RNA, while the large subunit mediates formation of peptide bonds in the nascent
protein [23]. An analysis of the relative positioning and orientation of ribosomes
imaged using CET and localized using subtomogram analysis revealed the compact
organization of bacterial and eukaryotic cytosolic polyribosomes [24–28]. In these
studies, neighboring ribosomes exhibit preferred arrangements, resulting in pseudoplanar or pseudohelical organizations. While the transcript is sequestered on the
inside of the polyribosome, polypeptide exit sites of individual ribosomes are well
separated and face the cytosol, presumably to limit aggregation of nascent peptides
and to promote productive folding. Another specific macromolecular arrangement
that has been structurally characterized using CET and subtomogram analysis is the
100S ribosome pair that transiently forms in bacterial cells under nutritional stress
[29]. The 3D configuration of these ribosome pairs is well preserved and results in
close contact between the small ribosomal subunits.
CET and subtomogram analysis furthermore provided an unprecedented view on
the structure and molecular architecture of the native ribosome-associated protein
translocon in the ER membrane [30] and further studies allowed identification of
the oligosaccharyl-transferase (OST) complex, the translocon associated protein
complex (TRAP) and the protein-conducting channel Sec61 in a map at 20 Å
resolution [31]. Subtomogram classification approaches furthermore uncovered
compositional heterogeneity with respect to translocon constituents; TRAP is present on all ribosome-bound translocon complexes, while OST is highly substoichiometric and found on only 40–70% of translocon complexes. Recently, technical
developments in the field of cryo-EM (direct detectors, acquisition and image
processing software) enabled us to visualize the mammalian ribosome-bound
translocon in a native membrane environment at subnanometer resolution [32].
Secondary structure elements were clearly resolved in the density for the ribosome
and the directly associated Sec61 protein-conducting channel. The visibility of
secondary structure elements allowed for a detailed analysis of the Sec61 conformational state in a native lipid environment. Finally, an analysis of the structure and
supramolecular organization of mitochondrial ribosomes in intact yeast mitochondria complements the growing compendium of cellular translation machineries
studied using CET and subtomogram analysis [33].
9.7.2 Other Cytosolic Complexes
A recent study imaged intact neuronal cells with CET and analysed the structure,
variability and distribution of a central component of the cytosolic protein degradation
machinery, the 26S proteasome, using subtomogram analysis [3]. Subtomogram
classification approaches allowed separating populations of proteasomes with one or
two regulatory 19S caps bound to the 20S core proteasomes, and visualized the
regulatory caps in different functional states of substrate degradation that have been
previously characterized using single particle cryo-EM analysis [34]. Based on the
low abundance of the substrate processing state of the proteasome, the authors
9 Structural Biology in Situ Using Cryo-Electron …
249
protein [23]. An analysis of the relative positioning and orientation of ribosomes
imaged using CET and localized using subtomogram analysis revealed the compact
organization of bacterial and eukaryotic cytosolic polyribosomes [24–28]. In these
studies, neighboring ribosomes exhibit preferred arrangements, resulting in pseudoplanar or pseudohelical organizations. While the transcript is sequestered on the
inside of the polyribosome, polypeptide exit sites of individual ribosomes are well
separated and face the cytosol, presumably to limit aggregation of nascent peptides
and to promote productive folding. Another specific macromolecular arrangement
that has been structurally characterized using CET and subtomogram analysis is the
100S ribosome pair that transiently forms in bacterial cells under nutritional stress
[29]. The 3D configuration of these ribosome pairs is well preserved and results in
close contact between the small ribosomal subunits.
CET and subtomogram analysis furthermore provided an unprecedented view on
the structure and molecular architecture of the native ribosome-associated protein
translocon in the ER membrane [30] and further studies allowed identification of
the oligosaccharyl-transferase (OST) complex, the translocon associated protein
complex (TRAP) and the protein-conducting channel Sec61 in a map at 20 Å
resolution [31]. Subtomogram classification approaches furthermore uncovered
compositional heterogeneity with respect to translocon constituents; TRAP is present on all ribosome-bound translocon complexes, while OST is highly substoichiometric and found on only 40–70% of translocon complexes. Recently, technical
developments in the field of cryo-EM (direct detectors, acquisition and image
processing software) enabled us to visualize the mammalian ribosome-bound
translocon in a native membrane environment at subnanometer resolution [32].
Secondary structure elements were clearly resolved in the density for the ribosome
and the directly associated Sec61 protein-conducting channel. The visibility of
secondary structure elements allowed for a detailed analysis of the Sec61 conformational state in a native lipid environment. Finally, an analysis of the structure and
supramolecular organization of mitochondrial ribosomes in intact yeast mitochondria complements the growing compendium of cellular translation machineries
studied using CET and subtomogram analysis [33].
9.7.2 Other Cytosolic Complexes
A recent study imaged intact neuronal cells with CET and analysed the structure,
variability and distribution of a central component of the cytosolic protein degradation
machinery, the 26S proteasome, using subtomogram analysis [3]. Subtomogram
classification approaches allowed separating populations of proteasomes with one or
two regulatory 19S caps bound to the 20S core proteasomes, and visualized the
regulatory caps in different functional states of substrate degradation that have been
previously characterized using single particle cryo-EM analysis [34]. Based on the
low abundance of the substrate processing state of the proteasome, the authors
9 Structural Biology in Situ Using Cryo-Electron …
249