PubReading [37] - High resolution single particle cryo-electron microscopy using beam-image shift - A. Cheng, B. Carragher et al.

<p>Automated data acquisition is used widely for <strong>single-particle reconstruction</strong> of three-dimensional (<strong>3D</strong>) volumes of biological complexes preserved in vitreous ice and imaged in a transmission electron microscope. Automation has become integral to this method because of the very large number of particle images required in order to overcome the typically low signal-to-noise ratio of these images. For optimal efficiency, automated data acquisition software packages typically employ some <strong>beam-image shift</strong> targeting as this method is both fast and accurate ( ± 0.1 μm). In contrast, using only stage movement, relocation to a targeted area under low-dose conditions can only be achieved in combination with multiple iterations or long relaxation times, both reducing efficiency. Nevertheless it is well known that applying beam- image shift induces beam-tilt and with it a potential structure phase error with a phase error π/4 the highest acceptable value. This theory has been used as an argument against beam-image shift for <strong>high resolution</strong> data collection. Nevertheless, in practice many small beam-image shift datasets have resulted in 3D reconstructions beyond the π/4 phase error limit. To address this apparent contradiction, we performed <strong>cryo-EM</strong> 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 <strong>FSC values</strong>, and examined the water density peaks in the 3D map. We conclude that the phase error does not limit the validity of the 3D reconstruction from single-particle averaging beyond the π/4 resolution limit. - doi.org/10.1016/j.jsb.2018.07.015 - 2018</p>