Abstract
This article documents a keynote seminar presented at the IUCr Congress in Prague, 2021. The cryo-EM method microcrystal electron diffraction is described and put in the context of macromolecular electron crystallography from its origins in 2D crystals of membrane proteins to today's application to 3D crystals a millionth the size of that needed for X-ray crystallography. Milestones in method development and applications are described with an outlook to the future.
Highlights
Working with biological specimens in cryo-electron microscope (EM) requires that the proteins are preserved in their native hydrated state and that any sample irradiation is minimal or very brief to preserve the structural integrity of the protein
The structure was solved by electron crystallography exclusively using electron diffraction patterns recorded at different tilt angles and phased by molecular replacement revealing the structure of the AQP0 tetramer, water molecules and the surrounding membrane (Fig. 6)
In 2013, the structure of hen egg-white lysozyme was determined from a 3D crystal by microcrystal electron diffraction (MicroED) using still diffraction patterns recorded at discrete tilt steps (Shi et al, 2013)
Summary
Electron cryomicroscopy (cryo-EM) is an important technique in structural biology. Electrons are scattered very efficiently by the sample, at the cost of only a modest amount of radiation damage, compared with X-rays and neutrons (Henderson, 1995). In cryo-EM imaging-based techniques, the electrons scattered by the sample are focused into a real-space image. In single-particle cryo-EM, high-resolution images are recorded of many individual protein complexes captured in random orientations (Cheng, 2015). These 2D projection images can be combined in Fourier space based on their various angular contributions to reconstruct a 3D real space model (De Rosier & Klug, 1968). An initial 3D structural model of the protein is reconstructed using phases extracted from the Fourier transforms of images, which are extended to higher resolution using intensities obtained from electron diffraction patterns (Wisedchaisri & Gonen, 2011).
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