ChimeraX cryoEM Visualization Tutorial: Bacterial ATP Synthase

Tom Goddard
January 15, 2020

This ChimeraX tutorial will look at how to visualize atomic models and maps of three conformations of a bacterial ATP synthase determined by cryoEM at 3.0 and 3.2 Anstrom resolution. We will create views of the data similar to those found in

      Structure of a bacterial ATP synthase.
      Guo H, Suzuki T, Rubinstein JL.
      Elife. 2019 Feb 6;8. pii: e43128. doi: 10.7554/eLife.43128.

Atomic models PDB 6n2y, 6n2z, 6n30. EMDB maps 9333, 9334, 9335. ChimeraX can fetch these data files directly from the PDB and EMDB or you can download the files for offline use.

Command links in this page will execute in ChimeraX if this web page is displayed within ChimeraX. To show this tutorial in ChimeraX use menu entry Help / Documentation Index, choose Tutorials, and select the January 2020 cryoEM tutorial.

Topics

Opening PDB models
Display styles
Morphing between 3 conformations
Viewing cryoEM map near ADP
Inspecting side-chain density using zones
Fitting atomic model into map
Look at proton channels
Look at unidirectional rotation

Opening PDB models

Open atomic model 6n2y from PDB
```
	  open 6n2y
	
```
Log chain and ligand tables: click letter to select atoms, click name to show sequence.
```
	  sequence chain #1/A
	
```
Hover mouse to show chain, residue name, number and atom name.
Movement: Mac trackpad, 2-finger rotate, 3-finger translate, 4-finger zoom (or pinch).
Turn off 3-finger mission control on Mac in system preferences Trackpad / More Gestures.
Mouse, left rotate, right translate, scroll to zoom.
Center of rotation: on surface close up, or at center of model when zoomed out.
More info... log link, shows publication, species, experimental method and resolution.
```
	  log metadata #1
	
```

Display styles

Show cartoon style. Use Toolbar icons or commands.
```
	  hide atoms
	  show cartoons
	
```
Show ADP. Click ADP in Log ligand table to select, then show selected atoms and change to sphere style with Toolbar.
```
	  select :ADP
	  show sel atoms
	  style sel sphere
	
```
Color a protein red. Select gray shaft protein with ctrl-click on ribbon, then up arrow key. Then color red with command.
```
	  select /H
	  color sel red
	
```
Show helices as cylinders with menu Presets / Cylinders.
Save image with Toolbar Snapshot icon.
Restore normal ribbons with Presets / Ribbons.
```
	  preset cylinders
	  save ~/Desktop/image1.png
	  preset ribbons
	
```

Morphing between 3 conformations

Open second and third ATP synthase conformations.
```
	  open 6n2z
	  open 6n30
	
```
Model Panel show/hide buttons to show or hide each model.
Morph between first two structures.
```
	  morph #1,2
	
```

Try play button on slider. Use atom style and simple lighting for faster playback.

	  hide cartoon
	  show atoms
	  light simple
	  coordset #4 1,21

Close morph, click on model 4 in Model Panel and press close button.
```
	  close #4
	
```
Now morph between all 3 conformations and end at the starting one.
```
	  morph #1,2,3 wrap true same true
	
```
The "wrap true" option returns to starting model.
The "same true" option means don't use sequence alignment algorithm, instead assume same sequences for faster calculation.

Press red circle to right of slider to record movie.

	  movie record ; coordset #4 1,61 ; wait 61 ; movie encode

Close morph and all but first conformation.
```
	  close #2-4
	  show #1 model
	
```

Viewing cryoEM map near ADP

Open cryoEM map from EM Databank
```
	  open 9333 from emdb
	
```
Show atoms in stick style. Toolbar, Molecule Display tab, Stick.
```
	  show atoms
	  style stick
	
```
Change volume display to show full resolution, step 1.
```
	  volume #2 step 1
	
```
Make surface transparent with color button above histogram.
```
	  transparency 50
	
```
Center on ADP and use clip planes
```
	  view :ADP
	
```
Hand adjust threshold on Volume Viewer panel histogram.
```
	  volume #2 level 3
	
```
Mesh style using menu above histogram.
```
	  volume #2 style mesh
	
```
Look at density around "tentatively assigned" phosphate
```
	  view :PO4
	
```
Density only seen at lower contour level.
```
	  volume #2 level 1
	
```
Show everything.
```
	  view
	
```

Inspecting side-chain density using zones

Figure 1 supplement 4 image of eLife publications shows example cryoEM density of side chains.
Show density near chain A residues 404-441 like in figure.
```
	  surface zone #2 near #1/A:404-441
	
```
Show only desired atoms. Hide all atoms with toolbar icon, then use show command
```
	  hide #1
	  show #1/A:404-441
	  view
	
```
Color oxygens and nitrogens using Molecule toolbar, color "heteroatom".
```
	  color byhetero
	
```
Show mesh with Volume Viewer menu above histogram.
```
	  volume #2 style mesh
	
```

To make finer mesh, subdivide triangles with command

	  volume #2 subdivide true
	  volume #2 subdivide true subdivisionlevels 2

Adjust contour level
```
	  volume #2 level 1.5
	
```
Make background white using Toolbar icon
```
	  set bg white
	
```

Turn off finer mesh to avoid slow surface calculations

	  vol #2 subdivide false
	  surface unzone #2

Fitting atomic model into map

The starting point for model building is often fitting existing models into map.
Fit 3.9A x-ray structure 4xd7 (F1 half of complex) into map.
```
	  open 4xd7
	
```
Select any atom of 4xd7 by ctrl-click on it.
```
	  select #3
	
```

Use translate and rotate selection mouse modes from toolbar Right Mouse tab to place model in map.

	  mousemode right "translate selected models"
	  mousemode right "rotate selected models"
	  view matrix models #3,0.15538,-0.72458,0.67144,118.99,0.67504,-0.41837,-0.60769,155.34,0.72124,0.54767,0.42412,175.3

Use menu Tools / Volume Data / Fit in Map to optimize fit of 4xd7 into 9333.
```
	  fit #3 in #2
	
```
Fine detail in map causes it not to find good global fit. So fit into smoothed map.
```
	  volume gaussian #2 sdev 3
	  fit #3 in #4
	
```
Fitting into this map has much larger radius of convergence.
Still need to get correct 6-fold symmetry alignment to match shaft subunits.

Sometimes useful to color map to match nearby atoms.

	  close #4
	  show #2 model
	  transparency 0
	  color zone #2 near #3 dist 8
	  hide #3 model

Look at proton channels

Look at proton path that drives motor, figure 4 in eLife paper.

Proton intake channel. Proton outlet channel.
Close X-ray model and cryoEM map.
```
	  close #2,3
	
```
Transmembrane drum carries protons around on glutamic acid E56 (chains c0,c1,...,c9). Show as sphere.
```
	  show #1 atoms,model
	  select /c*:56
	  style sel sphere
	
```
Drum is in membrane and proton from lower side goes up channel between side "a" subunit, onto drum, carried around one rotation, and released into another channel to exit on other side of membrane.
Show molecular surface. Molecule toolbar.
```
	  surface #1
	
```
Color E56 red.
```
	  color sel red
	
```
Orient view with connector rods on left.
Enable clip mouse mode in Right Mouse toolbar tab.
```
	  mousemode right clip
	
```
Use simple lighting for faster clip plane motion.
```
	  light simple
	
```

Clip with mouse to see channel from below to E56.

	  clip position 163,163,194 axis -.99,-.08,-.13 coord #1

Hold alt-key to clip far side to show channel releasing proton above.
```
	  clip back 0 position 147,162,192 axis .99,.08,.13 coord #1
	
```
After positioning clip plane turn on full lighting for darker channel cavity.
```
	  light soft
	
```

Look at unidirectional rotation

Figure 3 of eLife paper suggests mechanism why motor turns in one direction that produces ATP and is blocked in reverse direction that hydrolizes ATP and pumps protons.
Turn off clipping by click without drag in clip mouse mode, or by command
```
	  clip off
	  light simple
	
```
Orient structure axis perpendicular to screen with F1 extra-cellular hexamer at front. Color epsilon shaft subunit red.
```
	  color /H red
	
```

Clip with clip mouse mode.

	  clip axis 0.03,0.13,-0.99 position 164,161,206 coord #1

Red epsilon subunit is blocked from clockwise rotation by collision with beta subunit (gray color) of hexamer.


Proton intake channel.	Proton outlet channel.