with an overall resolution of approximately 9 Å (Fig. 9.3b), as estimated by Fourier
cross-resolution (FCR) and FSC of averages from two halves of the data. Rod-like
densities co-localizing with alpha helices in a superposed atomic model of the
ribosome as well as the presence of clearly resolved transmembrane helices for the
protein translocon are consistent with the estimated overall resolution. Local resolution estimation reveals that the ribosome and membrane-embedded parts of the
protein translocon are better resolved than segments protruding into the lumen of
the ER (Fig. 9.3c). In line with this observation, subtomogram classification
independently indicates that ribosome binding to the translocon is not entirely
homogeneous. The translocon allows for minor tilting of the ribosome, which
results in increasing alignment error of lumenal translocon segments.
Fig. 9.3 Examples for subtomogram averaging of the ER membrane-associated ribosome in
PyTom. a 377 ribosome-containing subtomograms originating from one tomogram were aligned
without prior knowledge using FRM. The subtomogram average evolves from a featureless sphere
used as a starting reference (left) to a defined ribosome with density for the ER membrane within
10 alignment iterations (right). 40S subunit: yellow, 60S subunit: blue, translation elongation
factors: magenta. b 17,500 subtomograms depicting ribosomes bound to the fully assembled
protein translocon were aligned using simultaneous translational and restricted rotational search in
real space, yielding a density with an overall resolution of 9 Å. Secondary structure elements are
clearly resolved for the ribosome (Coloring as in a) and membrane-embedded parts of the protein
translocon (Sec61: dark blue, TRAP: green, OST: red). c Local resolution estimation reveals that
the ribosome and membrane-embedded parts of the protein translocon are better resolved than
segments protruding into the lumen of the endoplasmic reticulum
9 Structural Biology in Situ Using Cryo-Electron …
245
cross-resolution (FCR) and FSC of averages from two halves of the data. Rod-like
densities co-localizing with alpha helices in a superposed atomic model of the
ribosome as well as the presence of clearly resolved transmembrane helices for the
protein translocon are consistent with the estimated overall resolution. Local resolution estimation reveals that the ribosome and membrane-embedded parts of the
protein translocon are better resolved than segments protruding into the lumen of
the ER (Fig. 9.3c). In line with this observation, subtomogram classification
independently indicates that ribosome binding to the translocon is not entirely
homogeneous. The translocon allows for minor tilting of the ribosome, which
results in increasing alignment error of lumenal translocon segments.
Fig. 9.3 Examples for subtomogram averaging of the ER membrane-associated ribosome in
PyTom. a 377 ribosome-containing subtomograms originating from one tomogram were aligned
without prior knowledge using FRM. The subtomogram average evolves from a featureless sphere
used as a starting reference (left) to a defined ribosome with density for the ER membrane within
10 alignment iterations (right). 40S subunit: yellow, 60S subunit: blue, translation elongation
factors: magenta. b 17,500 subtomograms depicting ribosomes bound to the fully assembled
protein translocon were aligned using simultaneous translational and restricted rotational search in
real space, yielding a density with an overall resolution of 9 Å. Secondary structure elements are
clearly resolved for the ribosome (Coloring as in a) and membrane-embedded parts of the protein
translocon (Sec61: dark blue, TRAP: green, OST: red). c Local resolution estimation reveals that
the ribosome and membrane-embedded parts of the protein translocon are better resolved than
segments protruding into the lumen of the endoplasmic reticulum
9 Structural Biology in Situ Using Cryo-Electron …
245