From electron crystallography of 2D crystals to MicroED of 3D crystals

Abstract

Electron crystallography is widespread in material science applications, but for biological samples its use has been restricted to a handful of examples where two-dimensional (2D) crystals or helical samples were studied either by electron diffraction and/or imaging. Electron crystallography in cryoEM, was developed in the mid-1970s and used to solve the structure of several membrane proteins and some soluble proteins. In 2013, a new method for cryoEM was unveiled and named Micro-crystal Electron Diffraction, or MicroED, which is essentially three-dimensional (3D) electron crystallography of microscopic crystals. This method uses truly 3D crystals, that are about a billion times smaller than those typically used for X-ray crystallography, for electron diffraction studies. There are several important differences and some similarities between electron crystallography of 2D crystals and MicroED. In this review, we describe the development of these techniques, their similarities and differences, and offer our opinion of future directions in both fields.

Keywords: Electron diffraction; MicroED; cryoEM (electorn cryo-microscopy); electron crystallography.

Fig. 1

(A) Structure solution of Bacteriorhopsin at 3.5 Å resolution determined by electron crystallography of 2D crystals. Density contoured at 1.5σ level. (B, C) Biological assembly showing the bacteriorhodoprin trimer arrangement in the membrane.

Fig. 2

The structure solution of Aquaporin-0 from electron crystallography at 1.9 Å resolution adapted from [35]. (A) Density for waters in the pore area, and (B) the full biological assembly showing resolved lipids (grey) surrounding the channel tetramer.

Fig. 3

Phase extension in electron crystallography from low resolution scheme adapted from [37]. Low resolution phases and amplitudes are collected from cryo-EM images and combined with phases from initially placed fragments. Density modification is done with phases from the combined phases and the electron diffraction intensities. Density modification then allows for final building and refinements.

Fig. 4

Lysozyme structure (A) and densities from MicroED with data collected from (B - 3J4G at 2.9 Å) stills, (C - 3J6K at 2.5 Å) continuous rotation, and (D - 5K7O at 1.8 Å) continuous rotation with fragmentation.

Fig. 5

(A) MicroED pattern from nanocrystals of the NACore of α-synuclein, inset with low-dose micrograph of typical crystals; adapted from [49]. (B) The structure solution of the asymmetric unit, and (C) arrangement of the biological assembly.

Fig. 6

(A) Sub-atomic resolution MicroED data breaking the 1 Å barrier from crystals of a segment of the Sup35 prior protein with (B) corresponding ab initio direct methods solution to the structure. (C) Diffraction from Au146 crystals extending beyond 0.8 Å, and (D) ab initio direct method solutions showing clear density for all gold and sulfur atoms. Figures adapted from [51,53^•].