Tutorial - Tomographic Reconstructions in Priism (1/2)
This tutorial will guide you through the reconstruction process. There is a
cryo dataset (473 megabytes)
available as an example. Start by downloading the dataset and taking it
through the entire process. This page only covers the first part of the
tutorial; for the second part, visit
1. Assemble the Raw Data
outputs an ordered stack of data starting from the most negative tilt through
to the most positive tilt (Movie 1). Contained within the header of this file
are the tilt angles and the tilt axis and shifts used to collect the data.
Note, the values of the tilt axis and shifts, while quite good, may need to be
further refined as described below. If your data is coming from some other
source, you can use the programs
to reorder and put the proper tilt angles in for the rest of the process. You
in place of CopyRegion. As a side note, the example dataset was preprocessed
with CopyRegion to throw away a 24 pixel wide border on each side that
contained camera artifacts; such preprocessing should not be necessary with
any datasets recently collected on the microscopes in the Keck Advanced
Movie 1. This movie shows a Drosophila centrosome imaged at 300kV, +/- 60
degree tilt range with projections every 2 degrees, and a 20eV slit. Total
electron does is 200 electrons per square angstrom. Note at one point the
slit becomes visible. The QuickTime movie can be downloaded from
It is 7.8 megabytes long.
2. Append Lower Resolutions
We usually being working with a reduced size version of the dataset, say
512 x 512, rather than the full size of the collected data (2048 x 2048 when
the data is collected with a binning factor of two on a 4096 x 4096 camera) to
reduce processing time and to improve the signal to noise ratio for the
cross-correlation alignment. The most convenient way to work with different
versions of the dataset at different resolutions is to use Priism's
(from the Priism menus, first select "DataViews", then "Resolution", and
finally "AppendRes). To use AppendRes, follow this recipe:
- In the AppendRes dialog, press the "Dataset" button to open a file
browser to select the input tilt series; alternatively, you can enter the
path to the file in the text field immediately adjacent to the "Dataset"
- Enter the number of resolutions you want in the "# of Resolutions" field.
For the example dataset, you'll want to enter three in the
"# of Resolutions" field (one for the original data, one for a 1000 x 1000
version, and a final one for a 500 x 500 version).
- Enter one in the "Z Scaling" field because you do not want the lower
resolutions to combine projections at different tilt angles.
- Press the "DoIt" button at the bottom of AppendRes's dialog to modify
Image 1. Shows a screenshot of Priism and AppendRes graphical user interface
with the AppendRes parameters appropriate for this example.
3. Use EMTAR for the Initial Alignment.
The EMTAR dialog (in Priism's menus, select "Processing", then
"EM Processing", and finally select "Alignment + Reconstruction") is the
graphical interface you will use for aligning and reconstructing the tilt
series. That dialog provides a way to run the different steps in the process
either one at a time or several at once in a fixed order. This tutorial will
use the steps one at a time: that is convenient for learning what the steps
do and for checking on the results before proceeding with subsequent steps.
Once you are familiar with the stages and have some confidence that the
selected parameters are appropriate for your data, you turn on multiple
stages and run them at once.
For move information about EMTAR beyond what this tutorial provides look at
Much of the process is computationally expensive. For the reconstructions,
you can speed them up by using a machine with multiple processors or a cluster
of several machines: see the notes on
parallel processing. For the
Macromolecular Structure Group at UCSF, the clusters, ec3 and ec6, are
available for performing reconstructions.
For the sample dataset, these steps will extract the alignment parameters
computed by UCSF_TOMO:
- In the EMTAR dialog, use the "TiltSeries" button to bring up a file
browser and select the tilt series file; alternatively, enter the path
to the file in the adjacent text field.
- Enter the resolution you want to use for calculations in the "Resolution"
field about halfway down EMTAR's main dialog. For the sample data set,
enter two in that field to select the second lower resolution (500 x 500
in this case). The alignment parameter results will be in the full
resolution coordinate system to make it easier to reuse those results when
reprocessing at a higher resolution. Other results, like the aligned and
vvv mass-normalized images or the final reconstruction volume, will be at the
lower resolution. Size-related parameters in the EMTAR dialogs are in
full resolution units
- Press the small button immediately to the left of the larger
"Rough alignment" button. That turns on the rough alignment
stage of processing and opens the parameter dialog for that processing
stage. In this case, the default parameters (which are to extract the
alignment parameters from the extended header) do not need to be modified
and you can close the dialog that appears. If you want to see that dialog
again without turning on or off the rough alignment processing stage,
press the "Rough alignment" button in EMTAR's main dialog. Note
that some controls are toggle buttons which are either on or off; if a
toggle button is on it will appear darker with a light shadow on one side
(to make it look like the button has been pushed inwards).
- Press the "Do It" at the buttom of EMTAR's main dialog. That runs
all the processing stages you've selected (in this case that is only
the rough alignment). The rough alignment for this example will generate
with a log file containing notes about the calculation and the script file
used to perform the calculation) in the same directory as your input tilt
series. Cent_ufix_001.bprmMn contains the shifts used by UCSF_TOMO to
track the sample during data collection. If your data did not come from
UCSF_TOMO, the rough alignment will compute a cross-correlation based
alignment: in order for those calculations to work well you should enter
your best estimate of the orientation of the tilt axis in the rough
alignment's "Axis orientation" field. To see that field, open the parameter
dialog for the rough alignment and then press the toggle button next
to "EMROUGH: special parameters"; the "Axis orientation" field will be at
the top of the dialog that appears after you press the toggle button.
- Turn off the rough alignment step in the EMTAR dialog by pressing
the small button immediately to the right of the "Rough alignment" button.
Leave the EMTAR dialog open; you'll be using it for the later steps
in this tutorial.
Image 2. Shows a screenshot of the EMTAR dialog with the rough alignment
stage enabled and the rough alignment parameter dialog with the default
4. Use EMTAR to Compute Mass-normalization Parameters
To compute the mass-normalization parameters for the sample dataset:
- Return to the EMTAR dialog you had opened for the rough alignment
and turn on the mass normalization processing step.
- In the mass-normalization parameter dialog, change the menu next
to "Quantity to fit" to "average intensity" rather than "background
intensity". Look at documentation for the mass-normalization step
if you want more information about when to use the average intensity
rather than an estimate of the background intensity level as the
basis for the mass-normalization calculations.
- In the special parameters dialog for the mass-normalization, change
the menu that reads "save only" to be "show and save". That will
cause the mass-normalization calculations to automatically display
diagnostic graphs. Once the mass-normalization calculations complete,
you can also use the "MASSNORM fit" option from the "Display" menu in
EMTAR's main dialog to view those graphs.
- Press the "Do It" button at the bottom of EMTAR's main dialog. The
alignment parameter file, Cent_ufix_001.bprmMn, will be updated with
the calculated mass-normalization parameters and you should see a graph
of the mass-normalization fit (Image 4 shows a screenshot of that graph).
- In the EMTAR dialog, turn off the mass-normalization step.
Image 3. Shows a screenshot of the EMTAR dialog with the mass-normalization
stage enabled and the mass-normalization parameter dialogs.
Image 4. Shows a screenshot of the diagnostic plot for the
Part 2 of the tutorial