Sample deposition onto cryo-EM grids: from sprays to jets and back.

David P Klebl,Dimitrios Kontziampasis,Martin Trebbin,Stephen P Muench,Florian Kopf,Frank Sobott,Howard D White,Diana C F Monteiro

doi:10.1107/s2059798320002958

David P Klebl, Dimitrios Kontziampasis + Show 6 more

Open Access

https://doi.org/10.1107/s2059798320002958

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Abstract

Despite the great strides made in the field of single-particle cryogenic electron microscopy (cryo-EM) in microscope design, direct electron detectors and new processing suites, the area of sample preparation is still far from ideal. Traditionally, sample preparation involves blotting, which has been used to achieve high resolution, particularly for well behaved samples such as apoferritin. However, this approach is flawed since the blotting process can have adverse effects on some proteins and protein complexes, and the long blot time increases exposure to the damaging air-water interface. To overcome these problems, new blotless approaches have been designed for the direct deposition of the sample on the grid. Here, different methods of producing droplets for sample deposition are compared. Using gas dynamic virtual nozzles, small and high-velocity droplets were deposited on cryo-EM grids, which spread sufficiently for high-resolution cryo-EM imaging. For those wishing to pursue a similar approach, an overview is given of the current use of spray technology for cryo-EM grid preparation and areas for enhancement are pointed out. It is further shown how the broad aspects of sprayer design and operation conditions can be utilized to improve grid quality reproducibly.

Highlights

Over the last decade, cryo-electron microscopy has emerged as a major technique for the high-resolution structure determination of proteins and protein complexes (Callaway, 2015; Cheng, 2018)
We have previously reported the use of another variation of cryo-electron microscopy (cryo-EM) grid preparation, voltage-assisted spraying, to enable rapid mixing and freezing for time-resolved cryo-EM studies (TrEM; Kontziampasis et al, 2019)
To generate the spray in the voltage-assisted approach, three main mechanisms are used. (i) Liquid exits the nozzle at a high flow rate, leading to the formation of a liquid jet which eventually breaks up into droplets, (ii) a sheath of N2 gas flow is used to aid the breakup of the liquid jet and accelerate the droplets and (iii) an electric potential of 5 kV is applied to the liquid, destabilizing the jet and dispersing the droplets

Summary

Introduction

Cryo-electron microscopy (cryo-EM) has emerged as a major technique for the high-resolution structure determination of proteins and protein complexes (Callaway, 2015; Cheng, 2018). Typically 3 ml of protein solution is applied to a cryo-EM grid, with subsequent blotting leaving only a thin liquid film on the grid ( 100–200 nm thick). The thin film is vitrified, typically by plunging into liquid ethane, and can be transferred for imaging in the electron microscope. While the blotting method has undoubtedly been very successful in producing high-resolution sub-2 Aresolution structures (Zivanov et al, 2018), its widespread use, and the increasing popularity of cryo-EM in general, have revealed its shortcomings. A large amount of sample is wasted through blotting, with 99.9% of the sample being removed by the blotting paper

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