FSC curve and preferred orientation?

Hi all,

My 2D result is quite beautiful. After hetero/homo refinement, the FSC curve can’t drop to 0. I’ve tried to remove the duplicate particles and it didn’t really help. All suggestions are welcomed.

Also any comments on the particle distribution?

Thanks,
Shasha

Hi Shasha,

What is your pixel size, and are you binning (Fourier cropping) your particles? It appears you are limited by sampling, so if you unbin you may see better results.

Re the orientation distribution, there is clearly a degree of preferred orientation, but to judge whether it is significant just look at the map - do you see clear, interpretable density, or is it stretched/streaky?

Cheers
Oli

Hi Oli,

Thanks for your reply!
Images are collect at 0.7888Å/pix (super-res 0.394Å/pix). When extracted particle, I binned 2X to 1.576Å/pix. Will try without binning.

Here I attached a figure of the map. It is not bad?

Thanks,
Shasha

It’s hard to tell from this snapshot, but I think yes it is stretched a bit - is that an alpha helix on the left? It looks like it is kind of smeared into the surrounding density, which would be consistent with the kind of artefacts seen with preferred orientation. To quantify, you can calculate a 3D-FSC.

Cheers
Oli