Notes for tomography demo

How long does this take?

Five basic parts: IMOD, planes, resample, mask, tomo plane.

Open ctl map.
Explain: parts of two cells, show boundary, human cytotoxic t-cell above
  killing target cell below.  Lytic granules (bright white) containing serine
  proteases being delivered to interface induce apoptosis in the target cell.
Analysis often starts by drawing surfaces around interesting objects.
  Chimera doesn't do that, but can import surfaces from IMOD.
  Open imod surfaces.
  Orient looking from 45 degrees above from t-cell side.
IMOD is from another NCRR center, Boulder Laboratory for 3-D Electron
   Microscopy of Cells.
Hover mouse over surfaces to see names.
Basic manipulations of surfaces.
Color cell membrane orange to distinguish from white map color (shortcut co).
Hide and reshow lytic granules (hs, sf).
Note all granules selected as group, exported from IMOD as one object.
  Split granules into disconnected pieces (Sc), note 18 granules, status line
Note different numbering in balloons, select far right.
Color that one light blue to distinguish it.
Measure its volume (mv), note 2 holes.  Hole handling added -- maybe not worth
  mentioning.

Now look at some map visualization capabilities.
Hide imod surfaces, zoom out to see full square, press Orient button.
Looking at single plane, show edge on, face on, move plane slider, slow
  because planes are large 2048^2.
Turn off planes and show thickness.  Note that step size jumps to 8, and
  fast read from disk is from precomputed subsamples.
Look at smaller region.  While displaying all planes box mitochondrion above
  lowest bright vesicle.
Lower middle histogram node and move left some to remove saturated bright
  parts.
Rock about x-axis with mouse.  Keep green outline box for 3-d effect.
Hard to see fine details.  Turn on plane display, press Play, then Stop after
  one trip center plane to center, and move slider by hand.
Single particle reconstructions typically viewed with contour surface.  Turn
  off planes, switch to surface, lower threshold, too much dust.
Easy plane by plane display added for tomography because high noise levels
  are the norm for tomograms.

Another aspect of tomograms requiring new capabilities is that objects are
  not aligned with the data axes as they are in single particle reconstructions.
For analysis it is useful if they are approximately aligned with data axes.
Example: Suppose we are interested in structures between the two cell
  membranes.
Just saw how we can box a subregion around mitochondrion aligned with data axes.
Press subregion panel Full button to get full map, Orient.
Drag horz box for 1/4 width piece of cell-cell interface toward left side.
Click on rotate, and rotate box parallel, then translate to enclose
  inter-cellular zone.
Turn off rotate and rotate everything about x-axis to show cell membrane is
  not perpendicular to z.  Rotate to see dark band between cells.
Turn on rotate and tilt box to face on.
Could check that box has not been rotated out of plane -- better to skip if
  possible to keep demo moving.
Because box is rotated, its grid is not aligned with original grid and data
  must be interpolated on new grid if we wish to save the boxed submap.
Can change grid spacing.  Leave the same.  Press Resample.
Note new map histogram.  Hide original map. View all (va).  Show original map.
Remove green box by turning off subregion selection.
Note new smaller map is shown at full resolution.
Change color of middle node to yellow, zoom out.
When we save this to a new map file want to preserve orientation so it realigns
  with original map in future sessions.  None of the standard map files
  support this.  For MRC format we added a Chimera specific comment in the map
  header to record orientation.

Besides extracting rotated boxes it is useful to extract density just for
  one object.
Close resampled map.
Show imod surfaces with model panel.  Select light blue vesicle.  Zoom on it.
Command "mask #1 sel axis 1,0,0".
Because the surface has holes I need to specify a projection axis for masking.
Note new map histogram appeared.
Switch to plane display and move middle histogram node too top to brighten
  and move plane slider to show range of planes.
You might do this if you wanted to analyze packaging of proteins within the
  vesicle.
Reshow full map, switch on planes, zoom out.  Another use of masking would be
  to separate the two cells using a surface dividing the cell membranes into
  two separate maps.  This would improve visibility of the cell membranes if
  structures embedded in the membranes were of interest.

I'll show you another technique to look at an interesting membrane feature.
Hide imod surfaces.  Close masked vesicle.
Point out nuclear envelope of t-cell.
Look at a small stretch close up, subregion selection on finger on right side
  pointing up and left, crop, view all, turn off subregion selection.
Double membrane.
Scan planes and point out break.  Scan further and point out another break.
Mark a few with path tracer.  Turn on drop on empty space, turn off link
  new marker.  Place marker at widest point inside nucleus near pore.  Set
  radius to 250 (pore diameter is ~50nm), color red.  Mark 3 nearby pores.
  Turn off placement.  Set plane to show middle pore at mid-point
What are these breaks?  This is a quiz!  Nuclear pore.
Say that sphere diameter is 50 nm, the typical pore hole size.
Will use a tool called Tomo Plane to show a face on pore view.
Tomo Plane is a chimera extension developed by Karin Gross and Christoph Best
  in Wolfgang Baumeister's lab at Max Planck Institute for Biochemistry.  Shows
  volume slices at arbitray angles.
Show Tomo Plane, set size to 256 by 256, press Create, view all.
Move tomo plane over to pore, model panel deactivate tomo plane,
  active reverse (ar), view all, raise middle histogram node, color light blue,
  move plane to show that it shows density.
Resize plane to step 10A -- gives higher quality.
Center square on pore, rotate about x about 90 degrees, active toggle (at),
  rotate scene 90 degrees about x.
Adjust tilt and depth (ctrl button 2), and thresholds to get nice donut.
  This is tricky.
Hide red balls, select one, up arrow, hide atoms (ha).
Orient for 45 degree from above view to give context of cross-section
  slice and xy plane boundary.
(With improved masking command could have a central imod nuclear envelope
 surface, pad a specified amount in and out and extract just the membrane.
 That would be cool.)

Those are some of the tools added specifically for tomography.