Center tube-like samples during 2D classification?

Hello All,

I am trying to center tube-like samples during 2D classification. The issue I face constantly is that the classes slowly drift from decently centered helices to only converging on one “strand” of the filament (see image. The image is a mix of different 2D classifications, but the general trend is always the same).

I wonder if there are any settings that I could try out. I have been trying to play around with:

  • Increasing the size of the particle extraction box size
  • Window inner & outer radius values
  • Enabling “Align Filament Classes Vertically”
  • Disabling “Use Circular mask on 2D classes”
  • Disabling “Re-Center 2D classes”
  • Enabling “Min over Scale after first iteration”

Do you have any other tips that could help centering these tubes? Going 3D is difficult without helical parameters, and I need good 2D classes to find twist/rise.

Thanks in advance!
Tom

Try switching on “Recenter mask binary”, then tweak the mask threshold based on the output masks (which will be displayed below the class averages during classification).

Dear Oli,
thanks for your quick response! I have played around a lot with binary mask thresholds and a value of 0.037 seems to get the best results for this sample.
Nevertheless the 2D classes still converge on one strand of the helix. Is this preventable at all? I am trying to re-center the tubes to be able to sort out different diameters via 2D classification.

Thanks!