Always get weird 2D averages

Hi guys, I am totally a freshman in cryo-EM and data processing but I have been watching all the tutorials all the time and trying to understand each and every parameter.
I am working with a membrane protein atm, and it seems very small. So I have been doing the 2D a lot by changing parameter etc. And I am starting to get some too symmetry averages that I am not sure whether they are real particles or just background noise that came up because of the parameters that I set or the algorithm behind it. Before I was getting the circular noise, and now they are flora mostly. From the particle extraction it looks fine but just the 2D that I am getting more and more confused.

By the way, can I upload pictures here?

Many thanks for any coming advice~~~~
Best regards,

Hi Yichao - yes, you can upload pictures (just copy and paste directly into the message, or there is also a picture icon to select). Without pictures it is difficult to judge your 2D averages.

Hiiii, Thanks a lot~~~~ So these are my junky 2Ds…

Basically 70-80 percent are like these.

Hi Yichao,

These look like noise - how do the individual particles look? Have you tried altering 2D classification parameters (e.g. switching off Force/max over poses/shifts and doing more iterations)


The most important factor for success in 2D classification is actually that you have great particle picking. Do you? Try this: Pick 100 particles manually, 2D with 2 classes. Use both 2 classes as templates for template picking (even if they look terrible). Repeat large scale 2D with template picked particles.