Cannot align side views of membrane protein

Hi all,

I have been struggling to get good 2D classifications, in particular the side views of my interested membrane protein.
The membrane protein is a trimeric beta-barrel, stablised in detergent micelles. Trimeric size is 50x3=~150kDa, with a particle diameter of ~100A.
After reading multiple threads in the forum, particles were extracted with the box size of 250A (after motion correction and CTF find) for 2D classifications.
I got very good top-down view that clearly indicated the presence of the trimer (with three distinct holes). However, I could only get a blob with limited details which I assumed them to be side views and I want to improve this.

I’ve tried the following parameter settings for 2D classification:

  • Circular mask diamter (A): 120, 130, 150, 180, 200
  • Re-center mask threshold: 0.2, 0.75
  • Force max over poses/shifts: OFF
  • Number of final full iterations: 1, 2, 5
  • Number of online-EM iterations: 20, 40, 50
  • Batchsize per class: 150, 200, 400
  • Enforce non-negativity: ON
  • Use clamp-solvent to solve 2D classes: ON

This is an example of attempted 2D classifications.

I have also tried to select only 2 classes (guessing to be side views) of ~100k particles for 2D classification.
And they don’t look much like beta-barrel… I’m not sure if it’s due to the front and rear layer of beta-sheets crossing of the barrel that makes it so difficult to align?

Any idea what else to try? Any feedback would be helpful and appreciated!

Hi there,

I’m facing the same problem. Did you get something to work? Thank you!

Not really, still pretty stuck in this situation…