• Download and Installation 
• Data Flow and Design 
• Converting Volumetric Bead Maps 
• Exhaustive Search Docking
• Visualization 
• Manual Docking and Refinement
• Outlook: Alternative Fitting Strategies 

Follow first the simple registration and download steps .

In addition to the executables, the Situs_2.3_saxs_tutorial/bin directory contains four data files:

• 0_tnc.pdb: Atomic coordinates of troponin C.
• 0_rib.pdb: Atomic coordinates of ribonuclease inhibitor.
• 0_tnc_sph.pdb: Bead model of troponin C.
• 0_rib_sph.pdb: Bead model of ribonuclease inhibitor.

Schematic diagram of SAXS related routines. Major Situs components (blue) are classified by their functionality. The main data flow is indicated by brown arrows. The visualization (orange) for the rendering of the bead models requires a molecular graphics viewer (we recommend the free VMD graphics program, version 1.8.4 or higher; Chimera and Sculptor also support Situs format). 

Visualization and modeling of atomic structures into SAXS bead models are supported through a conversion into 3D volumes with the pdb2vol kernel convolution tool. A docking between atomic structures to 3D maps can be achieved with a number of approaches (see below). The resulting docked complex can be inspected using a molecular graphics viewer. The data can be prepared further for the visualization using a variety of analysis and editing tools. Kernel convolution facilitates the smoothing of bead surfaces for their visualization in the form of density isocontours. 

Select no mass-weighting (enter 1) and no B-factor thresholding (enter 1), then enter the desired voxel spacing of the output map. Given the dimensions of the structure and the bead radius (6 Å), 1Å appears to be a good compromise between bead sampling accuracy and storage requirement (you may have to adjust this value upwards for other cases to avoid oversampling of beads and resulting large maps). Next, enter the kernel width (bead radius): +6 Å. This is the half-max radius where the kernel amplitude drops to its half maximal value. Next, select the "Hard Sphere" smoothing kernel (enter 5). Turn lattice correction off (enter 2), and enter the maximum amplitude of the kernel (enter 1 and keep a note of this).

The program projects the atomic structure to the lattice, computes the hard sphere kernel, and carries out the real-space convolution, writing the resulting map to the file 1_rib_sph.situs.

Here is the full pdblur session for file 1_rib_sph.situs:  

The colores program performs an exhaustive search of all rigid-body degrees of freedom. This approach can be used for SAXS bead models, but care must be taken in the case of SAXS to select volumetric correlation (option "-corr 0"), since the default option (Laplacian filter) would amplify the segmentation of the data caused by the SAXS beads. The use of colores with EM data has already been shown elsewhere, in the correlation-based docking tutorial.

As an example we perform here an exhaustive rigid body docking of the above data with colores. For this particular docking case it will be sufficient to perform a reduced angular search (sampled at the default 30°step size), and we set the target resolution to the resolution of the bead model returned above by pdb2vol:  option "-res 9.356". After the exhaustive search is done, the best 6 on-lattice maxima (option "-explor 6")  will be refined (off-lattice) using Powell optimization. 

The output of colores has already been described in detail in the corresponding tutorial. We list here the output of the entire calculation as a reference:

Note that 4 of the found results were redundant so only two solutions remain. Note that each run of colores or colacor overwrites the col_* files in your directory. Therefore, you should save these files after each run into a separate subdirectory:

As an additional exercise you may do the same for troponin C.

One of the problems with SAXS bead models is the rendering of an outer contour that does not occlude the interior. In the following, we use again pdb2vol to create a low-resolution map from the bead model, but this time we apply a "soft" smoothing kernel that will allow us to render a smooth envelope of the bead model surface without cluttering up the image with individal spheres. For this we use the VMD graphics program (version 1.8.4 or higher). Chimera and Sculptor also support Situs format.

At the command prompt, enter:

The following sequence of commands in the VMD text console (cf. VMD user guide ) will load the docked structure 2_rib_1.pdb and render it in cartoon representation, coded by color. The script then instructs VMD to render the file 3_rib_sph.situs:
 

Now inspect the alternative solution col_best_002.pdb. The second ribonuclease inhibitor structure is "flipped" about the pseudo-symmetry axis of the U-shaped molecule. This kind of degeneracy of the best fit can be expected in the case of symmetric shapes. One should expect unique fits only in cases where the shape of the molecule clearly determines the registration.

The program output shows how the initial correlation of 0.1 increases to 0.6 while the structure is matched. The output is again written to the file col_best_001.pdb. As before, we create a directory "manual" and move the col_* files into it.

When inspected with VMD as above the results should look similar to the following figure:

Using simulated markers (feature points) for docking

Flexible docking

It is well known that proteins can adopt a solution conformations that deviate from a known crystal structure. Consider e.g. the case of calmodulin which was first shown by SAXS to compact in solution (Heidorn & Trewhella, Biochemistry 1988 Feb 9; 27(3): 909-15) and whose first solved crystal structure was not in the "physiologically relevant" conformation. For cases like this the flexible docking tools described in the flexible docking tutorial, although developed with electron microscopy in mind, may also become very useful to SAXS modelers.