How to Obtain Clear Views of Objects in Tomograms
January 23, 2009
- Interactive visualization program Chimera shows EM maps, single particle
reconstructions and tomography.
- Much of Chimera is dedicated to looking at atomic models and sequences.
Atomic models and sequences
Single particle reconstructions
- When is subtomogram averaging feasible?
- Demonstration of some new and old Chimera capabilities for
looking at tomogram objects.
- Is the 8-fold symmetry of nuclear pores visible?
- Can 8-fold averaging be done to see more detail?
- Are nuclear pore shapes uniform enough to be averaged?
SIV Virus Spikes
- Can 3-fold symmetry of individual spikes be seen?
- Can spikes be averaged to improve signal to noise?
Clathrin Coated Vesicles
- Do some observed clathrin cages exhibit identical topology?
- Are some cages symmetric?
- Can cages or parts of cages be averaged?
- Where are vesicles positioned within cage?
- Improve signal to noise by averaging multiple objects to so that
individual molecules can be discerned.
- Objects do not have uniform shape.
- Objects are uniform but too noisy to align.
- Objects are often symmetric, more copies to average and helps alignment.
- Missing wedge produces anisotropic data quality and complicates averaging.
- How to assess if averaging is feasible using interactive visualization
- Chimera is for interactive analysis -- data triaging. It does not
automate the computational steps: classifying, aligning and averaging.
- Probably will only have time to look at nuclear pores, not clathrin cages
or SIV spikes.
- Point out human T-cell outline.
- Point out nuclear envelope.
- Flip through planes and mark 3 pores. 250 A diameter, color blue.
- Use volume dialog atom box to show box around center marker. 500 A pad.
- Surface display style.
- Color orange to see white specular highlights better.
- Hide dust. Move slider to show effect.
- For unstained samples that have more dust this is especially useful.
- Lower threshold obscures view.
- Adjust box tighter (and wider) so less density blocks view.
- Rotate box to align with pore, allows tighter cropping.
- Rotating makes a new map interpolated from original map on rotated grid.
- Extract circular region of pore to examine 8-fold symmetry.
- Center marker, color zone, split map. 450 A radius.
- Gaussian filter pore.
- Use interactive update and adjust Gaussian width with slider. 45 A good.
- Find symmetry axis of pore.
- Close unneeded maps, duplicate gaussian map, rotate by hand ~180, fit map in map, "measure rotation #2 #3".
To extract density new many membrane embedded objects like 100 virus spikes
rotating a box around each is time consuming. Better to extract density
near membrane surface for the entire membrane.
- Plot correlation of map with rotated copy of itself.
- "measure corr #3 #2 rot #4 plot t ang 0,360,2"
- Trace nuclear envelope surface.
- Set mouse mode, new marker set, trace top/bottom plane curves, surface.
- "mask #0 sel slab 700", show as surface
To show pores in their environment make a fly-through animation.
- Save 4 camera views: high above, one of each pore.
- "fly 100 p1 p2 p3 p4"
Chimera Setup and Support
- Chimera is free for academic use. Funded by NIH through 2012.
- Runs on Windows, Mac, Linux.
- Nightly builds have the latest features, production releases every
6 months for stability.
- 5 developers enable fast user support by email, and active development of
- How-to style documentation for map display:
Guide to Volume Data Display,
see link on
Chimera home page.