Fig. 3: Cryo-EM structure of Abo1-Walker B mutant in the ATP state shows unique AAA+ structural organization.

a An atomic model (left) and cryo-EM map (right) of ATP-Abo1 Walker B mutant viewed from “bottom” (AAA2 side, top panel) and “side” of the AAA+ ring (bottom panel). The AAA1 ___domain is colored in light green, the AAA2 ___domain in teal, and the C-terminal ___domain in magenta. From the side view, the bromodomain, AAA1, AAA2, and the C-terminal form four tiers. The scale bars indicate 2 nm (20 Å). b The structure of a single Abo1 monomer within the Abo1 hexamer. Helices of AAA1 and AAA2 NBD and HBD are labeled according to AAA+ structural convention. Approximate bromodomain position based on electron density maps is depicted as a cartoon oval. The connectivity of the AAA+ ___domain, bromodomain, and C-terminal ___domain based on the structure and cryo-EM map is indicated as dotted lines. c Superposition of the Cα’s of the Abo1 backbone (dark blue) with the AAA+ ATPases p97 (light green, PDB ID: 5FTM) and NSF (pink, PDB ID: 3J94), aligned with respect to AAA1. The unique inserts of Abo1—the AAA2 knob and the linker arm proceeding AAA2 α5—are highlighted by shaded ovals. The AAA2 domains do not align well due to the variable angle between AAA1 and AAA2. d The “knob and hole” packing of Abo1 subunits where the knob of one subunit inserts into the hole of the adjacent subunit (black arrowheads and dotted circles). Abo1 subunits are colored by chain. e The “split” AAA2 helical bundle ___domain (HBD) of Abo1, where α5 of AAA2 (teal, aa 734–750) interacts with three helices (α6–α8) of the C-terminal ___domain (magenta, aa1129–1185). AAA2 HBD is superposed onto its closest structural relative, 26 S proteasome AAA-ATPase subunit 6B (tan, PDB ID: 5ln3), showing that the three-dimensional structure of Abo1 HBD is conserved with other AAA+ HBD’s despite unique helix connectivity.