Experimental Assessment of Cryo Single Particle Data Acquisition by “Beam-image Shift”

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.

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Beam image-shift accelerated data acquisition for near-atomic resolution single-particle cryo-electron tomography

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s0108767321097907 ◽

2021 ◽

Vol 77 (a1) ◽

pp. a209-a209

Author(s):

Jonathan Bouvette ◽

Mario Borgnia ◽

Hsuan-Fu Liu ◽

Alberto Bartesaghi ◽

Roel Schaaper ◽

...

Keyword(s):

Data Acquisition ◽

Single Particle ◽

Electron Tomography ◽

Atomic Resolution ◽

Beam Image ◽

Cryo Electron Tomography ◽

Resolution Single

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Advances in Single Particle Analysis Data Acquisition

Microscopy and Microanalysis ◽

10.1017/s1431927619005798 ◽

2019 ◽

Vol 25 (S2) ◽

pp. 1012-1013 ◽

Cited By ~ 4

Author(s):

Stan Konings ◽

Maarten Kuijper ◽

Jeroen Keizer ◽

Fanis Grollios ◽

Tjerk Spanjer ◽

...

Keyword(s):

Data Acquisition ◽

Single Particle ◽

Analysis Data ◽

Particle Analysis ◽

Single Particle Analysis

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Using the Volta phase plate with defocus for cryo-EM single particle analysis

eLife ◽

10.7554/elife.23006 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 79

Author(s):

Radostin Danev ◽

Dimitry Tegunov ◽

Wolfgang Baumeister

Keyword(s):

Data Acquisition ◽

Single Particle ◽

Hybrid Approach ◽

Particle Analysis ◽

Phase Plate ◽

Single Particle Analysis ◽

Resolution Limit ◽

Contrast Transfer Function ◽

Acquisition Speed ◽

Acquisition Method

Previously, we reported an in-focus data acquisition method for cryo-EM single-particle analysis with the Volta phase plate (Danev and Baumeister, 2016). Here, we extend the technique to include a small amount of defocus which enables contrast transfer function measurement and correction. This hybrid approach simplifies the experiment and increases the data acquisition speed. It also removes the resolution limit inherent to the in-focus method thus allowing 3D reconstructions with resolutions better than 3 Å.

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Hardware Considerations to Optimize Zernike Phase Contrast TEM for Cryo-tomography and Single Particle Data Acquisition

Microscopy and Microanalysis ◽

10.1017/s1431927610054188 ◽

2010 ◽

Vol 16 (S2) ◽

pp. 554-555

Author(s):

B Armbruster ◽

J Brink ◽

R Danev ◽

TC Isabell ◽

M Kawasaki ◽

...

Keyword(s):

Data Acquisition ◽

Single Particle ◽

Phase Contrast

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

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High-resolution cryo-EM reconstructions in the presence of substantial aberrations

10.1101/798280 ◽

2019 ◽

Cited By ~ 2

Author(s):

Raquel Bromberg ◽

Yirui Guo ◽

Dominika Borek ◽

Zbyszek Otwinowski

Keyword(s):

High Resolution ◽

Single Particle ◽

Analytical Formula ◽

Information Loss ◽

Optical Aberrations ◽

Shift Method ◽

Single Particle Reconstruction ◽

Beam Image ◽

The Cost ◽

Particle Reconstruction

The beam-image shift method accelerates data acquisition in cryo-EM single particle reconstruction (cryo-EM SPR) by fast repositioning of the imaging area, but at the cost of more severe and complex optical aberrations.We analyze here how uncorrected anti-symmetric aberrations, such as coma and trefoil, affect cryo-EM SPR results, and then infer an analytical formula quantifying information loss due to their presence that explains why Fourier-shell coefficient (FSC)-based statistics may report significantly overestimated resolution if these aberrations are not fully corrected. We validate our analysis with reference-based aberration refinement for two cryo-EM SPR datasets acquired with a 200 kV microscope in the presence of coma exceeding 40 µm, and obtained 2.3 and 2.7 Å reconstructions for 144 and 173 kDa particles, respectively.Our results provide a description of an efficient approach for assessing information loss in cryo-EM SPR data acquired in the presence of higher-order aberrations and address inconsistent guidelines regarding the level of aberrations acceptable in cryo-EM SPR experiments.

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High-resolution cryo-EM using beam-image shift at 200 keV

10.1101/2020.01.21.914507 ◽

2020 ◽

Author(s):

Jennifer N. Cash ◽

Sarah Kearns ◽

Yilai Li ◽

Michael A. Cianfrocco

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Data Collection ◽

Data Quality ◽

Single Particle ◽

Aberration Correction ◽

Cryo Electron Microscopy ◽

Beam Image ◽

Recent Advances ◽

Shift Data

ABSTRACTRecent advances in single-particle cryo-electron microscopy (cryo-EM) data collection utilizes beam-image shift to improve throughput. Despite implementation on 300 keV cryo-EM instruments, it remains unknown how well beam-image shift data collection affects data quality on 200 keV instruments and how much aberrations can be computationally corrected. To test this, we collected and analyzed a cryo-EM dataset of aldolase at 200 keV using beam-image shift. This analysis shows that beam tilt on the instrument initially limited the resolution of aldolase to 4.9Å. After iterative rounds of aberration correction and particle polishing in RELION, we were able to obtain a 2.8Å structure. This analysis demonstrates that software correction of microscope aberrations can provide a significant improvement in resolution at 200 keV.

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Cryo-EM Performance Testing of Hardware and Data Acquisition Strategies

Microscopy ◽

10.1093/jmicro/dfab016 ◽

2021 ◽

Author(s):

Radostin Danev ◽

Haruaki Yanagisawa ◽

Masahide Kikkawa

Keyword(s):

Data Acquisition ◽

Performance Testing ◽

Test Sample ◽

Critical Parameters ◽

Site Specific ◽

Cryo Electron Microscopy ◽

Beam Image ◽

Acquisition Strategies ◽

Limited Experience ◽

Coma Aberration

Abstract The increasing popularity and adoption rate of cryo-electron microscopy is evidenced by a growing number of new microscope installations around the world. The quality and reliability of the instruments improved dramatically in recent years, but site-specific issues or unnoticed problems during installation could undermine productivity. Newcomers to the field may also have limited experience and/or low confidence in the capabilities of the equipment or their own skills. Therefore, it is recommended to perform an initial test of the complete cryo-EM workflow with an ‘easy’ test sample, such as apoferritin, before starting work with real and challenging samples. Analogous test experiments are also recommended for quantification of new data acquisition approaches or imaging hardware. Here, we present the results from our initial tests of a recently installed Krios G4 electron microscope equipped with two latest generation direct electron detector cameras—Gatan K3 and Falcon 4. Three beam-image shift-based data acquisition strategies were also tested. We detail the methodology and discuss the critical parameters and steps for performance testing. The two cameras performed equally, and the single and multi-shot per-hole acquisition schemes produced comparable results. We also evaluated the effects of environmental factors and optical flaws on data quality. Our results reaffirmed the exceptional performance of the software aberration correction in Relion in dealing with severe coma aberration. We hope that this work will help cryo-EM teams in their testing and troubleshooting of hardware and data collection approaches.

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High-resolution cryo-EM using beam-image shift at 200 keV

IUCrJ ◽

10.1107/s2052252520013482 ◽

2020 ◽

Vol 7 (6) ◽

pp. 1179-1187 ◽

Cited By ~ 1

Author(s):

Jennifer N. Cash ◽

Sarah Kearns ◽

Yilai Li ◽

Michael A. Cianfrocco

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Data Collection ◽

Single Particle ◽

Particle Motion ◽

Data Set ◽

Cryo Electron Microscopy ◽

Beam Image ◽

Shift Data ◽

Resolution Structure

Recent advances in single-particle cryo-electron microscopy (cryo-EM) data collection utilize beam-image shift to improve throughput. Despite implementation on 300 keV cryo-EM instruments, it remains unknown how well beam-image-shift data collection affects data quality on 200 keV instruments and the extent to which aberrations can be computationally corrected. To test this, a cryo-EM data set for aldolase was collected at 200 keV using beam-image shift and analyzed. This analysis shows that the instrument beam tilt and particle motion initially limited the resolution to 4.9 Å. After particle polishing and iterative rounds of aberration correction in RELION, a 2.8 Å resolution structure could be obtained. This analysis demonstrates that software correction of microscope aberrations can provide a significant improvement in resolution at 200 keV.

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