4. If images on either high-tilt end of the series need to be
removed use the “trimvol” command to generate a trimmed
stack:
trimvol – z <# of starting image>,<# of end image>

For example: if you need to remove images 1–10 of a
120-image stack, use the command:
trimvol -z 11,120

5. Begin the reconstruction with the command “etomo”, then
select the “Build Tomogram” option in the pop-up window.
6. A new dialogue box will open. Select the tilt-series file in the
“dataset name” field.
7. Set the “System template” to “cryoSample.adoc”.
8. Select “Scan Header” to have the program extract metadata
from the file header.
9. Fill in the fiducial diameter in nanometers (see Note 14).
10. Select “Create Com Scripts”. A new dialogue box will open
showing the reconstruction workflow.
3.7.1 Pre-processing
The preprocessing step finds individual pixels that are very far from
the mean value and replaces them with the local average. In nearly
all cases, the default values work perfectly, but they can be changed
if too many or too few pixels are being targeted for removal. Select
“Create Fixed Stack”. Once done, select “View Fixed Stack”. If the
fixed stack is adequate, close the 3dmod window and select “Use
Fixed Stack”. Select “Done”.
3.7.2 Coarse Alignment
Select “Calculate Cross-Correlation”. Then, select “Generate
Coarse Aligned Stack”. View the aligned stack in 3dmod to ensure
the alignment is satisfactory. Close the 3dmod window and select
“Done”.
3.7.3 Fiducial Model
Generation
Under “Seed Model” tab, select “Make Seed and Track” and
“Generate Seed Model Automatically”. Select between 10 and
20 fiducial points to track. If there are less than ten fiducials within
the field of view, use the number of fiducials visible (see Note 15).
Click “Generate Seed Model”. Open the seed model and ensure
that fiducials were selected correctly and evenly across the field of
view. Under the “Track Beads” tab, select “Track Seed Model”.
The project log window will display how many missing points are
left after the automatic tracking.
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Ryan K. Hylton et al.