Collecting large datasets of rotational electron diffraction with ParallEM and SerialEM

Abstract

A semi-automated protocol has been developed for rotational data collection of electron diffraction patterns by combined use of SerialEM and ParallEM, where SerialEM is used for positioning of sample crystals and ParallEM for rotational data collection. ParallEM calls standard camera control software through an AutoIt script, which adapts to software operational changes and to new GUI programs guiding other cameras. Development included periodic flashing and pausing of data collection during overnight or day-long recording with a cold field-emission beam. The protocol proved to be efficient and accurate in data collection of large-scale rotational series from two JEOL electron microscopes, a general-purpose JEM-2100 and a high-end CRYO ARM 300. Efficiency resulted from simpler steps and task specialization. It is possible to collect 12-20 rotational series from ~ -68º to ~ 68º at a rotation speed of 1º /s in one hour without human supervision.

Hydrogen properties in an organic molecule revealed by XFEL and electron crystallography

“…This hydrofluorocarbon solvent is effective for yielding a good distribution of crystals over the grid. After drying the solution, the grid was directly cooled with liquid nitrogen, and transferred into a CRYO ARM 300 electron microscope (JEOL) operated at an accelerating voltage of 300 kV and maintained at a specimen temperature of ~ 300 or 98 K. A semi-automated data collection of rotational ED patterns was carried out by combined use of SerialEM 38 and ParallEM 39,40 as previously described 18,41 .…”

Cryo-EM structure of the human TACAN in a closed state

“…The grid was blotted at 8 C and 100% humidity and plunge-frozen in liquid ethane using FEI Vitrobot Mark IV (Thermo Fisher Scientific). Cryo-EM data were collected on a Titan Krios electron microscope operated at an acceleration voltage of 300 kV with a defocus range of -1.0 mm to -2.0 mm using SerialEM software (Takaba et al, 2020). Micrographs were recorded by a Gatan K2 Summit direct electron detector in super-resolution mode with a pixel size of 0.507 A ˚(a physical pixel size of 1.014 A ˚) with a dose rate of 7.2 electrons per pixel per second for a total exposure time of 8 s, resulting in an image stack with 40 frames.…”