Extended Data Fig. 3: Workflow of 3D classification of the CCAN–Cenp-A^Nuc cryo-EM dataset.

a, After initial 2D classification, about 1.4 million particles were sorted by 3D classification into apo-CCAN (52%) and the CCAN–Cenp-A^Nuc complex (48%). For apo-CCAN, 4% existed as dimers (black box) and 19% showed an ordered head-group (Cenp-HIK^Head) for the Cenp-HIK–TW subcomplex (blue box). A mask was applied to the CCAN–Cenp-A^Nuc cryo-EM map to exclude the structurally variable Cenp-HIK^Head domain for reconstruction of the 4.15 Å structure. b, Details of the four masks used for MBR. c, A small 3D class of CCAN–Cenp-A^Nuc, revealing density attached to Cenp-HIK^Head contacting the DNA gyre of Cenp-A^Nuc was assigned as Cenp-T^HFDW.