Pymol– Tutorial

Exercise 1: Introduction

“File –> Open” 3OG7

Save your session by doing “File –> Save Session As...” (don’t forget to save your work regularly by “File –> Save Session”

PyMol can open more than one molecule at a time, or separate complex PDB files into individual components. Each opened or loaded molecule is given a name within the “Names Panel” (right part of the window). The first name is always “all.” Clicking on the name itself will undisplay the corresponding molecule(s) (temporarily invisible). Clicking again on the name will display the molecule.

The ASHLC menu is abbreviated for Action, Show, Hide, Label and Color.

For example, for a better overview “S”“Show as”“cartoon”. This hides everything and shows the protein’s backbone representation.

Go to the following menu cascade: Setting > Cartoon > Cylindrical Helices Select this option again to remove its effect and do the following: Setting > Cartoon > Fancy Helices

PyMol is optimized for a 3-button mouse:

Rotation around the X or Y axis: (left) click and drag Rotation around the Z axis: (left) click on the top left or right corner Translate (move sideways) X or Y: click middle button and drag Zoom (move along Z axis): click right button and drag up or down

Now start to “experiment” with your mouse. BIOCHEMISTRY 660 / 712 – FALL 2006 J.Y. SGRO

6) Final image(s)

If you still see the selection dots over the ligand from the previous section simply click anywhere on the white background to unselect. Alternatively click on the “Hide-Sele” button at the top right hand side of the “external GUI.” BIOCHEMISTRY 660 / 712 – FALL 2006 J.Y. SGRO Rotate the molecule to find a perspective that you deem instructive of the conformation of the protein2BIW.pdb2 and it’s bound> A > ligand.preset > default Explore the “preset” options of the Action set of the ASHLC FollowThis menus:thiscommand menu cascadehas a similar to save effect the but image is not currently the withinsame the- simple as Viewer: the following cascade: hide everything and show- simple (no solvent)lines: File > Save- ball and stick Image… 2BIW.pdb2- b factor putty > H > everything Thenand replace…. the default word “pymol” to give a name to the2BIW.pdb2 file -you publication want > toS save, > lines e.g. image1 The image will be saved as a PNG image on the desktop Note: the “preset” options will set some variables that are specific to these views and may change further drawings. To remove the effect of these presets affecting an object representation, use the HoweverSave this final images: image is rather crude in terms of graphics and resolution. PyMol offers an internalA>preset>default “ray tracer” to menu create cascade stunning reset rendered parameters. images with a high visual quality much more pleasant to the eye and ideal for publication. Note: “toFile get–> Save Images As... back to the original” –> “ PNG…opening” view simply type reset at the PyMOL> line command. To create a standard ray-traced image of the current Viewer scene,2) The image will be saved as a PNG image. However, this image is rather clickExploring the “Ray” more button at the top right of the “external GUI.” Thiscrude will in take terms a of few graphics seconds and to resolution. a few minutes PyMol offers depending an internal on theExplore complexity“ray tracer” to create rendered images with a high visual qual the other of the menus PDB file of and this the series. the chosen display, andity ideal willThe also cascadefor publication. depend menu on the 2BIW.pdb2 speed of the > A computer > preset CPU.is assumed Once in the following commands: rendered, the image appears within the Viewer. To save the file, use theTo create a standard ray save cascade as above:-traced image of the current Viewer scene, File > Save Image… click the “Ray” button and save the simple image as PNG. The tracing can then be made thicker by unselecting the smoothType the commands option with the following menu cascade: Setting“set ray_trace_mode, 1 > Ribbon > Smooth” Note: “aset ray_trace_color, black set of 3 histidines is ”also shown. for a black line. This is not very useful for a ball and stick large protein such as this.

b factor putty

The segments with the highest temperature factor are shown as thicker cylinders. Regions of better resolution have thinner diameterZoomed and side-by-side are usually comparison found between at the corethe pymol of the image protein. and the ray-traced image: note the jagginess Mostlyof the loops original in image the outside and the smoothof the appearanceprotein wobble: of the ray-traced the core image, with shadows as a bonus. portions of the proteins usually appear more stable than the external loops. This is mostly useful for crystallographers but is a cool representation. L02 – page 7 Color domains in separate rainbow colors and shows backbone and side chains. technical Note that a subset name appears in the Names Panel (2BIW.pdb2_pol_co) that control the dashed-line hydrogen bonds.

L02 – page 9 When you made yourself familiar with PyMOL and its mouse movements, click on the “S” in the bottom-right corner. This shows you the protein’s sequence. At the beginning of the protein sequence, there is a compound, which was crystallized together with the structure of the protein. Select the compound “O32”.

You now see a second panel called “(sele)” under “3OG7” which represents the currently selected part of the structure. Use it to show the ligand “O32” in sticks.

Next we change the color of the ligand. Click “(sele):C –> by element –> and select a color that you like”.

Zoom to the ligand using your mouse. You can center the ligand (or any other part of the structure) by selecting it and then pressing the mouse wheel or type “zoom sele”.

We are now interested in the amino acids around the ligand – the so-called “binding site”. To explore this further, select the ligand, click “(sele): A –> modify – > around –> residues within 4 A”. You have now all residues within 4 Angstrom selected. Without clicking anywhere else (this would undo your selection) click “(sele): S –> side chain –> sticks” and “(sele): C –> by element –> and select a color different than the ligand”

Your window should look somewhat like this:

Now we want to look at an important interaction between the ligand and the protein: The ligand O32 is forming hydrogen bonds interactions with the backbone of Gln530 and Cys532. Scroll in the sequence at the top to find this residue and select it. To confirm that you have selected the right residue, click “(sele): L –> Residues”. You will find your residue labeled by name and residue number. Change size and font in “Setting > Label” “(sele): S –> Sticks” Furthermore there are two command line options you should know for selecting residues: “sele resn gln” which selects all aspartic acids and “sele resi 530” which selects residue number 530

Next we want to confirm that there is a strong interaction between Lys483, Gln530 and Cys532 and the ligand O32. We do this by measuring the distance. Therefore use the small GUI window, click “Wizard –> Measurement” and then in the main window click the two atoms whose distance you want to be measured.

Which atoms of O32 and Gln530 are interacting through a hydrogen bond? What is the distance? Which atoms of O32 and Cys532 are interacting through a hydrogen bond? What is the distance? Which atoms of O32 and Lys483 are interacting through a hydrogen bond? What is the distance?

Reproduce (more or less) the following pictures, which display amino acids interacting with the ligand O32

Select “Movie > Camera loop > Nutate > 30 deg. Over 4 sec.” It creates 120 states of the figure … Click the play button. “File > Save Movie As > MPEG” You can save the movie with high quality “Ray” figures. Click Ray and save again. Open the file in powerpoint.

Exercise 2: Cavities and molecular electrostatic potential

Open 5P21

Hide everything

Show cartoon

Copy residues GNP167 and MG168 to a new object

Remove GNP167 and MG168

Rename the new object to “GNP”

Set Surface Cavities & Pockets (Culled)

Show surface

Show GNP as spheres

Set Surface Exterior normal

Hide/Show GNP

Calculation of Electrostatic Surface with APBS, via PyMol

ABPS (Adaptive Poisson-Boltzmann Solver, http://www.poissonboltzmann.org/apbs/) calculates the electrostatic properties at the molecular surface by solving the Poisson-Boltzmann equation (http://en.wikipedia.org/wiki/Poisson–Boltzmann_equation). PyMol has an integrated plugin that calculate ABPS.

PyMol>Plugin open “APBS tool”

In the “Main” menu select “Use PyMOL generated PQR and existing Hydrogens and termini”

In the “Configuration” menu select “Set grid” ( the x,y,z values of the grid is define) (protein and solvent dielectric are defined for soluble proteins, and ion concentration is setup by default)

Select “Run APBS” and wait …… until the “Visualization” menu automatically shows up.

In the “Visualization menu” select “Color by potential on sol. acc. surf” and select “Update”

Change the electrostatic range value by modifying “Low” to -6.0, “Middle” to 0.0 and “High” to 4.0 and select “Update”. Play with these values.

Hide/Show GNP Exercise 3: Superimposition of structures

We want to compare the 2Y03 structure with 3PWH.

Open 2Y03 (β1-adrenergic receptor) Display 2Y03 as a cartoon. It is a dimer (contains chains A and B). Remove atoms of chain B type “sele chain B” “(sele): A –> remove atoms” Be careful, PyMOL has no “undo” function. Removed atoms can’t be brought back!

Open 3PWH (adenosine A2A receptor) Display 3PWH as cartoon.

Now we want to compare the adrenergic and the dopamine receptor. You will see that they are in different positions and comparison between them is impossible.

Click “3PWH: A –> align –> to molecule –> 2Y03”.

In the GUI window you can see the rmsd value between both structures. What is it?

What is the rmsd value of the superimposition? Which was the better approach in this case?

Is the ligand of β1- (5FW) binding at the same cavity as the ligand of A2A (ZMA)?

Exercise 4: Build an α-helix

Build the following sequence “ERARSTLQKEVHAAKSLAIIVGLFALCWLPLHIINCFTFFC” as a α-helix using the following command fab ERARSTLQKEVHAAKSLAIIVGLFALCWLPLHIINCFTFFC, TM6, ss=1

Display TM6 as a cartoon

Open 3PWH

Display only helix 6 of 3PWH from E219 to C259

Superimpose E1-C41 of the build helix “TM6” to E219-C259 of 3PWH

We can superimpose only certain amino acids of both molecules (only accessible via the command line):

Type “super tm6 and resi 1-41, 3PWH and resi 219-259”

Do they superimpose properly? What is the rmsd?

Superimpose E1-L23 of the build helix “TM6” to E219-L241 of 3PWH

Do they superimpose properly? What is the rmsd?

Are both helices identical?