Abstract
MicroED is a method which combines cryo-EM sample preparation and instrumentation, with electron and X-ray crystallography data analysis, and it has been employed to solve many protein crystal structures at high resolution. Initially, the main doubts of this method for structure determination were the dynamic scattering of electrons, which would cause severe inaccuracies in the measured intensities. In this paper, we will review the evolution of MicroED data collection and processing, the major differences of multiple scattering effects in protein crystals and inorganic material, and the advantages of continuous rotation data collection. Additionally, because of the periodic nature of the crystalline sample, radiation doses can be kept significantly lower than those used in single particle data collection. We review the work where this was used to assess the radiation damage of a high-energy electron beam on the protein molecules at much lower dose ranges compared to imaging.
Highlights
Cryo-EM has become one of the most powerful tools in structural biology after nearly four decades of improvements in electron optics, direct electron detectors, and software (Frank, 2016)
The rotation electron diffraction (RED) method was developed to determine the structures of inorganic crystals using fine step rotation and small-angle beam tilting (Wan et al, 2013), in which the diffraction data was off-zone axis patterns and might contain less overall dynamic scattering events
The signal of a diffraction experiment increases with the square of the number of unit cells in the crystal; MicroED can obtain accurate 3D density maps at high resolution using total doses of as low as ∼1 e/Å2, which allows the analysis of radiation damage effects in both reciprocal space and real space (Hattne et al, 2018)
Summary
Cryo-EM has become one of the most powerful tools in structural biology after nearly four decades of improvements in electron optics, direct electron detectors, and software (Frank, 2016). The general applicability of electron diffraction techniques to all these samples has been made possible by continued method development and optimization.
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