How does cryo-EM enable structure determination and what are the key considerations?

Cryo-electron microscopy determines structures by imaging frozen-hydrated samples and computationally reconstructing 3D density maps. Sample preparation involves rapid freezing in vitrified ice to preserve native state. Data collection on modern detectors (K3, Falcon) with dose fractionation and motion correction (MotionCor2) achieves high resolution. Image processing includes CTF estimation, particle picking (template or neural network-based), 2D/3D classification, and refinement (RELION, cryoSPARC). Challenges include preferred orientation, sample heterogeneity, small particle size (<150 kDa typically difficult), and beam-induced motion. Resolution is validated by FSC curves and map-model correlation. Cryo-EM excels for large complexes, membrane proteins, and conformational heterogeneity analysis, complementing X-ray crystallography.