Streaking issues in data

Hi all,

Reaching out to the community because I have been having streaky artifacts with a lot of separate datasets recently that have me kind of stumped. For a little bit of background: I have this issue with data collected on two different Krioses (each with a K3 camera). I work nearly exclusively with ribosomes on UltrAuFoil grids that I float my own amorphous carbon onto (one exception where the holey film was carbon instead of gold). My typical workflow jumps around between relion and cryosparc and I see issues regardless of software, but cryosparc provides a bit more visual feedback that I thought may be helpful to share here.

(I could only attach one image so I put a bunch in one file, here are details:)
(1) Example: CS-refined map around the edge of the ribosome into the solvent region, when I push the contour very low to where noise is prominent

(2) Unfiltered half-map from the same job (a bit more zoomed out)

(3) I notice I often get these features in the Fourier space slices: sometimes they are on the negative side of the spectrum, sometimes positive, sometimes areas of both (left, center, right). (some contrast adjustments applied to help bring it out. From different projects)

(3.2) Also, as you can see above it has almost always had a pretty consistent directionality. However, recently we had one that has changed direction

(4) The noise model: this also varies between datasets, where I usually see at least one of these 3 features: 1. a bump around ~3.5 Å (but there is some variation here), 2. a trend up instead of down (least common), and 3. a sort of “hook” at the end where the curve sharply turns down.

(5) During 3D refine jobs, there are noisy features in the region outside the ribosome that have a different “texture” in different planes. (this is from the final iter, going to 2.5 Å. Middle panel has different-looking noise, hope it is visible here)

(6) At the end I am generally seeing effects mostly at high resolution (as above), but sometimes intermediate steps with binned particles show lower-res striped patterns too (dark background, left: one slice from an ab initio job @ 4x bin; light background, right: 4 classes from a 3D hetero refine job @ 4x bin, where the 3rd contains particles I proceed with).

And some further comment:
We have had a couple cases where we suspected some contamination either from ice or possibly from the gold foil - in those cases we reject micrographs with strong high-res rings in the FFT but it isn’t always feasible to completely reject any hint of a ring. I know this isn’t ideal but I’d be a little surprised if this was the problem, given that in well-ordered map regions we clearly get many ribosome particles aligning very well to each other (base density really clear, many ions, solvation, etc. I am also generally putting these through ~4-6 rounds of classification between 2D and 3D). Plus I have at least one case where contamination was very minor.

If you have any thoughts or suggestions or questions about what all I have examined, I’d appreciate any help! I have tried reaching out to some others at my facility but no one I shared with seemed to be plagued with this the way I am. I have tried to keep this post focused on observation so as not to inundate you further with information on all the things I have checked. FWIW I typically get pretty believable global resolutions in the mid-low 2’s (as mentioned above). But for some of the weaker map features I am interested in this becomes a problem for interpretation in maps where it is really pronounced.

Hi @zlwatson,

We discussed this internally and came up with a few thoughts:

  • Can you post the orientation distribution plots from the hetero refinement job? Perhaps these streaks are indicative of preferential orientation (though your plot (3) doesn’t show anything obvious)
  • Can you provide an example of a micrograph? (output of the exposure curation job)
  • Have you checked for anisotropic magnification? You can do this in cryoSPARC using the global CTF refinement job (turning everything off except aniso mag)
  • Does this happen for one particular ribosome or for many different ones? Does the ribosome density also exhibit similar streaks?


Hi Valentin,

Thanks for getting back to me. I am unable to gather more screenshots at the moment and will have to answer some of your questions later, but here is what I can answer via text:

The ribosomes are always E. coli ribosome complexes with varying mRNAs and tRNAs, and we prepare them in the same way. I generally use the same synthetic reference from pdb for initial 3D volumes (at the beginning), but I have tried rotating it or using other references and I don’t think that is the problem (which makes sense to me since they are highly lowpassed anyway).

I know preferential orientation is in general a problem for ribosomes and I always have some obvious hotspots, but I wasn’t strongly suspecting it since I didn’t think it would manifest as streaks that extend to the edge of the box (outside the particle, almost like they are overlaid on the map). I did have one dataset (with grids I don’t typically use) where I did think it was clear that preferred orientation actually was a significant problem early on, but that was an outlier case. There have also been a couple occasions where a colleague has seen relion volumes where the ribosome itself is very streaky before post-processing, but I would say this is also not the norm (I didn’t really handle these much myself, either).

I’ve wondered a lot about whether or not this may be different issues causing something that looks similar between the datasets, I don’t know.

Usually I use MotionCor2 in relion to later do Bayesian polishing so I don’t have the exposure curation result, but can add snapshots of micrographs later. Anisotropic magnification is also something I typically do in relion but I can try to test this sometime.