Better Cryo-EM Specimen Preparation: How to Deal with the Air–Water Interface?

Liu, Nan; Wang, Hongwei

doi:10.1016/j.jmb.2022.167926

Cited by 22 publications

(10 citation statements)

References 105 publications

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“…The ‘resolution revolution’ of cryogenic-electron microscopy (cryo-EM) marks a significant shift in the field of structural biology 1 . However, the continued growth of cryo-EM single particle analysis (SPA) structural biology faces an unexpected problem at the step of sample grid preparation — sample damage 1–4 1,5,6 . The standard method for cryo-EM SPA sample grid preparation is plunge freezing, where small volumes of sample are applied onto a pretreated cryo-EM grid, blotted, and rapidly plunge-frozen into a cryogen such as liquid ethane.…”

Section: Mainmentioning

confidence: 99%

“…To mitigate the AWI problem, researchers have developed a variety of solutions. These include the addition of mild detergents to the sample 6 , the use of chemical crosslinking 3,13,14 , and adsorbing or tethering samples to surfaces to prevent sample contact with the AWI 12,15–24 . More sophisticated advanced techniques involve rapidly freezing the sample before the proteins can diffuse to the AWIs 25– 27 .…”

Section: Mainmentioning

confidence: 99%

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Small LEA proteins as an effective air-water interface protectant for fragile samples during cryo-EM grid plunge freezing

Abe,

Lim

2024

Preprint

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Sample loss due to air-water interface (AWI) interactions is a significant challenge during cryo-electron microscopy (cryo-EM) sample grid plunge freezing. We report that small Late Embryogenesis Abundant (LEA) proteins, which naturally bind to AWI, can protect samples from AWI damage during plunge freezing. This protection is demonstrated with two LEA proteins from nematodes and tardigrades, which rescued the cryo-EM structural determination outcome of two fragile multisubunit protein complexes.

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Section: Mainmentioning

confidence: 99%

Section: Mainmentioning

confidence: 99%

Small LEA proteins as an effective air-water interface protectant for fragile samples during cryo-EM grid plunge freezing

Abe,

Lim

2024

Preprint

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“…To address these issues, other groups have developed methods to prevent nucleosome-protein complexes from contacting the AWI [15][16][17][18][19][20][21] . However, these methods either require the use of sophisticated equipment and/or materials for grid preparation.…”

Section: Introductionmentioning

confidence: 99%

Functionalized graphene-oxide grids enable high-resolution cryo-EM structures of the SNF2h-nucleosome complex without crosslinking

Chio

Palovcak

Autzen

et al. 2023

Preprint

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Single-particle cryo-EM is widely used to determine enzyme-nucleosome complex structures. However, cryo-EM sample preparation remains challenging and inconsistent due to complex denaturation at the air-water interface (AWI). To address this issue, we developed graphene-oxide-coated EM grids functionalized with either single-stranded DNA (ssDNA) or thiol-poly(acrylic acid-co-styrene) (TAASTY) co-polymer. These grids protect complexes between the chromatin remodeler SNF2h and nucleosomes from the AWI and facilitated collection of high-quality micrographs of intact SNF2h-nucleosome complexes in the absence of crosslinking. The data yields maps ranging from 2.3 to 3 angstrom in resolution. 3D variability analysis reveals nucleotide-state linked conformational changes in SNF2h bound to a nucleosome. In addition, the analysis provides structural evidence for asymmetric coordination between two SNF2h protomers acting on the same nucleosome. We envision these grids will enable similar detailed structural analyses for other enzyme-nucleosome complexes and possibly other protein-nucleic acid complexes in general.

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“…It has been shown through recent studies that during freezing, very often macromolecules can interact with the air-water interface (AWI). This interaction can cause the macromolecules to denature, dissociate, adapt preferential orientations, or avoid the holes completely and stick to the carbon support (D’Imprima et al, 2019; Drulyte et al, 2018; Kampjut et al, 2021; Liu & Wang, 2023; Noble, Dandey, et al, 2018; Noble, Wei, et al, 2018). Along with protein biochemistry, the propensity of macromolecules to adopt preferential orientations is perhaps a common rate-limiting step in high-resolution structure determination.…”

Section: Introductionmentioning

confidence: 99%

Factors affecting macromolecule orientations in thin films formed in cryo-EM

Yadav,

Vinothkumar

2023

Preprint

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The formation of vitrified thin film embedded with randomly oriented macromolecules is an essential prerequisite for cryogenic sample electron microscopy. Most commonly, this is achieved using the plunge freeze method described nearly 40 years ago. Although this is a robust method, the behaviour of different macromolecules shows great variation upon freezing and often needs to be optimized to obtain an isotropic, high-resolution reconstruction. For a macromolecule in such a film, the probability of encountering the air-water interface in the time between blotting and freezing and adopting preferred orientations is very high. 3D reconstruction using preferentially oriented particles often leads to anisotropic and uninterpretable maps. Currently, there are no general solutions to this prevalent issue, but several approaches largely focusing on sample preparation with the use of additives and novel grid modifications have been attempted. In this study, through an analysis of selected well-studied macromolecules, the effect of physical and chemical factors on the orientations of macromolecules was investigated, and important parameters that determine the behaviour of proteins on cryo-EM grids were revealed. These insights highlight the nature of interactions that cause preferred orientations and can be utilized to systematically address orientation bias for any given macromolecule and provide a framework to design small-molecule additives to enhance sample stability and behaviour.

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Better Cryo-EM Specimen Preparation: How to Deal with the Air–Water Interface?

Cited by 22 publications

References 105 publications

Small LEA proteins as an effective air-water interface protectant for fragile samples during cryo-EM grid plunge freezing

Small LEA proteins as an effective air-water interface protectant for fragile samples during cryo-EM grid plunge freezing

Functionalized graphene-oxide grids enable high-resolution cryo-EM structures of the SNF2h-nucleosome complex without crosslinking

Factors affecting macromolecule orientations in thin films formed in cryo-EM

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