Template picking artifacts for long rod particle

Particle Picking
1

Hello,

I have a relatively elongated rod-shaped particle (roughly 350Å x 70Å) and blob picker is not great at picking my particles so I decided to give template picking a go. I used classes from my initial 2D classification job (ran using blob-picked particles) to set up template picking (masked to 40Å), and I was able to pick about 700,000 more particles this way. The issue is that my 2D classification job using the template picked particles has some kind of artifact: for all classes that represent the long-sided view of my particle, the particle spans the top right corner to the bottom left corner of the box, ie they are all oriented in the same diagonal direction. The parameters I used for this 2D classification job are: 100 classes, 40 online EM iterations, uncertainty factor set to 6, circular mask on (no diameter set), and everything else was left as default. Does anyone know why this happened and how I can resolve it? Happy to provide any additional information needed!

J77_2d_classes_for_iteration_40
J77_2d_classes_for_iteration_401920×1920 663 KB

2

Likely was run with the similarity sort method (default in v5) :

image
image1470×1364 192 KB

Although this shouldn’t (in the most recent version) affect anything except the orientation of your 2D classes. You can switch it to size or class index though, if you don’t want this effect.

3

Thank you for the information, I haven’t used cryoSPARC since it was updated to v5, so I didn’t know this was an option. Thank you so much!