Fig 2: Sequence alignments of CCTs and PDCD5.a, Sequence alignments of PDCD5 from various organisms using ClustalO in Jalview. b, Evolutionary conservation analysis of PDCD5 using ConSurf Web Server. c, Sequence alignments of the stem loops of CCT1 from various organisms. d, Sequence alignments of the stem loops of human CCT1-CCT8. e, Open TRiC (PDB 7NVO) and PDCD5 (helices α3 to α5, residues Q50 to T103) was shown as surface coloured by electrostatic potential. Black circles highlighted the stem loops of CCT1-CCT8. f, Binding of PDCD5 to TRiC was measured by co-immunoprecipitation (co-IP) from HEK 293F cells transfected with PDCD5-Flag constructs (WT: wild-type; IL: I93G, L96G; RKK: R55A, K63A, K66A). The experiment was repeated independently five times with similar results. g, Quantification of co-eluted CCT1 with PDCD5-Flag in (f). The percentage was calculated with IPCCT1/IPPDCD5 and normalized to WT (set at 100%). The data represent the mean ± SD of five biologically independent experiments. Statistical analysis was performed using two-tailed unpaired t-tests. Significantly different (P < 0.05). h, Native gels show western blots analysis of the binding of increasing amounts of recombinant WT PDCD5 (top) and RKK PDCD5 (bottom) to 300 nM TRiC. Interaction was measured after 20 min of interaction at 25 °C in buffer (50 mM Tris pH 7.4, 100 mM KCl, 5mM MgCl2, 10% glycerol, and 1mM TCEP). left panel: anti-PDCD5, right panel: anti-CCT8. The experiment was repeated independently two times with similar results. i, Native gels show the binding of PDCD5 WT or IL (3 μM) to TRiC (300 nM). The interaction was measured in ATPase buffer at 25 °C after 10 to 30 min. j, Quantification of the band intensity in (i). n = 2 biologically independent experiments. Data are presented as mean values ± standard error of the mean (SEM). Source Data

Fig 3: PDCD5 structures and densities of the open TRiC map.a, NMR structure of PDCD5 (1 – 112 a.a.). The five helices of PDCD 5 were labelled as α1 to α5. b, Overlay of PDCD5 at different conformations (PDB 2K6B). c, Overlay of the AlphaFold predicted model of PDCD5 (PDCD5, AF-O14737-F1, full length, 1 – 125 a.a.) with PDB 2K6B coloured in grey. AlphaFold produces a per-residue model confidence score (predicted local distance difference test, pLDDT) between 0 and 100. Very high (blue, pLDDT > 90), High (light blue, 90 > pLDDT > 70), Low (yellow, 70 > pLDDT > 50), Very low (orange, pLDDT < 50). d, The additional density that was not modelled by in vitro studies of TRiC. The density was fitted with the AlphaFold predicted model of PDCD5.

Fig 4: Atomic models were fitted into TRiC structures in HHT-treated cells.a, Cryo-ET map of open TRiC without PFD, fitted with PDB 7X3J in treated cells. b, Overlaid maps (at similar contour level) of untreated and treated TRiC without PFD. The opacity of the untreated map was set at 60%. The predicted model of PDCD5-CCT3-CCT1-CCT4 was fitted into the map in (a). c, PDB 7WU7 was fitted into the open TRiC structure bound with 1 PFD. d, PDB 7WU7 was fitted into the PFD density segmented from (c). e, Overlaid maps (at similar contour level) of untreated and treated TRiC with one PFD. The densities inside the TRiC chamber were Gaussian-filtered (sDev = 4) in (b) and (e) for visualization. The predicted model of PDCD5-CCT3-1-4 was fitted into the map in (c). f, The open TRiC structure bound with 2 PFDs was fitted with PDB 7WU7. g, PFD densities segmented from (f) were fitted with PDB 7WU7. h, Overlaid maps (at similar contour level) of untreated and treated TRiC with two PFDs. The predicted model of PDCD5-CCT3-CCT1-CCT4 was fitted into the map in (f). i, Overlaid maps (at similar contour level) of untreated and treated TRiC at the closed conformation. The opacity of untreated maps was set at 60%.

Fig 5: PDCD5 was not associated with closed TRiC.a, The atomic model of PDCD5-CCT3-CCT1-CCT4 (AlphaFold-Multimer) was fitted into two rings of the open TRiC map (C1 symmetry). PDB 7NVO, grey. b, The closed TRiC map (C1 symmetry) was fitted with equatorial domains of closed TRiC (PDB 7NVL). c,d, PDCD5-CCT3-1-4 (AlphaFold-Multimer) was superimposed with open TRiC (PDB 7X3J) and closed TRiC (PDB 7NVL). The black arrow indicates a potential clash between the C-terminus of PDCD5 and the helix in CCT4 (N394 to V416) in the closed TRiC but not in the open TRiC. e,f, PDCD5-CCT3-1-4 (AlphaFold-Multimer) was overlaid with closed TRiC associated with actin (PDB 7NVM) and tubulin (PDB 7NVN). g, CCT3 antibody (rabbit) pulldown endogenous PDCD5 in HEK293F cells. Rabbit IgG (mock) as a control. h, The schematic of induction of TRiC closure during co-IP in two conditions. i, As illustrated in (h), beads bound with TRiC-PDCD5-Flag (from co-IP) were incubated in buffer with ATP/AlFx or without ATP/AlFx (control) for 1 h at 37 °C. The supernatant (containing released TRiC and PDCD5) and beads (bound with TRiC-PDCD5-Flag) were detected by western blotting. The experiment was repeated independently four times with similar results. j, The ratio of PDCD5 (left two columns) in supernatant compared to PDCD5 remaining bound to beads after ATP/AlFx incubation in (i). The ratio of TRiC (right two columns) in supernatant compared to TRiC bound to beads after ATP/AlFx incubation in (i). The data represent the mean ± SD of four biologically independent experiments. Statistical analysis was performed using two-tailed unpaired t-tests. Significantly different (P < 0.05). ns, not significant. (k) Native gels of the interaction of 300 nM TRiC to 3 μM WT PDCD5 in buffer containing 1 mM of different ATP analogues, which induce different TRiC conformational states, analysed by immunoblotting with anti PDCD5 or CCT8 antibodies. The experiment was repeated two times with similar results. Source Data