Extended Data Fig. 9: Biochemical validation of higher oligomeric TOM complexes.
From: Cryo-EM structure of the mitochondrial protein-import channel TOM complex at near-atomic resolution
a, Left, overview (angled cytosolic view) of the tetrameric TOM complex. Monomeric units B and C are shown in color, and A and D are in gray. The region in the black dashed box is magnified and shown in the right panel. Right, the cryo-EM density map (semitransparent gray) and the atomic model (in color) are shown for the B–C interface. The blue dashed arrows indicate the directions of the unmodeled N-terminal segments (residues 1–24) of the Tom6C and Tom6B subunits. The black dotted oval indicates the hydrophobic patch HP2. The green dashed lines indicate the unmodeled loop (L14-15; residues 277–294) between β14 and β15 of Tom40. b, Mitochondria were treated with BM-PEG2 and analyzed by SDS-PAGE and immunoblotting (IB). Tom40 contained no or an indicated single cysteine. c, Mitochondria were purified from cells expressing Tom40Strep (M287C) from the chromosomal locus and Tom40His (M287C) from a CEN plasmid. After treating with BM-PEG2, mitochondria were solubilized with octyl glucoside and subjected to immunoprecipitation (IP) using anti-Strep-tag antibodies (mock: IP without anti-Strep-tag antibodies). d, Mitochondria with Tom40Strep (M287C) expressed from the endogenous promoter were solubilized in 0.5% LMNG and 0.1% CHS and then injected to Superose 6 column. Fractions were treated with BM-PEG2 before SDS-PAGE and immunoblotting. e, As in Fig. 4e, but with mitochondria isolated from the tom6Δ mutant background. “T” and “D” indicate the peak positions of tetramers and dimers, respectively. Yeast were grown in YPEG (b and e) or YPD (c and d). Source data for panels b–e are available with the paper online. The experiments in b–e were repeated at least twice with similar results.
