Images of capsids of native virions (33,612 particles) from the 3D classification of the asymmetric reconstruction were used as the initial dataset for fivefold symmetrized reconstruction. Refinement with imposed C5 symmetry was followed by 3D classification without alignment. After 3D classification, 28,826 particles were retained for further reconstructions. These particles were re-extracted in boxes of 600 × 600 pixels. A mask that included the capsid was created using the programs UCSF Chimera, Segger, and (4-pixel expansion and 5-pixel soft edges) (45, 49, 50). Masked 3D refinement with local searches around the previously determined orientations was performed using the RELION auto refine procedure (45). Identical strategies were used for the reconstructions of fivefold symmetrized capsids of genome release intermediates and empty particles.

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