.starfile included has a few corrupted micrographs, and the following file can be used to get the valid subset of particles. The original investigation of this compound was carried out by Nguyen et al., and the resulting reconstructions from their process can be found on EMDB. In this case study, we will show how the new Local Refinement toolkit in cryoSPARC will produce results that improve upon the published results.
map_locresvolume, as well as the final map (
map_sharp) from the Homogenous Refinement job. Open both volumes in Chimera. Using the Surface Color tool (
Tools > Volume Tools > Surface Color), you can color the refined map by the values of the local resolution and manually set the color thresholds.
Favorites > Command Line), type the following two commands:
vop threshold #0 minimum 0.18 set 0and
vop threshold #1 maximum 0.17 set 1. Change #0 for the model ID in your session, and 0.18 with your optimal viewing level. These two commands will map every voxel with a density below 0.18 to 0, and every voxel above 0.17 to 1. Make sure you run the second command on the output of the first one. (Note: we've occasinally encountered a bug where the maximum gets set at some value lower than 1. This is OK, you should make sure you use the correct threshold in later steps when using Volume Tools in cryoSPARC. You can also use Chimera's
vop multiplycommand to get the values to 1.)
Tools > Volume Tools > Volume Eraser. You will now see a transparent sphere floating next to your structure. You can use the middle mouse button to move the sphere, and press
Eraseto delete chunks of the binarized volume until you are left with the desired region. Note that Chimera will subtract from the currently active volume only, and it will generate a new subtracted volume instead of overwriting the original.
vop subtract #2 #3, where #2 is the binarized overall structure, and #3 is the mask you've created. The result of the subtraction will be the negative of the mask you created. At this point, you should have two contiguous volumes generated from the original map. The masks we generated for the head look like this:
Import 3D Volumestool to import the newly-made masks into cryoSPARC - make sure you set the map type to
maskin the parameters before you run the job.
maskinput slot of each. You can use the following parameters. This job will also fill the holes inside your structure, and give you a solid map. Make sure to specify the output type as
Volume Tracertool in Chimera. We will place our fulcrum at the bottom of the head region, around the marker you see pictured in the image below. Go to
Tools > Volume Data > Volume Tracerto open up the Volume Tracer dialog. From mouse, select
Place markers on high density. Navigate to the part of the structure you want to place the fulcrum on, and use the middle mouse to drop a spherical marker.
getcrd selinto the command line (assuming you still have the marker selected). Note that these coordinates are in Angstroms, while the local refinement job expects a value in voxels. Simply divide the values by the pixel size, which is 1.4 for this dataset, to get the correct fulcrum. The fulcrum we found was (196, 197, 211).
alignment3Dfields are required for Particle Subtraction.
-150, we can obtain a map that improves the visibility of the high-resolution features.