charles Bowman和Gabriel Lander博士帮助设计，组装和测试视频中显示的手动柱塞。

Materials

4 slot grid storage box Ted Pella 160-40
14 gauge flat metal dispensing tip Amazon B07M7YWWLT
22×22 mm square glass coverslip Sigma C9802-1PAK
60 mm glass Petri dish to store grids Fisher 08-747A
100 mm glass Petri dish to store Whatman paper Fisher 08-747D
150 mm glass Petri dish to store Whatman paper Fisher 08-747F
250 mL beaker Fisher 02-555-25B
Blue styrofoam dewar Spear Lab FD-500
Brass ethane vessel Lasco 17-4075
Clamping tweezers Ted Pella 38825
Delicate task wipes Fisher 06-666
Dual-stage regulator with control valve Airgas Y12N245D580-AG
Dewer grid base UCSD
Ethane platform UCSD
Ethane propane tank Praxair ET PR50ZU-G ethane (50%) : propane (50%) in a high-pressure tank
Ethane tank Praxair UN1035 ethane (100%)
Flexible arm task light Amscope LED-11CR
Grids (UltrAufoil R 1.2/1.3 300 mesh) Electron Microscopy Sciences Q325AR1.3
Humidifier Target 719438
Hygrometer ThermoPro B01H1R0K68
Lab coat UCSD
Liquid Nitrogen dewar Worthington LD4
Liquid Nitrogen gloves Fisher 19-059-925
Manual plunger stand (black stand + foot pedal) UCSD
Mark 5 (plunging platform) UCSD
Nitrile gloves VWR 82026-424
P20 pipette Eppendorf 13-690-029
PCR tubes Eppendorf E0030124286
Pipette tips ibis scientific 63300005
Ring lamp Amazon B07HMR4H8G
Safety glasses UCSD
Scissors Amazon Fiskars 01-004761J
Screw driver Ironside 354711
Tape Fisher 15-901-10R
Tweezer to transfer grid box Amazon LTS-3
Tygon tubing Fisher 14-171-130
Whatman blotting paper Fisher 1001-090
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Tags

Keywords: Manual Blot-and-plunge FreezingBiological SpecimensSingle-particle Cryogenic Electron MicroscopyTransmission Electron MicroscopyCryogen DewarManual PlungerEthane VesselPlunging ArmCryogen PreparationLiquid NitrogenEthane Gas
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Nguyen, H. P. M., McGuire, K. L., Cook, B. D., Herzik, Jr., M. A. Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy. J. Vis. Exp. (180), e62765, doi:10.3791/62765 (2022).
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