Combining high throughput and high quality for cryo-electron microscopy data collection - PubMed
- ️Wed Jan 01 2020
Review
Combining high throughput and high quality for cryo-electron microscopy data collection
Felix Weis et al. Acta Crystallogr D Struct Biol. 2020.
Abstract
Cryo-electron microscopy (cryo-EM) can be used to elucidate the 3D structure of macromolecular complexes. Driven by technological breakthroughs in electron-microscope and electron-detector development, coupled with improved image-processing procedures, it is now possible to reach high resolution both in single-particle analysis and in cryo-electron tomography and subtomogram-averaging approaches. As a consequence, the way in which cryo-EM data are collected has changed and new challenges have arisen in terms of microscope alignment, aberration correction and imaging parameters. This review describes how high-end data collection is performed at the EMBL Heidelberg cryo-EM platform, presenting recent microscope implementations that allow an increase in throughput while maintaining aberration-free imaging and the optimization of acquisition parameters to collect high-resolution data.
Keywords: coma-free imaging; cryo-electron microscopy; fringe-free imaging; high-end data collection.
open access.
Figures

Fringe-free illumination. (a, b) Image of the beam without (a) and with (b) fringe-free illumination. The beam diameter is 460 nm and the images were recorded at spot 9 over a 20 s exposure with a pixel size of 1.34 Å. (c, d) Acquisition scheme without (c) and with (d) fringe-free illumination. The sample is embedded in a thin layer of ice over a holey carbon film with 2 µm diameter holes. The blue squares represent the imaged area in the context of a 1.04 Å pixel. Without fringe-free illumination (c) the beam size, depicted by an orange circle, needs to be ∼1 µm in diameter in order to avoid seeing fringes within the imaged area, limiting the number of acquisitions to five within the hole. In the case of fringe-free illumination (d), a beam size of 600 nm is enough to cover the camera, allowing up to ten acquisitions.
Similar articles
-
Subtomogram averaging from cryo-electron tomograms.
Leigh KE, Navarro PP, Scaramuzza S, Chen W, Zhang Y, Castaño-Díez D, Kudryashev M. Leigh KE, et al. Methods Cell Biol. 2019;152:217-259. doi: 10.1016/bs.mcb.2019.04.003. Epub 2019 May 15. Methods Cell Biol. 2019. PMID: 31326022 Review.
-
Cryo-electron Tomography Remote Data Collection and Subtomogram Averaging.
Sheng Y, Morris K, Radecke J, Zhang P. Sheng Y, et al. J Vis Exp. 2022 Jul 12;(185):10.3791/63923. doi: 10.3791/63923. J Vis Exp. 2022. PMID: 35913165 Free PMC article.
-
Cryo-Electron Tomography and Subtomogram Averaging.
Wan W, Briggs JA. Wan W, et al. Methods Enzymol. 2016;579:329-67. doi: 10.1016/bs.mie.2016.04.014. Epub 2016 Jun 22. Methods Enzymol. 2016. PMID: 27572733 Review.
-
Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography.
Morado DR, Hu B, Liu J. Morado DR, et al. J Vis Exp. 2016 Jan 30;(107):e53608. doi: 10.3791/53608. J Vis Exp. 2016. PMID: 26863591 Free PMC article.
-
In Situ Studies of Mitochondrial Translation by Cryo-Electron Tomography.
Englmeier R, Förster F. Englmeier R, et al. Methods Mol Biol. 2021;2192:243-268. doi: 10.1007/978-1-0716-0834-0_18. Methods Mol Biol. 2021. PMID: 33230778
Cited by
-
Burley SK, Berman HM, Chiu W, Dai W, Flatt JW, Hudson BP, Kaelber JT, Khare SD, Kulczyk AW, Lawson CL, Pintilie GD, Sali A, Vallat B, Westbrook JD, Young JY, Zardecki C. Burley SK, et al. Biophys Rev. 2022 Dec 2;14(6):1281-1301. doi: 10.1007/s12551-022-01013-w. eCollection 2022 Dec. Biophys Rev. 2022. PMID: 36474933 Free PMC article. Review.
-
Structure of the human 20S U5 snRNP.
Schneider S, Brandina I, Peter D, Lagad S, Fraudeau A, Portell-Montserrat J, Tholen J, Zhao J, Galej WP. Schneider S, et al. Nat Struct Mol Biol. 2024 May;31(5):752-756. doi: 10.1038/s41594-024-01250-5. Epub 2024 Mar 11. Nat Struct Mol Biol. 2024. PMID: 38467877 Free PMC article.
-
Square beams for optimal tiling in TEM.
Chua EY, Alink LM, Kopylov M, Johnston J, Eisenstein F, de Marco A. Chua EY, et al. bioRxiv [Preprint]. 2023 Oct 9:2023.07.29.551095. doi: 10.1101/2023.07.29.551095. bioRxiv. 2023. PMID: 37873376 Free PMC article. Updated. Preprint.
-
Recent advances and current trends in cryo-electron microscopy.
Guaita M, Watters SC, Loerch S. Guaita M, et al. Curr Opin Struct Biol. 2022 Dec;77:102484. doi: 10.1016/j.sbi.2022.102484. Epub 2022 Oct 28. Curr Opin Struct Biol. 2022. PMID: 36323134 Free PMC article. Review.
-
Application of super-resolution and correlative double sampling in cryo-electron microscopy.
Sheng Y, Harrison PJ, Vogirala V, Yang Z, Strain-Damerell C, Frosio T, Himes BA, Siebert CA, Zhang P, Clare DK. Sheng Y, et al. Faraday Discuss. 2022 Nov 8;240(0):261-276. doi: 10.1039/d2fd00049k. Faraday Discuss. 2022. PMID: 35938521 Free PMC article.
References
-
- Christenson, K. K. & Eades, J. A. (1986). Ultramicroscopy, 19, 191–194.
-
- Eades, A. (2006). Ultramicroscopy, 106, 432–438. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Research Materials