Alignment and classification of identified ribosomes in tomograms from starved E. coli lysates. (A–E) Reference-based alignment and classification using constrained correlation. (A) Slices of the average structure derived from 1,232 subtomograms (Pool II) containing 70S ribosomes. (B) Mask used for classification excluding the aligned central ribosome. (C and D) Slices of average structures containing 100S ribosomes with the two ribosomes in a preferred orientation derived from 43 classified subtomograms from Pool II and 35 classified subtomograms from Pool I, respectively. (E) Schematic slices of the two ribosomes oriented as in C (50S, blue; 30S, yellow; light colors for central ribosome excluded during classification; dark colors adjacent particle revealed after classification). (F and G) Unsupervised alignment and classification using a maximum likelihood approach. (F) Isosurface representation of 3D maps obtained during iterative reference-free alignment of 601 subtomograms (Pool I) that were windowed to contain only a single ribosome particle. Iteration 0 (iter 0) corresponds to the initial, unbiased reference that was obtained by averaging over all sub-tomograms in random orientations. The final map (iter 15) has a resolution of 38 Å according to the FSC = 0.5 criterion and is readily identified as a 70S ribosome particle. (G) Simultaneous alignment and classification of the same particles used in F, but without windowing in order to include neighboring ribosomes. Three initial reference structures (class 1–3, iter 0) were refined simultaneously during 25 iterations. Note that during the first 5 iterations similarity between the three references was imposed. The final averages (iter 25) were interpreted as class 1 trimers (or larger clusters, 74 particles), class 2 dimers (158 particles), and class 3 monomers of 70S ribosome (369 particles).