In extracto cryo-EM reveals eEF2 as a major hibernation factor on 60S and 80S particles

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 or 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 A 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 ribosomes and, unexpectedly, to 60S subunits. eEF2.GDP is stabilized by interactions with the sarcin-ricin loop and protein uL14. Hibernating ribosomes also feature 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 cryo-EM to discover native cellular complexes and mechanisms at near-atomic resolution.