a, Comparison of the cryo-EM structure of Tom22-containing dimer (D) and crosslinked-based structure model of Tom22-containing trimer (T). b, The residues whose proximity relationships are inconsistent between the trimer (crosslinking) and the dimer (EM structure) are mapped on the EM structure. Top view from the cytosolic side (left) and side view (right) of the EM dimer structure (cartoon model) with the side chains (space-filling model) of the residues that were crosslinked to other subunits4 but were not within a distance of 4.5 Å from the crosslinked partners in the EM structure (Supplementary Table 1), and therefore judged to be important for formation of the mature trimer4. c, The residues suggesting the possible difference in the subunit interfaces between the trimer and dimer are mapped on the EM structure. Although Tom22 A P112 and Tom40 B Y309 face each other in the EM structure, the Tom22 transmembrane helix was crosslinked to two Tom40 molecules through Tom22 P112 and Tom40 Y3094, suggesting that these two residues should not face each other in the trimer. Tom22 E120 and Tom40 R310 are evolutionary conserved (Extended Data Fig. 7 and Supplementary Table 2) and electrostatic interaction between the two residues would contribute to stabilization of the dimer. d, e, Upon formation of the trimer from the dimer, the Tom22 transmembrane helix in the EM structure may rotate (with a possible conformational change), so that Tom22 A P112 faces the Tom40 A molecule and Tom22 A S114 interacts with the Tom40 B molecule. This rotation would lead to a switch of the electrostatic-interaction pairs from Tom22 E120 and Tom40 R310 to Tom22 E120 and Tom40 K90 with possible concomitant movement of the Tom7 C-terminal segment; the second short C-terminal helix of Tom7 appears to be stabilized by the hydrogen bond between the carbonyl O of L51 in Tom7 and the side-chain NH of K90 in Tom40 in the dimer, which is probably lost in the trimer. The rotation of the Tom22 transmembrane helix would also allow K94 and R89 of Tom22 to interact with D350 of the neighbouring Tom40 molecule. Basic and acidic residues are coloured in blue and red, respectively.