of a Cryo Tilt Series
University of Colorado,
This example data set illustrates how
to use the interface in Etomo for reconstruction with SIRT (Simultaneous
Iterative Reconstruction Technique). This method involves three steps at
Reprojecting from the current estimate of the tomogram by adding
up densities along projection lines through the tomogram (blue ray line in first
Taking the difference between the original projection data and
this reprojection at each pixel; this difference represents the amount of
error in the current estimate
Distributing this difference among the pixels along the ray
contributing to it, essentially adding the error to the tomogram by a
Because SIRT is relatively
time-consuming and there is no clear point at which to stop iterating, the
strategy is to do a trial reconstruction on a subarea and examine the results
after a selected set of iterations. One can then pick the number of
iterations to use for the full reconstruction, a number that may be suitable for
other similar data sets.
The example tilt series is of a preparation of bovine papilloma virus (BPV), taken
by Mary Morphew on
an F20 with a US4000 CCD camera at a defocus of -3.5 microns.
If you already unpacked the data set for the CTF tutorial, skip to "Enter the
data set directory".
Download the sample data set from our web site.
Move the data set file "CTF-SIRT-Data.tar.bz2" to the directory where you
want to work on it. Its contents will unpack into a subdirectory named
cd to the directory with the downloaded file
- Enter the command:
or, anywhere except on Windows without Cygwin, you can use
tar -xjf CTF-SIRT-Data.tar.bz2
- Enter the data set directory with:
- Start Etomo with the command
- Open the Final Aligned Stack page.
- If you did not make a CTF corrected stack in the CTF exercise, press Create
Full Aligned Stack.
If you have gone through the exercise of finding the defocus with Ctfplotter for
this data set but did not make a CTF corrected stack, you can select the Correct CTF tab, press Correct CTF,
and press Use CTF Correction when done.
Press Done to go on to Tomogram Generation.
Uncheck Take logarithm of densities with offset. (This dataset was
started before there were templates; the cryoTemplate turns this off for new
Select Reconstruct subarea.
Set the Size in X and Y to 300,300 and the Offset in Y to
-500. The subarea can be offset only in Y through Etomo; offsetting
to a general location is possible but considerably more complicated.
Set Iteration #'s to retain to 3,5,8,11,14. The reconstruction
program will write out the results at each of these iterations.
Select Use GPU if you have a GPU available, and only one GPU for this
step if you have more than one. Otherwise, select the number of CPUs to
use in the parallel processing table.
Press Run SIRT.
When done, press View Tomograms(s) in 3dmod. This brings up a file
chooser with the available reconstructions. To select all of them, click
in the list and type Ctrl-A.
Note that the early iterations are dominated by low frequencies and that the
reconstructions become progressively noisier as high frequencies are added in
during later iterations.
Pick a number of iterations. If you wanted this reconstruction just for
picking particles, you might use 5 or even 3; otherwise 8 is reasonable.
Uncheck Reconstruct subarea and change Iteration #'s to retain to
your selected value.
If you are using a GPU, it is useful to select up to 3 if they are available.
If you are using CPUs, you may want to select more at this stage.
Press Run SIRT.
When done, press View Tomograms(s) in 3dmod. You will still need to
select the file in a file chooser.
If you want to apply post-processing to this reconstruction, press Use SIRT
Output File, which will rename it to the standard name for a tomogram at