Jochen Heyd and Stefan Birmanns,
Beyond the Black Box: Interactive Global Docking of Protein Complexes,
Microscopy Today, Vol: 16 # 4, July 2008. [Article - PDF].

Obtaining exhaustive search data

"Interactive global docking" (IGD) is an immersive peak search technique for exploring the results of an exhaustive search based multi-resolution docking run performed with Situs / colores. Instead of inspecting dozens or even hundreds of solutions by hand, the user can interactively explore the docking landscape by moving the atomic probe structure through the low resolution volumetric data set. This tutorial covers all the main features of IGD, using a simulated data set as an example.

Make sure to specify the '-sculptor' option when using colores, so that the program automatically generates the necessary files (the option is available in Situs 2.5 and higher). For details, please see the Situs documentation on how to carry out exhaustive search docking runs (correlation-based docking tutorial).

For this tutorial we will use a small heat shock protein RecA.

Download the hexamer structure from the PDB database:

    http://www.rcsb.org/pdb/files/2rec.pdb.gz

Uncompress the structure

gunzip 2REC.pdb.gz

In the menu, select “Structure -> Blur...”

Choose a resolution of 15A

and a 3A voxel size

Sculptor will blur the structure and generate a new document with the simulated volume data set:

Now, save the document by selecting it and choosing “File -> Save As” from the menu:

Save it under the name '2REC.sit' . Click here to download file.

This generates a map called ‘2REC.sit’ with  3.0A voxel width and 15.0A resolution. Now, we need to extract one monomer to use as the probe structure. This can be done using a text editor or via the ‘extract’ functionality of Sculptor. For the latter approach, start Sculptor using

sculptor 2REC.pdb

We can split the hexamer structure via “Structure -> Extract”

Now, we have the monomers as individual documents

and can save chain A to a file:

Click here to download the file.

This generates a simulated map with 3.0A voxel width and 15.0A resolution, which can be used to perform an exhaustive search in Colores:

colores 2REC.sit 2REC-mono.pdb -res 15.0 -deg 9.0 -sculptor -explor 6 -nopowell -nopeaksharp -nprocs 4

The options after ‘-sculptor’ are not essential for the current pupose but they save time.

Click here to download the file.

Loading exhaustive search data into Sculptor

The exhaustive search data sets comprise several files. Colores will generate the files in the current directory (i.e. where the best_fit* solutions and other output files appear).

Loading a data set in Sculptor is accomplished by loading the main description file of the set, which ends in ‘.eli’:

Sculptor then loads the full data set and displays the target map and the probe structure:

Recommended display properties

Before starting the actual docking, it is convenient to first set up all the display options. Select the map in the document panel

Now click into the histogram to select a lower iso-surface value (here we used 20).

Next, open the color chooser dialog via the “Set Color” button

and select white.

To enable us to see the probe structure inside the map, we now need to lower the transparency of the iso-surface:

Next, select the probe structure.

Change its rendering mode to “Cartoon”.

Finally, we can maximize our work area by first dragging down the top edge of the console window until it disappears

and then either dragging down the properties window on the left or by double clicking on any document name.

Having done all the setup work, now is a good time to save a scene file.

Exploring the docking landscape

To start the Interactive Global Docking (IGD), just select the probe structure with the move tool.

Since IGD automatically reorients the probe, the left mouse button is used for X and Y translations and the middle button (or wheel press) is performs Z translations.

As the structure is moved, the current cross-correlation (CC) score is displayed both numerically and via the color of the central sphere. An arrow points towards regions of higher CC scores and its size scales with the gradient of the CC landscape.

Local maxima in the scoring landscape are highlighted via a wireframe around the central sphere:

We are looking at three criteria when docking the first structure:

• Score from exhaustive search

• Local maximum

• Good visual and biological fit of the subunit

The last criterion is the one which sets IGD apart from other docking techniques. Besides a good fit with the density map, one can also check known or suspected positions of certain residues by highlighting these residues in the probe structure.

Remembering solution candidates

Having found a good initial position for one monomer, we can now save it as a candidate solution. This is accomplished either via:

A) While having the probe selected in the document list, click on the green solution candidate icon in the tool bar

or B) double-clicking the probe icon in the document list, or the keyboard shortcut ‘x’.

This adds a new document with a green icon to the list, signifying a solution candidate.

As can be seen in the next screen shot, the solution candidate structure is colored green to make it readily identifiable in the main display window.

Visualizing potential protein-protein interactions

Turn on steric clashing via the tool bar button

or with the keyboard shortcut ‘c’.

The toolbar icon will be activated and the move tool also changes to signify that steric interactions are being calculated. Favorable backbone-backbone distances are highlighted with green spheres while clashes are shown in red:

Using the steric information as an additional guide, we obtain the following position for the second monomer:

Now, we can easily place the remaining monomers.

Interactive adjustment of candidate solutions

Typically, IGD is an iterative process where candidate solutions are first placed approximately and later adjusted using steric interactions as a guide. To simplify this task, Sculptor supports rapid switching between the probe and solutions. The color scheme of the original probe can be set to follow the currently active structure. To enable this feature, select “Auto Adjust Colors” from the docking menu:

Now, any solution can be selected and the previous probe structure will become a green solution and the solution candidate will become the probe. The new probe structure will be displayed using the color scheme of the old one. As an example, color the probe by residue name:

Now, when we switch to a solution, the currently moved solution will be shown in the residue name coloring scheme.

Saving your work

As you build up your list of candidate solutions, it is prudent to save a scene file periodically. Please go to File->Save State to save the current state of Sculptor into a single file. The scene file stores all locations and properties of the candidate solutions (but not the actual experimental data! Please always move the model and structure files together with the scene file and do not change their location relative to each other).

Once all subunits have been placed satisfactorily, they can be saved as normal PDB files.

Refining the docking positions

To move the structure freely, IGD can be turned off via double clicking the move icon:

The IGD display turns off and the structure can be moved freely with the mouse, as usual.

When the move icon is double clicked again, IGD is reactivated and the structure rotates into the orientation determined by the exhaustive search.

Another refinement option is to use the standard ‘Refinement’ dialog:

If IGD is turned on when the refinement is performed, Sculptor automatically turns it off and shows a warning with how to reactivate IGD (if so desired).

IGD options

The behavior of IGD can be influenced via numerous options. To access the options dialog, select “Force Feedback Properties” from the “Haptic Docking” menu or use keyboard shortcut ‘g’:

“Scale Translations” determines how much the probe moves relative to the mouse.

“Central Sphere” adjusts the diameter of the central sphere.

“Scale Arrow” scales the force arrow.

The threshold for good (favorable) interactions and clashes can be adjusted. The values are in Angstrom and refer to backbone-backbone distances.

The way the central sphere color changes depending on the score can be specified via a simple color function. In the above screenshot, any score below 200 will show as white. Between 200 and 800, a gradient between red and blue will be used. Between 800 and 950, a blue-green gradient is employed. Above a 950, the sphere will be bright green.

A movie of an example session using experimental data

The movie below shows IGD applied to fitting atomic structures of capsid proteins into an experimental map of the coxsackie virus B3 (M strain) at 27A resolution. The volume data set by Hafenstein et al. is available from the EMDB (EMDB-1411). The virus is a member of the picorna family and its icosahedral, non-enveloped shell is approximately 300A in diameter. The capsid is arranged in a T=1 (P=3) lattice which contains 60 copies each of the four viral proteins

VP1 (31.4 kDa), VP2 (28.9 kDa), VP3 (26.2 kDa), and VP4 (7.4 kDa). The total weight of the asymmetric unit is 93.8 kDa. An exhaustive search using the PDB structure of the complete asymmetric unit 1COV (Muckelbauer et al., 1995) was performed (Laplacian filtering was used). The movie (recorded in real time) clearly shows the high scoring but incorrect solution in the center of the face and then proceeds to demonstrate the docking of the three subunits in their correct positions using IGD.

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Using a haptic device

IGD fully supports all haptic devices that work with Sculptor. The force feedback is based on the gradient of the CC landscape. Since the probe orientations are set automatically, only the three translational degrees of freedom of the haptic device are used. This also allows the use of inexpensive devices such as the Novint Falcon.

User interface features for haptic devices

To turn haptic rendering on and off, use the keyboard shortcut ‘f’ or click on the lightning bold toolbar button. If haptic rendering is off, documents can be moved with the mouse.

When haptic rendering is active, the scene can be moved with the mouse (without having to select the scene with the move tool).

Using the Novint Falcon

Start Sculptor via ‘sculptor -falcon’. This will set all the necessary defaults and prompt the user (in a text window) to calibrate the Falcon. The calibration is only necessary the first time the Falcon is accessed in a windows session.

Whenever a structure is moved via IGD, forces are output to the Falcon. Press the round central button on the grip to move the structure. The strength of the forces can be adjusted via the ‘Scale CC Forces’ slider in the options window. The middle (“lightning”) button on the Falcon switches between two sensitivity levels for the translations. Press it for 0.5 sec and release it to switch to the higher accuracy mode. Repeat to get back to normal sensitivity.

Should you experience some ‘jitter’ around the best docking positions, try a lower ‘Scale CC Forces’ setting.

Known issue: The Windows Falcon driver sometimes has issues with high display resolutions. If you experience strange forces, move the Sculptor window to the lower left corner of the screen. If the inconsistent forces persist, make the window smaller.