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!