3.4 Tilt Series
Alignment
and Tomographic
Reconstruction
Using IMOD
1. After data collection, the raw tilt series are saved as new stack
file with the suffix “.st” using the newstack command of IMOD
software.
2. Type “etomo” for starting the IMOD software graphical user
interface (GUI).
3. Open the new stack file and select appropriate data type.
4. Click the “Scan Header” for reading pixel size and image
rotation from header of new stack file. Type the diameter of
fiducial gold maker.
5. Align tilt series based on cross-correlation. Click “Calculate
Cross-Correlation” and “Generate Coarse Aligned Stack.”
Click “View Aligned Stack in 3dmod” for checking the result
of coarse alignment.
6. Select ~20 gold fiducial makers and make seed model. Click
“Track Seed Model” for tracing gold fiducial makers at each tilt
angle. If you find any untracked or mis-tracked makers, you can
manually fix them using “Fix Fiducial Model.”
7. Click “Compute Alignment” for calculating fine alignment
using seed model and “View/Edit Fiducial Model” for checking this result.
8. Type appropriate tomogram thickness and select “Use whole
tomogram” with “Binning 2.” Click “Create Whole Tomogram” and “Create Boundary Model” for selecting boundary
lines and then save it. Finally, click “Create Final Alignment”
for calculating final alignment parameter.
9. Click “Create Full Aligned Stack” for making aligned stack file
(see Note 7).
10. Select “Weighted Back Projection” or “SIRT (simultaneous
iterative reconstruction technique)” for generating final tomogram (see Note 8). Click “View Tomogram(s) in 3dmod” for
checking the generated tomogram.
Fig. 1 Representative cryo-EM image of Leptospira biflexa. Target region is a cell
tip area of the cell in the hole of grid. Red arrowheads indicate the region
selected to collect a tilt series
Visualization of Intact Periplasmic Flagella
135
Alignment
and Tomographic
Reconstruction
Using IMOD
1. After data collection, the raw tilt series are saved as new stack
file with the suffix “.st” using the newstack command of IMOD
software.
2. Type “etomo” for starting the IMOD software graphical user
interface (GUI).
3. Open the new stack file and select appropriate data type.
4. Click the “Scan Header” for reading pixel size and image
rotation from header of new stack file. Type the diameter of
fiducial gold maker.
5. Align tilt series based on cross-correlation. Click “Calculate
Cross-Correlation” and “Generate Coarse Aligned Stack.”
Click “View Aligned Stack in 3dmod” for checking the result
of coarse alignment.
6. Select ~20 gold fiducial makers and make seed model. Click
“Track Seed Model” for tracing gold fiducial makers at each tilt
angle. If you find any untracked or mis-tracked makers, you can
manually fix them using “Fix Fiducial Model.”
7. Click “Compute Alignment” for calculating fine alignment
using seed model and “View/Edit Fiducial Model” for checking this result.
8. Type appropriate tomogram thickness and select “Use whole
tomogram” with “Binning 2.” Click “Create Whole Tomogram” and “Create Boundary Model” for selecting boundary
lines and then save it. Finally, click “Create Final Alignment”
for calculating final alignment parameter.
9. Click “Create Full Aligned Stack” for making aligned stack file
(see Note 7).
10. Select “Weighted Back Projection” or “SIRT (simultaneous
iterative reconstruction technique)” for generating final tomogram (see Note 8). Click “View Tomogram(s) in 3dmod” for
checking the generated tomogram.
Fig. 1 Representative cryo-EM image of Leptospira biflexa. Target region is a cell
tip area of the cell in the hole of grid. Red arrowheads indicate the region
selected to collect a tilt series
Visualization of Intact Periplasmic Flagella
135