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Prepare Spline Configuration for Your Structure of Interest

The default spline configuration on startup is optimized for microtubules. If you want to analyze other cylindrical structures, you need to define a custom spline configuration. This is a tedious task, but is very important for successful analysis.

A sample containing tobacco mosaic virus (TMV) is used to make the example images below.

1. Manually fit spline

To define a custom configuration, you will have to prepare a well-fitted spline first.

  1. Draw a spline along your structure of interest. The length of the spline should be 50 - 200 nm, depending on the density of your structure.
  2. Open the spline fitter widget from Splines > Open spline fitter.
  3. Click "Auto-center all" button. If the quality of your tomogram is good enough, you should see the center of the overlay accurately follows the center of your structure. If the auto-centering fails, you need to manually click the center of your structure slice by slice.
  4. Click "Fit" button to apply the shift to the spline. This will update the spline coordinates.

If the spline is fitted correctly, you should see the spline follows the center of the structure in the napari viewer.

2. Measure cylinder radius and thickness

Building a cylindrical coordinate system requires the radius and the thickness. Radius can be measured for every spline, but the thickness values must be properly predefined in the config.

  1. Open the spline slicer widget from Splines > Open spline slicer. Because the radius is not known yet, no image will be shown in the canvas.
  2. Click the "Measure radius" button. This will automatically measure the radius the spline by detecting the peak of the radial profile around the spline. Cross-sectional slice will be shown in the canvas, based on the radius. If the measured value largely deviates from your expectation, you can manually adjust the "Radius (nm)" value in the GUI.
  3. In the upper area of the widget, change the bin size and filter so that the cross-sectional slice clearly displays the structure.
  4. Adjust the "Inner thickness" and "Outer thickness" parameters so that the circles shown in the canvas accurately represent the inner and outer surfaces of your structure. Cylindrical coordinate system will be constructed between these two surfaces.
  5. Click "Apply radius and thickness" button to update spline radius and config.

3. Measure lattice parameters

Now, we can accurately build the cylindrical coordinate system to measure the lattice parameters.

  1. Open the spectra inspector widget from Analysis > Open spectra inspector.
  2. By default, the global-CFT power spectrum of your structure is shown in the canvas. If you think the resolution of power spectrum is low, you can switch to "Upsampled global-CFT" mode.
  3. Enable "Select axial peak" mode, and click the axial peak in the power spectrum.
  4. Enable "Select angular peak" mode, and click the angular peak in the power spectrum.
  5. The measured lattice parameters will be shown in the "Measured parameters" section. For later use, you can log these parameters to the console.

4. Determine config parameters

You now have lattice parameters of a representative segment of your structure. Based on these values, you can determine the config parameters. Open the config editor widget by following these steps:

  1. Open the spline slicer widget as in 2.
  2. thickness_inner and thickness_outer ... Set to the same value as the values you set in the spline slicer.

  3. clockwise ... Click "Measure CW/CCW" button. This method will measure whether the current segment has a clockwise (CW) or counter-clockwise (CCW) slew and log the result in the logger widget. If the result is "clockwise" and the current segment visually appears to have the "PlusToMinus" polarity, set this parameter to "PlusToMinus". Do the same for other cases.

  4. Open the spectra inspector widget as in 3.

  5. npf_range, spacing_range and twist_range ... Set to a range that covers the measured parameters in the spectra inspector. There is no standard way to determine the range, as the actual achievable range depends on both the heterogeneity of the structure and the noise level of the reconstructed tomograms. The best way is to try several segments from different tomograms and get a sense of the distribution.
  6. rise_range and rise_sign ... If the "rise" value is positive, leave rise_sign as is and set rise_range to a range that covers the measured value. If the "rise" value is negative, invert rise_sign and set rise_range to a range that covers the absolute value of the measured value. For example, if the measured "rise" is -10.5° and "rise_sign" is -1, you can set rise_sign to 1 and rise_range to [8, 12].

Range of nPF

Although in some cases the number of protofilaments (nPF) is well known to be a fixed value, it is recommended to set a small range. This will help you notice the bad fitting results.

Sign of rise

The reason why we have a rise_sign parameter is that the rise_angle and start does not span the full minus to plus range for most type of structures, so we can just keep in mind that we always use the positive rise_angle and start — with rise_sign = 1, the lattice type of microtubule would be "13_-3", which is not what we usually see in the literature. Therefore, setting rise_sign does not have any mathematical meaning and will not affect the fitting result.

5. Save as a config preset

Click "Save as new config" button to save the current config as a new preset. You can load this preset in the future analyses using the "Load preset" on the left panel.