Classification and refinement procedure of an improved model of the human EMC. (A) A representative micrograph with several particles highlighted with yellow circles. Scale bar = 75 nm. (B) Representative 2D class averages generated during data processing. Scale bar = 5 nm. The number of particles for each class and its resolution are indicated. (C) Flowchart highlighting the data processing pipeline used to obtain an improved structure of the EMC. The 3D Variability Analysis (3DVA) enabled the exploration of the heterogeneity of the sample and allowed to parse out a subset of particles that lack the subunit EMC10, which provided unique insights into the placement of EMC10’s TMD. Particles with all nine subunits, or those missing EMC10 (dashed boxes) were combined separately. Particles with poorly defined or low-resolution features were discarded (see Materials and methods). (D) Final EM density maps colored by local resolution in Å. For clarity, a dust filter was applied in ChimeraX. (E) Gold-standard FSC curves for the consensus, nine-, and eight-subunit complex maps generated by cryoSPARC V4.0.