kryoEM

kryoEM, often written as cryo-EM, refers to cryogenic electron microscopy, a set of imaging methods used to determine the structures of biological macromolecules at near-native conditions. Specimens are rapidly frozen to form vitreous ice, preserving their structure without chemical fixation or staining. Using direct electron detectors and advanced software, kryoEM can reveal shapes and internal features at resolutions from a few tens of angstroms to sub-angstrom levels for suitable samples.

Two main modalities are single-particle cryo-EM (SPA) and cryo-electron tomography (cryo-ET). In SPA, purified macromolecules are dispersed in thin ice and imaged from many random orientations; computational alignment and averaging yield a 3D density map. In cryo-ET, thicker specimens or cellular contexts are imaged through multiple tilt angles to reconstruct volumes reflecting spatial organization in situ.

Sample preparation involves applying a small volume to a perforated grid, blotting to achieve a thin film, and plunge-freezing into liquid ethane cooled by liquid nitrogen. The vitrified specimens are then imaged under low electron dose to minimize radiation damage. Data collection benefits from direct detectors, dose fractionation, and motion correction, with estimation of the contrast transfer function to recover high-resolution information.

Reconstruction converts many two-dimensional projections into three-dimensional maps, with resolution assessed by Fourier shell correlation. CryoEM has enabled near-atomic models of protein complexes, membrane proteins, and viruses, supporting structural biology, mechanistic studies, and drug discovery. The field rose to prominence through advances in detectors and computation, culminating in the 2017 Nobel Prize in Chemistry for pioneers of cryo-EM. Limitations include sample heterogeneity, preferred orientation, and the need for specialized equipment and expertise.