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Microtubule Seam-search

Microtubule seam-search is very important to separate α- and β-tubulins.

What the seam-search algorithms in cylindra do is simply adding a new column named "isotype-id" to the molecules features. As seam-search is usually combined with subtomogram averaging and alignment, these methods are located in the STA widget.

Seam-search Based on Cross-correlation

API: seam_search

GUI: STA widget > Analysis > Seam search > Seam search by correlation

Although the structure of α/β-tubulins are very similar, it is known that trying all the possible patterns and comparing the cross-correlation is useful to distinguish them.

Seam search

List of parameters
  1. "layer" is the molecules layer to be seam-searched.
  2. Set the template image and mask parameters in the STA widget.
  3. "anti-template path" is the path to the anti-template image.
  4. "interpolation" is the interpolation method used for resampling the sub- volumes.
  5. "npf" is the number of protofilaments. If molecules are created in cylindra, this value should already be determined by CFT.
  6. You can select how to (or not to) show all the averages for different seam locations by the "show averages as" combobox.
  7. "cutoff" is the relative cutoff frequency of the low-pass filter. This value is usually smaller than the value used for subtomogram alignment.

Molecules labeled with 0 are the molecules that should be considered as the template, and 1 the anti-template.

Seam-search in the Tilt Series

API: seam_search_on_tilt

GUI: STA widget > Analysis > Seam search > Seam search on tilt series

If the tomogram is opened from a tilt series, the molecules can be scored directly in the tilt images instead of the reconstructed subvolumes. The template (and the anti-template) is projected to each tilt image at the position and orientation of each molecule, and compared with the tilt image by ZNCC. The score of each molecule is the mean over the tilts, and the mean scores of the two groups of molecules are compared for all the seam locations. The location with the largest difference is the seam.

List of parameters
  1. "layer", the template, the mask, "anti-template path", "npf" and "cutoff" are the same as seam_search.
  2. "bin size" is the bin size of the projections.
  3. If "apply CTF" is checked, the CTF is applied to the projections, with the defocus of each molecule in each tilt. The tilt series must have the CTF parameters.

API: seam_search_manually

GUI: STA widget > Analysis > Seam search > Seam search manually

This method labels the molecules by the given seam location. location=0 will label molecules with

[0, 0, ..., 0,
 1, 1, ..., 1,
 0, 0, ..., 0,
 1, 1, ..., 1,]

and location=1 will label molecules with

[1, 0, ..., 0,
 0, 1, ..., 1,
 1, 0, ..., 0,
 0, 1, ..., 1,]

Seam-search by Features

API: seam_search_by_feature

GUI: STA widget > Analysis > Seam search > Seam search by feature

This method labels the molecules by the given feature. The feature should be such that approximately label the α- or β-tubulins. Usually, molecules should be labeled by classification using microtubule-associated proteins as the fiducials.