In extracto cryo-EM reveals eEF2 as a major hibernation factor on 60S and 80S particles
Abstract
Cryogenic electron microscopy (cryo-EM) made impressive progress in resolving cellular macromolecules and their detailed interactions. Single-particle cryo-EM traditionally relies on purified macromolecules and lacks the complexity of cellular environments, whereas in situ cryo-EM and cryogenic electron tomography (cryo-ET) require extensive sample preparation and data acquisition, presenting challenges in achieving high resolution. We describe cryo-EM of cellular lysates-in extracto cryo-EM-allowing the flexibility and high-resolution of cryo-EM in the context of cellular components. High-resolution 2D template matching (2DTM) yields ~2.2 Å maps of the mammalian translational apparatus. Elongating ribosome abundances in primate cell lines (MCF-7 and BSC-1) and rabbit reticulocyte lysates range from ~70% to ~10%, reflecting translational stress responses. Non-translating (hibernating) ribosomes carrying no mRNA feature numerous proteins shielding ribosomal functional centers. Elongation factor 2 (eEF2) is the most abundant hibernation factor bound to >95% of 80S ribosomes and, unexpectedly, to 60S subunits. eEF2•GDP is stabilized by interactions with the sarcin-ricin loop and protein uL14. Hibernating ribosomes also feature La-related protein 1 (LARP1) involved in initiation and mTOR signaling, eIF5A implicated in elongation and termination, and other factors, exposing the variety of hibernation scenarios. Our work underscores the efficiency and potential of in extracto…
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