Chimera Hands-On Tutorial: Fitting a ParM Filament Map
|Presenter:||Tom Goddard, Chimera developer
|Date:||Tuesday, November 30, 2011
|Time:||10:15 - 11:45 AM
|Room:||MRGN 129, Purdue University
Bring your laptop with the following Chimera version and data files.
Older Chimera versions won't work. They do not have many new features
we will use.
- Fitting molecules in maps with global search.
- Calculating map symmetry and creating symmetric molecule copies.
- Symmetric fitting to avoid molecular clashes.
- Calculating and displaying difference maps.
Open Question and Answer Session
30 minutes will be reserved for answering any Chimera questions with live
demonstration. Questions need not be related to the tutorial.
What does ParM do?
- The ParM protein forms filaments that segregates DNA plasmids
prior to cell division.
- To partition low copy number DNA plasmids in E coli evenly during
cell division between the two daughter cells, a plasmid is attached to each
end of a growing ParM filament that pushes them to opposite sides of the
- ParM filaments look similar to actin filaments.
- Filament growth is driven by ATP and filaments have dynamic instability
Modeling a ParM Filament
- ParM monomers bind ATP and can have the binding cleft open or closed.
- We'll build a model of the open state filament with the ATP binding site
empty using x-ray structure
and cryoEM map EMDB
5129 (19 Angstroms).
- Closed state data is also available: x-ray model
and map EMDB
(17 Angstroms). Won't have time to look at those.
|Show Molecule and Map
||Fit Molecule in Map
- Open PDB 1mwk, EMDB 5129.
- Show Command Line (Favorites menu).
- Deactivate map (model 0) below command-line to move 1mwk away from map.
- Command "rainbow chain" to color two ParM monomers.
- Delete chain B. Use menu Select / Chains / B, then menu Actions / Atoms / Delete.
- Fit 1mwk in map.
- Move 1mwk into map.
- Press Fit button in Fit in Map dialog (volume dialog Tools menu).
- Fit using correlation: Fit dialog Options button, enable "Use map
simulated from atoms..." resolution 19. Press Fit.
- Show simulated map (Volume dialog eye icon) as mesh.
- Correlation depends on domain of calculation. Change simulated map threshold and press Update in Fit dialog.
- Spend a few minutes trying alternative fit positions.
|Global Fit Search
||Calculate Map Symmetry
||Fit Asymmetric Unit
- Search for best fit using 30 tries with command "fit #1 #0 search 30"
- Fits appear all along filament, many are equivalent due to symmetry
of the filament.
- Determine map symmetry to eliminate equivalent fits.
- Command "measure symmetry #0 helix 20,180,opt minimumCorrelation 0.95".
- The "helix" option gives Chimera a hint about helical parameters.
- The "minimumCorrelation" option accounts for this unusual map where
the helix does not extend to the edges of the volume box.
- View symmetry copies of molecule with command "sym #1 group #0 surf true".
- Remove symmetry copies with "~sym #1" (note leading tilde character
which means "undo" in Chimera commands).
- Clear fit list.
- Rerun previous fitting command "fit #1 #0 search 50".
- Clear fit list.
- Use correlation optimization "fit #1 #0 search 50 res 19".
|Make Filament Model
||Reduce Molecular Clashes
||Calculate Predicted Map
- Show symmetric molecule copies for best fit.
Command "sym #1 group #0 update true".
- Color molecules distinctly. Command "rainbow model".
- Inspect clashes between adjacent ParM molecules.
- Fit asymmetric unit including all overlapped symmetric molecules.
Command "fit #1 #0 sym true res 19"
- Note increased space between molecules.
- Compute difference map between experimental map and predicted map
for molecular model.
- First delete extra ParM molecules outside experimental map.
Ctrl-drag mouse to select outside molecules. Press up-arrow key to extend
selection to full molecules. Menu Actions / Atoms / Delete to delete.
- Calculate predicted map. Command "molmap #1,2, 19"
||Compare "Open" and "Closed" Filaments.