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

Summary

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

Abstract

Imaging biological specimens with electrons for high-resolution structure determination by single-particle cryogenic electron microscopy (cryoEM) requires a thin layer of vitreous ice containing the biomolecules of interest. Despite numerous technological advances in recent years that have propelled single-particle cryoEM to the forefront of structural biology, the methods by which specimens are vitrified for high-resolution imaging often remain the rate-limiting step. Although numerous recent efforts have provided means to overcome hurdles frequently encountered during specimen vitrification, including the development of novel sample supports and innovative vitrification instrumentation, the traditional manually operated plunger remains a staple in the cryoEM community due to the low cost to purchase and ease of operation. Here, we provide detailed methods for using a standard, guillotine-style manually operated blot-and-plunge device for the vitrification of biological specimens for high-resolution imaging by single-particle cryoEM. Additionally, commonly encountered issues and troubleshooting recommendations for when a standard preparation fails to yield a suitable specimen are also described.

Introduction

Single-particle cryogenic electron microscopy (cryoEM) is a powerful structural technique that can be used to solve structures of dynamic biological specimens to near-atomic resolution^1,2,3,4. Indeed, recent advances in direct electron detector technologies^{4,5,6,7,8,9,10}, improvements in electron sources

Protocol

1. Prepare the manual plunging environment

NOTE: Estimated operating time: 5-30 minutes

1. Locate the manual plunger in a 4 °C cold room where a humidifier can be co-located to maintain the room close to 100% relative humidity (RH) (Figure 1A).
   CAUTION: Please consult with the institution's Environmental Health and Safety guidelines for the safe location of the manual plunger and recommended operations.
2. Prior to grid preparation, turn on the humidifier in the cold room to ensure the RH of the cold room is ≥ 95 %.
   NOTE: Grid preparation in low humidity can result i....

Results

Successful execution of the blot-and-plunge protocol described here will result in a thin, uniform layer of vitreous ice that is free of any hexagonal ice, contaminants, and large gradients of unusable ice which can be observed under the electron microscope (Figure 3). Inconsistent contact of the blotting paper with the grid surface, prematurely removing the blotting paper, or moving the blotting paper during grid contact can decrease the quality of the vitreous ice and lead to inconsistent .......

Discussion

The vitrification of biological specimens for imaging by single-particle cryogenic electron microscopy (cryoEM) remains a critically important step for successful structure determination. The manual blot-and-plunge method described in this protocol represents a cost-effective, reliable, and robust method for quickly freezing biological samples in thin films of vitreous ice for cryoEM imaging. Using the methods outlined in the manuscript, researchers will be able to assemble and operate the manual plunger, prepare cryogen.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4 slot grid storage box	Ted Pella	160-40
14 gauge flat metal dispensing tip	Amazon	B07M7YWWLT
22x22 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