Visualizing translating dynamics in situ at high spatial and temporal resolution in eukaryotic cells

Author:

Cheng JingORCID,Wu Chunling,Li Junxi,Yang Qi,Zhang XinzhengORCID

Abstract

SummaryCryo-electron microscopy can determine the structures of complexes within their native cellular environments. Because many of these complexes are highly dynamic in cells, characterizing their conformational changes is crucial for unraveling their biological functions. Here, we used translating ribosomes as a model system to investigate protein dynamic processes by GisSPA. We obtained 451,700 particles within two-days data collection from cryo-FIB milledSaccharomyces cerevisiaecells and solved the ribosome 60S region to 2.9 Å resolution. Multiple conformations with resolutions typically higher than 4 Å were identified by improving the 3D classification workflow. Some of these conformations revealed previously unobserved intermediates of peptidyl transfer and tRNA translocation. Thus, our work provides unprecedented high spatial and temporal resolution of the ribosome elongation cycle and demonstrates the power of GisSPA to visualize protein dynamics in atomic detail inside cells, highlighting its potential for studying structural dynamics in biological systems.

Publisher

Cold Spring Harbor Laboratory

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