(a) From the left: apoferritin structure, template at 220 nm underfocus, image of a single apoferritin in ice at 220 nm underfocus with 1200 electrons/nm2. (b) Cross-correlograms (CCGs), left to right: template five degrees rotated around z-axis from best orientation, template at best orientation, maximum intensity projection (MIP) across all template orientations. (c) GroEL (decoy) template at 220 nm underfocus, single CCG, and MIP. (d) Image at 2200 nm underfocus. Orange octahedrons indicate the orientation and location of CCG peak values from a full search of an image of the same area at 220 nm underfocus taken before the displayed image. Traces along right and top edge: horizontal and vertical projections of the maximum across orientations (orange: apoferritin, blue: GroEL; blue traces offset by 1.5 SNR units for clarity). Note the dark rings, which presumably correspond to apoferritin particles (the large round objects are gold particles). The boxed region indicates the image region used for (a), (b), and (c). (e) CCG value survival histograms (number of CCG values above a given SNR) for apoferritin (orange), GroEL (blue), and as expected for Gaussian noise (crosses). Scale bars are 5 nm for images and 1 nm for surface plots in (a)-(c), 5 SNR units for surface plots in (b), and 10 nm and 3 SNR units in (d). The top and bottom ends of the amplitude scale bar in (d) correspond to SNRs of 10 and 13, respectively.