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

2018 ◽  
Author(s):  
Anchi Cheng ◽  
Edward T. Eng ◽  
Lambertus Alink ◽  
William J. Rice ◽  
Kelsey D. Jordan ◽  
...  
Keyword(s):  
High Resolution ◽  
Data Acquisition ◽  
Single Particle ◽  
Phase Error ◽  
Relaxation Times ◽  
Resolution Limit ◽  
High Resolution Data ◽  
Density Peaks ◽  
Beam Image ◽  
Automated Data Acquisition

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.

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

eLife ◽  
2017 ◽  
Vol 6 ◽  
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.

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

2019 ◽  
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.

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

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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