Hi,
I have a query regarding the Fourier slice view shown during each iteration of NU refinement.
Initially I was using 3x binned particles which gave me 5.5 A map with Fourier slices looking like this:
However, when I re scaled the particles to actual pixel size and ran NU refinement again I could get 4A map with Fourier slices looking like following:
I’m not sure why the final Fourier slices looks different between downscaled and re-scaled maps. Please shed some light on this for better understanding if something wrong is happening.
Thank you.
When you say rescaled, do you mean you took binned data and literally scaled it back to the original size, or did you use the original data?
if you did the latter which I imagined you did, the difference is that your resolution limit based on your sample is at about 4A where as based on your magnification it could probably be lower but you are sample limited. Although the trend between resolution and magnification is not linear (binning most readily resembles a fold change in magnification used), you would expect to see a marked change in the resolution attainable due to the change in A/px attained.
The bottom Fourier slices are masked using the calculated FSC during refinement and since your cut off is near 4A for tight masking, the fourier shells beyond 4A are truncate and masked (violet). In the binned data the same is true but the masking is much closer to the theoretical limit which is why the shells go all the way to the edge of image.
You’ll notice that the slices are almost identical view for view but with finer detail in the unbinned data. With that said since 5.5-4A is not a large change in fourier shells but your image size is tripled, you’ll notice that the actual Fourier shells shown are approximately the same size, but the bottom is nested in the 350px image whereas in the top the dimensions are 133px but the fourier sphere is approximately the same size due to the mask based on the FSC limit which is 5.5 in the top and only improved to 4 in the bottom.
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Another aspect is the colour can make it look different when vs if it was on a gray scale. Certain colours pop out more to the eye when they are colour, but the same trend in relative magnitude may be not that different.
