nuclear import (an import-defective control revealed accumulation on the cytoplasmic face and in the pore only) (Fig. 3.2n) [121]. In addition to being a strikingly
direct proof-of-principle of visualization of molecular function in situ, this suggested an important possible mechanistic insight: nuclear import is through an
off-axis channel, while export passes through the centre of the nuclear pore channel.
ECT has also provided mechanistic insights into disassembly of nuclear pore
complexes triggered at mitosis to enable chromosomal segregation. By purifying
nuclei from human cells, structures have been determined to resolutions as high as
23 Å [24, 126], enabling fitting of a structure of the Y complex and identification of
protein interfaces. These protein interfaces are enriched for phosphorylation sites
pivotal in controlling nuclear pore complex disassembly, an insight not possible
without an overview of the entire intact structure provided by ECT.
3.7 Future Challenges: What Do We Need from ECT
for Better Mechanistic Insights into Biology?
ECT is uniquely capable of making in situ insights into biological mechanisms.
What advances will further push its capabilities? We finish this chapter by covering
two broad areas: higher resolution tomograms, and methods to interpret them.
Higher resolution tomograms and subtomogram averages will clearly enable
exciting biological insights at molecular and cellular scales. Many areas of ECT can
be developed towards higher resolution tomograms, and advances will be synergistic: higher quality images are needed; better processing is needed, and more data
is needed.
Higher quality images from the electron microscope will provide higher resolution tomograms and subtomogram averages. The key advantage of higher signal
will be the ability to perform better tilt series alignments during tomogram
reconstruction and better alignment of subtomograms for averaging. Better cameras
with improved DQEs combined with robust and comprehensively characterized
phase plates would provide the most obvious single resolution improvement, enable
imaging specimens with lowered electron damage yet higher contrast. This would
be further augmented by higher coherence electron optics: ultra-high coherence
electron sources, monochromators, and spherical aberration correctors. To take full
advantage of improved imaging systems, improved specimen stability will reduce
attenuation of higher resolutions in high-quality images. This could be achieved
with a combination of further improved ultra-stable supports [26], additional
approaches to reduce specimen charging such as paraxial charge compensators
[127] or routine platinum sputtering [32], development of as-yet-unforeseen
approaches to stabilize the specimen analogous to graphene supports for single
particle analysis [128, 129], or more stable stages capable of faster and more stable
data collection. Finally, sample thickness remains a primary problem toward
3 Electron Cryo-Tomography
85
direct proof-of-principle of visualization of molecular function in situ, this suggested an important possible mechanistic insight: nuclear import is through an
off-axis channel, while export passes through the centre of the nuclear pore channel.
ECT has also provided mechanistic insights into disassembly of nuclear pore
complexes triggered at mitosis to enable chromosomal segregation. By purifying
nuclei from human cells, structures have been determined to resolutions as high as
23 Å [24, 126], enabling fitting of a structure of the Y complex and identification of
protein interfaces. These protein interfaces are enriched for phosphorylation sites
pivotal in controlling nuclear pore complex disassembly, an insight not possible
without an overview of the entire intact structure provided by ECT.
3.7 Future Challenges: What Do We Need from ECT
for Better Mechanistic Insights into Biology?
ECT is uniquely capable of making in situ insights into biological mechanisms.
What advances will further push its capabilities? We finish this chapter by covering
two broad areas: higher resolution tomograms, and methods to interpret them.
Higher resolution tomograms and subtomogram averages will clearly enable
exciting biological insights at molecular and cellular scales. Many areas of ECT can
be developed towards higher resolution tomograms, and advances will be synergistic: higher quality images are needed; better processing is needed, and more data
is needed.
Higher quality images from the electron microscope will provide higher resolution tomograms and subtomogram averages. The key advantage of higher signal
will be the ability to perform better tilt series alignments during tomogram
reconstruction and better alignment of subtomograms for averaging. Better cameras
with improved DQEs combined with robust and comprehensively characterized
phase plates would provide the most obvious single resolution improvement, enable
imaging specimens with lowered electron damage yet higher contrast. This would
be further augmented by higher coherence electron optics: ultra-high coherence
electron sources, monochromators, and spherical aberration correctors. To take full
advantage of improved imaging systems, improved specimen stability will reduce
attenuation of higher resolutions in high-quality images. This could be achieved
with a combination of further improved ultra-stable supports [26], additional
approaches to reduce specimen charging such as paraxial charge compensators
[127] or routine platinum sputtering [32], development of as-yet-unforeseen
approaches to stabilize the specimen analogous to graphene supports for single
particle analysis [128, 129], or more stable stages capable of faster and more stable
data collection. Finally, sample thickness remains a primary problem toward
3 Electron Cryo-Tomography
85