Cryoprotectant-free buffer containing ∼1 mg ml −1 of the biomolecule of interest is dispensed onto a glow-discharge cleaned and charged, 10–50 nm-thick carbon or gold `foil' supported by a 200–400 mesh copper or gold grid. Biomolecule samples must be expressed, isolated and purified. The basic principles and methods in current use were developed in the 1980s (Dubochet et al., 1988 ), and many recent advances in sample preparation technology are rooted in ideas and methods developed at that time. It allows the structural study of systems that have been intractable to crystallization and is becoming a go-to method for initial attempts at structure determination.Īs in cryo-crystallography, the key challenges in single-particle cryo-EM are associated with sample preparation and handling. Unlike X-ray crystallography, cryo-EM requires only a small amount of biomolecular sample dispersed in solution.
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Major investments in new cryo-EM facilities and development of easy-to-use software (Punjani et al., 2017 Zivanov et al., 2018 ) have greatly expanded access, especially to non-experts. Development of high-efficiency, high frame rate direct electron detectors (Faruqi & McMullan, 2018 ), algorithms for correcting acquired movies for electron-beam-induced motion (Zheng et al., 2017 ), and computational tools for classifying and averaging 10 5–10 6 molecular images have dramatically increased achievable resolution and throughput. Single-particle cryo-electron microscopy (cryo-EM) (Frank, 2002 ) has emerged as a powerful approach to obtaining near atomic resolution structures of large biomolecular complexes, membrane proteins, and other targets of major scientific, pharmaceutical and biotechnological interest (Cheng, 2015, 2018 Glaeser, 2016 b, 2019 Vinothkumar & Henderson, 2016 Lyumkis, 2019 ).
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The use of liquid nitrogen as the primary coolant may allow manual and automated workflows to be simplified and may reduce sample stresses that contribute to beam-induced motion.
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Using an automated cryocooling instrument developed for cryocrystallography that combines high plunge speeds with efficient removal of cold gas, we show that single-particle cryo-EM samples on commercial grids can be routinely vitrified using only boiling nitrogen and obtain apoferritin datasets and refined structures with 2.65 Å resolution. Experiments over the last 15 years have shown that cooling rates required to vitrify pure water are only ∼250 000 K s −1, at the low end of earlier estimates, and that the dominant factor that has limited cooling rates of small samples in liquid nitrogen is sample precooling in cold gas present above the liquid cryogen surface, not the Leidenfrost effect.
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However, ethane and propane are flammable, they must be liquified in liquid nitrogen immediately before cryo-EM sample preparation, and cryocooled samples must be transferred to liquid nitrogen for storage, complicating workflows and increasing the chance of sample damage during handling.
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These liquid cryogens have a large difference between their melting and boiling temperatures and so can absorb substantial heat without formation of an insulating vapor layer adjacent to a cooling sample. Based on work by Dubochet and others in the 1980s and 1990s, samples for single-particle cryo-electron microscopy (cryo-EM) have been vitrified using ethane, propane or ethane/propane mixtures.