❄️ Modern Imaging: Cryo-Electron Microscopy (Cryo-EM)
Cryo-EM single-particle analysis (SPA) determines macromolecular structures at near-atomic resolution (<2 Å) in vitreous ice without the requirement for crystallization, capturing flexible and heterogeneous functional states.
1. The Cryo-EM Workflow
- Vitrification: Macromolecular solution is applied to a holey carbon or gold grid, blotted to a thin film, and plunged into liquid ethane cooled by liquid nitrogen (-180°C) at cooling rates >10⁶ °C/s. Water vitrifies into amorphous ice, avoiding destructive hexagonal ice crystal lattice formation.
- Low-Dose TEM Imaging: Grids are imaged in a 300 kV transmission electron microscope (e.g. Titan Krios) with direct electron detectors (DED) using movie mode to correct for beam-induced motion.
- Single-Particle Reconstruction: Hundreds of thousands of individual particle projections are identified, aligned, classified into 2D class averages, and merged through iterative angular refinement into a 3D Coulomb potential map. Resolution is validated via the Fourier Shell Correlation (FSC = 0.143 gold-standard criterion).