Principle of cryo-EM and single-particle reconstruction. Molecules (in this case ribosomes) lying in random orientations are embedded in a thin layer of ice. Exposure to a low-dose electron beam in the transmission electron microscope produces a projection image ('electron micrograph'). A typical electron micrograph shows E. coli ribosomes as low-contrast single particles on a noisy background. After the orientations of the particles have been determined, usually by matching them with a reference, they are used to reconstruct a density map by a back-projection or a similar reconstruction algorithm. This density map is segmented into the different components (subunits, ligands), and the different components are displayed using different colours in a surface representation (bottom panel; small and large subunits are shown in yellow and blue, respectively. A- and P-site tRNAs are coloured pink and green, respectively).