Fig. 3: Dimeric cryo-EM structure dimer interface.

a Comparison of the stalk orientations in the crystal structure dimer interface, (PDB code: 4BEJ, dark blue - chain A, light blue - chain B) and the dimeric cryo-EM structure (dark orange - chain A, light orange - chain B). b Alignment of each helix comprising the stalk domain (PDB code: 4BEJ, blue; cryo-EM structure, orange). Y493 is highlighted in light green. c Helices forming the dimer interface are fit within the cryo-EM density (chain A, orange; chain B, yellow). Y493 (green) bookends the interface. d Alignment of crystal structure dimer and dimeric cryo-EM structure stalks, reference chain is chain A. Zoomed in view, Y493 in light green. e Negative stain electron microscopy characterization of WT and Y493A Drp1 in the presence and absence of non-hydrolysable GMP-PCP and CL-containing nanotubes (CLnts). Multiple grids were made and several images were collected for each condition (WT apo=8, WT + GMP-PCP = 38, WT+CLNTs = 44, Y493A apo = 9, Y493A + GMP-PCP = 25, Y493A+CLNTs=46). Scale bar = 100 nm. f, g Sedimentation assays were used to quantify changes in polymerization based on the relative percent of protein detected in the supernatant (S) and pellet (P) fractions. Data are presented as mean values +/− SEM, and a two-tail t-test was used to determine statistical significance. Each dot represents an experimental replicate (WT (blue) = 9, Y493A (green) = 8.***P = 0.0005; *P = 0.04). h SEC-MALS analysis of Drp1 WT (blue) and Drp1 Y493A (green) is presented with multimeric states indicated. i GTPase activity was assessed for WT (blue) and Drp1 Y493A (green) alone and stimulated with CLnts. Data are presented as mean values +/− SEM, and a two-tail t-test was used to determine statistical significance. Each dot represents an experimental replicate (WT (apo) = 12, WT (nts) = 10, Y493A (apo) = 12, Y493A (nts)=11. ****P < 0.0001). j, k MitoTracker Orange was used assess mitochondrial morphology in WT MEFs compared to Drp1 knock-out MEFs transfected with an empty pCMV vector (EV) and pCMV vectors containing Drp1 WT and Drp1 Y493A. Scale bar 5 µm. Inset 10 µm. Mitochondrial morphology was quantified using a blinded assessment described in the methods section. Two independent experiments were performed to assess the percentage of cells with defined mitochondrial morphologies in each sample (Total cells counted: WT MEF (dark gray) = 60, EV (light grey) = 64, KO+Drp1 WT (blue) = 30, KO + Y493A (green) = 29).