Microcrystal electron diffraction methodology and applications
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Introduction Microcrystal electron diffraction (MicroED) uses crystals that are a billionth of the size typically needed for x-ray crystallography.1 For small molecules and natural products (molecules produced by natural sources such as natural drugs and metabolites), these crystals may have a powder-like appearance, which may make them seem to be amorphous to the eye.2,3 These types of samples are typically placed onto an electron microscopy grid and plunged into ethane cooled by liquid nitrogen prior to loading into the cryo-transmission electron microscope to facilitate investigation under cryogenic conditions. Although sufficient data can be collected from small molecules without cooling, the cryogenic conditions improve the obtained data by reducing the effects of radiation damage. For proteins, peptides, natural products, and samples susceptible to radiation damage, cryogenic conditions, typically liquid nitrogen or liquid ethane, are imperative for the collection of high-quality data. Once frozen, the grid is transferred under cryogenic conditions into a cryo-TEM and investigated under low-dose conditions. Nanocrystals in these observations appear as dark shapes, often with sharp edges.4 When a target is identified, the electron beam is focused onto the sample, and continuous rotation MicroED data are collected by using a fast camera as the sample is continuously rotated in a single direction4,5 (Figure 1). It is important to note that for screening and data collection, the
microscope is always operating in diffraction mode or lowmagnification mode, never in higher magnification imaging modes, to limit the exposure of the sample to the damaging electron beam.6 MicroED is analogous to the rotation method used in x-ray crystallography, and the data are processed using x-ray data processing software (e.g., MOSFLM, XDS, DIALS7–9) after conversion using various tools.10 Refinement proceeds as with any crystallography experiment and is described in greater detail next.
MicroED methodology and procedures In this section, we present an overview of the MicroED procedures—from sample preparation to structure determination. Because the descriptions are only briefly addressed, we encourage readers to refer to more detailed previously published literature.4,10–12 The procedure for MicroED analysis begins with the preparation of the crystalline material on a carbon-coated electron microscopy (EM) grid. For many samples, especially biological samples, it is important that the hydration of the sample be preserved; therefore, samples are applied to the carbon-coated EM grid, blotted to remove excess solution, and immediately plunged into liquid ethane to vitrify the sample. These procedures are similar to what is commonly used in single particle cryo-EM.13 For samples where maintaining hydration is not important (e.g., crystals of inorganic materials or some small
Brent L. Nannenga, School for Engineering Matter, Transport and Energy, Arizona State University, USA; [email protected] Tamir Gonen, Howard Hughes Medical In
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