Cryo-EM Structure Determination Workflow

Reconstruction was performed using cryoSPARC^{25 (link)}. For each particle set, we first generated an ab initio structure with a single class. These structures were then refined using cryoSPARC’s “homogenous refinement” option with symmetry specified depending on the dataset (T20S proteasome: D7, 80S ribosome: C1, aldolase: D2). For the aldolase dataset, we used C2 symmetry for ab initio structure determination. Otherwise, all other parameters were left in the default setting. When evaluating the quality of Topaz particle sets for decreasing score thresholds, each particle set was selected by taking all particles predicted by the Topaz model with scores greater than or equal to the given threshold. Reconstructions were calculated for each of these sets independently as described above.