Hi, I have recently started to use Cryosparc so sorry if my question appears trivial. But maybe someone can explain my observation. During homogeneous refinement, I have observed dark blue (negative density) showing up in the (real space slices) around my protein. Is the dynamic mask too narrow?
Negative densities in the reconstructions are possible where there are regions that scatter less than solvent, perhaps in between packed helices, etc. But moreover they are actually guaranteed because our volumes are usually mean-centered (mean is set to 0).
You can visualize the negative densities in Chimera by unchecking “cap high values at surface faces” in the Surface and Mesh Options of the Volume Viewer. That allows the program to render iso-surfaces (instead of the cube) when the slider is below 0.
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Thanks @DanielAsarnow - as you say, negative density is generally expected due to the “floating” (mean zero) of particle images before processing. We also expect negative density (“true” negative density) as mentioned in regions where the density is less than solvent. We also expect generally a “halo” of negative density in a 3D reconstruction around the actual particle, typically due to crowding of particles in the micrograph. If there are often secondary particles in the corners of particle images, then the mean-zero floating will further set the background density level of the central particles to a slightly negative value. This is normal and does not affect results in terms of resolution or quality.
If you are seeing something other than the above (eg. very large magnitude negative density, or very sharp transitions between positive/negative density) there may be something else going on.
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