研究目的

To present a standard operating procedure for single-particle data collection with Thermo Fisher Scientific EPU software, using the two most common direct electron detectors (the Thermo Fisher Scientific Falcon 3 (F3EC) and the Gatan K2), and to provide a strategy for structuring these data to enable efficient pre-processing and on-the-fly monitoring of data collection.

研究成果

By following this protocol, the user should be able to produce high-quality electron micrographs for single-particle data analysis and pre-process the results in close to real time. Monitoring the micrograph analysis output permits visualization of the estimated defocus, resolution in the micrographs, astigmatism and, where relevant, the phase shift. The user can then make informed decisions about alterations to defocus range and objective stigmation. For phase plate data, useful phase shifts are between 20° and 120°, therefore, on-the-fly analysis of the phase shift allows a user to ensure that data are collected in the optimal range by altering the frequency with which the phase plate position is changed.

研究不足

The protocol describes how the authors utilize their specific hardware setup at ABSL using the Titan Krios microscope equipped with an F3EC or an integrated K2 with their specific data storage systems and processing hardware. This will need to be adapted for each individual hardware setup at new facilities. The protocol describes a ‘standard’ single-particle data collection. In some cases in which the specimen creates specific challenges, such as a preferred orientation, the method could be easily altered to include collection of tilted data. There are a range of other software packages that can be used for automated data collection for single-particle analysis, notably SerialEM and Leginon.