Macromolecular crystallography using microcrystal electron diffraction.

Hongyi Xu,Max T. B. Clabbers

doi:10.1107/s2059798320016368

Abstract

Microcrystal electron diffraction (MicroED) has recently emerged as a promising method for macromolecular structure determination in structural biology. Since the first protein structure was determined in 2013, the method has been evolving rapidly. Several protein structures have been determined and various studies indicate that MicroED is capable of (i) revealing atomic structures with charges, (ii) solving new protein structures by molecular replacement, (iii) visualizing ligand-binding interactions and (iv) determining membrane-protein structures from microcrystals embedded in lipidic mesophases. However, further development and optimization is required to make MicroED experiments more accurate and more accessible to the structural biology community. Here, we provide an overview of the current status of the field, and highlight the ongoing development, to provide an indication of where the field may be going in the coming years. We anticipate that MicroED will become a robust method for macromolecular structure determination, complementing existing methods in structural biology.

Highlights

For the past several decades, X-ray crystallography has been the most prominent method for protein structure determination in structural biology
In 2D electron crystallography, a 3D reconstruction of the protein structure can be obtained from 2D projection images recorded at different tilt angles by combining diffraction intensities extracted from electron diffraction patterns and crystallographic structure-factor phases extracted from electron micrographs (De Rosier & Klug, 1968)
These results demonstrate that many potentially difficult-to-crystallize membrane proteins embedded in lipid mesophases can become targets for structure determination by Microcrystal electron diffraction (MicroED)

Summary

Introduction

For the past several decades, X-ray crystallography has been the most prominent method for protein structure determination in structural biology. In 2D electron crystallography, a 3D reconstruction of the protein structure can be obtained from 2D projection images recorded at different tilt angles by combining diffraction intensities extracted from electron diffraction patterns and crystallographic structure-factor phases extracted from electron micrographs (De Rosier & Klug, 1968). Using this reconstruction method, several protein structures were solved from thin 2D crystals (Unwin & Henderson, 1975; Henderson et al, 1990; Grigorieff et al, 1996; Mitsuoka et al, 1999; Gonen et al, 2005). It becomes increasingly challenging to obtain sufficient contrast from cryo-EM imaging for protein complexes smaller than 50 kDa (Henderson, 1995; Glaeser, 1999; Khoshouei et al, 2017; Fan et al, 2019; Herzik et al, 2019)

Electron diffraction data collection

Data processing

Structure determination

Drug discovery and fragment screening

Crystal size

Crystallization

Specimen preparation

Cryo-FIB milling

Data quality

Dynamical scattering

Modelling the electrostatic potential

Isomorphous replacement