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

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.\n\nFor optimal efficiency, all automated data acquisition software packages employ some degree of beam-image shift because this method is fast and accurate (+/- 0.1 m). 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 {pi}/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.\n\nIn 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 {pi}/4 phase error does not limit the validity of the 3D reconstruction from single-particle averaging beyond the {pi}/4 resolution limit.