ChimeraX cryoEM Visualization Tutorial: Bacterial ATP Synthase

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 the February 2019 publication.

      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.

Topics

• Basics: open, movement, styles, chains...
• Morphing between 3 conformations.
• Viewing bound ADP, phosphate, ATP.
• Inspecting side-chain density, zones.
• Fitting atomic model into map.
• Look at proton channels.
• Look at unidirectional rotation.

Basics: open, movement, styles, chains...

• Command "open 6n2y", or toolbar Open button, or File/Open menu to load file from local drive.
• Log chain and ligand tables: click letter to select atoms, click name to show sequence.
• 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). 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.

Miscellaneous - not sure how much of this to show at the start versus later on.

• Toolbar icons: Save image. hide/show atoms/cartoon, styles, bg color, lighting modes, mol display, coloring, hbonds.
• Selection with mouse, or Select menu, up arrow promotion, toolbar actions on selection.
• Presets. Cylinder display is cool, plus show ligands and save image. Let them try different styles.
• Help menu, user's guide.

Morphing between 3 conformations

• open 6n2z ; open 6n30
• Model Panel show/hide buttons to show or hide each model.
• morph #1,2 to morph between first two structures. Try play button on slider. May need to use simple lighting for speed.
• Close morph, click on model 4 in model panel and press close button.
• 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.
• morph made 20 steps between each structure. To increase this see documentation using command "help morph".
• Press red circle to right of slider to record movie.
• Close morph.

Viewing bound ADP, phosphate, ATP

• Open cryoEM map, "open 9333 from emdb", or Toolbar Open button or File / Open for file on local drive.
• Toolbar, molecule display, stick style.
• Volume step 2 to step 1.
• Hand adjust threshold on histogram. May want simple lighting for faster updates.
• Make surface transparent with color button above histogram.
• Center on ADP and use clip planes command: "view :ADP"
• Mesh style using menu above histogram.
• Show everything: view all
• Where is phosphate? Command "sel :ADP:PO4" shows they are in different binding sites. Not sure why.
• Look at density around "tentatively assigned" phosphate: "view :PO4" Only seen at lower contour level.
• view all

Inspecting side-chain density, 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.
• Command "surface zone #4 near #1/A:404-441"
• Hide all atoms with toolbar icon or command "hide #1"
• Show atoms: "show #1/A:404-441"
• Molecule toolbar, color "heteroatom".
• To make finer mesh: "volume #4 subdivide true", finer mesh "volume #4 subdivide true subdivisionlevels 2"
• volume #4 level 1.5
• Graphics toolbar white background.
• "vol #4 subdivide false" Avoid future slow surface calculations

Fitting atomic model into map

• 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.
• Command: "open 4xd7"
• Select an atom of 4xd7 by ctrl-click on it.
• Use translate and rotate selection mouse modes from toolbar to place model in map.
• Use menu Tools / Volume Data / Fit in Map to optimize fit of 4xd7 into 9333.
• Fine detail in map causes it not to find good global fit. So fit into smoothed map.
• Command "volume gaussian #4 sdev 3"
• 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: "color zone #4 near #5 dist 8"

Proton intake channel.	Proton outlet channel.

Look at proton channels

• Look at proton path that drives motor, figure 4 in eLife paper.
• Transmembrane drum carries protons around on glutamic acid E56: "select /c*:56". Show as 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.
• Molecule toolbar, show surface.
• Orient view with long helical rods on left.
• Enable clip mouse mode in toolbar. Use simple lighting for faster clip plane motion.
• Clip to see channel from below to E56.
• Hold alt-key to clip far side to show channel releasing proton above.
• After positioning clip plane turn on full lighting for darker channel cavity.

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.
• Turn off clipping, command: "clip off"
• Orient structure axis perpendicular to screen with F1 hexamer at front.
• Make motor shaft epsilon subunit (chain H) more visible: "color /H red surface"
• Clip with clip mouse mode.
• Red epsilon subunit blocked from clockwise rotation by collision with beta subunit (gray color) of hexamer.