Fig. 2: The BSE lock.

a, b Comparing the previous crystal structure⁹ (PBD ID: 4BEJ, blue) to the dimeric cryo-EM structure (orange) based on overlay of the stalk region. c Loop 3 (L3^s) interacts with BSE helix α2^b and contains R456. d Negative stain electron microscopy characterization of WT and R456E 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, R456E apo = 4, R456E + GMP-PCP = 21, R456E+CLNTs=48). Scale bar = 100 nm. e, f 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, R456E(pink) = 8. ** P = 0.006). g SEC-MALS analysis of Drp1 WT (blue) and Drp1 R456E (pink) is presented with multimeric states indicated. h GTPase activity was determined for WT (blue) and Drp1 R456E (pink) 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 (CLnts)=10, R456E (apo and CLnts)=12. ****P < 0.0001). i, j 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 R456E. Scale bar 5 µm, inset 10 × 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 gray) = 64, KO+Drp1 (WT, blue) = 30, KO + R456E (pink) = 33).