The diameters of Dnm1-lipid tubes recover after an initial constriction. (a–d) Cryo-EM images of Dnm1-lipid tubes in the absence of GTP (a & b) and 5 seconds after addition of 0.1 mM GTP (c & d). Scale bar, 100 nm. (e–g) Distribution of Dnm1 tube diameters before (e) and after (f, 5 sec and g, 30 sec) addition of 0.1 mM GTP were measured. Corresponding outer tube diameters are highlighted by dashed blue lines (no GTP) in panels a (110 nm) and b (150 nm) and by dashed red lines (0.1 mM GTP, 5 sec) in c (65 nm) and d (50 nm).

A model for mitochondrial fission. (a) An active contractile force is proposed to play a role in mitochondrial fission, where Dnm1 is recruited to mitochondria and constricts the underlying membrane(s), which leads to fission and release of the protein. (b) Differences in helical packing and GTP-induced conformational changes between Dnm1 and Dyn-1 are compared. Unlike Dyn-1, Dnm1 assembles as a two-start helix and no axial compression is observed upon addition of GTP. The inner lumen of Dnm1 tubes decrease from ~80 nm to ~25 nm, while dynamin decreases from ~20 nm to ~10 nm.

Dnm1-lipid tubes constrict upon addition of GTP. (a–i) Dnm1-lipid tubes were imaged using negative stain EM (a–c) and cryo-EM (d–i). Dnm1 tubes in the absence of nucleotide (a, d & g), in the presence of GMP-PCP (b & e), and after addition of 1 mM GTP (c, f, h & i). Scale bars, 50 nm (a–f) and 100 nm (g–i). Bare lipid tubes (h, *), regions where Dnm1 filaments are loosely packed (i, arrows), and Dnm1 filaments that have dissociated from the membrane (i, arrowheads and insets) are highlighted. (j) Distributions of tube diameters for Dnm1 tubes treated with or without 1 mM GTP for 5 seconds. (k) 90° light scattering of Dnm1 tubes decreased upon addition of 1 mM GTP (red arrow).

An analysis of Dnm1 helical packing. (a) Comparison between Dnm1 and ΔPRD-dynamin 1 (Dyn-1) structures37. The helical pitch is 28.8 nm for Dnm1 and 10.6 nm for dynamin. The axial spacing between the two starts of the Dnm1 helix is 14.4 nm. The outer diameters (129 nm and 50 nm, respectively), radial path lengths (16.9 nm and 11.1 nm, respectively), ridge and cleft features are also indicated. The outer radial density (green) and the inner radial density (blue) are where the GTPase domains and the middle/GED domains are predicted to reside, respectively. (b) Four GTPase domain crystal structures from dyn A (PDB ID: 1JX2, chain B) were manually fitted to one asymmetric subunit of the Dnm1 helical structure. Two dimers found in separate helical starts of the asymmetric subunit are colored purple and orange, respectively. Two density thresholds are presented (low threshold, blue-green; high threshold, yellow). The dashed box highlights the region presented in c (left panel) after a 90° rotation. (c) Fittings of GTPase domains are compared between Dnm1 (left) and Dyn-139 (right). A 3–4 nm gap between Dnm1 and the lipid bilayer (grey) exists where the PH domain (orange ribbon, yellow mesh) of Dyn-1 resides.

3D reconstructions of Dnm1 helices. (a) The primary sequence of Dnm1 contains 4 domains: GTPase, middle, B-insert and GTPase-effector (GED). (b) A cryo-ET reconstruction of Dnm1-lipid tubes is shown from two orthogonal perspectives. The cylindrical shape of the helices is highlighted adjacent to a central z-slice by an end view of the tomogram of Dnm1 (blue box). Scale bar, 100 nm. (c) The 3D structure of the Dnm1 tube is presented with a wedge of the helix removed to show a representative cross-section. The protein is colored with a radial gradient (blue near the lipid to green at the periphery). The lipid bilayer is grey. The outer diameter (129 nm), inner lumen (89 nm), helical pitch (28.8 nm), the two helical starts (labeled 1 and 2) and the spacing between each start (14.4 nm) are highlighted. The lipid bilayer, ridge and cleft features are indicated. (d) A representative raw image of the Dnm1 tubes. (e) One side of the raw image highlights the gap between lipid and protein. (f) An average 2D projection of the final Dnm1 reconstruction. (g) One side of the 2D projection. (h), End view of the final 3D structure. Scale bar, 20 nm. (i) A cross-section of the final 3D map highlights the gap between Dnm1 and the lipid bilayer (compare with e & g). Scale bar, 10 nm.