How to do focus classification - phonchi/ASCEP GitHub Wiki

Using Chimera

CisTEM

A focus mask is defined as a sphere specified by radius and x,y,z coordinates of the sphere center, all given in Å. In order to find x,y,z, in CisTEM

  1. Open the 3D map in Chimera. The map should be assigned model #0.
  2. Display the command line by selecting Tools > General Controls > Command Line.
  3. Execute vop threshold #0 maximum -1000 setMaximum 1.0 (this assumes that the minimum voxel value in the 3D map is larger than 1000). This will create a new model #1.
  4. Execute shape sphere radius 30 color red mesh true on the Command Line. This will generate a sphere with a 30 Å radius, numbered model #2 (change the radius as needed).
  5. Open Tools > General Controls > Model Panel and deactivate models #0 and #1. Then move the sphere (model #2) into model #1 until it is fully contained in that volume.
  6. Execute mask #1 #2 on the Command Line. This will generate a new model #3 of a solid sphere with a 30 Å radius.
  7. Close models #1 and #2 in Model Panel and make sure model #0 is visible but deactive.
  8. Select model #3 (the solid sphere) in Volume Viewer and change its color to red (or something easily distiguishable from the original 3D map) and make it transparent.
  9. Move model #3 to the desired location of the focus mask in the 3D map.
  10. Execute vop resample #3 onGrid #0 on the Command Line. This will generate a copy of the solid sphere as a new model #1, now resampled in the same coordinate system as the original 3D map.
  11. Execute measure center #1 on the Command Line. This will display the x,y,z coordinates of the mask in pixel coordinates below the command line. These coordinates have to be converted to Å by multiplying them with the pixel size of the 3D map before they can be used in cisTEM's Manual Refine panel.

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