Fitting Molecular Models in Single-Particle EM Maps
July 12, 2012
- 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.
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.