Examples of tilt-pair plots from two different samples, used to validate determination of the single-particle orientations that are needed to calculate the 3D structure. In (a) the change in the angle of the Haliotis diversicolor hemocyanin particles is clustered at 10°, which was the angular difference between the two images, indicating the validity of the 3D model. The structure of this hemocyanin was solved subsequently to a resolution of 4.5 Å (Zhang et al., 2013). In (b) a similar experiment on Norwalk virus-like particles with a 5° change in tilt angle between the two images is shown. In this experiment, three pairs of images were recorded, with particles from different image pairs shown in different colours. With molecular weights of 8 and 10 MDa, respectively, these two additional tilt-pair parameter plots fill in the gap in particle size, between 3 and 50 MDa, that was missing from an earlier publication (PDB code 1ihm; Prasad et al., 1999) on a range of different specimens.