In extracto cryo-EM workflow for sample preparation and data collection.

A) Sample and grid preparation from primate semi-permeabilized cells. Examples of an MCF-7 cell culture (left) and grid with cell lysate (right) are shown. B) Representative lysate micrograph (left) and 2DTM processing yielding positions and orientations of 60S subunits (right). C) Examples of averaged maps (prior to classification) from typical single-particle picking pipelines (cyan) and from 2DTM processing (middle and gray). The close-up view highlights densities of 28S rRNA nucleotides (gray) and ribosomal protein residues (cyan). Also see Fig. S2.

Ribosome and 60S particle distributions in control MCF-7 and starved MCF-7 cells.

A-H) Eight cryo-EM maps correspond to elongating ribosomes (A-E): codon sampling with eEF1A and A/T tRNA (A), non-rotated with A/A, P/P,E/E tRNAs and putative extended eEF1A (B), non-rotated pre-translocation with A/A, P/P,E/E tRNAs (C), rotated pre-translocation with hybrid-state A/P and P/E tRNAs (D), and post-translocation ribosome with eEF2, P and E tRNAs (E); hibernating rotated ribosome with eEF2, SERBP1 and P/E tRNA (F); 60S subunits with eEF2 and E-tRNA (G); and vacant 60S subunits (H). I-P) Eight cryo-EM maps corresponding to nutrient-deprived MCF-7 cell lysates comprise elongation states (I-L) similar to those in panels A-D; hibernating head-swiveled state with eEF2, CCDC124, pe/E tRNA and putative LARP1 (M), hibernating rotated ribosome with eEF2, SERBP1 and P/E tRNA (N); 60S with eEF2 and E-tRNA (O); 60S with eIF6 (P).

In extracto cryo-EM of rabbit reticulocyte lysates.

A) NLuc mRNA construct used to monitor NanoLuciferase translation (top) and real-time translation kinetics (bottom). The graph represents the mean ± SEM from three independent experiments. B-L) Cryo-EM maps of the major classes resulting from 3D classification: (B) initiation complex with eIF5B; C-F) elongating ribosomes: codon-sampling with eEF1A (C), post-translocation with eEF2 ap/P and pe/E tRNAs (D), pre-translocation hybrid-state (E), and with GTPase density putatively assigned to DRG1/DRG2 next to A/A tRNA (F). G-J) Hibernating ribosomes: 40S-rotated ribosome with eEF2, eIF5A, and SERBP1 (G); 40S-head-swiveled ribosome with eEF2, CCDC124, and LARP1 in the mRNA tunnel (H); 40S head-swiveled with eEF2, IFRD2, eEF2 and LARP1 in the mRNA tunnel (I); 40S-head-swiveled with IFRD2 and LARP1 (J); 60S subunit with eEF2 domain IV open (K); 60S with eEF2 domain IV closed (L): the extent of domain IV movement in 60S-bound eEF2.

The occupancy of the mRNA tunnel in hibernating ribosomes from RRL.

A) The 80S ribosome with eEF2, eIF5A and SERBP1 (DC: decoding center). (B) Close-up view of cryo-EM density for SERBP1 in the mRNA tunnel. (C) Density for SERBP1 interaction with eEF2. (D) The 80S ribosome with eEF2, IFRD2 and LARP1. (E) Close-up view of LARP1 density in the mRNA tunnel. F-G) Views of the density for the LARP1 helix interacting with 40S ribosomal proteins and RNA. H-J) Similar densities for LARP1 in CCDC124- and IFRD2-bound ribosomes.

Interactions of eEF2 with the GTPase-activating center in hibernating ribosomes.

A) Overall view of the 80S structure with eEF2, eIF5A, and SERBP1. B) Cryo-EM density and model of the eEF2 GTPase center at the SRL. C) GDP density in the GTPase center of the predominant hibernating ribosomes. D, E) Distinct conformations of the N-terminal tail of uL14, highlighted in red, in ribosomes bound with eEF2•eIF5A•SERBP1 (D) and with eEF1A•GDP (PDB ID, 5LZS) (E).

Comparison of in extracto cryo-EM with traditional in vitro and in situ cryo-ET approaches to structural biology.

Binding sites of hibernation factors overlap with ribosomal functional centers.

A-D) Comparison of a translating ribosome (PDB 5LZS and mRNA from PDB 4V6F; panel A) with hibernating ribosomes identified in this work. E-G) Superposition of translating and hibernating ribosomes illustrates that the key ribosomal functional centers all shielded by hibernation factors (eEF1A, A/T-tRNA, P-tRNA and E-tRNA are from PDB 5LZS and mRNA from PDB 4V6F). Panel E shows the view from panels B-D rotated by 36°.