PVY image processing. A total number of 726 micrographs of the PVY sample were collected, and 235 micrographs were subsequently selected for determination of PVY CP structure. Straight segments of the filaments were manually picked using e2helixboxer.py in the EMAN2.1 package (31). The subsequent steps of the data analysis workflow were performed in the RELION2.1 package (32). The data analysis comprised the following steps: A subset of 215,123 segments of PVY was extracted from the micrographs (boxes of 360 pixels × 360 pixels). Two rounds of reference-free two-dimensional (2D) classification with 25 iterations were performed, and only class averages showing high-resolution structural features were selected after each round. Initial estimate of the helical parameters was obtained from indexing the power spectra (fig. S1D). The helical parameters were found to be very similar to those of PepMV (7). The helical_toolbox program (RELION2.1) was then used to generate the initial geometrical model of the one-start left-handed helix with rise of 4 Å and twist of 40°. The 3D classification using 162,213 segments revealed minimal structural variability at 4 Å resolution. Therefore, all these segments were used for the final 3D refinement of the model, with a local search of helical parameters, which yielded values of −40.95° for helical twist and 3.95 Å for helical rise. The reconstructed 3D map of PVY was sharpened using the relion_postprocess program (RELION2.1), and helical symmetry was imposed using the relion_helix_toolbox program (RELION2.1). The overall resolution of 3.4 Å was determined on the basis of gold-standard fourier shell correlation (FSC) at 0.143 criterion (fig. S1F) (33). Local resolution was calculated using MonoRes (34), and cryo-EM densities were visualized in UCSF Chimera (35).

VLP image processing. A total number of 5344 micrographs of the VLP sample were collected, and 3759 micrographs were subsequently used for data analysis. Straight segments of VLP filaments were manually picked from the micrographs using e2helixboxer.py in the EMAN2.1 package (31). Subsequently, helical reconstruction based on single-particle analysis was achieved using the RELION2.0 and RELION2.1 packages (32). A subset of 244,988 segments of VLPs was extracted from the micrographs (boxes of 360 pixels × 360 pixels). Two rounds of reference-free 2D classification with 25 iterations were performed, and only class averages showing high-resolution structural features were selected after each round.

Additional analysis of the micrographs revealed the population of circular particles corresponding to the orthogonal projections (top views) of the single VLP ring. Reference-free 2D classification revealed the presence of eightfold symmetry within the ring. Therefore, C8 symmetry was imposed during subsequent steps of structure refinement. A featureless cylinder was used as the initial 3D model for 3D refinement. No particles were removed on the basis of 3D classification; therefore, 148,876 segments of VLPs were used in 3D autorefinement with an applied mask with a diameter of 184 Å. A local search of helical parameters during 3D autorefinement yielded values of 13.24° for helical twist and 42.65 Å for helical rise. The reconstructed 3D map of VLP was sharpened using the relion_postprocess program (RELION2.1), and helical symmetry was imposed using the relion_helix_toolbox program (RELION2.1). Overall, resolution of 4.1 Å was determined on the basis of gold-standard FSC at 0.143 criterion (fig. S7F) (33). The local resolutions were calculated using MonoRes (34), and cryo-EM densities were visualized in UCSF Chimera (35).

ΔN49 deletion mutant image processing. A total of 23 micrographs of ΔN49 deletion mutant were collected to obtain 2D class averages of the oligomer. In total, 2132 particles were extracted using e2boxer.py in EMAN2.1 package (31). After reference-free 2D classification with 25 iterations and three classes in RELION2.1 (36), the best 2D class image with 1907 particles indicated octameric ring assembly of the ΔN49 CPs.

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