(A)/(B) Typical 300 kV image of ice embedded Hc in the resting/activated state recorded in a JEM3200FSC electron cryomicroscope. Representative particles are highlighted by white boxes. Scale bar represents 245 Å.
(C)/(D) Top view of Hc in the resting/activated state at a threshold corresponding to Hc molecular weight. Different subunits are illustrated in different colors, and labelled. The resolution is 6.8 Å and 8.0 Å for resting and activated states, respectively. The dotted circles highlight the new bridging densities absent/formed in the resting/activated state. The four hexamers are labelled as 1, 2, 1', and 2'. 2-fold symmetry was imposed in the reconstruction. Scale bar in (D) represents 60 Å, which is applicable for (C)–(H).
(E)/(F) Hexamer 3-fold view of Hc in resting/activated state.
(G)/(H) Bottom bow tie view of Hc in resting/activate state.

(A) SSEs of the linker subunit C1 in the resting state identified by SSEHunter. Eleven α-helices longer than 2-turns (cyan cylinders) and three large β-sheets (green surfaces) have been identified in this subunit.
(B) SSEs of the same subunit C1 in the activated state. Ten α-helices (orange cylinders) and three β-sheets (pink surfaces) are identified.
(C)/(D) Pseudo atomic model of C1 subunit fitted into the corresponding map of the resting/activated state where blue/yellow coloring represents the resting/activated state. The same color scheme is utilized in the rest of the figures.

(A) 3-D variance map (purple) among the 12 subunit map segments within an asymmetric unit of the resting state illustrating their conformational variations. The variance map is shown in the frame of the corresponding average-map, with a model in light blue ribbon shown to orient the figure. Notably, domain I (highlighted by dotted circle) as a whole shows more pronounced variation than the other two domains. Overall, the outer exposed area tends to have more variations as compared to the interior, especially at the inter-subunit contacting regions indicated by arrowheads (brown, blue, and red).
(B) Variance map among the 12 subunits within an asymmetric unit of the activated state with the same visualization style and labels as in (A).

(A) Conformational variation analysis using deduced SSEs of the two states (as in Figure 2A and 2B, same colour code adopted), which are aligned together and illustrated in the frame of the resting state map. The black dotted circle highlights the domain I region. Black arrows show the twisting direction of domain I as induced by SDS activation. The cyan and magenta spheres demonstrate the putative locations of the Cu and oxygen atoms forming the active site.
(B) Cα movement between the averaged models of the two states, mapped onto the activated state averaged Cα model. Red demonstrates larger variance (Cα movement 3.8 Å and larger) while blue demonstrates smaller variance.
(C) The aligned averaged Cα models of resting state (light blue) and activated state (gold). The same illustration style as in (A) (dotted circle, black arrow and active site) is adopted. Location of the Cα of the placeholder residue PHE49 is illustrated using blue (resting state) and red (activated state) spheres.
(D) Zoom in view of the active site with solid line circle illustrating the location of the loop region containing PHE49, which also connects to one of the domain I α-helices (indicated by black straight arrow). The small curved arrow indicates the moving direction of PHE49 as a result of SDS activation.

(A) Overall view of the four bridges (three types) in the interface between hexamers 1 and 2' in the resting state. The visualization angle and location with respect to the entire Hc complex are illustrated using a small reference panel in the middle column.
(B) View of the four interaction bridges at the same location as in (A), but for the activated state. Maps for both states are visualized at a threshold corresponding to the molecular mass of Hc.
(D) Bridge IV (front bridge) formed across the central cavity connecting the opposite linker subunits C1 and C1' in the activated state, whereas no such bridge exists in the resting state as illustrated in (C). The red stars in (C) and (D) indicate the conformational change loop regions involved in the formation of bridge IV due to activation.
(F) Bridge V (back bridge) in the activated state across the central cavity connecting the N-terminal ends of the opposite two linker subunits C1 and C1'. The black arrows in (E) and (F) point to the N-terminal ends of subunits C1 and C1'. This bridge is extremely weak and almost invisible in the resting state (E).