The ring-shaped chaperonin T-complex protein ring complex (TRiC; also known as chaperonin containing TCP-1, CCT) is an ATP-driven protein-folding machine that is essential for maintenance of cellular homeostasis^1,2. Its dysfunction is related to cancer and neurodegenerative disease^3,4. Despite its importance, how TRiC works in the cell remains unclear. Here we structurally analysed the architecture, conformational dynamics and spatial organization of the chaperonin TRiC in human cells using cryo-electron tomography. We resolved distinctive open, closed, substrate-bound and prefoldin-associated states of TRiC, and reconstructed its duty cycle in situ. The substrate-bound open and symmetrically closed TRiC states were equally abundant. Closed TRiC containing substrate forms distinctive clusters, indicative of spatial organization. Translation inhibition did not fundamentally change the distribution of duty cycle intermediates, but reduced substrate binding for all states as well as cluster formation. From our in-cell structures, we identified the programmed cell death protein 5 (PDCD5) as an interactor that specifically binds to almost all open but not closed TRiC, in a position that is compatible with both substrate and prefoldin binding. Our data support a model in which TRiC functions at near full occupancy to fold newly synthesized proteins inside cells. Defining the TRiC cycle and function inside cells lays the foundation to understand its dysfunction during cancer and neurodegeneration.

环形伴侣蛋白 T 复合体蛋白环复合体（TRiC;也称为含 TCP-1、CCT 的伴侣蛋白）是一种 ATP 驱动的蛋白质折叠机器，对维持细胞稳态至关重要^1,2。其功能障碍与癌症和神经退行性疾病有关^3,4。尽管 TRiC 很重要，但 TRiC 在细胞中的工作原理仍不清楚。在这里，我们使用冷冻电子断层扫描结构分析了人类细胞中伴侣蛋白 TRiC 的结构、构象动力学和空间组织。我们解析了 TRiC 独特的开放、闭合、底物结合和前折叠蛋白相关状态，并原位重建了其占空比。底物结合的开放和对称闭合 TRiC 状态相同。含有 TRiC 的封闭底物形成独特的簇，指示空间组织。翻译抑制并没有从根本上改变占空比中间体的分布，但减少了所有状态的底物结合以及簇的形成。从我们的细胞内结构中，我们确定了程序性细胞死亡蛋白 5 （PDCD5） 是一种相互作用物，它与几乎所有开放但不闭合的 TRiC 特异性结合，其位置与底物和前折叠蛋白结合兼容。我们的数据支持一个模型，在该模型中，TRiC 在接近完全占据的情况下发挥作用，以折叠细胞内新合成的蛋白质。定义细胞内的 TRiC 循环和功能为了解其在癌症和神经退行性变期间的功能障碍奠定了基础。