Abstract
The cryoEM method Microcrystal Electron Diffraction (MicroED) involves transmission electron microscope (TEM) and electron detector working in synchrony to collect electron diffraction data by continuous rotation. We previously reported several protein, peptide, and small molecule structures by MicroED using manual control of the microscope and detector to collect data. Here we present a procedure to automate this process using a script developed for the popular open-source software package SerialEM. With this approach, SerialEM coordinates stage rotation, microscope operation, and camera functions for automated continuous-rotation MicroED data collection. Depending on crystal and substrate geometry, more than 300 datasets can be collected overnight in this way, facilitating high-throughput MicroED data collection for large-scale data analyses.
Highlights
The cryoEM method Microcrystal Electron Diffraction (MicroED) involves transmission electron microscope (TEM) and electron detector working in synchrony to collect electron diffraction data by continuous rotation
MicroED could benefit from automation, where multiple targets are selected for acquisition in a single run, as is commonly done with other cryoEM modalities such as electron tomography (ET) [9] and single particle analysis (SPA) [10]
We developed a procedure using a script for SerialEM which enables large-scale MicroED data collection on TEMs by Thermo Fisher Scientific coupled to electron detectors from various manufacturers
Summary
We developed a procedure using a script for SerialEM (http://bio3d.colorado.edu/SerialEM) which enables large-scale MicroED data collection on TEMs by Thermo Fisher Scientific (formerly FEI Company, Philips Electron Optics) coupled to electron detectors from various manufacturers. The operator can collect a sample diffraction pattern from each of the marked crystals to narrow down the selection of crystals for complete data collection (Figure 2) This step can be done either manually or automatically in SerialEM using its Navigator tools. SerialEM continues to the crystal location in the job list The relevant metadata, such as rotation rate and camera configuration, are automatically stored with the diffraction images in directories on the filesystem of the data collection host. These output files need to be converted to formats readable by typical crystallography integration packages such as DIALS [18], iMosflm/MOSFLM [19], or XDS [20] using our conversion software [7], which is freely available from https://cryoem.ucla.edu/pages/MicroED. A stepby-step protocol of SerialEM setup and instructions for MicroED data collection is presented in Supplementary Material
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