General question about increasing the box size of our particles.
Would it be better to perform general ctf refinement / local ctf refinement on big box-extracted particles (3 times particle"s size) ? Or rather on the normal box size (~ 2 times particle’s size) ?
Same question for 3D variability jobs, i.e 3d classification and 3DVA.
Im a little confused because I obtained different results regarding the box size, which can be over-interpreted at the end
I feel like the big box particle extraction should be handle at the very end of the processing. I suspect some noise-related bias when I used the particles extracted in a bigger box. My protein of interest is about 120 Å length.
Thanks for your post! I’ll take a swing at helping you out here.
My understanding is that the CTF refinement should work more-or-less similarly for your two box sizes, provided they both have the same pixel size. Have you tried refining with either or both of the boxes?
I’m also interested in the differences you’ve observed. Could you share a few images of the differences so we can figure out what might be going on and help you interpret them moving forward?
I also want to point you to this very helpful in-depth discussion of the various effects people have observed with large and small box sizes. I frequently go back and read it myself! Why does smaller box size give better resolution?
Have you tried refining with either or both of the boxes?
Yes, I did, and obviously I had a better map with big box, but because I reached ~2.5-2.7 Å im not sure whether to use anisotropic, or 4th order aberration refinement. Those parameters didn’t improve the map, sometimes it even worsens some regions.
I’m also interested in the differences you’ve observed. Could you share a few images of the differences so we can figure out what might be going on and help you interpret them moving forward?
I can share the most obvious differences I noticed with 3DVA jobs. Here are some pictures with 2 modes, 5A filter resolution. 330 pix vs 220 pix, particle’s size 120 Å, pixel size 0.87 Å/pix
