one-size-fits-all tomography tag akin to GFP has not yet been developed, fluorescent proteins can be used by correlating light and electron cryo-microscopies
(Correlated Light and Electron Microscopy, or CLEM) in which the location of a
fluorescent protein in a cryo-preserved specimen is determined using light
cryo-microscopy, followed by ECT and correlation of the two. This technique
works best with high resolution imaging such as cryo-PALM (PhotoActivated Light
Microscopy) [92]. Alternative work towards a general-purpose tag for ECT has
been to develop a ferritin-based fusion tag, although because ferritin is an oligomer,
aggregative artefacts are associated with this approach [93]. In the case of identifying objects in subtomogram averages, contrast is sufficiently high to obviate the
need for a particularly dense metal tag and densities may be identified by removing
components and monitoring resulting subtomogram averages for density losses [94,
35, 95], or by fusing additional domains to components and monitoring for additional densities in the subtomogram average of these mutants [96].
The final stage of the project workflow is the backup and archival of data. Local
data should be robustly backed up. Published data can be deposited in the EMDB,
which serves as a repository for published electron cryo-microscopy data [97],
while raw images used to generate published data can be deposited in the EMPIAR
public repository [98].
3.6 Examples of ECT
ECT’s unique ability to span cellular and structural biology has lead to important
insights in a number of areas. Many of these successes have been in biological
systems that are integral to, or associated with, cells and membranes. The reason for
this is clear: the pleomorphic nature of membranes makes traditional structural
biology techniques difficult or intractable, but poses no such problems for tomographic imaging and analysis. Here we illustrate ECT’s unique abilities to provide
mechanistic insights into large macromolecular complexes and dynamic cellular
processes by highlighting its contributions to two fields: bacterial cell shape
maintenance, and the eukaryotic nuclear pore complex. ECT has also provided
pivotal advances in a number of other fields such as bacterial pathogenesis [54, 99,
100], proteasome structure [101], membrane trafficking and remodelling [100, 103],
and virus infection [102–104, 105, 106] beyond the scope of this chapter.
3.6.1 Bacterial Cell Shape Maintenance
Bacteria shape themselves, grow, and divide in a deterministic and orchestrated
manner. What mechanisms underly these fundamental processes of bacterial cell
biology? ECT has made a number of crucial contributions in this area (Fig. 3.2a–i).
3 Electron Cryo-Tomography
81
(Correlated Light and Electron Microscopy, or CLEM) in which the location of a
fluorescent protein in a cryo-preserved specimen is determined using light
cryo-microscopy, followed by ECT and correlation of the two. This technique
works best with high resolution imaging such as cryo-PALM (PhotoActivated Light
Microscopy) [92]. Alternative work towards a general-purpose tag for ECT has
been to develop a ferritin-based fusion tag, although because ferritin is an oligomer,
aggregative artefacts are associated with this approach [93]. In the case of identifying objects in subtomogram averages, contrast is sufficiently high to obviate the
need for a particularly dense metal tag and densities may be identified by removing
components and monitoring resulting subtomogram averages for density losses [94,
35, 95], or by fusing additional domains to components and monitoring for additional densities in the subtomogram average of these mutants [96].
The final stage of the project workflow is the backup and archival of data. Local
data should be robustly backed up. Published data can be deposited in the EMDB,
which serves as a repository for published electron cryo-microscopy data [97],
while raw images used to generate published data can be deposited in the EMPIAR
public repository [98].
3.6 Examples of ECT
ECT’s unique ability to span cellular and structural biology has lead to important
insights in a number of areas. Many of these successes have been in biological
systems that are integral to, or associated with, cells and membranes. The reason for
this is clear: the pleomorphic nature of membranes makes traditional structural
biology techniques difficult or intractable, but poses no such problems for tomographic imaging and analysis. Here we illustrate ECT’s unique abilities to provide
mechanistic insights into large macromolecular complexes and dynamic cellular
processes by highlighting its contributions to two fields: bacterial cell shape
maintenance, and the eukaryotic nuclear pore complex. ECT has also provided
pivotal advances in a number of other fields such as bacterial pathogenesis [54, 99,
100], proteasome structure [101], membrane trafficking and remodelling [100, 103],
and virus infection [102–104, 105, 106] beyond the scope of this chapter.
3.6.1 Bacterial Cell Shape Maintenance
Bacteria shape themselves, grow, and divide in a deterministic and orchestrated
manner. What mechanisms underly these fundamental processes of bacterial cell
biology? ECT has made a number of crucial contributions in this area (Fig. 3.2a–i).
3 Electron Cryo-Tomography
81