Figure 1.
Cellular cryo-ET imaging and processing workflow captures cytoplasmic ribosomes positioned for protein import on mitochondrial membranes. (A)S. cerevisiae yeast expressing TIM50-GFP are grown in respiratory or fermentative conditions and treated with vehicle or CHX (100 μg/ml) prior to deposition on electron microscopy grids (black mesh circle) and vitrification via plunge freezing. (B) Vitrified yeast cells were imaged by cryo-FM to assess sample quality, cell density, and ice thickness. (C) Clumps of yeast were targeted for cryo-FIB milling to generate thin cellular sections (i.e., lamellae). (D) Cellular lamellae were imaged by standard cryo-ET acquisition procedures to generate tilt series that were further processed to generate 3D reconstructions (i.e., tomograms). Subcellular components such as mitochondria, the ER, the plasma membrane, and ribosomes are visible within the resulting tomograms. Scale bars = 250 nm. (E) Reconstructed tomograms were processed through “particle picking” software, which identified the initial positions and orientations of all visible cellular ribosomes. The positions and orientations were refined using subtomogram averaging to produce a consensus 8 Å 80S ribosome structure. (F) Mitochondrial membranes were traced, and separate three-dimensional voxel segmentations were generated for the OMM and IMM. These voxel segmentations were converted to surface mesh reconstructions using the surface morphometrics (Barad et al., 2023) pipeline such that the location of the membrane is represented by the coordinate of each triangle within the mesh. (G) The position and orientation of each ribosome relative to the OMM surface mesh reconstruction were calculated and rendered in the ArtiaX module of ChimeraX. The three-color arrows on ribosomes represent the Euler angles, with the yellow arrow representing the orientation of the ribosome peptide exit tunnel. (H) The cutoff for identifying cytoplasmic ribosomes engaged in protein import on the OMM was established by referring to the distance between the peptide exit tunnel of ER-translocon ribosome and the ER membrane. The optimal cutoff of the distance between the exit tunnel and OMM was identified as 0–95 Å in ArtiaX as we started to observe the exit tunnel pointed away from OMM in the expanded cutoff, either 0–110 or 0–120 Å. (I) Cytoplasmic ribosomes optimally positioned for protein import were identified as those with their exit tunnel closer than 95 Å from the OMM.

Cellular cryo- ET imaging and processing workflow captures cytoplasmic ribosomes positioned for protein import on mitochondrial membranes. (A) S. cerevisiae yeast expressing TIM50-GFP are grown in respiratory or fermentative conditions and treated with vehicle or CHX (100 μg/ml) prior to deposition on electron microscopy grids (black mesh circle) and vitrification via plunge freezing. (B) Vitrified yeast cells were imaged by cryo-FM to assess sample quality, cell density, and ice thickness. (C) Clumps of yeast were targeted for cryo-FIB milling to generate thin cellular sections (i.e., lamellae). (D) Cellular lamellae were imaged by standard cryo-ET acquisition procedures to generate tilt series that were further processed to generate 3D reconstructions (i.e., tomograms). Subcellular components such as mitochondria, the ER, the plasma membrane, and ribosomes are visible within the resulting tomograms. Scale bars = 250 nm. (E) Reconstructed tomograms were processed through “particle picking” software, which identified the initial positions and orientations of all visible cellular ribosomes. The positions and orientations were refined using subtomogram averaging to produce a consensus 8 Å 80S ribosome structure. (F) Mitochondrial membranes were traced, and separate three-dimensional voxel segmentations were generated for the OMM and IMM. These voxel segmentations were converted to surface mesh reconstructions using the surface morphometrics (Barad et al., 2023) pipeline such that the location of the membrane is represented by the coordinate of each triangle within the mesh. (G) The position and orientation of each ribosome relative to the OMM surface mesh reconstruction were calculated and rendered in the ArtiaX module of ChimeraX. The three-color arrows on ribosomes represent the Euler angles, with the yellow arrow representing the orientation of the ribosome peptide exit tunnel. (H) The cutoff for identifying cytoplasmic ribosomes engaged in protein import on the OMM was established by referring to the distance between the peptide exit tunnel of ER-translocon ribosome and the ER membrane. The optimal cutoff of the distance between the exit tunnel and OMM was identified as 0–95 Å in ArtiaX as we started to observe the exit tunnel pointed away from OMM in the expanded cutoff, either 0–110 or 0–120 Å. (I) Cytoplasmic ribosomes optimally positioned for protein import were identified as those with their exit tunnel closer than 95 Å from the OMM.

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