Cryo-EM Sample Preparation Techniques

Single particle analysis is an increasingly popular technique to study proteins, protein complexes, viruses and other biomolecules at near-atomic resolutions through fixation of them in a thin layer of vitreous ice. Once imaged, thousands of particles in various orientations are averaged allowing for reconstruction of a high-resolution structure of the isolated protein present in the sample. As sample preparation is a vital step in the single particle workflow, it is important to have a good understanding of the steps involved and to have the right reagents, tools, and instrumentation.

Cryo-electron tomography (cryo-ET) is a technique that allows the study of the 3D structure of cells and tissues at near-native conditions. Sample preparation is a critical step in cryo-ET, which can greatly affect the quality and resolution of the results. Cryo-ET has been applied to many different sample types, from single molecules to protein complexes, viruses, bacteria, cells, to tissue cells and large tissue samples.

Microcrystal electron diffraction (MicroED) enables fast, high-resolution, structural determination of small molecules and proteins. Atomic details can be extracted from individual nanocrystals (<200 nm in depth), even in a heterogeneous mixture. Sample preparation for MicroED varies between small molecule and protein samples.