Don't understand the Relative Signal in Orientation Diagnostics

Utilities
1

Hello Cryosparc,

I have been willing to ask this question for a while now.

We have an initial structure with encouraging initial resolution but some of the cFSCs are low rez (~7A) leading to very bad cFAR because of preferred orientation (structure generated from low amount of particles. We are hoping these views will populate with more data.

J270_summary_of_3072_conical_fscs
J270_summary_of_3072_conical_fscs577×288 30 KB

Anyways, when I run Orientation Diagnostics to get a better understand of the bias, I can never fully understand that grid plot that looks very useful.

J270_avg_relative_signal_amount_within_azimuth_elevation_viewing_regions
J270_avg_relative_signal_amount_within_azimuth_elevation_viewing_regions1780×880 354 KB

J270_3dfsc_volume_central_slices_clipped_at_2572_å
J270_3dfsc_volume_central_slices_clipped_at_2572_å2009×495 105 KB

I am expecting a few viewing regions to have resolutions around 7A with very bad relative signal such as the ones I saw in the documentation examples (around 0.3). According to this plot, they all look OK to me with resolutions all within the 3.3-2.8 region with descent relative signal values (around 0.6). It is then hard for me to identify the “missing” views

I clearly am not understanding everything about the generation of these plots.

Thanks for the help

2

Hi @wjnicol,

Thanks for sharing this! I think this might be a very similar case to what @olibclarke reported in this post.

cFSCs are computed by considering a cone in Fourier space, whereas relative signal uses a ‘wobbly slice’ (or what we refer to as a torus) so that we can link poor relative signal to viewing directions via the Fourier slice theorem.

If that ‘missing wedge’ of correlation in the 3DFSC plot above is much more evident with conical regions as opposed to slices, the relative signal plots will not contain the same worst-case correlations you see in the cFSC plot.

A couple questions:

  1. Is this a relatively small target (< 100 kDA?)? Does it have a micelle?
  2. Does the density itself show signs of preferred orientation?

We’re actively investigating this phenomenon so your input is very much appreciated!

Thanks,
Valentin

1 Like
3

Hello,

Thanks for quick reply.

Our complex is roughly 200kDa and has no micelle
I did not inspect the density. This is a quick structure from a low amound of exposures. Data processing with larger amount of data is undergoing.

I will read the post you pointed at. A quick look at it shows a very similar pattern.

Thanks!

1 Like