Pore complexes were separated into seven classes according to their shape and diameter by principal component analysis and clusterPCA in IMOD (Kremer et al., 1996). The best class with 844 pore complexes was used for further processing as described above. In the final pore complex average, individual subunits were clearly distinguished in one third of the ring, indicating some heterogeneity of ring size and symmetry (Figure 5B). The number of PLY monomers in the pore was determined as 34 from the angular distance between distinct subunits in the average volume and the ring diameter. As for the prepores, all averaging steps were performed in PEET (Nicastro et al., 2006). The resolution at FSC0.5 was 27 Å for the pore and 22 Å for the prepore (Figure 6c). At FSC0.3, the resolution of the pore and prepore complex would be 21 Å and 20 Å, respectively.