Cheng Jing, Wu Chunling, Li Junxi, Yang Qi, Zhao Mingjie, Zhang Xinzheng
Key Laboratory of Biomacromolecules (CAS), National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
University of Chinese Academy of Sciences, Beijing, China.
Nat Struct Mol Biol. 2025 Apr;32(4):698-708. doi: 10.1038/s41594-024-01454-9. Epub 2025 Jan 9.
Many protein complexes are highly dynamic in cells; thus, characterizing their conformational changes in cells is crucial for unraveling their functions. Here, using cryo-electron microscopy, 451,700 ribosome particles from Saccharomyces cerevisiae cell lamellae were obtained to solve the 60S region to 2.9-Å resolution by in situ single-particle analysis. Over 20 distinct conformations were identified by three-dimensional classification with resolutions typically higher than 4 Å. These conformations were used to reconstruct a complete elongation cycle of eukaryotic translation with elongation factors (eEFs). We found that compact eEF2 anchors to the partially rotated ribosome after subunit rolling and hypothesize that it stabilizes the local conformation for peptidyl transfer. Moreover, open-eEF3 binding to a fully rotated ribosome was observed, whose conformational change was coupled with head swiveling and body back-rotation of the 40S subunit.
许多蛋白质复合物在细胞中具有高度动态性；因此，表征它们在细胞中的构象变化对于阐明其功能至关重要。在这里，通过冷冻电子显微镜，从酿酒酵母细胞片层中获得了451,700个核糖体颗粒，通过原位单颗粒分析将60S区域解析到2.9埃分辨率。通过三维分类鉴定出超过20种不同的构象，分辨率通常高于4埃。这些构象被用于重建真核生物翻译与延伸因子（eEFs）的完整延伸循环。我们发现紧密的eEF2在亚基滚动后锚定到部分旋转的核糖体上，并推测它稳定了肽基转移的局部构象。此外，观察到开放的eEF3与完全旋转的核糖体结合，其构象变化与40S亚基的头部旋转和主体向后旋转相关联。
