For particle picking and initial rounds of sub-tomogram averaging (Frangakis and Hegerl, 2001), tomograms were filtered by nonlinear anisotropic diffusion to enhance contrast. Ring-shaped complexes were picked manually in 3dmod. A total of 752 prepore complexes and 2400 pore complexes were picked for averaging. Ring volumes were pre-aligned in one plane in 2 × 2 binned unfiltered tomograms. All prepores had roughly the same size and shape and were averaged in PEET (Nicastro et al., 2006). The average volume was used as an initial reference for symmetry determination by sub-tomogram averaging with IMOD (Kremer et al., 1996). Symmetries of 42-fold or lower were applied to each prepore volume on a trial-and-error basis. Volumes were aligned and averaged in an iterative process, which converged within about 15 iterations on an average volume with clear 34-fold symmetry. The best 70% of the prepore volumes contributed to the final average, in which 34 PLY monomers were resolved in the ring.