MicroED data collection with SerialEM

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.

Keywords: Electron diffraction; MicroED; Microcrystal; SerialEM; Transmission electron microscopy; cryoEM.

Fig. 1.

SerialEM automation workflow for MicroED. A cryoTEM grid containing protein nanocrystals is examined via a whole-grid atlas collected at low magnification. Crystal areas are then identified from this atlas and added into the SerialEM Navigator queue for subsequent diffraction screening. Diffracting crystals on this list are selected for subsequent MicroED data collection in batch. (a), (b), and (c) denote crystals selected for diffraction screening in Fig. 2.

Fig. 2.

Electron diffraction screening of selected crystals. (a), (b), and (c) are the resulting diffraction patterns of crystals (a), (b), and (c) in Fig. 1.