Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Staggered ATP binding mechanism of eukaryotic chaperonin TRiC (CCT) revealed through high-resolution cryo-EM


The eukaryotic chaperonin TRiC (or CCT) assists in the folding of 10% of cytosolic proteins. Here we present two cryo-EM structures of Saccharomyces cerevisiae TRiC in a newly identified nucleotide partially preloaded (NPP) state and in the ATP-bound state, at 4.7-Å and 4.6-Å resolution, respectively. Through inner-subunit eGFP tagging, we identified the subunit locations in open-state TRiC and found that the CCT2 subunit pair forms an unexpected Z shape. ATP binding induces a dramatic conformational change on the CCT2 side, thereby suggesting that CCT2 plays an essential role in TRiC allosteric cooperativity. Our structural and biochemical data reveal a staggered ATP binding mechanism of TRiC with preloaded nucleotide on the CCT6 side of NPP-TRiC and demonstrate that TRiC has evolved into a complex that is structurally divided into two sides. This work offers insight into how the TRiC nucleotide cycle coordinates with its mechanical cycle in preparing folding intermediates for further productive folding.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Cryo-EM maps of TRiC in the NPP and ATP-bound states.
Figure 2: Determination of subunit locations in NPP-TRiC and the match between the pseudoatomic model and the corresponding map.
Figure 3: Conformational change in TRiC triggered by ATP binding.
Figure 4: Nucleotide distribution analysis of different states of TRiC.
Figure 5: Changes in inter-ring and intraring interaction patterns induced by ATP binding.
Figure 6: Proposed staggered ATP binding and substrate-folding mechanisms for TRiC.

Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Referenced accessions

Protein Data Bank


  1. 1

    Balchin, D., Hayer-Hartl, M. & Hartl, F.U. In vivo aspects of protein folding and quality control. Science 353, aac4354 (2016).

    Article  Google Scholar 

  2. 2

    Balch, W.E., Morimoto, R.I., Dillin, A. & Kelly, J.W. Adapting proteostasis for disease intervention. Science 319, 916–919 (2008).

    CAS  Article  Google Scholar 

  3. 3

    Kim, Y.E., Hipp, M.S., Bracher, A., Hayer-Hartl, M. & Hartl, F.U. Molecular chaperone functions in protein folding and proteostasis. Annu. Rev. Biochem. 82, 323–355 (2013).

    CAS  Article  Google Scholar 

  4. 4

    Li, J. & Buchner, J. Structure, function and regulation of the hsp90 machinery. Biomed. J. 36, 106–117 (2013).

    Article  Google Scholar 

  5. 5

    Saibil, H. Chaperone machines for protein folding, unfolding and disaggregation. Nat. Rev. Mol. Cell Biol. 14, 630–642 (2013).

    CAS  Article  Google Scholar 

  6. 6

    Yam, A.Y. et al. Defining the TRiC/CCT interactome links chaperonin function to stabilization of newly made proteins with complex topologies. Nat. Struct. Mol. Biol. 15, 1255–1262 (2008).

    CAS  Article  Google Scholar 

  7. 7

    Llorca, O. et al. Eukaryotic type II chaperonin CCT interacts with actin through specific subunits. Nature 402, 693–696 (1999).

    CAS  Article  Google Scholar 

  8. 8

    Llorca, O. et al. Eukaryotic chaperonin CCT stabilizes actin and tubulin folding intermediates in open quasi-native conformations. EMBO J. 19, 5971–5979 (2000).

    CAS  Article  Google Scholar 

  9. 9

    Camasses, A., Bogdanova, A., Shevchenko, A. & Zachariae, W. The CCT chaperonin promotes activation of the anaphase-promoting complex through the generation of functional Cdc20. Mol. Cell 12, 87–100 (2003).

    CAS  Article  Google Scholar 

  10. 10

    Trinidad, A.G. et al. Interaction of p53 with the CCT complex promotes protein folding and wild-type p53 activity. Mol. Cell 50, 805–817 (2013).

    CAS  Article  Google Scholar 

  11. 11

    Kasembeli, M. et al. Modulation of STAT3 folding and function by TRiC/CCT chaperonin. PLoS Biol. 12, e1001844 (2014).

    Article  Google Scholar 

  12. 12

    McClellan, A.J., Scott, M.D. & Frydman, J. Folding and quality control of the VHL tumor suppressor proceed through distinct chaperone pathways. Cell 121, 739–748 (2005).

    CAS  Article  Google Scholar 

  13. 13

    Roh, S.H., Kasembeli, M., Bakthavatsalam, D., Chiu, W. & Tweardy, D.J. Contribution of the type II chaperonin, TRiC/CCT, to oncogenesis. Int. J. Mol. Sci. 16, 26706–26720 (2015).

    CAS  Article  Google Scholar 

  14. 14

    Tam, S. et al. The chaperonin TRiC blocks a huntingtin sequence element that promotes the conformational switch to aggregation. Nat. Struct. Mol. Biol. 16, 1279–1285 (2009).

    CAS  Article  Google Scholar 

  15. 15

    Khabirova, E. et al. The TRiC/CCT chaperone is implicated in Alzheimer's disease based on patient GWAS and an RNAi screen in Aβ-expressing Caenorhabditis elegans. PLoS One 9, e102985 (2014).

    Article  Google Scholar 

  16. 16

    Booth, C.R. et al. Mechanism of lid closure in the eukaryotic chaperonin TRiC/CCT. Nat. Struct. Mol. Biol. 15, 746–753 (2008).

    CAS  Article  Google Scholar 

  17. 17

    Cong, Y. et al. Symmetry-free cryo-EM structures of the chaperonin TRiC along its ATPase-driven conformational cycle. EMBO J. 31, 720–730 (2012).

    CAS  Article  Google Scholar 

  18. 18

    Dekker, C. et al. The crystal structure of yeast CCT reveals intrinsic asymmetry of eukaryotic cytosolic chaperonins. EMBO J. 30, 3078–3090 (2011).

    CAS  Article  Google Scholar 

  19. 19

    Cong, Y. et al. 4.0-A resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement. Proc. Natl. Acad. Sci. USA 107, 4967–4972 (2010).

    CAS  Article  Google Scholar 

  20. 20

    Muñoz, I.G. et al. Crystal structure of the open conformation of the mammalian chaperonin CCT in complex with tubulin. Nat. Struct. Mol. Biol. 18, 14–19 (2011).

    Article  Google Scholar 

  21. 21

    Clare, D.K. et al. Multiple states of a nucleotide-bound group 2 chaperonin. Structure 16, 528–534 (2008).

    CAS  Article  Google Scholar 

  22. 22

    Zhang, J. et al. Mechanism of folding chamber closure in a group II chaperonin. Nature 463, 379–383 (2010).

    CAS  Article  Google Scholar 

  23. 23

    Zhang, J. et al. Cryo-EM structure of a group II chaperonin in the prehydrolysis ATP-bound state leading to lid closure. Structure 19, 633–639 (2011).

    CAS  Article  Google Scholar 

  24. 24

    Douglas, N.R. et al. Dual action of ATP hydrolysis couples lid closure to substrate release into the group II chaperonin chamber. Cell 144, 240–252 (2011).

    CAS  Article  Google Scholar 

  25. 25

    Pereira, J.H. et al. Mechanism of nucleotide sensing in group II chaperonins. EMBO J. 31, 731–740 (2012).

    CAS  Article  Google Scholar 

  26. 26

    Pereira, J.H. et al. Crystal structures of a group II chaperonin reveal the open and closed states associated with the protein folding cycle. J. Biol. Chem. 285, 27958–27966 (2010).

    CAS  Article  Google Scholar 

  27. 27

    Liou, A.K. & Willison, K.R. Elucidation of the subunit orientation in CCT (chaperonin containing TCP1) from the subunit composition of CCT micro-complexes. EMBO J. 16, 4311–4316 (1997).

    CAS  Article  Google Scholar 

  28. 28

    Kalisman, N., Adams, C.M. & Levitt, M. Subunit order of eukaryotic TRiC/CCT chaperonin by cross-linking, mass spectrometry, and combinatorial homology modeling. Proc. Natl. Acad. Sci. USA 109, 2884–2889 (2012).

    CAS  Article  Google Scholar 

  29. 29

    Leitner, A. et al. The molecular architecture of the eukaryotic chaperonin TRiC/CCT. Structure 20, 814–825 (2012).

    CAS  Article  Google Scholar 

  30. 30

    Martín-Benito, J. et al. The inter-ring arrangement of the cytosolic chaperonin CCT. EMBO Rep. 8, 252–257 (2007).

    Article  Google Scholar 

  31. 31

    Kalisman, N., Schröder, G.F. & Levitt, M. The crystal structures of the eukaryotic chaperonin CCT reveal its functional partitioning. Structure 21, 540–549 (2013).

    CAS  Article  Google Scholar 

  32. 32

    Jiang, Y. et al. Sensing cooperativity in ATP hydrolysis for single multisubunit enzymes in solution. Proc. Natl. Acad. Sci. USA 108, 16962–16967 (2011).

    CAS  Article  Google Scholar 

  33. 33

    Reissmann, S. et al. A gradient of ATP affinities generates an asymmetric power stroke driving the chaperonin TRIC/CCT folding cycle. Cell Rep. 2, 866–877 (2012).

    CAS  Article  Google Scholar 

  34. 34

    Amit, M. et al. Equivalent mutations in the eight subunits of the chaperonin CCT produce dramatically different cellular and gene expression phenotypes. J. Mol. Biol. 401, 532–543 (2010).

    CAS  Article  Google Scholar 

  35. 35

    Herzog, F. et al. Structural probing of a protein phosphatase 2A network by chemical cross-linking and mass spectrometry. Science 337, 1348–1352 (2012).

    CAS  Article  Google Scholar 

  36. 36

    DiMaio, F. et al. Atomic-accuracy models from 4.5-Å cryo-electron microscopy data with density-guided iterative local refinement. Nat. Methods 12, 361–365 (2015).

    CAS  Article  Google Scholar 

  37. 37

    Sigler, P.B. et al. Structure and function in GroEL-mediated protein folding. Annu. Rev. Biochem. 67, 581–608 (1998).

    CAS  Article  Google Scholar 

  38. 38

    Joachimiak, L.A., Walzthoeni, T., Liu, C.W., Aebersold, R. & Frydman, J. The structural basis of substrate recognition by the eukaryotic chaperonin TRiC/CCT. Cell 159, 1042–1055 (2014).

    CAS  Article  Google Scholar 

  39. 39

    Stewart, M.A., Franks-Skiba, K., Chen, S. & Cooke, R. Myosin ATP turnover rate is a mechanism involved in thermogenesis in resting skeletal muscle fibers. Proc. Natl. Acad. Sci. USA 107, 430–435 (2010).

    CAS  Article  Google Scholar 

  40. 40

    Naber, N., Cooke, R. & Pate, E. Slow myosin ATP turnover in the super-relaxed state in tarantula muscle. J. Mol. Biol. 411, 943–950 (2011).

    CAS  Article  Google Scholar 

  41. 41

    Rye, H.S. et al. Distinct actions of cis and trans ATP within the double ring of the chaperonin GroEL. Nature 388, 792–798 (1997).

    CAS  Article  Google Scholar 

  42. 42

    Polletta, L. et al. SIRT5 regulation of ammonia-induced autophagy and mitophagy. Autophagy 11, 253–270 (2015).

    Article  Google Scholar 

  43. 43

    Hartl, F.U., Bracher, A. & Hayer-Hartl, M. Molecular chaperones in protein folding and proteostasis. Nature 475, 324–332 (2011).

    CAS  Article  Google Scholar 

  44. 44

    Shiau, A.K., Harris, S.F., Southworth, D.R. & Agard, D.A. Structural Analysis of E. coli hsp90 reveals dramatic nucleotide-dependent conformational rearrangements. Cell 127, 329–340 (2006).

    CAS  Article  Google Scholar 

  45. 45

    Zhuravleva, A. & Gierasch, L.M. Substrate-binding domain conformational dynamics mediate Hsp70 allostery. Proc. Natl. Acad. Sci. USA 112, E2865–E2873 (2015).

    CAS  Article  Google Scholar 

  46. 46

    Pappenberger, G., McCormack, E.A. & Willison, K.R. Quantitative actin folding reactions using yeast CCT purified via an internal tag in the CCT3/gamma subunit. J. Mol. Biol. 360, 484–496 (2006).

    CAS  Article  Google Scholar 

  47. 47

    Lander, G.C. et al. Complete subunit architecture of the proteasome regulatory particle. Nature 482, 186–191 (2012).

    CAS  Article  Google Scholar 

  48. 48

    Meyer, A.S. et al. Closing the folding chamber of the eukaryotic chaperonin requires the transition state of ATP hydrolysis. Cell 113, 369–381 (2003).

    CAS  Article  Google Scholar 

  49. 49

    Horst, M., Oppliger, W., Feifel, B., Schatz, G. & Glick, B.S. The mitochondrial protein import motor: dissociation of mitochondrial hsp70 from its membrane anchor requires ATP binding rather than ATP hydrolysis. Protein Sci. 5, 759–767 (1996).

    CAS  Article  Google Scholar 

  50. 50

    Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013).

    CAS  Article  Google Scholar 

  51. 51

    Mindell, J.A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003).

    Article  Google Scholar 

  52. 52

    Scheres, S.H. Semi-automated selection of cryo-EM particles in RELION-1.3. J. Struct. Biol. 189, 114–122 (2015).

    CAS  Article  Google Scholar 

  53. 53

    Scheres, S.H. Beam-induced motion correction for sub-megadalton cryo-EM particles. eLife 3, e03665 (2014).

    Article  Google Scholar 

  54. 54

    Kucukelbir, A., Sigworth, F.J. & Tagare, H.D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).

    CAS  Article  Google Scholar 

  55. 55

    Ludtke, S.J., Baldwin, P.R. & Chiu, W. EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999).

    CAS  Article  Google Scholar 

  56. 56

    Yang, Z. et al. UCSF Chimera, MODELLER, and IMP: an integrated modeling system. J. Struct. Biol. 179, 269–278 (2012).

    CAS  Article  Google Scholar 

  57. 57

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  58. 58

    Szpikowska, B.K., Swiderek, K.M., Sherman, M.A. & Mas, M.T. MgATP binding to the nucleotide-binding domains of the eukaryotic cytoplasmic chaperonin induces conformational changes in the putative substrate-binding domains. Protein Sci. 7, 1524–1530 (1998).

    CAS  Article  Google Scholar 

Download references


We thank W. Chiu (Baylor College of Medicine) for critical discussion of this work, and J. Zhou and Z. Zhou (Institute of Biochemistry and Cell Biology) for their generous support. We thank J. Li, Y. Zhang, and M. Cao from the EM facility and managers from the Database and Computing facility of NCPSS for their assistance with the EM instruments and parallel computing. We are grateful to the NCPSS Protein Expression and Purification Facility, Mass Spectrometry Facility, Nuclear Magnetic Resonance Facility, and Integrated Laser Microscopy Facility for instrument support and technical assistance. This work was supported by grants to Y.C. from the CAS Pilot Strategic Science and Technology Projects B (XDB08030201), the National Basic Research Program of China (2013CB910401), the National Natural Science Foundation of China (31270771 and 31222016), the STS program of the CAS (KFJ-EW-STS-098), and the CAS–Shanghai Science Research Center (CAS-SSRC-YH-2015-01).

Author information




Y.C. and Y.Z. designed the experiments; Y.Z. purified the proteins and optimized the cryo-EM sample-preparation conditions, and M.J., H.W., Z.C., and C.L. were also involved in the protein purification; Y.Z., M.J., and H.W. collected the cryo-EM data with assistance from L.K.; Y.Z. and M.J. performed the cryo-EM reconstruction; Y.Z., M.J., and Y.C. analyzed the data and wrote the paper.

Corresponding author

Correspondence to Yao Cong.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Purification and characterization of the TRiC complex from S. cerevisiae.

(a) SDS-PAGE of the purified TRiC sample stained with Coomassie Brilliant Blue. The red rectangle highlights the bands belonging to the TRiC subunits. (b) The purified yeast TRiC remained active in a luciferase refolding assay. Yeast TRiC refolded an unfolded luciferase more efficiently than did BSA or buffer controls. (c) Representative cryo-EM images of NPP-TRiC and TRiC-AMP-PNP, respectively. Scales are labeled. (d) Reference-free 2D class averages of NPP-TRiC and TRiC-AMP-PNP, respectively.

Supplementary Figure 2 Resolution evaluation of the NPP-TRiC and TRiC–AMP-PNP cryo-EM maps.

(a) Resolution estimation of the NPP-TRiC (blue curve) and TRiC-AMP-PNP (red curve) maps according to the gold-standard FSC criterion of 0.143. (b) Local resolution estimation of the NPP-TRiC map by Resmap. The resolution color bar (in Å) is also labeled. (c) Local resolution estimation of the TRiC-AMP-PNP map.

Supplementary Figure 3 Comparison of the yeast and bovine TRiC conformations and visualization of the extra densities in the E-domain region of yeast TRiC.

(a) Alignment of the yeast NPP-TRiC (gold) and bovine1 apo-TRiC (gray) maps revealed distinct conformations in several subunits. (b) Alignment of the yeast (dodger blue) and bovine (gray) TRiC-AMP-PNP maps indicated similar conformations. The two yeast TRiC maps were low pass filtered to 5.5 Å resolution. (c,d) Zoom in view of the extra densities in the E domain region of the NPP-TRiC (c) and TRiC-AMP-PNP (d) maps in both the end-on view (left panel) and the side view (right panel). The two maps were both low pass filtered to 10 Å resolution to better visualize the two pieces of extra densities (in blue) in the E domain region, forming a blockage between the two chambers of TRiC. The maps are colored according to its cylinder radius following the same color scheme as in Fig. 1a,b. The connecting region between the extra density and the inner surface of TRiC is indicated by red dotted circle. In both maps, we can observe that each of the extra densities could potentially connect to the N-terminal region of the CCT3, CCT5′, and CCT7′ subunits.

1. Cong, Y. et al. Symmetry-free cryo-EM structures of the chaperonin TRiC along its ATPase-driven conformational cycle. EMBO J 31, 720-30 (2012).

Supplementary Figure 4 Analysis of the relative orientations of subunits CCT1, CCT4, and CCT6 between the two rings of TRiC, and CCT6 subunit identification in the CCT6-eGFP TRiC map in the open state.

The top row three 3D maps are of ATP-AlFx TRiC in the both-ring closed conformation. eGFP was used to tag (a) the E domain of CCT1, (b) I domain of CCT4, or (c) I domain of CCT6. The TRiC density is in gray, and the extra density protruding from the complex corresponding to the eGFP tag is colored in green throughout this figure. Three subunit pairs were found to separate the CCT1 subunits from each ring, whereas only one subunit pair was determined to separate the CCT4 subunits from each ring. The CCT6 subunits of the cis-ring and trans-ring directly contacted each other. These geometrical relationships between the two eGFP tags (one from each ring) are in line with that proposed in the XL-MS studies. (d) The end-on view and two side views of the CCT6-eGFP TRiC map in the open state. Two extra pieces of exposed densities were observed to be located in the I domain of the on-axis subunit a5 and a5′ positions, indicating a5 to be CCT6.

Supplementary Figure 5 Schematic drawing of the secondary-structure elements of a TRiC subunit.

Supplementary Figure 6 Identification of the residual nucleotide forms in the NPP-TRiC and TRiC–AMP-PNP samples on the basis of the luciferin-luciferase reaction with the ADP/ATP ratio assay.

(a) Nucleotide form identification of the NPP-TRiC sample. The relative light unit (RLU) values were measured for NPP-TRiC sample with or without proteinase K digestion, with NPP-TRiC sample buffer as negative control (NC) and 1 μM ATP and 1 μM ADP as positive control. This set of experiments demonstrated the presence of both ATP and ADP in the NPP-TRiC sample, with the ADP form being the much dominant population. Moreover, the proteinase K digested sample showed much higher RLU value compared to the undigested sample, indicating proteinase K digestion did help to release the residual nucleotide from the binding pockets. Error bars indicate SD; n=3 technical replicates of a representative experiment (out of three experiments). (b) Nucleotide form identification of the TRiC-AMP-PNP sample. TRiC sample was incubated with 10 mM AMP-PNP, then the free AMP-PNP as well as the potentially replaced nucleotide from the TRiC sample were removed. We tested the RLU values for the AMP-PNP incubated sample with or without proteinase K digestion, as well as the AMP-PNP un-incubated sample with or without proteinase K digestion, with the AMP-PNP incubation buffer as negative control (NC). This experiment suggested the presence of both ATP and ADP in the TRiC-AMP-PNP sample, with the ADP form being the much dominant population. Additionally, the AMP-PNP incubated sample showed lower RLU value compared to the AMP-PNP un-incubated sample, indicating that part of the residual nucleotide can be replaced by the excess AMP-PNP. Moreover, there was detectable ADP in the filtrate, which could come from the broken TRiC particles caused by the repeated dilution and concentration in the buffer exchange process in all the experiments in b. Error bars indicate SD; n=3 technical replicates of a representative experiment (out of three experiments). (c) As a control experiment, the RLU values of ATP and ADP at different concentrations were also measured using the same assay. Error bars indicate SD; n=3 technical replicates of a representative experiment (out of three experiments).

Supplementary Figure 7 Conformation comparison between the 3D reconstructions with a fully asymmetric initial model or a C8-symmetry-imposed initial model.

The resulting 3D map (light green) from the completely asymmetric initial model showed a conformation almost identical to that of our 4.7 Å resolution map (gold) using the C8 symmetrized map as the initial model.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Table 1 (PDF 1231 kb)

Conformational transition of TRiC triggered by ATP binding.

Conformational changes of TRiC complex triggered by ATP-binding are show in the top and four different side views. (MOV 13284 kb)

Conformational change of CCT2, CCT5, and CCT7 triggered by ATP binding.

The conformational change of TRiC subunit CCT2, CCT5, CCT7 triggered by ATP-binding is shown in the side view, respectively. (MOV 13638 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zang, Y., Jin, M., Wang, H. et al. Staggered ATP binding mechanism of eukaryotic chaperonin TRiC (CCT) revealed through high-resolution cryo-EM. Nat Struct Mol Biol 23, 1083–1091 (2016).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing