Cryo-EM study of TRiC conformational cycle. Cartoon diagram illustrates the TRiC conformational transitions in the apo state and four distinct nucleotide biochemical states throughout the ATPase cycle. In addition, a representative micrograph of ice-embedded TRiC in each state is shown. Scale bar is 320 Å.

Symmetry-free cryo-EM maps of TRiC in the three open conformational states. (A) Top, bottom, and side views of apo-TRiC. In the top ring, the subunits were labelled as a₁–a₈; whereas in the bottom ring, the subunits were labelled as a′₁–a′₈. The location of the pseudo two-fold axis was labelled in the side view. The extra density seen in the equatorial domain region is indicated by a black arrow. (B) Equivalent views of TRiC–AMP-PNP (ATP-bound state). (C) Equivalent views of TRiC–ADP (ADP-bound state). The same radial colour scheme from the centre of a cylinder is used throughout.

Symmetry-free cryo-EM density maps of TRiC in the ADP-AlFx and ATP-AlFx states. The same colour scheme is adopted as in Figure 2. (A) Top, bottom, and side views of TRiC–ADP-AlFx in an asymmetric conformation with the cis-ring closed while the trans-ring still open. There is a small opening in the lid around subunits a₃ (indicated by a black arrow). (B) Views of TRiC–ATP-AlFx in a both ring closed conformation (Cong et al, 2010). (C) TRiC–ADP-AlFx closed cis-ring chamber inter-volume diameter (∼100 Å) compared with that of TRiC–ATP-AlFx (∼92 Å). The diameter was measured at the equivalent location of the intermediate domain of the two rotational averaged maps. The high-resolution TRiC–ATP-AlFx map was blurred to ∼10 Å, in a comparable manner to the ADP-AlFx map. (D) Overlapping of the closed ring of TRiC–ADP-AlFx (in purple) and TRiC–ATP-AlFx (in dark blue) showing different extents of TRiC chamber closure, that is, the closed ring in TRiC–ATP-AlFx is more compact than that in TRiC–ADP-AlFx.

Unwrapped maps of TRiC in different biochemical states. By cutting the map and unrolling it onto a planar surface, we can see all of the subunits and their positional relationships more clearly. (A) Side view of the apo-TRiC unwrapped map, visualized from outside of the complex. The upper ring subunits are rendered in blue and the lower ring subunits in red, while the interacting equatorial domain regions in both rings are white. The location of the pseudo two-fold axis is labelled as a green ellipsoid. The top ring subunits are labelled as a₁–a₈, while the bottom ring subunits labelled as a′₁–a′₈. The tilt angle of the characteristic subunits a₃ and its connecting subunit a′₇ is highlighted with a green dotted line, as a comparison the tilt angles of subunit pairs a₂–a′₈ and a₄–a′₆ are also labelled (cyan dotted lines). (B) Side view of the unwrapped map of TRiC–AMP-PNP. The tilt angle of the characteristic subunits pair a₃–a′₇ is comparable to that in apo-TRiC and still highlighted with a green dotted line. A similar rendering style as in (A) was adopted hereafter. (C) Side view of the unwrapped map of TRiC–ADP-AlFx, with cis-ring (upper) in blue and trans-ring (lower) in red. The tilt angle of subunit pair a₃–a′₇ is labelled in yellow dotted line, which is reduced ∼10° as compared with that in the AMP-PNP state (green dotted line). (D, E) Side view of the unwrapped map of TRiC–ADP/TRiC–ATP-AlFx. To render the features of the maps in a comparable manner, the high-resolution TRiC–ATP-AlFx map was blurred into a similar resolution as the other maps. In the unwrapping process, due to oversampling in the region close to the cylindrical axis, there might be slight distortions in the unwrapped map especially in the inward tilting upper apical domain regions, similar to a Mercator Projection of a global map of the Earth.

TRiC conformational comparison and transition in the ATPase cycle. (A) Rotational correlation analysis between the two rings of apo-TRiC, with the top ring fixed and bottom ring rotated. At 0°, the two rings have the best correlation score, indicating the location of the two-fold axis as illustrated in Figure 2A. Among the eight peaks, four of them have higher value and were highlighted by orange stars indicating the existence of a pseudo four-fold symmetry in the complex. (B) Conformational comparison between the two rings of apo-TRiC (top ring in grey and bottom ring in yellow). (C) Conformational comparison between the two rings of TRiC–AMP-PNP (top ring in grey and bottom ring in cyan). (D) Conformational variation between the two rings of TRiC–ADP-AlFx (cis-ring in grey and trans-ring in purple). (E) Rotational correlation analysis between TRiC–AMP-PNP and TRiC–ADP-AlFx. At 0°, the two maps have the best correlation score, indicating the best alignment angle between the two maps as illustrated in (G, H). As in (A), among the eight peaks, four of them have higher value and were labelled by orange stars. (F) Conformational variation between apo-TRiC (in yellow) and TRiC–AMP-PNP (in cyan). (G, H) Conformational transition from the ATP-bound state to the ATP hydrolysis transition state with (G) showing the closed cis-ring of TRiC–ADP-AlFx overlaid with the open top ring of TRiC–AMP-PNP, and (H) showing the open trans-ring of TRiC–ADP-AlFx aligned with the other open ring of TRiC–AMP-PNP.

The structural mechanism of TRiC negative inter-ring cooperativity. (A) Conformational comparison between TRiC–AMP-PNP (in light blue) and TRiC–ADP-AlFx (in purple). Only the side view of a central slide of the two maps was shown. The direction of the inward/outward tilt motion in the cis- and trans-rings of TRiC–ADP-AlFx is indicated by black arrows. (B) The plot of the density map mean intensity versus the height of TRiC. The plot for TRiC–AMP-PNP is in light blue, and that for TRiC–ADP-AlFx in purple. The top two ranking peaks (indicated by dotted lines) correspond to the location of the most condensed region in the equatorial domain in each ring. There is a detectable distance enlargement (4–5 Å) between the two rings of TRiC induced by ATP hydrolysis.