Extended Data Fig. 7: 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.

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