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ChimeraX Tutorial: Protein-Ligand Binding Sites

H-bonds
lipophilicity
electrostatics

This tutorial explores some ways to examine the complementarity between proteins and their ligands, including drug molecules. Related tutorials: Coloring by Sequence Conservation, Delta-Opioid Receptor, Structure Analysis and Comparison, Coloring CRM1 by Electrostatic Potential

** ChimeraX 1.2 (production release May 2021) or newer should be used to follow this tutorial. **

Opening a Structure and Surveying Its Parts

Start ChimeraX. If you want to use the click-to-execute links in this tutorial, view this page in the ChimeraX Browser. One way to do so is by entering the ChimeraX command:

Commandopen help:user/tutorials/binding-sites.html
All of the click-to-execute links in this tutorial are indented and appear after “Command:” Other links are simply informative and can be clicked or not, as you prefer.

ChimeraX can fetch a structure directly from the Protein Data Bank (PDB) given its 4-character ID. Open 2hyy, a structure of the human Abl kinase domain in complex with the anticancer drug imatinib:

Commandopen 2hyy

The Model Panel (lower right side of the ChimeraX window) lists 2hyy as model 1, and the  eye icon  checkbox indicates that the model is displayed.

The Log (right side of window) shows several kinds of information associated with the structure:

In the Chain information table, try clicking the chain IDs (A, etc.); see that the corresponding part of the structure becomes selected in the graphics window, as indicated with green outlines. The structure includes four copies of the kinase as chains A, B, C, and D.

Note: Selections are used to limit the scope of using the Actions menu or clicking certain icons. The  hand icon checkbox in the Model Panel shows when a model is selected, with three possible states: checked, unchecked, and partially checked (shown with a dash on Mac).

In the Non-standard residues table, click the residue name STI (imatinib) to see where it is in the structure. In this context, “nonstandard” means residues other than water and the types of amino acids and nucleic acids typically found in biomolecules. Imatinib is a kinase inhibitor, and each copy of the kinase has its own imatinib binding site.

In the 2hyy mmCIF Assemblies table, click 1 to generate “assembly 1,” a single copy of the protein-ligand complex. As can be seen in the Model Panel, this opens the assembly as model 2 and hides the original structure (model 1, eye icon  box unchecked). Close the original structure:

Commandclose #1
Note: In this case, the assembly has fewer chains than the original structure. For assemblies with more than 12 copies of the original structure, clicking a link in the Assemblies table adds copies as graphical clones for efficient visualization. However, if the plan is to analyze atomic interactions between the copies or to display them differently from one another, the assembly should be generated instead as full atomic copies with the sym command and copies true, for example:
     sym #1 assembly 1 copies true
Alternatively, entire assemblies can be fetched directly from the PDB with the open command, as shown in the section on Surface Coloring by Electrostatic Potential.

If the model information becomes inconvenient to access by scrolling, or gets removed by clearing the Log, it can be re-shown with commands (details...).

Manipulation

Try moving the structure around. Default mouse/trackpad assignments:

rotate
left mouse button
trackpad click-drag
translate in 2D
middle or right mouse button
trackpad + Alt
+ Shift switches from rotate to translate and vice versa
zoom
mouse scroll wheel
trackpad 2-finger drag (except pinch on Mac as per Trackpad preferences)
select
left mouse button + Ctrl, + Shift to toggle or add to selection
trackpad + Ctrl, + Shift to toggle or add to selection

To list the current assignments in the Log:

Commandmousemode

If anything is selected, Ctrl-click in “empty space” in the graphics window (away from any atoms) to clear the selection. Alternatively, you can use the menu: Select... Clear or the command:

Commandselect clear

Atomic Display Styles

The initial display has cartoons (ribbons) for the protein, with most atoms hidden. Only the nonstandard residue and nearby sidechains are shown in atomic detail.

Many actions can be done in several different ways. One way to change the display is with presets. For example, all atoms as sticks can be shown with menu: Presets... Sticks or the command:

Commandpreset sticks

...and the original appearance or at least something similar (in this case, the color is different) can be restored with menu: Presets... Original Look or the command:

Commandpreset original

Of course, you can change the style of any subset of the atoms. Atoms to change can be specified by chain ID, residue number, residue name, atom name, etc., or by selection (indicated as sel in the command line). If nothing is specified, all atoms are changed, even though they might not be displayed at the time.

Commandstyle ball
Commandstyle :STI sphere
Commandstyle stick

The style icons in the Molecule Display tab of the Toolbar apply to the currently selected atoms, or if none are selected, all atoms.

imatinib binding site

Make imatinib more obvious by changing the color of its carbon atoms:

Command color :sti & C salmon

Capitalization of residue names in the command line is optional, but element symbols always need to be capitalized. To list color names in the Log:

Command color list

(A more organized table is also available.)

H-Bonds and Contacts

To find only the H-bonds involving imatinib, Ctrl-click to select any atom in that residue, then press the keyboard up arrow to promote the selection to the whole residue. If the initial selection is a bond rather than an atom, the up-arrow key will need to be pressed twice. If you overshoot and select more than the drug molecule, simply press the keyboard down arrow to go back.

Alternatively, the imatinib residue could be selected with the menu: Select... Residues... STI or a command:

Commandselect :sti

(You could also scroll back up in the Log to the Non-standard residues table and click its name there.) Focus the view on the selection and hide all other atoms:

Commandview sel
Commandhide ~sel

Use the H-Bonds tool (menu: Tools... Structure Analysis... H-Bonds). Keep the default settings, except turn on (check) the following if they are not already:

Make sure that the imatinib residue is still selected, then run the calculation by clicking Apply or OK (the difference between those two is that OK closes the dialog).

imatinib H-bonds

The Log lists five H-bonds involving imatinib. If the wrong settings were used by mistake, no worries: just change the settings and run the calculation again. The same calculation could be done with the following command:

Command hbonds :sti reveal true log true

The structure does not include hydrogens, but their inferred positions were used in the calculation.

The H-bonds are shown in the graphics window as dashed lines. Some of the dashed lines just go to the ribbon. By default, ribbon display suppresses the display of backbone atoms. Enable showing backbone atoms at the same time as the ribbon with command:

Commandcartoon suppress false

Now you can see two backbone nitrogen atoms donating H-bonds to imatinib, and a backbone carbonyl oxygen accepting an H-bond from imatinib. The backbone atoms can still be shown and hidden individually, and most backbone atoms are still hidden. The backbone atoms of these specific residues were displayed by the Reveal atoms... option in the H-bonds calculation.

Use the Contacts tool (menu: Tools... Structure Analysis... Contacts). Keep the default settings, except turn on (check) the following if they are not already:

...and turn off (uncheck) Display as pseudobonds, Make sure that the imatinib residue is selected as described above, then click Apply or OK.

The Log lists 87 atomic contacts involving imatinib. The same calculation could be done with the following command:

Commandcontacts :sti makePseudobonds false reveal true log true

Label the residues with displayed atoms:

Commandlabel @@display

A couple of the displayed residues are water (residue name HOH). Hide water and clear the selection:

Commandhide solvent
Commandselect clear

Resistance Mutations and Sequence Viewer

Several mutations in kinase domain of the Bcr-Abl fusion protein have been implicated in cancer resistance to imatinib treatment. According to Chandrasekhar et al. Sci Rep 9:2412 (2019), these include G250E, Y253H, V256G, L301I, T315I, F317L, Y320H, M351T, E373D, and D381N.

Coloring the carbon atoms and residue labels at these 10 positions shows that five of them are already displayed due to their interactions with imatinib:

Commandname resist :250,253,256,301,315,317,320,351,373,381
Commandcolor resist & C gold target al

The five residues 253, 256, 315, 317, and 381 are among those listed in the Log as forming H-bonds and/or contact interactions with imatinib. Atoms at the other five positions implicated in resistance to this drug are not yet displayed. To see them all:

Commandshow resist

The newly displayed residues have gold carbons but are not labeled. One way to make the 10 residues more obvious is to change their style, for example:

Commandstyle resist ball
Commandview resist clip false

The latter command zooms out to make the view contain all of the specified residues and turns off clipping. Clipping had been turned on by the use of view in the previous section. If you like, make the labels bigger (default height 0.7 Å):

Commandlabel height 1.0

Residues not directly involved in binding might contribute to drug resistance by changing in the conformation, flexibility, or stability of the protein.

Show the protein sequence in the Sequence Viewer:

Commandseq chain /A

If the sequence window seems vertically scrunched, you can click and drag its top margin upwards to increase its size within the overall window.

A selection in the 3D structure is shown as green boxes in the sequence, for example:

Commandselect resist

Conversely, you can change the 3D structure selection by clicking an existing box or dragging a new box in the Sequence Viewer window. The other colored boxes indicate information from the structure: structure helices, structure strands, missing from structure.

When done with this structure, close the session (both structure and sequence) and clear the Log:

Commandclose session
Commandlog clear

Surface Coloring by Lipophilicity

Coloring a protein surface by lipophilicity (hydrophobicity) may help to identify interaction sites for nonpolar molecules or the lipid membrane.

Open 1fpp, a structure of the protein farnesyltransferase in complex with farnesyl diphosphate (residue name FPP):

Commandopen 1fpp

From the interactive tables of information in the Log, you can see that the structure is a heterodimer (two different protein chains) bound to a single molecule of FPP. Remember that clicking a residue name in the Non-standard residues table selects the corresponding residue(s). Clear the selection by Ctrl-clicking in empty space or with the command:

Commandselect clear

A “molecular lipophilicity potential” (MLP) is calculated using atomic coordinates, atomic lipophilicity parameters, and a distance-dependent function. ChimeraX can calculate MLP maps for proteins and color their surfaces by the values. This can be done with the command:

Commandmlp
hydrophobic pocket

...or by clicking the icon in the Molecule Display tab of the Toolbar. The default coloring is from dark cyan for most hydrophilic through white to dark goldenrod for most hydrophobic:
. Different options for the functional form of the calculation and the coloring scheme are available with the mlp command.

If you rotate the structure, you may be able to see that FPP occupies a deep pocket that is mostly hydrophobic. The pocket is so deep, however, it may be difficult to see the coloring. A better view is obtained by limiting the surface to a zone around FPP and hiding ribbons and atoms other than FPP:

Commandsurface zone #1 near :fpp dist 8 max 1
Commandhide ~:fpp target ar

The pocket surface can now be viewed from the protein side as well as the ligand side.

In the surface command, max 1 limits the display to the largest surface component, hiding any disconnected bits. Both protein chains contribute to the pocket. Using the disclosure triangle for 1fpp in the Model Panel (lower right side of the ChimeraX window) shows that there are separate models for the surfaces of chain A and chain B. You can hide/show them individually with the checkboxes under the eye icon  eye icon.

When finished viewing this pocket, close the structure:

Commandclose

Coloring by lipophilicity is also used to identify or highlight membrane-embedded regions. Optionally, try using the icon or the command mlp on a transmembrane protein (many examples: 1pho, 4xt3, 1eys, etc.). A “belt” of hydrophobicity may be visible where a protein sits in the membrane. For example:

Commandopen 4xt3
Commandmlp
4xt3: MLP vs. Kyte-Doolittle coloring
4xt3 MLP vs. kdHydrophobicity

An alternative to calculating the MLP is to use a constant lookup value for each type of amino acid residue. A ChimeraX command script to assign Kyte-Doolittle hydrophobicity values and color the protein surface from dodger blue for the most hydrophilic through white to orange red for the most hydrophobic
is provided here: kyte-doolittle_hydrophobicity.cxc. Saving the file as plain text and opening it in ChimeraX will execute the commands on any proteins being viewed at the time. The script can also be used to define a custom preset in ChimeraX similar to the preset that was built into Chimera. (Of course, a different coloring scheme could be used, including the same as used by mlp.)

Coloring by a lookup value for each residue type often gives a similar impression to MLP coloring, but can be quite different in specific cases because all types of amino acids contain both polar and nonpolar atoms. In other words, even the most hydrophobic residues include some hydrophilic atoms, and vice versa.

If any structures are open, close them:

Commandclose

Coloring a Clipped Surface

clipped surface colored by MLP

The protein in structure 1g74 has an oleic acid residue OLA in an interior pocket. The following commands open the structure (model #1), show its molecular surface (model #1.1), and turn on clipping to slice the structure (creating a planar surface cap, model #1.1.1):

Commandopen 1g74
Commandsurface
Commandclip

The model hierarchy can be viewed in the Model Panel.

Next, turn off clipping of the atomic model so that only the surface is clipped (per-model clipping), hide ribbons and atoms other than oleic acid, and color the carbon atoms in oleic acid green:

Commandclip model #!1 off
Commandhide ~:ola targ ar
Commandcolor :ola & C green

The clipping plane can be translated and rotated interactively with the mouse .

Color the molecular surface by molecular lipophilicity potential (MLP). The planar surface cap is not automatically colored along with the outer surface, but can be colored with an additional command in which the atoms to use (here, all protein atoms) are specified separately from the surface to color:

Commandmlp
Commandmlp protein surface #1.1.1

The cap can be made transparent with another command, for example:

Commandtransparency #1.1.1 30
Analogously, to color the surface cap by Coulombic electrostatic potential (more about electrostatic potential in the next section):

Commandcoulombic protein
Commandcoulombic protein surf #1.1.1 offset 0

The Coulombic cap coloring would need to be reapplied if the clipping plane were subsequently moved, however (unless you are using ChimeraX 1.3 from 5/16/2021 or newer).

When finished with this structure, close the session:

Commandclose session

Surface Coloring by Electrostatic Potential

Coloring a protein surface by electrostatic potential (ESP) may help to identify binding sites for charged or polar molecules. Regions of positive potential are complementary to negative charges, whereas regions of negative potential complement positive charges.

ESP is calculated from atomic coordinates and partial charges. ChimeraX can calculate a simple Coulombic ESP on the fly, or it can read in an ESP map file calculated with another program, such as APBS. This tutorial uses the first (simple) method, as it does not require a separate calculation.

Open an assembly of 3d6y, the bacterial gene regulator BmrR:

Commandopen 3d6y from rcsb_bio maxAssemblies 1
3d6y assembly

BmrR regulates multidrug resistance. In this structure, it is bound to DNA and the positively charged alkaloid berberine. The Non-standard residues table in the Log lists berberine and glycerol; click the residue names BER and GOL to see where they are in the structure. Hide glycerol, water, and protein atoms:

Commandhide :gol
Commandhide solvent
Commandhide protein

To avoid any confusion from a hidden selection, clear it by Ctrl-clicking in empty space or using the command:

Commandselect clear

The following shows surfaces for the protein chains and colors them by Coulombic ESP calculated on the fly:

Commandcoulombic protein

The default color scheme is red for negative potential, white at zero, and blue for positive:
. There are positive patches for binding the negatively charged DNA, and a deep pocket of negative potential for binding the positively charged small molecule berberine.

The Molecule Display tab of the Toolbar includes an icon for Coulombic coloring, but it will also apply to the DNA unless protein (only) is selected before the icon is clicked.

The remaining commands in this section are just examples of display settings that might be useful for figures.

The nucleotides are color-coded by base type:
A T G C
. Make them a single color instead, so as not to distract from the surface coloring. You can also change the DNA ribbon to a tube shape and show nucleotide sidechains as stubs (broken ladder rungs):

Commandcolor nucleic tan
Commandcartoon style nucleic xsection round width 1.5 thick 1.5
Commandnuc stubs

The nucleotide change to stubs can also be done by clicking in the Nucleotides tab of the Toolbar.

Make the berberine stand out with fatter sticks and bright green carbons:

Commandsize :ber stickrad .7
Commandcolor C & :ber limegreen

Use icons in the Graphics tab of the Toolbar to:

Coulombic ESP flat lighting

Color Key and 2D Label

When making a figure, it is usually best to compose the rest of the image before adding any color key and/or 2D labels, since their sizes and positions will need to be adjusted to fit the image layout.

A color key can be added with the key option of the coloring command, in this case, coulombic:

Commandcoulombic protein key true

(If you already did the coloring, just repeat the same command except with key true appended.) This draws an initial color key, but also opens the Color Key tool and enables redrawing and moving the key with the mouse (the Adjust key... option in the tool). The Adjust key... option can be unchecked to restore the mouse to moving the structure instead of the key. Closing the tool also restores the mouse function to moving the structure.

When the color key is in place, a text label can be added with a command, for example:

Command2dlab text 'electrostatic potential'

...and then dragged interactively to the desired location with the move label mouse mode: in the Right Mouse tab of the Toolbar, click the icon and then use the right mouse button or trackpad + Alt on Windows, trackpad + on Mac to drag the label.

Although it is usually most convenient to position the key and label interactively as described above, and to set color-key colors and values by using the key option of the coloring command, these could be done explicitly with commands instead. For example:

Command2dlab text 'electrostatic potential' xpos .53 ypos .15
Commandkey red:-10 white:0 blue:10 pos .51,.09 size .32,.04

If you now have multiple copies of the label, you can hide or delete the extras. Each label is added as a submodel of the 2D labels model. One way to show/hide/close them is by using the Model Panel; in that tool, use the disclosure triangle next to a model's name to show its submodels.

To quit from ChimeraX, just close its main window or use a command:

Commandexit

UCSF Resource for Biocomputing, Visualization, and Informatics / July 2021