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

Abstract

Automated 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.

New tools for automated high-resolution cryo-EM structure determination in RELION-3

“…Because it is much faster to shift the electron beam instead, one could achieve higher data acquisition rates by using beam-shift to image multiple holes before shifting the stage. Beam-shift is already used extensively to take multiple images within a single hole, but because it introduces beam tilt, its use for imaging multiple holes has only recently attracted attention ( Cheng et al, 2018 ). Although it is possible to actively compensate for the introduced beam tilt using the microscope’s deflection coils ( Eades, 2006 ; Glaeser et al, 2011 ), this procedure puts more stringent requirements on microscope alignment.…”

Structural basis of redox modulation on chloroplast ATP synthase

“…The exposure time was 6 s with a subframe rate of 200 ms for each movie, accumulating to a total dosage of 43.5 e − /Å 2 . For the images of oxidized sample, the beam-image shift was applied to promote the data acquisition speed 58 . The data collection was automated using SerialEM program (version 3.7) with customized macros 59 .…”

Structure of the bacterial ribosome at 2 Å resolution

“…Movies were collected with the defocus range -0.6 to -1.5 µm and the total dose was 39.89 e -/Å 2 split over 40 frames. One movie was collected for each hole, with image shift used to collect a series of 3x3 holes for faster data collection (Cheng et al, 2018), and stage shift used to move to the center hole. Based on the 1.2/1.3 grid hole specification, this should correspond to a maximum image shift of ~1.8 μm, although the true image shift used was not measured.…”