Advances and applications of microcrystal electron diffraction (MicroED)

Abstract

Microcrystal electron diffraction, commonly referred to as MicroED, has become a powerful tool for high-resolution structure determination. The method makes use of cryogenic transmission electron microscopes to collect electron diffraction data from crystals that are several orders of magnitude smaller than those used by other conventional diffraction techniques. MicroED has been used on a variety of samples including soluble proteins, membrane proteins, small organic molecules, and materials. Here we will review the MicroED method and highlight recent advancements to the methodology, as well as describe applications of MicroED within the fields of structural biology and chemical crystallography.

Keywords: Cryo-EM; Cryo-electron microscopy; Crystallography; MicoED; Microcrystal electron diffraction; Protein crystallography; Small molecule crystallography.

Figure 1.. The MicroED workflow.

The workflow for macromolecular crystals begins by transferring the crystals from crystallization experiments (e.g. hanging drop, sitting drop, batch crystallization) to the surface of a carbon-coated EM grid. The samples are then blotted and vitrified using manual or automatic plunge freezing setups. At this point, the microcrystals may be loaded into the cryo-TEM for MicroED data collection, or can be further processed using cryo-FIB milling (green arrows) prior to transfer into the cryo-TEM. For small molecule samples, the microcrystals are transferred to the grid by different approaches depending on the form of the sample. Dried powder can be directly applied to the surface of the grids. Microcrystals in suspension are applied to the grids and the solvent is then blotted away or allowed to evaporate from the surface. Finally, samples may be crystallized directly on the grid by applying the sample in solution and allowing evaporation to occur. For both macromolecular and small molecule crystals, data are collected by continuous-rotation MicroED data collection. The resulting data are processed, and structures are determined and refined, using standard crystallographic programs.

Figure 2.. MicroED methods advancement has greatly improved structure quality.

(A) The first structure determined by MicroED was of tetragonal hen egg-white lysozyme in 2013. Data was collected using discrete tilts of the stage and the structure was determined to 2.90 Å. A short time later in 2014, continuous-rotation MicroED data collection was introduced, which improved data quality and allowed data processing to be performed using standard crystallographic programs. This advancement led the lysozyme structure to now be determined to 2.50 Å. Following continued MicroED methodology improvements, the structure of lysozyme was determined to 1.80 Å in 2017. (B) Recently, the powerful combined effect of several new MicroED method improvements, including cryo-FIB milling and data collection with direct electron detectors, was used on triclinic lysozyme crystals, which are known to diffract to higher resolution. The use of improved methodology and very high-quality crystals facilitated the structure determination of triclinic lysozyme to 0.87 Å resolution. Together these lysozyme studies show how critical methods development has been, and will continue to be, for MicroED. The structures displayed in (A) are of lysozyme residues 19 to 37 and residues 58 to 70 in (B), and the potential maps have been contoured to 1.5σ.