Initial maps of the trimer and dimer were generated by hand-picking ~60 particles from a single tomogram (defocus value of 9 μm), assigning initial Euler angles in reference to the tube backbone as defined above, and performing one round of azimuthal refinement with three- or twofold symmetry, as appropriate. These maps were used as initial search templates for the each of the seven tubes, as described below.

For each tube, the optimized tube backbone was defined in reference to the refined CA hexamer positions. This, in turn, was used to generate a tubular mesh with a 50-px radius; this mesh oversampled the TRIM lattice by at least 25× for the dimer and 35× for the trimer. Subvolumes of 64 × 64 × 64 px whose centers were uniformly distributed on this mesh were extracted from 2× binned tomograms. Initial polar angles were assigned in reference to the tube backbone, whereas azimuthal angles were randomized. One iteration of azimuthal and positional refinement was performed, using the far-from-focus trimer and dimer models as search templates, again applying three- or twofold symmetry as appropriate and a low-pass filter of 40 Å (fig. S3A). The averaged maps from these first rounds were then used as search template in all subsequent refinements. In the first three iterations, refined positions that were within 4 px of each other were averaged, reextracted from the tomograms, and reassigned Euler angles as above. On the fourth iteration, lattice maps were examined, and particles that migrated to unrealistic positions and/or had very low cross-correlation values were discarded. Subvolumes were then reextracted (128 × 128 × 128 px) from unbinned tomograms (3204 dimers and 2108 trimers from seven tubes) and split into even/odd subsets, which were treated independently from this point forward. Refinement iterations were performed until convergence (which required six iterations for the dimer and eight for the trimer), with progressively narrower angular and positional search ranges and a low-pass filter set at 30 Å. Soft-edged tubular and ellipsoidal masks were used for the dimer and trimer, respectively.

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