Structural Dynamics of Ribosome Subunit Association Studied by Mixing-Spraying Time-Resolved Cryogenic Electron Microscopy
Structural Dynamics of Ribosome Subunit Association Studied by Mixing-Spraying Time-Resolved Cryogenic Electron Microscopy
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DOI：
10.1016/j.str.2015.04.007
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发表时间：
2015-06-02
期刊：
影响因子：
5.7
通讯作者：
Frank, Joachim
中科院分区：
文献类型：
--
作者：
Chen, Bo;Kaledhonkar, Sandip;Frank, Joachim
核糖体亚基结合是翻译起始的关键检查点，但其结构动力学尚不清楚。在这里，我们使用了最近开发的混合喷涂，时间分辨，低温电子显微镜（cryo-EM）方法来研究亚秒时间范围内核糖体亚基的关联。我们改进了这种方法，并将低温电镜数据收率提高了十倍。通过核糖体亚基混合物反应60ms和140ms，捕获结合反应的前平衡状态。我们还在相关的核糖体中发现了三种不同的核糖体构象。在这两个时间点所观察到的这三种构象的比例是相同的，这表明核糖体在形成后不到60 ms的时间内就在三种构象之间达到了平衡。我们的研究结果表明，混合喷射方法可以在亚秒反应中捕获大分子的多种状态。其他快速过程，如翻译起始、解码和核糖体再循环，也适用于用这种方法研究。
Ribosomal subunit association is a key checkpoint in translation initiation but its structural dynamics are poorly understood. Here, we used a recently developed mixing-spraying, time-resolved, cryogenic electron microscopy (cryo-EM) method to study ribosomal subunit association in the sub-second time range. We have improved this method and increased the cryo-EM data yield by tenfold. Pre-equilibrium states of the association reaction were captured by reacting the mixture of ribosomal sub-units for 60 ms and 140 ms. We also identified three distinct ribosome conformations in the associated ribosomes. The observed proportions of these conformations are the same in these two time points, suggesting that ribosomes equilibrate among the three conformations within less than 60 ms upon formation. Our results demonstrate that the mixing-spraying method can capture multiple states of macromolecules during a sub-second reaction. Other fast processes, such as translation initiation, decoding, and ribosome recycling, are amenable to study with this method.