<i>In extracto</i> 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 <i>in situ</i> 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-<i>in extracto</i> 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 <i>in extracto</i> cryo-EM to discover native cellular complexes and mechanisms at near-atomic resolution.