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Single-particle cryo-EM traditionally relies on purified macromolecules and lacks the complexity of cellular environments, whereas\n                    <jats:italic>in situ<\/jats:italic>\n                    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\u2014\n                    <jats:italic>in extracto<\/jats:italic>\n                    cryo-EM\u2014allowing the flexibility and high-resolution of cryo-EM in the context of cellular components. High-resolution 2D template matching (2DTM) yields ~2.2 \u00c5 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 &gt;95% of 80S ribosomes and, unexpectedly, to 60S subunits. eEF2\u2022GDP 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\n                    <jats:italic>in extracto<\/jats:italic>\n                    cryo-EM to discover native cellular complexes and mechanisms at near-atomic resolution.\n                  <\/jats:p>","DOI":"10.7554\/elife.110114","type":"journal-article","created":{"date-parts":[[2026,1,29]],"date-time":"2026-01-29T20:30:37Z","timestamp":1769718637000},"update-policy":"https:\/\/doi.org\/10.7554\/elife.110114","source":"Crossref","is-referenced-by-count":2,"title":["In extracto cryo-EM reveals eEF2 as a major hibernation factor on 60S and 80S particles"],"prefix":"10.7554","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2405-6212","authenticated-orcid":true,"given":"Zahra","family":"Seraj","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/0464eyp60","id-type":"ROR","asserted-by":"publisher"}],"name":"RNA Therapeutics Institute, UMass Chan Medical School","place":["Worcester, United 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