Nanobody ChimeraX Tutorial

Tom Goddard
February 17, 2021, updated March 23, 2021
For UCSF Methods in Macromolecular Structure course.

This tutorial is to get to know some basics of using ChimeraX software to look at atomic models of proteins and X-ray density maps and electron microscopy density maps. There is some material on other pages showing molecular dynamics trajectories and NMR chemical shift perturbations. The steps below use a March 2021 ChimeraX daily build. A few of the toolbar buttons are not available in the older ChimeraX 1.1 release but those steps can be done with typed commands.

Nanobody Atomic Model

We will look at nanobodies that bind to SARS-CoV-2 spike proteins and neutralize the virus. These nanobodies were used to make a therapeutic aerosol that when sprayed in the nose can block SARS-CoV-2 infection. Aashish Manglik and many others at UCSF created this therapeutic.

    An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike.
    Schoof M, Faust B, Saunders RA, Sangwan S, Rezelj V, Hoppe N, Boone M, Billesbolle CB,
      Puchades C, Azumaya CM, Kratochvil HT, Zimanyi M, Deshpande I, Liang J, Dickinson S,
      Nguyen HC, Chio CM, Merz GE, Thompson MC, Diwanji D, Schaefer K, Anand AA, Dobzinski N,
      Zha BS, Simoneau CR, Leon K, White KM, Chio US, Gupta M, Jin M, Li F, Liu Y, Zhang K,
      Bulkley D, Sun M, Smith AM, Rizo AN, Moss F, Brilot AF, Pourmal S, Trenker R, Pospiech T,
      Gupta S, Barsi-Rhyne B, Belyy V, Barile-Hill AW, Nock S, Liu Y, Krogan NJ, Ralston CY,
      Swaney DL, GarcĂ­a-Sastre A, Ott M, Vignuzzi M; QCRG Structural Biology Consortium, Walter P, Manglik A.
    Science. 2020 Dec 18;370(6523):1473-1479. doi: 10.1126/science.abe3255. Epub 2020 Nov 5. PMID: 33154106.
    

Nanobodies are much smaller type of antibody, a single protein domain, found in camels, llamas, alpaca ("camelids"), and sharks. They are much easier to engineer than full-size antibodies.

Open PDB structure, move, rotate and zoom

ActionNotes
Type command "open 7kkj" Fetches Protein DataBank nanobody structure with PDB id 7kkj.
2-finger trackpad drag
to rotate
Works on Mac laptop. Or use left mouse button, or trackpad click and drag.
3-finger trackpad drag
to translate
Works on Mac laptop. Or use right mouse button, or Alt or Cmd modifier key plus mouse drag.
4-finger trackpad drag or pinch to zoom Works on Mac laptop. Or use mouse scroll wheel, or two-finger trackpad scroll on Windows.

Chains, ligands and assembly tables in Log panel

ActionNotes
Click chain A or C links in Log panel This selects each copy of the nanobody, outlining it in green.
Click CL chloride ion link Selects ion. To show it click Show Atoms in Toolbar.
Toolbar buttons operate on selected atoms.
Click SO4 sulfate ion link Sulfate probably helps crystallization. This structure was solved by X-ray crystallography.
Click more info... link Log shows literature citation and X-ray diffraction resolution 2.05 Angstroms.
Click 1 link in mmCIF Assemblies table. Shows just one copy of nanobody. Sometimes assemblies create large multimers like a virus capsid structures from a single capsid protein.
Click assembly line in Models panel and Close button Closes the single nanobody copy made in the previous step.
Click Models panel checkbutton under the eye icon for model #1 This checkbutton hides and shows the atomic model.

Display styles using the Toolbar

Try toolbar buttons.

ActionNotes
Click Show Atoms toolbar button. Notice there are hydrogen atoms (white) -- usually not seen in a 2 Angstrom resolution structure.
Hide Cartoon toolbar button.
Try Sphere and Ball and Stick styles.
Soft lighting This casts shadows from 64 directions. With atom spheres it makes crevices dark.
Full lighting This is like soft with an added spot light that casts a sharp shadow,

Selecting Atoms

Change display style of just one protein.

ActionNotes
Click with ctrl key down one atom. Ctrl-click select an atom. Shows green outline around atom.
Press up-arrow keyboard key. Extends to select whole residue.
Press up-arrow again. Selects whole beta-strand. Another press selects whole chain.
Hide atoms, show cartoon. Toolbar buttons only act on selected atoms.
Press down-arrow. Reduces the selection.
Ctrl-click not on an atom. Clears the selection. Or menu Select / Clear. Or command "select clear"

Coloring using the Toolbar

Different coloring to highlight different aspects of structure.

ActionNotes
Click Molecule Display tab of toolbar The toolbar has many tabs to provide different sets of operations.
Color Chain Useful to see boundaries between proteins.
Color Heteroatom Makes oxygen red, nitrogen blue, leaves carbon colors unchanged.
Color Rainbow Blue at N-terminus to red at C-terminus, usually used with ribbon display to see path of the backbone. Note 4-strand beta sheets are not a contiguous span of residues.
Menu Presets / Original Look To restore original structure appearance, including color, styles and what atoms shown.

Surface Coloring using the Toolbar

Coloring surfaces by electrostatic potential, hydrophobicity or B-factor.

ActionNotes
Click Surface Show icon in Molecule Display toolbar Surface shown for each chain. Does not enclose water or ligands. Solvent excluded surface defined by rolling water-size sphere over protein.
Click Electrostatic coloring. Uses calculated charges on atoms. Red is negative, blue is positive. Notice the two bright red/blue interfaces between the two nanobodies -- opposite charges attract.
Click Hydrophobic coloring. Blue is hydrophilic, yellow is hydrophobic. Notice the paired yellow hydrophobic interfaces between the two proteins. Interfaces like to bury hydrophobic regions.
Click B-factor coloring. Red indicates mobile atoms in X-ray data -- usually at periphery of structure. Blue indicates immobile regions usually in core.

Amino Acid Sequence

Display sequence of protein.

ActionNotes
Click Sequence icon in Molecule Display toolbar Shows amino acid sequence in separate panel. Beta strands blue, alpha helices yellow
Click and drag on sequence to select Green outline of selected residues shown on structure.
See six histidines at end of sequence Black outline box means these residues do not have atom coordinates. Histidine tag for purifying protein.

X-ray Density Map

ActionNotes
Type command "open 7kkj from eds" Fetches X-ray density from the Electron Density Server 7kkj.
Hide cartoons, show atoms with toolbar Want to see atoms to compare to X-ray density
Ctrl-click Volume Viewer histogram, menu Show Outline Box This shows the unit cell of the x-ray map, a skewed box.
Click Zone icon in Map toolbar Show only the density mesh near the atomic model.
Typed command "hide H" Hide hydrogen atoms to reduce visual clutter. They are not visible in density.
Menu Tools / General / Side View Shows view of molecule from side with eye and clip planes (vertical lines).
Drag vertical bars in Side View to clip Sets near/far clipping to look at a thin slab.
Drag vertical bar on Volume Viewer histogram This changes the density level for showing the mesh surface to see weaker or stonger signals.
Click Clip in Right Mouse toolbar Enables moving clip planes in graphics window with right mouse button, or Option key + trackpad on Mac.
Option+Shift key drag Moves near and far planes together to see different depths in density.

SARS-CoV-2 spike structure

ActionNotes
Type command "open 7kkl" Open Protein DataBank SARS-CoV-2 spike with bound nanobody, PDB id 7kkl.
Hover mouse over nanobodies to find chain identifiers. Chains B, E and F are 3 bound nanobodies shown in popup window when hovering mouse.
Type command color /B,E,F gold Color nanobodies gold.

Save an image

To make an image for a presentation often use white background, lighting, silhouettes.

ActionNotes
White background from Home toolbar White background is usually better than black in articles and talk slides.
Silhouettes from Graphics toolbar Puts thin black lines around edges of structure, helps see overlaps.
Soft lighting from Graphics toolbar Shadows show depth in a static image.
Snapshot from Home toolbar Saves an image file to the Desktop matching the size shown on the screen.
"save spike.jpg width 1000 transparent true" typed command Saves a higher resolution image. When overlaying on top of another image use transparent background.

Hydrogen bonds

Look at hydrogen bonds between nanobody and spike.

ActionNotes
Click H-bonds in Molecule Display toolbar Shows predicted hydrogen bonds in structures.
Select nanobody and click H-bonds Show only hydrogen bonds with nanobody.
Hide atoms and cartoons with toolbar
Typed command "show /D,F cartoon" Visually simplify by only showing one nanobody, one spike protein.
Tools / Structure Analysis / H-Bonds menu More options for showing hydrogen bonds.
Hydrogen bonds panel options:
Limit selection with exactly one end selected
Show hyrdogen bonds only between nanobody and spike.
Press Ok or Apply button in H-bonds panel. 9 hydrogen bonds found, table shown in Log panel. 6 are to spike chain D and 3 to undisplayed chain A.
9 H-bonds
H-bonds (donor, acceptor, hydrogen, D..A dist, D-H..A dist):
/A ASN 440 ND2  /F GLN 39 OE1   /A ASN 440 HD22  3.004  2.048
/D TYR 489 OH   /F ASP 99 O     /D TYR 489 HH    2.868  1.876
/D SER 494 N    /F GLY 30 O     /D SER 494 H     2.936  1.929
/F GLY 26 N     /D GLN 498 OE1  /F GLY 26 H      2.865  1.851
/F ARG 31 NE    /D GLN 493 OE1  /F ARG 31 HE     3.071  2.213
/F ASN 32 N     /D GLN 493 OE1  /F ASN 32 H      2.907  1.902
/F ASN 32 ND2   /D PHE 490 O    /F ASN 32 HD22   2.939  1.936
/F ASN 77 ND2   /D GLY 446 O    /F ASN 77 HD21   3.084  2.067
/F GLN 111 NE2  /A PRO 499 O    /F GLN 111 HE21  2.854  1.838

Electron Microscopy of SARS-CoV-2 Spike and Nanobody

Spike structure with bound nanobody came from electron microscopy, EMD 22910 as shown on the PDB web page for 7kkl.

Here is a smaller EM map file emd_22910_trimmed.mrc (7 Mbytes, smaller box, subsample step 2) while the database map is 226 Mbytes and can be slow to download.

ActionNotes
Open downloaded map emd_22910_trimmed.mrc

or

Type command "open 22910 from emdb"

You can double click the file icon, or use the Open toolbar button or File / Open... menu entry to open the downloaded reduced size map file.

or

With fast network (> 100 Mbits/sec) just fetch the 226 Mbyte file directly from the EM Databank.

Fit SARS-CoV-2 spike atomic model into map

To start building an atomic model from electron microscopy we fit in previously know atomic structures.

ActionNotes
Type command "open 6vxx" Open Protein DataBank SARS-CoV-2 spike, PDB id 6vxx.
Click color chain in molecule toolbar The spike is a trimer, each protein copy a separate color.
Click Move model in Right Mouse toolbar We will move the trimer by hand into the correct position in the EM map.
Ctrl-click an atom to select it The selected model will be moved while the map stays still.
Drag to move spike into map. Hold Option-key on Mac with trackpad, or use right mouse button, or Alt-key on Windows.
Shift-key + drag to rotate spike. Pressing or releasing the shift key switches between translating and rotating the model.

Optimize fit of spike in map

Do a local rigid motion to optimize the manually fit position.

ActionNotes
Click Fit icon in Map toolbar Do local rigid fit optimization moving spike atomic model, takes less than a second.
Show atoms as spheres Molecule display toolbar, unselect (ctrl-click background), Show atoms, Sphere style.

Find nanobody location in map

Color the map near the fit spike atomic model. The place not colored is where the nanobody binds.

ActionNotes
Click Color near atoms icon in Map toolbar Color map to match atomic model colors

Fit nanobody atomic model in EM map

Same procedure as fitting spike. Place manually, optimize fit. Need to place 3 copies of nanobody. Getting the right orientation is very challenging.

ActionNotes
Typed command "delete #1/C" First delete one of the two nanobody copies in X-ray model.
Typed command "delete solvent" Water molecules in nanobody are distracting. Delete them.
Use Move model in Right Mouse toolbar Move the nanobody by hand into the correct position in the EM map.
Ctrl-click an atom to select nanobody and drag nanobody into map Hold Option-key on Mac with trackpad, or use right mouse button, or Alt-key on Windows. Pressing or releasing the shift key switches between translating and rotating the model.
Type command "fit #1 in #4" Command to rigidly optimize fit of nanobody in map.

Compare bound nanobody shape to unbound

The nanobody bound to the spike adopts a different conformation than the unbound x-ray nanobody atomic model. Superimpose them.

The loops that interact with the spike move a great deal.

ActionNotes
Show x-ray nanobody and 7KKL spike/nanobody models Use Models panel show/hide checkbuttons under eye icon.
Show in cartoon style Use Molecule Display toolbar, Hide Atoms, Show Cartoon.
Type command "color #1 red" Make x-ray nanobody a contrasting color.
Type command "color #3/F gold" Make EM nanobody a contrasting color.
Menu Tools / Structure Analysis / Matchmaker This panel will align the two nanobodies. Rigid motion to minimize corresponding C-alpha atom positions.
Click Specific chain in reference structure... We want to tell which of 3 spike nanobodies to align to.
Click 7kkl #3/F as Reference chain This is the target we align to.
Click 7kkj #1 as Structure to match This is the x-ray model.
Click Apply in Matchmaker panel Does the alignment
Type command "matchmaker #1 to #3/F" This command does the same thing as all those previous steps with the Matchmaker panel.
Type command "hide #3/A,B,C,E cartoon" Hide extra chains of spike model to get clearer view.

Next step: Model refinement with ISOLDE

Next steps would be to use the ISOLDE flexible atomic model refinement package using manual tugging and molecular dynamics to make the spike and nanobody agree with the EM density.