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Conventional Methods in Cryo-ET Studies

cylindra provides several common methods used in cryo-ET studies.

Subtomogram Averaging

API: average_all

GUI: STA widget > Averaging > Average all molecules

This method uses one or more molecules-layer to calculate the subtomogram average.

Average all

List of parameters
  1. Select the template image in the "template" combobox of STA widget.
  2. "layers" is the molecules layer(s) that will be averaged. If you selected multiple layers, all the molecules will be averaged to a single image.
  3. "size (nm)" is the size of the output image. If not given, it tries to use the size of the template image. It will raise an error if the size cannot be unambiguously determined.
  4. "interpolation" is the interpolation method used for resampling the sub- volumes.
  5. "bin size" is the binning factor for resampling.

After running the method, a new napari viewer will be opened to show the average image. This viewer can be accessed via ui.sta.sub_viewer.

Averaged tubulin

Note

There are more options for averaging molecules in the Averaging menu:

Subtomogram Alignment

API: align_all

GUI: STA widget > Alignment > Align all molecules

Align all

Alignment is a step in which each coordinate of a molecules layer is updated to maximize the correlation score between the subtomogram around the molecule and the template image. New molecules layer will be suffixed with "ALN1", "ALN2", etc.

List of parameters
  1. Set the template image and mask parameters in the STA widget.
  2. "layers" is the molecules layer(s) that will be averaged. If you selected multiple layers, all the molecules will be averaged to a single image.
  3. "Max shifts (nm)" is the maximum allowed shift in (z, y, x) direction.
  4. "rotations" is the maximum allowed rotation angles in degree around each axis. Two float values are expected for each axis. (a, b) means the rotation angle will be between -a and a with step b. For example, (4, 2) means the rotation angles will be -4, -2, 0, 2, 4.
  5. "cutoff" is the relative cutoff frequency of the low-pass filter.
  6. "interpolation" is the interpolation method used for resampling the sub- volumes.
  7. "method" is the method used to calculate cross correlation.
  8. "bin size" is the binning factor for resampling.

Align Averaged Image

API: align_averaged

GUI: STA widget > Alignment > Align average to template

This method is mostly used after molecules are initialized by map_monomers. It first calculates the average, then fit the average to the template image. The optimal fitting parameters are then used to shift and rotate all the molecules. It means that if you run average_all on the aligned molecules, the average image should be almost well fitted to the template image.

Align averaged

List of parameters
  1. Select the template image and mask parameters in the STA widget. The mask is usually not needed, as the average image does not have much noise outside.
  2. "layers" is the molecules layer(s) that will be averaged. If you selected multiple layers, all the molecules will be averaged to a single image.
  3. "Max shifts (nm)" is the maximum allowed shift in (z, y, x) direction. If not lattice parameters will be used to infer the maximum shift, so you don't have to set this parameter in most cases.
  4. "rotations" is the maximum allowed rotation angles in degree around each axis. Two float values are expected for each axis. (a, b) means the rotation angle will be between -a and a with step b. For example, (4, 2) means the rotation angles will be -4, -2, 0, 2, 4.
  5. "bin size" is the binning factor for resampling.
  6. "method" is the method used to calculate cross correlation.

Fourier Shell Correlation (FSC)

API: calculate_fsc

GUI: STA widget > Analysis > Calculate FSC

This method automatically split the selected molecules into two groups, and calculate the Fourier Shell Correlation (FSC) between the two groups. The FSC curve will be shown in the logger widget. Since the average image can easily be calculated after FSC, this method also adds the average image to the sub-viewer as average_all does. The FSC results will also be stored in the metadata attribute of the image layer for the average image.

Calculate FSC

List of parameters
  1. Set the mask parameters (and the template image if you are going to create the mask from the template) in the STA widget.
  2. "layers" is the molecules layer(s) that will be averaged. If you selected multiple layers, all the molecules will be averaged to a single image.
  3. "size (nm)" is the size of the output image. If not given, it tries to use the size of the template image. It will raise an error if the size cannot be unambiguously determined.
  4. "seed" is the random seed value used to split the molecules into two groups.
  5. "interpolation" is the interpolation method used for resampling the sub- volumes.
  6. You can set "number of image pairs" if you want to split molecules many times to get a more accurate FSC curve.
  7. Uncheck "show average" if it is not needed.
  8. "Frequency precision" will be automatically determined but you can set it manually.

Note

If you need the two average images separately, you can run:

API: split_and_average

GUI: STA widget > Averaging > Split and average molecules

PCA/K-means Classification

API: classify_pca

GUI: STA widget > Analysis > PCA/K-means classification

PCA/K-means classification