A 20-μl aliquot of the coassembled sample was mixed with an equal volume of 10-nm BSA Gold Tracer (Electron Microscopy Sciences); 3.5 μl was applied on glow-discharged C-flat grids (Protochips) and then plunge-frozen into liquid ethane. Cryotomograms were acquired using an FEI Titan Krios electron microscope operating at 300 kV and equipped with a Falcon II camera. Tilt series were collected using the data collection software Tomography 3.0 (FEI) with an angular range of −60° to +60°, an angular increment of 1°, defocus values of 2.5 to 4 μm, and a nominal magnification of ×29,000, which corresponds to a pixel (px) size of 2.92 Å. One dataset was collected at a defocus value of 9 μm and used to generate initial reference-free maps for the TRIM5α trimer and dimer. Tilt series were aligned by using IMOD (56). Weighted back-projection was used to reconstruct tomograms, and the contrast transfer function was applied in IMOD. Subtomogram averaging was carried out using the Dynamo software package (57).

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