Bump on FSC after NU-refinement

3D Refinement
1

Hi,

I’m getting an extra bump in FSC and I’ve seen that this is normal for membrane proteins (which is my case) [Extra bump in FSC?] However, the one that I’m getting looks like a little bit different from the ones that I’ve found on the forum.

Is that normal too or am I doing something wrong?
What I’ve done:

  1. Rounds 2D classification
  2. Ab initio reconstruction - 4 classes
  3. Heterogeneous refinement - 4 classes/all particles
  4. NU-refinement of the best class/best particles
  5. 2D classification of the particles from (4)
  6. Re-run NU-refinement with volume from (4) and best particles from (5)
    All refinements I’m running on default parameters.

I’m working with an asymmetric membrane protein.

Thanks in advance!

FSC

1 Like
2

I’ve been able to reduce this bump in my data (not membrane protein) by reducing the Inner and Outer window radius. You might also want to consider using Topaz train to pick your particles. That alone doubled my particle count and improved my resolutions almost 0.6A.

1 Like
3

By “reduce the bump” do you mean that the FSC curve stays up more and doesn’t go down near ~6.2 A (in this FSC)?

4

Yup, that’s what I meant. Mine went from looking like this
1

to this
2.

Of course, I was also imposing symmetry in the first one and that can be a big factor. Also, if you have a lot of overlapping boxes, you tend to see this kind of behavior (i.e. a bump in the FSC at a lower res.)

A better explanation is here

5

@fernandesj from my reading, I think everything you are doing is correct. The “bump” (as discussed in the other post you linked) is a relatively common occurrence and definitely can be helped by having better picks and more particles, as @vamsee described

1 Like
6

@vamsee thank you! I’ll try here to see if it helps in my case

7

@apunjani ok, thank you!