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Last HMEDit: 20210412.


The following are simply my and other's shared experiences and may well not work for you and should not displace discussing face-to-face with other members of the community. 

Small particles

If you are having issues with 2D classification alignment, I would first try setting the batch size to 200 or even 400...this gives cryosparc more particles to play with initially. If you are still struggling, try setting the force/max over poses/shifts to off and up the number of iterations say to 40.

Check that you have a sensible box size too (see the relevant pages about this) and play around with the number of classes. 

mask creation for local refinements

1. Load map to be masked in chimera. Fit in model initially by inactivating the map (via command line or model panel) and then Tools->Volume Data->Fit in map.
2. Select the part of the model that you are interested in via command line (to display the command line Tools->General Controls-> command line, bar at the bottom should appear ready to be commanded). Note that the syntax for selection is different to chimeraX and best checked online. e.g. select #4:1-541.A , where # is the model/map number, other nums are the atom nums and .A is the chain.

Your selection should be surrounded by a beautiful luminous green border.

3. On the command line, type:
color <insert colour name here> selection

4. Volume Viewer->Tools->Color Zone. Check that the map that you want to crop is selected. Fiddle with the colouring radius and color/uncolor until your region of interest is adequately covered. When you are happy, split map.

###Split map will create two maps, one half with your region of interest, the other might be worth saving if you are considering doing density subtraction-sometimes essential for decent local refinements. Note that sometimes this causes my laptop to have a mini panic attack [probs all of those browser windows etc]...just leave it to do its thing and it will be fine. This new map should have the same box size as the parent map (just check on the volume viewer...if it does not, can rectify with vop onGrid later).####

5. Apply a gaussian filter to your new volume with region of interest.
Tools->VolumeData->Volume filter . This will result in yet another new map. Use the volume viewer window to toggle this map to figure out a sensible contour level.

6. Select this final map in volume viewer and in the volume viwere hit file->save map as... . I tend to include the contour level in the name.

7. [yet more faff...nearly there, promise] Use relion_mask_create to make a mask from this volume-remember to insert the correct contour. Since it already has a gaussian filter applied, you do not need to really extend the map much, but I would recommend a lill bit of a soft edge of say 3 pix. Should run pretty quick even if you have a monster map.
Repeat steps 5-7 for the unwanted bit of density too if you are planning on trying out a subtraction job.
8.[nearly there...] Import your newly created masks using the Volume import module in cryosparc, ensuring that you tell it that it is a mask. Look at the output from this job to check that it kinda looks sensible.