further implicated other bacterial cytoskeletal proteins in shape determination. For
example, in Caulobacter crescentus, cryo-tomographic imaging revealed multiple
classes of cytoplasmic filamentous bundles involved in cell shape determination
[119]. Further work established the major filament bundle to be composed of CTP
synthase which together with crescentin (an intermediate filament-like protein)
dictates C. crescentus’s distinctive curved shape [120] (Fig. 3.2i).
In imaging peptidoglycan and the bacterial cytoskeleton, ECT was uniquely able
to provide insights into the architecture of large macromolecular complexes that
were intractable to traditional structural biology techniques, yet too small for
accurate insights from AFM, and too dynamic, have too many components, and too
finely-structured to study using super-resolution light microscopy.
3.6.2 The Eukaryotic Nuclear Pore Complex
The *100 Mda,*450-protein eukaryotic nuclear pore regulates high-speed,
bidirectional and highly selective transport of a wide range of molecules in and out
of the nucleus. Its size, complexity, its location deep within the eukaryotic cell, and
membrane association make it a formidable specimen to study structurally. ECT,
therefore, has been uniquely able to contextualize results from more reductionist
approaches, providing considerable insights into the structure and mechanism of the
nuclear pore from a range of species, and enabling construction of comprehensive
pseudoatomic models [121, 122] (Fig. 3.2j-n).
The power of cryo-FIB milling was first demonstrated on whole Dictyostelium
discoideum cells, providing a subtomogram average of nuclear pore complexes
in situ [123]. With cryo-FIB milling, structures can be observed in their native
environment, yet with a specimen thickness as thin as (or thinner than) isolated
organelles. Using cryo-tomographic data collected from a thin cryo-FIB-milled
lamellum, 67 protomers from the ten octameric nuclear pore complexes were
extracted and used to generate a subtomogram average to 79 Å resolution.
A follow-up study to this involved cryo-FIB milling human cells to visualize the
nuclear envelope using a Volta phase plate, enabling a lower electron dosage on the
specimen due to higher contrast (Fig. 3.2l, m) [32]. Here, specimen charging was
minimized by sputtering the specimen with platinum both before and after cryo-FIB
milling. The resulting high signal-to-noise ratio in individual particles enabled the
authors to observe differences in nuclear pore complex diameters between nuclear
pore complexes from different nuclei, suggesting that different conformations are
related to the physiological state of the cell.
High resolution subtomogram averages of the Xenopus laevis oocyte nuclear
pore complex to 20 Å resolution have directly visualized the structure in situ. One
insight enabled by this was the visualization of two possible transport routes
through the complex [124, 125]. This was consistent with ECT snapshots of the
positions of hundreds of gold-labelled proteins with nuclear localization signals
providing information on the dynamics of pathways followed by proteins during
84
J. L. Ferreira et al.
example, in Caulobacter crescentus, cryo-tomographic imaging revealed multiple
classes of cytoplasmic filamentous bundles involved in cell shape determination
[119]. Further work established the major filament bundle to be composed of CTP
synthase which together with crescentin (an intermediate filament-like protein)
dictates C. crescentus’s distinctive curved shape [120] (Fig. 3.2i).
In imaging peptidoglycan and the bacterial cytoskeleton, ECT was uniquely able
to provide insights into the architecture of large macromolecular complexes that
were intractable to traditional structural biology techniques, yet too small for
accurate insights from AFM, and too dynamic, have too many components, and too
finely-structured to study using super-resolution light microscopy.
3.6.2 The Eukaryotic Nuclear Pore Complex
The *100 Mda,*450-protein eukaryotic nuclear pore regulates high-speed,
bidirectional and highly selective transport of a wide range of molecules in and out
of the nucleus. Its size, complexity, its location deep within the eukaryotic cell, and
membrane association make it a formidable specimen to study structurally. ECT,
therefore, has been uniquely able to contextualize results from more reductionist
approaches, providing considerable insights into the structure and mechanism of the
nuclear pore from a range of species, and enabling construction of comprehensive
pseudoatomic models [121, 122] (Fig. 3.2j-n).
The power of cryo-FIB milling was first demonstrated on whole Dictyostelium
discoideum cells, providing a subtomogram average of nuclear pore complexes
in situ [123]. With cryo-FIB milling, structures can be observed in their native
environment, yet with a specimen thickness as thin as (or thinner than) isolated
organelles. Using cryo-tomographic data collected from a thin cryo-FIB-milled
lamellum, 67 protomers from the ten octameric nuclear pore complexes were
extracted and used to generate a subtomogram average to 79 Å resolution.
A follow-up study to this involved cryo-FIB milling human cells to visualize the
nuclear envelope using a Volta phase plate, enabling a lower electron dosage on the
specimen due to higher contrast (Fig. 3.2l, m) [32]. Here, specimen charging was
minimized by sputtering the specimen with platinum both before and after cryo-FIB
milling. The resulting high signal-to-noise ratio in individual particles enabled the
authors to observe differences in nuclear pore complex diameters between nuclear
pore complexes from different nuclei, suggesting that different conformations are
related to the physiological state of the cell.
High resolution subtomogram averages of the Xenopus laevis oocyte nuclear
pore complex to 20 Å resolution have directly visualized the structure in situ. One
insight enabled by this was the visualization of two possible transport routes
through the complex [124, 125]. This was consistent with ECT snapshots of the
positions of hundreds of gold-labelled proteins with nuclear localization signals
providing information on the dynamics of pathways followed by proteins during
84
J. L. Ferreira et al.