Hi,
I recently begun using Cryosparc 4.4.1, and tried polishing my map using reference based motion correction.
I was able to complete the job successfully.
However, when I used the outputted polished particles as input for a homogenous/non-uniform refinement job, although the nominal resolution improved nicely which was encouraging, I got unusually heavy maps (1.7 GB per map).
When trying to visualize these maps with Chimera, I was not able to see the complete map but only saw slices of z-sections. I’m not sure if this was because the file was too heavy for Chimera or some other problem, but Cryosparc viewer and Coot were able to display the complete map without a problem (though very slowly because of the large file).
I, however, obviously need to be able to visualize the map with Chimera for fitting, masking and visualization purposes…
Any idea why this happened? Is this considered normal? How could I make the map less heavy, and how could I visualize it in Chimera?
The particle stack was resampled to 8K pixel sampling if imported with upsampling 2.
I’ve not checked whether RBMC auto-resamples to original data if input is Fourier cropped, someone else can chime in if that is the case.
The Chimera issue is, as you surmise, related to Chimera defaulting to a visualisation scheme which cuts down graphical load. In the Volume Viewer, change from “Plane” to “Surface” (I forget the exact name, I usually load a settings file with the defaults adjusted to avoid this issue)
After RBMC, the map will be at unbinned pixel size. That’s why it is bigger if you use Fourier crop before. If you want to reduce the size, just use Volume Tool to reduce to the pixel size without affect the resolution of the map.
Reference based motion correction will output particles that are at the same (super)resolution as your original movie.
Fourier cropping needs to be applied at the RBMC level, just as you would do at the motioncorrection level.
The map is bigger because the particles are now at the same A/pixel as the unbinned movies.
You might want to downsample your particle stack as well, unless you really need the super resolution.
I have a map, the grid size showed in ChimeraX is 640, with pixel size 0.4. After Crop to box size (pix) 256 in Volume Tool, I found that the pixel size change to 1, and the size of volume was reduced. However, I found the density will also be worse and two maps look different in threshhold.
Another method is to Resample to box size (pix) 256 and without Crop to box size ,the out put file also have less features and the threshold exhibit a similar mode with the croped map.
So how to reset pixel size and without affect the resolution?
We tested the Crop to box size (pix) function of Volume Tools in CryosPARC version 4.7.1 and found it to be operating as expected by cropping the box in real space with no change to the map pixel size.
Depending on your map resolution and how generous the 640 box is relative to your particle diameter, you might achieve a box of 256 for your map using a combination of Crop to box size (pix) and Resample to box size (pix). For example Resample to box size (pix) 400 and Crop to box size (pix) 256 would still give you a Nyquist map resolution limitation of 1.28 Ă…, but the box will be tighter to the map.
If you continue to see unexpected results in the output pixel size from Volume tools after checking your settings, and are using an earlier version of CryoSPARC, then we suggest that you update to the latest version.
