Analysis of Nucleotide-Dependent Domain Movements
(A) A 15 Å section through the overlaid side view sections of Hsp104^N728A ATPγS and ATP. Colored lines indicate the long axes of the domains.
(B) Overlaid NBD1 cross-sections of Hsp104^N728A ATPγS and ATP. Section position is indicated by dashed lines in (A). Colored lines indicate the long axes of the domains.
(C) A 15 Å section through the overlaid side views of Hsp104^N728A ATP and ADP. Colored lines indicate the long axes of the domains.
(D) Overlaid NBD1 cross-sections of Hsp104^N728A ATP and ADP. Section position is indicated by dashed lines in (C). Colored lines indicate the long axes of the domains.
(E) Cross-section of Hsp104^N728A ATPγS NBD2. The position of the connection to NBD1 is indicated by a black ellipse. The long axis of the domain is represented by a black line.
(F) Overlaid NBD2 cross-sections of Hsp104^N728A ATPγS and ATP. The connection to NBD1 and the long axis of the domain are indicated as a green ellipse and line. Black line and ellipse are as in (E).
(G) Overlaid NBD2 cross-sections of Hsp104^N728A ATPγS and ADP. The connection to NBD1 and the long axis of the domain are indicated as an orange ellipse and line. Black line and ellipse are as in (E).

Surface Exposure of Hsp104 Coiled-Coil Domain and NBD2 Tyr Loops
(A) Monomeric or hexameric Hsp104 (1 μg or 0.25 μg) was applied to nitrocellulose and probed with monoclonal antibodies that recognize either the N-terminal domain (28B), middle domain (4G10, 4B, and 17B), or C-terminal domain (27B) of Hsp104.
(B) Hsp104^Y662W or Hsp104^Y662W:N728A (2 μM) was incubated in the absence or presence of ATPγS, ATP, or ADP (1 mM) for 10 min at 25°C. The change in tryptophan fluorescence (ΔF) was determined by comparing the fluorescence after 10 min (F) to the starting fluorescence (F₀), such that ΔF = (F/F₀) − 1. Values represent means ± SD (n = 4).

Model for AAA+ Cooperativity and Clockwise ATP Hydrolysis
(A) In the presence of ATP, Hsp104^N728A is mainly bound to ATP in NBD2, and NBD1 is in a posthydrolysis state (ADP.Pi or ADP). The tyrosine loops in NBD2 are accessible (TYR), whereas the ADP-bound NBD1 is incompetent to bind substrate and Tyr loops are inaccessible (tyr).
(B) ATP binding to NBD1 moves the Tyr loops from the center of the cavity to the N-terminal entrance of the cavity, enabling substrate binding in NBD1. NBD2 Tyr loops are rotated downward and toward the right-hand neighbor, where they are occluded in the interface between the subunits.
(C–F) Model of a hybrid Hsp104 hexamer consisting of one subunit in the T/T state (blue) and the other five in the D/T state (white). Shown are filtered surface representations of the Hsp104 homology-modeled subunits derived from the fits to the Hsp104^N728A ATPγS and ATP maps. In each subunit, Tyr 257 and Tyr 662 are colored in pink and red, respectively. For the side view representation (C), the front two ATP subunits are omitted for clarity. The positions of the tyrosine loops of the T/T subunit (T1, T4) and the D/T state (T2, T3) are indicated. The top view of the AAA+ double layer (D) shows the location of all tyrosine loops in the hexamer by displaying transparent NBDs. The NBD1 (E) and NBD2 (F) rings are displayed as top views.

Cryo-EM Reconstructions of Hsp104^N728A in the Presence of ATPγS, ATP, and ADP
(A) (Upper row) Class averages generated from the final data set obtained by multivariate statistical analysis in IMAGIC, containing an average of 15 images per class. (Lower row) Corresponding reprojections of the 3D structures.
(B) Three-dimensional reconstructions of Hsp104^N728A in the presence of ATPγS (blue), ATP (green), and ADP (yellow) as side view (upper row) and cut-open side view (lower row). Surface views show the density rendered at a threshold accounting for a molecular mass of 612 kDa. Red areas of the cut surfaces indicate high-density core regions. Measurements are given in Å.

Domain Docking of the Hsp104 Homology Model into EM Densities
Rigid body domain fitting into the Hsp104^N728A ATPγS (A, D, and G), ATP (B, E, and H), and ADP (C, F, and I) maps. Domains and subdomains are color-coded as follows: N, yellow-orange; NBD1, red/orange; NBD2, blue/cyan; coiled coil, green. The ATP-binding pocket is located at the interface between the NBD subdomains (red/orange, blue/cyan). The ClpB X-ray structure (PDB code:1QVR) was used for docking into the N-terminal density. Tyrosines 257 and 662 are depicted as magenta spheres.
Cross-sections through NBD1 of Hsp104^N728A ATPγS (A), ATP (B), and ADP (C), showing half of a ring. Equivalent views of NBD2 of Hsp104^N728A ATPγS (D), ATP (E), and ADP (F). The long axis of the domain and positioning of Tyr 257 and Tyr 662 are indicated in cartoons. Side view sections of an Hsp104^N728A ATPγS subunit (G), ATP (H), and ADP (I) subunit.

Asymmetric Cryo-EM Reconstructions of Hsp104 ATPγS and ATP
(A) Side view of the asymmetric 3D reconstruction of Hsp104^N728A ATPγS. Each subunit is colored differently.
(B) Overlaid cross-sections through NBD1 of 6-fold (transparent blue) and asymmetric maps of Hsp104^N728A ATPγS. Color code of the asymmetric reconstruction is as in (A).
(C) Overlaid cross-sections through NBD2 of 6-fold and asymmetric maps of Hsp104^N728A ATPγS. Color code of the asymmetric reconstruction is as in (A).
(D) Side view section through each subunit of the asymmetric reconstruction compared to sections of 6-fold symmetric Hsp104^N728A ATPγS (left) and Hsp104^N728A ATP (right). N, NBD1, and NBD2 indicate domain assignments.
(E) Side view of the asymmetric 3D reconstruction of Hsp104^N728A ATP. Each subunit is colored differently.
(F) Overlaid cross-sections through NBD1 of 6-fold (transparent green) and asymmetric maps of Hsp104^N728A ATP. Color code of the asymmetric reconstruction is as in (A).
(G) Overlaid cross-sections through NBD2 of 6-fold and asymmetric maps of Hsp104^N728A ATP. Color code of the asymmetric reconstruction is as in (A).
(H) Side view section through each subunit of the asymmetric reconstruction compared to sections of 6-fold symmetric Hsp104^N728A ATPγS (left) and Hsp104^N728A ATP (right). N, NBD1, and NBD2 indicate domain assignments.