Extended Data Fig. 3 |. The workflow of cryo-EM data processing for EDTA–CALHM2.

A total of 4,809 movies was collected using a Titan Krios equipped with K2. Particles were autopicked using Gautomatch, and visually examined in RELION to eradicate false-positive selections. After manual clean-up, particles were subjected to two rounds of 2D classification in RELION. Ab initio reconstruction with two classes was performed in CryoSPARC to separate hemichannel and gap-junction particles, and to generate initial models of the hemichannel and gap junction. Hemichannel and gap-junction particles were further cleaned up using 2D and 3D classification in RELION. Particles from 3D classes that showed high-resolution features and obvious C₁₁ symmetry were combined and refined in RELION. To assess the structural heterogeneity of the helix S1, an approach that combined symmetry expansion and signal subtraction was carried out, in which all the subunits were subtracted and classified without image alignment in RELION.