Reaching the potential of electron diffraction

Abstract

Microcrystal electron diffraction (MicroED) is an emerging structural technique in which submicron crystals are used to generate diffraction data for structural studies. Structures allow for the study of molecular-level architecture and drive hypotheses about modes of action, mechanisms, dynamics, and interactions with other molecules. Combining cryoelectron microscopy (cryo-EM) instrumentation with crystallographic techniques, MicroED has led to three-dimensional structural models of small molecules, peptides, and proteins and has generated tremendous interest due to its ability to use vanishingly small crystals. In this perspective, we describe the current state of the field for MicroED methodologies, including making and detecting crystals of the appropriate size for the technique, as well as ways to best handle and characterize these crystals. Our perspective provides insight into ways to unlock the full range of potential for MicroED to access previously intractable samples and describes areas of future development.

Figure 1.. Schematic showing different diffraction-based structural methods and crystal detection techniques for a range of crystal sizes

Different diffraction-based methods (left) are appropriate for crystals in different size ranges. Representative crystal size ranges (middle) and available detection techniques (right) are shown. Micrometer-sized crystals in the size range suitable for conventional X-ray diffraction experiments, at either a home source or a synchrotron, can be seen by eye or with readily available bright-field microscopes. Micron-sized crystals are used for more advanced techniques like serial synchrotron and XFELs. When the crystals are even smaller, in the submicron size regime, electron beam sources can be used to collect diffraction data. While larger crystals can be detected by eye or light microscopy, smaller crystals suitable for advanced X-ray and electron diffraction experiments often require other methods such as TEM or nonlinear optical imaging techniques.

Figure 2.. Comparison of X-ray and electron crystallography

When incident X-ray or electron waves interact with the crystal lattice planes and interfere constructively, they form diffraction spots (Bragg reflections, shown as blue dots in reciprocal space). The detector captures the signal from the reflections that intersect with the Ewald sphere, thus satisfying the Bragg condition (spots colored dark blue). The Ewald sphere radius (drawn as black line) is reciprocal of the incident wavelength. For X-rays from a copper source, the wavelength is 1.54 Å, producing diffraction patterns that include information about all three reciprocal space dimensions (h,k,l indices) in a single image. For electrons from a 200 kV TEM, the wavelength is 0.025 Å, resulting in diffraction patterns that only contain information froma single plane (capturing only two reciprocal space indices) on the flatter Ewald sphere.

Figure 3.. Sample preparation for electron diffraction experiments can be modified for different crystal samples

Small molecules are naturally more likely to form some nanocrystals. In general, the right conditions need to be found to form protein crystals. If the crystals are thin enough for an electron beam, they can be frozen on EM grids and used directly for data collection. FIB milling is a popular option for thinning larger crystals frozen on grids before collecting electron diffraction data.

Figure 4.. Schematic showing a pipeline for nanocrystal generation, detection, and deposition to the EM grid

A protein or peptide sample is incubated in batch or on vapor diffusion plates with the crystallization conditions to form small crystals. Protein-rich crystalline material can be detected using the combination of SHG and UV-TPEF (inset figure adapted from Miller et al.34; for scale, the diameter of the well and image field of view is 0.9 mm). The sample is loaded onto EM grids (depicted as a pipette transfer in the schematic; there are other transfer methods possible). The sample frozen on the EM grids could then be further screened with TEM to ascertain the size (scale bar: 5 μm) and quality distribution of the crystals before collecting electron diffraction data using the same cryo-EM.