Masks

A real-space mask is important to boost SNR by focusing the analysis on the region of interest.

Using the GUI: the Mask Wizard

To make a solvent or focus mask in the GUI, use the built-in Mask Wizard. Open any job's Volumes tab and click the green wand icon on a volume (usually the mean reconstruction) to launch it.

The wizard builds a mask from the volume and previews it live as you adjust:

• Threshold -- automatic (Otsu) or a manual density cutoff
• Dilation -- expand the mask outward by a number of voxels
• Soft edge -- width of the cosine falloff at the mask boundary
• Lowpass smoothing -- automatic or manual, to smooth the surface
• Cleanup -- fill interior holes and keep only the largest connected component
• Segments -- run connected-component analysis and keep the N largest blobs

Switch between the Slice view (scroll through X / Y / Z planes with the green mask overlaid) and the 3D isosurface view:

Click Save Mask to write the result to <project>/Masks/. It is then available as a solvent or focus mask in any new Pipeline job, and in the Masks library (sidebar), where you can combine masks with union / intersect / subtract.

The rest of this page covers the equivalent command-line options.

Solvent mask

Most consensus reconstruction software outputs a solvent mask. Use it directly:

recovar pipeline particles.star -o output --mask mask.mrc

Warning

Make sure the mask is not too tight. Use --mask-dilate-iter to expand it if needed.

Auto-generated masks

If you don't have a mask:

Option	Description
--mask=from_halfmaps	Estimate mask from the mean reconstruction (averages half-maps, low-pass filters, Otsu thresholds, cleans up, and softens)
--mask=sphere	Use a loose spherical mask
--mask=none	No mask (not recommended)

A good approach is to first run with --mask=sphere, inspect the variance map to see which regions have heterogeneity, then create a focused mask around those regions.

Focus mask

A focus mask restricts the heterogeneity analysis to a specific region of the molecule, such as a single domain or binding site.

recovar pipeline particles.star -o output \
    --mask mask.mrc --focus-mask focus_mask.mrc

If you only have a focus mask:

recovar pipeline particles.star -o output \
    --mask=sphere --focus-mask focus_mask.mrc

Creating a focus mask

Use the Mask Wizard above: open the mean reconstruction, set a threshold, and use the Segments control to keep only the connected region of interest.

You can also build one in UCSF Chimera or ChimeraX:

1. Open your consensus map
2. Select the region of interest
3. Create a mask around that region
4. Save as .mrc

See cryoSPARC's guide on mask generation for step-by-step instructions.

Mask dilation

To expand a mask by a few pixels:

recovar pipeline particles.star -o output \
    --mask mask.mrc --mask-dilate-iter 5

The --mask-dilate-iter flag applies to both the solvent mask and the focus mask.