vop thresholdcommand by setting all values below the level to 0. This can be done using the following command, with a level example of 0.18:
vop threshold #<volume-spec-0> minimum 0.18 set 0
<volume-spec-1>. Next, threshold the new volume by setting all values above the level to 1. Typically it's best to use a level number slightly less than what was used in the previous command, to ensure that every voxel is set to 0 or 1 and there are no voxels left sitting exactly at the level. So here, we will use a level of 0.17:
vop threshold #<volume-spec-1> maximum 0.17 setMaximum 1
Tools > Volume Eraser, this will open a transparent sphere, which can be moved using the indicated mouse control in the Volume Eraser panel. By moving the sphere and clicking the
Erasebutton on the Volume Eraser panel, you can progressively erase the sub-volume from the overall binarized mask. Doing so will generate a mask covering the volume, excluding the region to be locally refined, which will be useful in particle subtraction. Below shows an image of the Volume Eraser tool in use.
vop subtractcommand. Note that in some cases, Local Refinement may also be successful with using non-subtracted particle images, in which case only a mask covering the sub-volume will be needed. In either case, the mask covering only the sub-volume to be refined can be generated by subtracting the above mask from the whole binarized structure. For example, if volume
#1holds the thresholded mask, and volume
#2holds the mask covering everything except for the sub-volume, we can create a volume covering just the region of interest by using the command
vop subtract #1 #2.
vop resamplecommand. Then, you can save it as a
.mrcfile, and import the mask into cryoSPARC for subsequent processing.
.mrcfiles, we can run an Import 3D Volumes job to import the masks. This job requires the path to the volume, and the "Type of volume being imported" must be set to "mask".
Thresholdparameter should be set below the level value of the voxels within the imported mask. As well, the
Dilation Radiusshould be set to a value (in voxels) that results in an expanded mask that extends just beyond the original volume, and cuts through as minimal density as possible (although, cutting through density at the joint between the sub-volume and the rest of the volume cannot be avoided).
Soft padding widthparameter. We typically recommend setting the
Soft padding widthparameter to be a voxel value that ensures the resulting padding is several factors larger than the expected resolution of the complex. A good rule of thumb gives the minimum mask softness as
5 * res_angstroms / pixel_size_angstroms.For example, a complex reaching 3.0 Å in resolution with a pixel size of 1.0 Å should be padded to at least 5 * 3.0 / 1.0 = 15 voxels.