MicroED: conception, practice and future opportunities

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.

Keywords: MicroED; cryo-EM; crystallography; membrane proteins; microcrystal electron diffraction.

Figure 1

The MicroED method and examples of some structures. (a) Illustration of MicroED data collection where the crystal is continuously rotated and diffraction patterns are recorded as a movie. (b) Examples of several protein structures determined by MicroED are listed with their names and PDB identifiers. Proteins are shown in their biological assembly and membrane proteins are highlighted in yellow.

Figure 2

Differences between X-ray and electron diffraction. Diffraction patterns are shown for X-ray diffraction and MicroED. The much shorter wavelength of high-energy electrons means that the Ewald sphere construction is virtually flat. Indexing from a single diffraction frame is therefore not typically possible for electron diffraction, and a larger wedge of reciprocal space needs to be covered to find the unit-cell dimensions.

Figure 3

Sample preparation for macromolecular MicroED. (a) Several examples of crystallization conditions with protein microcrystals of different morphologies suitable for MicroED. Crystals can be identified using light microscopy as shown in the top row, corresponding images of negatively stained samples are shown on the bottom row. Scale bars for the top row are 500 nm and bottom row 400 nm. Images were adapted from the work by Shi et al. (2016 ▸). (b) Typical workflow involved in MicroED sample preparation for protein crystals. The crystals are pipetted from the crystallization drop and deposited onto the carbon-side of a glow-discharged EM-grid held between the tips of a tweezer. The crystals are allowed to settle on the grid before any excess liquid is blotted away from the back-side of the grid with filter paper. The grid is then rapidly plunged into liquid ethane for freezing and kept at cryogenic temperatures until use. The grid is either transferred directly to the TEM for MicroED data collection, or can be thinned by cryo-FIB milling into crystalline lamellae suitable for MicroED. (c) FIB milling of tetragonal lysozyme crystals, showing the SEM images (left) side by side with the FIB images (right) during a typical milling workflow as described previously by Martynowycz & Gonen (2021a ▸). After a suitable crystal is identified, rectangular boxes (blue) are drawn for coarse milling the bulk material (top). The resulting 3 µm-thick lamella is then further thinned by polishing using a lower current and smaller step sizes (middle). This results in a thin crystalline lamella of ideally 200–300 nm thickness and a width of 5 µm (bottom). Scale bars for the SEM images from top to bottom are 50, 25 and 25 µm, for the FIB images from top to bottom 10, 10 and 1 µm.

Figure 4

MicroED for small-molecule research. (a) Typical workflow involved for MicroED of small-molecule samples from powder to individual crystals on a standard EM grid, data collection, processing and structure solution. The solved structure in the last panel shows the model of the compound limaspermidine with the observed map showing the individual atoms (blue, top), and the difference map showing hydrogen atom positions (green, bottom). (b) Individual compounds can be identified from heterogeneous mixtures using MicroED. Resolution rings are shown at 0.8 Å (blue).

Figure 5

MicroED and drug discovery. (a) Ligand binding showing the heme group and NADP resolved by MicroED for bovine liver catalase (Nannenga, Shi, Hattne et al., 2014 ▸). (b) Drug binding of the inhibitor bevirimat (BVM) in complex with the C-terminal domain of the HIV Gag protein fragment (Purdy et al., 2018 ▸). (c) Drug-bound MicroED structure of human carbonic anhydrase isoform II (HCA II) complexed with the clinical drug acetazolamide (AZM) (Clabbers et al., 2020 ▸). Inset shows the active site where the ligand is coordinated to the active site zinc metal co-factor. (d) Efficient ligand soaking into microcrystals was demonstrated from lamellae of proteinase K that were briefly soaked on-grid with I3C compounds (Martynowycz & Gonen, 2021a ▸). Four I3C molecules could be identified in the difference map, each difference map is shown next to the observed map of the fitted and refined ligands. (e) Structure of the adenosine A_2A-receptor determined from LCP crystals using MicroED (Martynowycz, Shiriaeva et al., 2021 ▸). The ligand ZM241385 (ZMA) could be resolved in the orthosteric pocket, as well as four surrounding cholesterol molecules bound to the receptor on the extracellular side. Difference maps are shown next to the refined maps with the fitted ligand and the four numbered cholesterol molecules. In all panels, the positions of the ligand in the protein models are highlighted with yellow rectangular boxes.

Figure 6

Examples of membrane protein crystallization modalities and structures solved. (a) Membrane protein structure determination in MicroED in a lipid and/or detergent environment mimicking the lipid bilayer. The structure of the NaK ion channel was determined from detergent micelles (Liu & Gonen, 2018 ▸), mVDAC from lipid bicelles (Martynowycz et al., 2020 ▸), the A_2A-receptor from LCP crystals (Martynowycz, Shiriaeva et al., 2021 ▸), whereas aqua­porin-0 (AQP0) was determined from double-layered crystals of the protein embedded in lipid bilayers using electron diffraction (Gonen et al., 2005 ▸). (b) Top view showing the electron crystallography structure of AQP0 resolved lipid-protein packing interactions between the tetramer (yellow) and the surrounding lipid molecules (blue). (c) Top view showing the MicroED structure of mVDAC and the crystal packing of the monomers (yellow) where the map (blue) in between the barrels indicates the space is likely to be occupied by the lipid molecules.