Challenges in making ideal cryo-EM samples

Han, Bong-Gyoon; Avila-Sakar, Agustin; Remis, Jonathan; Glaeser, Robert M.

doi:10.1016/j.sbi.2023.102646

Cited by 20 publications

(5 citation statements)

References 52 publications

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“…Recent studies have shown that in the majority of cryo-EM grids, the sample is located at the air water interface(Noble, Dandey, et al 2018). Complicating the situation, every different sample whether its protein, nucleoprotein or large virus-like particle may behave differently while plunge-freezing (B.-G. Han et al 2023; B. G. Han et al 2022; Weissenberger, Henderikx, and Peters 2021).…”

Section: Introductionmentioning

confidence: 99%

SPOT-RASTR - a cryo-EM specimen preparation technique that overcomes problems with preferred orientation and the air/water interface

Esfahani,

Randolph,

Peng

et al. 2024

Preprint

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In cryogenic electron microscopy (cryo-EM), specimen preparation remains a bottleneck despite recent advancements. Classical plunge freezing methods often result in issues like aggregation and preferred orientations at the air/water interface. Many alternative methods have been proposed, but there remains a lack a universal solution, and multiple techniques are often required for challenging samples. Here, we demonstrate the use of lipid nanotubes with nickel NTA headgroups as a platform for cryo-EM sample preparation. His-tagged specimens of interest are added to the tubules, and they can be frozen by conventional plunge freezing. We show that the nanotubes protect samples from the air/water interface and promote a wider range of orientations. The reconstruction of average subtracted tubular regions (RASTR) method allows for the removal of the nanotubule signal from the cryo-EM images resulting in isolated images of specimens of interest. Testing with β-galactosidase validates the method's ability to capture particles at lower concentrations, overcome preferred orientations, and achieve near-atomic resolution reconstructions. Since the nanotubules can be identified and targeted automatically at low magnification, the method enables fully automated data collection. Furthermore, the particles on the tubes can be automatically identified and centered using 2D classification enabling particle picking without requiring prior information. Altogether, our approach that we call specimen preparation on a tube RASTR (SPOT-RASTR) holds promise for overcoming air-water interface and preferred orientation challenges and offers the potential for fully automated cryo-EM data collection and structure determination.

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Section: Introductionmentioning

confidence: 99%

SPOT-RASTR - a cryo-EM specimen preparation technique that overcomes problems with preferred orientation and the air/water interface

Esfahani,

Randolph,

Peng

et al. 2024

Preprint

View full text Add to dashboard Cite

show abstract

“…The optimal concentration must be determined empirically for each sample. For samples that cannot be concentrated, the use of support films (thin carbon, graphene, graphene oxide or streptavidin monolayers) on cryo-EM grids can facilitate the achievement of a better particle density, although some of these supports introduce additional background noise or processing challenges (Han et al, 2023).…”

Section: Optimizing the Sample Before Vitrificationmentioning

confidence: 99%

Cryo-EM sample preparation for high-resolution structure studies

Wang,

Zimanyi

2024

Acta Cryst Sect F

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High-resolution structures of biomolecules can be obtained using single-particle cryo-electron microscopy (SPA cryo-EM), and the rapidly growing number of structures solved by this method is encouraging more researchers to utilize this technique. As with other structural biology methods, sample preparation for an SPA cryo-EM data collection requires some expertise and an understanding of the strengths and limitations of the technique in order to make sensible decisions in the sample-preparation process. In this article, common strategies and pitfalls are described and practical advice is given to increase the chances of success when starting an SPA cryo-EM project.

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“…With the rapid progresses in instrumentation and data analysis methods/software, cryo-electron microscopy (cryo-EM) single-particle analysis has emerged as a powerful tool for the structural characterization of biomolecules, particularly macromolecular complexes [1][2][3][4][5][6] . However, for low abundance and/or fragile macromolecular complexes, as well as biomolecules exhibiting preferred orientation, disintegration or aggregation problems related to air-water interface (AWI) interference, purification and vitrification process has become a bottleneck for cryo-EM [7][8][9][10][11][12] . A promising approach involves the utilization of affinity grids for on-grid specimen purification [13][14][15][16][17] .…”

Section: Introductionmentioning

confidence: 99%

Development of immobilized antibody-based affinity grid strategy and application in on-grid purification of target proteins for high-resolution cryo-EM

Zhao,

Hong,

Wang

et al. 2024

Preprint

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In cryo-electron microscopy (cryo-EM), sample preparation, especially for rare or fragile macromolecular assemblies and those suffering from air-water interface denaturation and particle orientation distribution problems, is a major bottleneck. Here, we developed and characterized an immobilized antibody-based affinity grid (IAAG) strategy based on the high-affinity PA tag/NZ-1 antibody epitope tag system. We used Pyr-NHS as a linker to immobilize NZ-1 Fab on the graphene oxide or carbon covered grid surface. We showed that the IAAG grid can enrich the PA-tagged target proteins, and overcome preferred orientation problems. Furthermore, we demonstrated that our IAAG strategy can be utilized for on-grid purification of low-abundance target complexes from cell lysates and enables atomic resolution cryo-EM. This approach greatly streamlines the purification process, reduces the need for large quantities of biological samples, and addresses common challenges encountered in cryo-EM sample preparation. Collectively, our IAAG strategy provides an efficient and robust means for combined sample purification and vitrification feasible for high-resolution cryo-EM. This approach also has the potential for broader applicability in cryo-EM and cryo-ET.

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Challenges in making ideal cryo-EM samples

Cited by 20 publications

References 52 publications

SPOT-RASTR - a cryo-EM specimen preparation technique that overcomes problems with preferred orientation and the air/water interface

SPOT-RASTR - a cryo-EM specimen preparation technique that overcomes problems with preferred orientation and the air/water interface

Cryo-EM sample preparation for high-resolution structure studies

Development of immobilized antibody-based affinity grid strategy and application in on-grid purification of target proteins for high-resolution cryo-EM

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