DOI: 10.1101/306241. High Resolution Single Particle Cryo-Electron Microscopy using Beam-Image Shift

High Resolution Single Particle Cryo-Electron Microscopy using Beam-Image Shift

10.1101/306241

Crossref posted-content

Cold Spring Harbor Laboratory

[] (246)

Abstract

AbstractAutomated data acquisition is now used widely for the single-particle averaging approach to reconstruction of three-dimensional (3D) volumes of biological complexes preserved in vitreous ice and imaged in a transmission electron microscope (cryo-EM). Automation has become integral to this method because of the very large number of particle images required to be averaged in order to overcome the typically low signal-to-noise ratio of these images.For optimal efficiency, all automated data acquisition software packages employ some degree of beam-image shift because this method is fast and accurate (+/− 0.1 μm). Relocation to a targeted area under low-dose conditions can only be achieved using stage movements in combination with multiple iterations or long relaxation times, both reducing efficiency. It is, however, well known that applying beam-image shift induces beam-tilt and hence structure phase error. A π/4 phase error is considered as the worst that can be accepted, and is used as an argument against the use of any beam-image shift for high resolution data collection.In this study, we performed cryo-EM single-particle reconstructions on a T20S proteasome sample using applied beam-image shifts corresponding to beam tilts from 0 to 10 mrad. To evaluate the results we compared the FSC values, and examined the water density peaks in the 3D map. We conclude that the π/4 phase error does not limit the validity of the 3D reconstruction from single-particle averaging beyond the π/4 resolution limit.

Bibliography

Cheng, A., Eng, E. T., Alink, L., Rice, W. J., Jordan, K. D., Kim, L. Y., Potter, C. S., & Carragher, B. (2018). High Resolution Single Particle Cryo-Electron Microscopy using Beam-Image Shift.

Authors 8

Anchi Cheng (first)
Edward T. Eng (additional)
Lambertus Alink (additional)
William J. Rice (additional)
Kelsey D. Jordan (additional)
Laura Y. Kim (additional)
Clinton S. Potter (additional)
Bridget Carragher (additional)

References 17 Referenced 6

10.1016/bs.mie.2016.04.008 / Methods Enzym. / Strategies for Automated CryoEM Data Collection Using Direct Detectors (2016)
Crowther, R.A. , 2016. The Resolution Revolution: Recent Advances In cryoEM, Methods in Enzymology. Elsevier Science.
10.1016/j.jsb.2010.12.005
10.1038/nmeth.4461
10.1038/nmeth.2472
10.1016/j.jsb.2005.07.007
10.1002/jcc.20084
10.1038/nmeth.4169
10.1016/j.jsb.2015.08.008
10.1016/j.jsb.2003.11.007
10.1016/j.jmb.2003.07.013
10.1016/j.jsb.2012.09.006
10.1016/j.jsb.2005.03.010
10.1093/jmicro/dfv369 / Microsc. / Automated data collection in single particle electron microscopy (2016)
10.1016/S0304-3991(97)00102-2
10.1016/j.jsb.2009.01.004
10.1038/nmeth.4193

Dates

Type	When
Created	7 years, 3 months ago (April 23, 2018, 5:55 a.m.)
Deposited	5 years, 7 months ago (Jan. 17, 2020, 4:49 p.m.)
Indexed	1 year ago (Aug. 4, 2024, 12:37 a.m.)
Issued	7 years, 3 months ago (April 23, 2018)
Published	7 years, 3 months ago (April 23, 2018)

Funders 0

None

BibTeX

@article{Cheng_2018, title={High Resolution Single Particle Cryo-Electron Microscopy using Beam-Image Shift}, url={http://dx.doi.org/10.1101/306241}, DOI={10.1101/306241}, publisher={Cold Spring Harbor Laboratory}, author={Cheng, Anchi and Eng, Edward T. and Alink, Lambertus and Rice, William J. and Jordan, Kelsey D. and Kim, Laura Y. and Potter, Clinton S. and Carragher, Bridget}, year={2018}, month=apr }

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