subtomogram analysis. Dissection of macromolecular machinery in situ has met
with success by imaging deletion and truncation mutants and imaging the resulting
assembly intermediates [35, 95]. Recent developments suggest a powerful complementary approach in which proteins are tagged for localization. A generic
framework to rapidly design such tags would facilitate easy interpretation of densities in subtomograms [96].
Finally, these methods may provide a platform for integration of ECT imaging
with systems-perspective and cell-level simulations and promises to extract a
wealth of information from cryo-tomograms.
3.8 Concluding Remarks
In summary, ECT has become a fully-fledged technique capable of unique insights
into biological systems, bridging scales and perspectives from molecular to cellular.
Most exciting is the fact that the technique has clearly not yet reached its prime. The
future is bright for ECT.
3.9 Practical Guide
Technically, ECT currently requires considerable training. To competently carry
out all steps without supervision will typically require multiple months of exposure,
training, and experience, and years of additional experience will continue to expand
the user’s knowledge and abilities. Nevertheless, the fact that the initial stages of
ECT do not rely upon in-depth knowledge of Fourier transforms means that an
intuitive grasp of the basics of the technique is relatively easy to acquire, and the
learning curve to becoming a tomographer, though long, is rarely steep.
The workflow for a ECT project starts when the user identifies a biological
question that can be answered by ECT:
1. Identify the ECT experiment(s) required to address the question. Identify the
resources required. This may involve data-collection at a remote facility,
depending on local facility availability.
2. Identify optimal specimen: optimize genetics, growth conditions, species,
purification procedures, as appropriate. This is often the most challenging step.
3. Iteratively optimize vitrification of the specimen, a process that will take one or
more days for normal specimens. This process typically involves freezing using
a vitrification device starting with parameters and conditions that have been used
successfully in a previous project.
3 Electron Cryo-Tomography
87
with success by imaging deletion and truncation mutants and imaging the resulting
assembly intermediates [35, 95]. Recent developments suggest a powerful complementary approach in which proteins are tagged for localization. A generic
framework to rapidly design such tags would facilitate easy interpretation of densities in subtomograms [96].
Finally, these methods may provide a platform for integration of ECT imaging
with systems-perspective and cell-level simulations and promises to extract a
wealth of information from cryo-tomograms.
3.8 Concluding Remarks
In summary, ECT has become a fully-fledged technique capable of unique insights
into biological systems, bridging scales and perspectives from molecular to cellular.
Most exciting is the fact that the technique has clearly not yet reached its prime. The
future is bright for ECT.
3.9 Practical Guide
Technically, ECT currently requires considerable training. To competently carry
out all steps without supervision will typically require multiple months of exposure,
training, and experience, and years of additional experience will continue to expand
the user’s knowledge and abilities. Nevertheless, the fact that the initial stages of
ECT do not rely upon in-depth knowledge of Fourier transforms means that an
intuitive grasp of the basics of the technique is relatively easy to acquire, and the
learning curve to becoming a tomographer, though long, is rarely steep.
The workflow for a ECT project starts when the user identifies a biological
question that can be answered by ECT:
1. Identify the ECT experiment(s) required to address the question. Identify the
resources required. This may involve data-collection at a remote facility,
depending on local facility availability.
2. Identify optimal specimen: optimize genetics, growth conditions, species,
purification procedures, as appropriate. This is often the most challenging step.
3. Iteratively optimize vitrification of the specimen, a process that will take one or
more days for normal specimens. This process typically involves freezing using
a vitrification device starting with parameters and conditions that have been used
successfully in a previous project.
3 Electron Cryo-Tomography
87