Extra bump in FSC?

So I’ve been getting this extra bump (around 6.5 Å) in my FSCs from a variety of programs for my dataset.

I’ve seen this (GSFSC curve bilobed) and this (Tight, corrected and loose GSFSC curves) and Penzcek’s chapter about common FSC issues (https://books.google.com/books?id=D1PHZBdDkjEC&pg=PA98&lpg=PA98&dq=fsc+artifact+cryoem&source=bl&ots=CImN2pr1Wt&sig=d2SD2nrPH36s1Gc5UQ9JMjiMc6Q&hl=en&sa=X&ved=0ahUKEwi38av0gNzSAhVG9mMKHam4BhgQ6AEISjAG#v=onepage&q=fsc%20artifact%20cryoem&f=false)

Any ideas on why I see this bump? One of the links suggests crowded micrographs with overlapping boxes could cause this. I had a look at the FSC of the super crowded aldolase paper from Lander lab (https://www.biorxiv.org/content/biorxiv/early/2017/05/25/141994.full.pdf) and it doesn’t have a bump like this. I’ve checked to ensure I don’t have overlapping particles, but my micrographs are crowded. I suppose the issue could be related to estimating the noise, since some of the background in my boxes is other particles?

I can confidently assign alpha helices, in my density, so is this type of FSC still acceptable? I’ve use ab initio models to refine the dataset.

We think it’s a combination of a few possible factors - naturally lower scattering from protein at these frequencies, a loss of coherence due to the membrane (basically I assume it’s a membrane protein based on that FSC alone), persistent misalignment, or overlapping boxes combined with symmetry. The latter can even make FSC never fall to zero (including soluble protein).

Almost all (all?) published membrane protein structures have such a bump. In better refinements (for the same dataset) it will be less pronounced. It’s also affected by masking. I’ve been actively investigating it, but no conclusions so far.

Map-to-model FSC, EM ringer score, and subjective map quality assessment should guide you as well as GS-FSC. Usually the 0.143 FSC reported is close to the subjective assessment in such cases, so I wouldn’t worry about it if that remains the case.

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@DanielAsarnow is certainly the expert here but we also very often see this behaviour on membrane proteins or those with significant regions of flexibility/disorder/partial occupancy.