Asymmetric reconstruction of the portal complex of native P68 was determined using symmetry expansion from the C12 symmetrized localized reconstruction of the portal (21,702 particles). Initially, the particle orientations from the previous C12 reconstruction were symmetry expanded to C1 using the program (45). Mask covering the portal complex was generated from the C12 symmetrized structure. Focused 3D classification with 12 classes using the symmetry-expanded orientations was performed in RELION (45). The alignment step was omitted to prevent overfitting. Regularization factors in the range of 20 to 50 were tested to establish the optimal value to detect asymmetric features in the portal. The best reconstruction as judged by map interpretability was obtained with the regularization factor 33. The class from the 3D classification showing an asymmetric interaction between one of the portal protein subunits and dsDNA was chosen for further refinement. Duplicate particle images (C12 symmetry–related images of the same particle) were discarded from the reconstruction, resulting in 7568 unique particles. Final refinement was performed using RELION auto refine, with the application of a mask, but without the orientation search (45). The refinement strategy is summarized in fig. S6.

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