3. Using the command line, move into the directory containing
the tilt series for reconstruction.
4. Start Etomo by typing “etomo” in the command line.
5. When the “Front Page” window opens, select “Build
Tomogram”.
6. On the “Setup Tomogram” window, select the name of the
filename.mrc file in the dataset name bar and choose “cryoSample.adoc” as the “system template”.
7. Press “Scan Header”, which should automatically fill in “Pixel
size (nm)” and “Image rotation” (degrees). If not, fill in manually (see Note 37).
8. Add the value for “Fiducial diameter (nm)” (e.g. 10).
9. Enable “Parallel Processing” when working on multiworkstation clusters and “Graphics card processing”, if
available.
10. For bidirectional tilt series, ensure “Series was bidirectional
from” is activated under Axis A and the correct start angle is
displayed (e.g. 20.0).
11. Press “View Raw Image Stack” to open the tilt series.
12. Manually inspect the quality of all individual images in the tilt
series stack. Take note of images which are unfocused, blurry,
or too dark to detect gold particles. Add the numbers of these
images to the “Exclude views” section under Axis A and select
“remove excluded views” in the section above Axis A.
13. Press “Create Com Scripts”. This will generate the “align.
com” file inside the working directory and open the window
for the tomogram generation workflow. Close this window.
14. At the command line, open the “align.com” file in a text editor,
(e.g. “vi align.com”), go to line 52 and change the “XTiltOption” from 0 to 4. Save changes keeping original file name.
15. Restart Etomo by typing “etomo filename.edf” in the
command line.
16. Follow the tomogram generation workflow as outlined in the
online tutorial (https://bio3d.colorado.edu/imod/doc/
etomoTutorial.html). Ignore the steps for dual-tilt reconstruction and under “Fiducal Model gen.” choose “Make seed
model manually”.
17. For tomogram generation, you can choose either the back
projection or SIRT algorithm (~15 iteration should be sufficient). SIRT will give a higher contrast tomogram than back
projection but with the potential loss of high-resolution information. Thus, for subtomogram averaging, back projections
are usually used. The final tomogram will be saved as a filename.rec file in your working directory.
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Daniel Serwas and Karen M. Davies
the tilt series for reconstruction.
4. Start Etomo by typing “etomo” in the command line.
5. When the “Front Page” window opens, select “Build
Tomogram”.
6. On the “Setup Tomogram” window, select the name of the
filename.mrc file in the dataset name bar and choose “cryoSample.adoc” as the “system template”.
7. Press “Scan Header”, which should automatically fill in “Pixel
size (nm)” and “Image rotation” (degrees). If not, fill in manually (see Note 37).
8. Add the value for “Fiducial diameter (nm)” (e.g. 10).
9. Enable “Parallel Processing” when working on multiworkstation clusters and “Graphics card processing”, if
available.
10. For bidirectional tilt series, ensure “Series was bidirectional
from” is activated under Axis A and the correct start angle is
displayed (e.g. 20.0).
11. Press “View Raw Image Stack” to open the tilt series.
12. Manually inspect the quality of all individual images in the tilt
series stack. Take note of images which are unfocused, blurry,
or too dark to detect gold particles. Add the numbers of these
images to the “Exclude views” section under Axis A and select
“remove excluded views” in the section above Axis A.
13. Press “Create Com Scripts”. This will generate the “align.
com” file inside the working directory and open the window
for the tomogram generation workflow. Close this window.
14. At the command line, open the “align.com” file in a text editor,
(e.g. “vi align.com”), go to line 52 and change the “XTiltOption” from 0 to 4. Save changes keeping original file name.
15. Restart Etomo by typing “etomo filename.edf” in the
command line.
16. Follow the tomogram generation workflow as outlined in the
online tutorial (https://bio3d.colorado.edu/imod/doc/
etomoTutorial.html). Ignore the steps for dual-tilt reconstruction and under “Fiducal Model gen.” choose “Make seed
model manually”.
17. For tomogram generation, you can choose either the back
projection or SIRT algorithm (~15 iteration should be sufficient). SIRT will give a higher contrast tomogram than back
projection but with the potential loss of high-resolution information. Thus, for subtomogram averaging, back projections
are usually used. The final tomogram will be saved as a filename.rec file in your working directory.
14
Daniel Serwas and Karen M. Davies