possibility to measure the resolution for each micrograph based on the tilting angle.
Finally, if some protein complex with the known structure in the tomogram may be
unambiguously identified, FSC may be calculated between this structure and its
instance in the tomogram, assuming that CTF correction have been carried out.
Analysis of tomograms from HSV particles recorded on a CCD demonstrated
resolution *7 nm with low tilts corresponding to 5.5 nm and high tilts—10 nm.
Higher resolution in tomograms was observed closer to the tilting axis [54]. More
recent tomograms recorded with a Volta phase plate, a direct detector and an energy
filter were evaluated by a similarity of the 26S proteasome particles from the
tomograms and the average structure of the proteasome. The best subtomograms
had resolutions up to 3.5 nm [32].
Resolution anisotropy present in tomograms and sometimes in the average
structures from StA may be quantitatively estimated using FSC in the conical
segments of the Fourier space [55]. The median resolution of single- and dual-axis
cryo TEM tomograms recorded on CCD was *6 nm [56]. The highest resolution
for single tilt tomograms of *3 nm was measured along the tilting axis due to
oversampling of the projection information of all the micrographs; the resolution of
dual-axis tomograms was more isotropic as of single-axis tomograms [56].
Another quantitative resolution criterion was proposed based on the edge quality
of gold particles [57] which are used as fiducial markers for alignment of tilt series.
The intensity decay of the edges can be quantitatively assessed in three directions
and be used to monitor the convergence of SIRT reconstructions. The STEM
tomograms had resolution of 1.2–2.8 nm in different directions for gold particles
[57]. The method in principle may be used for defocused TEM tomography taking
CTF into account.
10.4 Limits of Subtomogram Averaging
StA described in the Chap. 9 of this book improves the signal to noise ratio of the
subtomograms containing repeating elements, located in cryo electron tomograms.
If the particles in subtomograms do not have preferential orientation to the electron
beam StA fills the missing and the Crowther criterion does not apply. Otherwise all
the aspects from imaging cryo-sample preparation, microscope alignments and
tomographic reconstruction apply for the resolution limits. Recently several groups
obtained reconstructions at sub-nanometer resolution including the structure of the
HIV protein GAG at an atomic resolution (Table 10.1). At the resolution protein
secondary structure may be seen and atomic structures may be fit by flexible fitting
with molecular dynamics [58]. The structures of the GAG protein in the intact HIV
virions [59] and of ribosomes on the ER membranes [60] directly implement the
in situ structural approach analysing the structure the molecular complex of interest
in the native context.
Several StA-specific parameters affect the resolution of the final maps.
10 Resolution in Electron Tomography
273
Finally, if some protein complex with the known structure in the tomogram may be
unambiguously identified, FSC may be calculated between this structure and its
instance in the tomogram, assuming that CTF correction have been carried out.
Analysis of tomograms from HSV particles recorded on a CCD demonstrated
resolution *7 nm with low tilts corresponding to 5.5 nm and high tilts—10 nm.
Higher resolution in tomograms was observed closer to the tilting axis [54]. More
recent tomograms recorded with a Volta phase plate, a direct detector and an energy
filter were evaluated by a similarity of the 26S proteasome particles from the
tomograms and the average structure of the proteasome. The best subtomograms
had resolutions up to 3.5 nm [32].
Resolution anisotropy present in tomograms and sometimes in the average
structures from StA may be quantitatively estimated using FSC in the conical
segments of the Fourier space [55]. The median resolution of single- and dual-axis
cryo TEM tomograms recorded on CCD was *6 nm [56]. The highest resolution
for single tilt tomograms of *3 nm was measured along the tilting axis due to
oversampling of the projection information of all the micrographs; the resolution of
dual-axis tomograms was more isotropic as of single-axis tomograms [56].
Another quantitative resolution criterion was proposed based on the edge quality
of gold particles [57] which are used as fiducial markers for alignment of tilt series.
The intensity decay of the edges can be quantitatively assessed in three directions
and be used to monitor the convergence of SIRT reconstructions. The STEM
tomograms had resolution of 1.2–2.8 nm in different directions for gold particles
[57]. The method in principle may be used for defocused TEM tomography taking
CTF into account.
10.4 Limits of Subtomogram Averaging
StA described in the Chap. 9 of this book improves the signal to noise ratio of the
subtomograms containing repeating elements, located in cryo electron tomograms.
If the particles in subtomograms do not have preferential orientation to the electron
beam StA fills the missing and the Crowther criterion does not apply. Otherwise all
the aspects from imaging cryo-sample preparation, microscope alignments and
tomographic reconstruction apply for the resolution limits. Recently several groups
obtained reconstructions at sub-nanometer resolution including the structure of the
HIV protein GAG at an atomic resolution (Table 10.1). At the resolution protein
secondary structure may be seen and atomic structures may be fit by flexible fitting
with molecular dynamics [58]. The structures of the GAG protein in the intact HIV
virions [59] and of ribosomes on the ER membranes [60] directly implement the
in situ structural approach analysing the structure the molecular complex of interest
in the native context.
Several StA-specific parameters affect the resolution of the final maps.
10 Resolution in Electron Tomography
273