• Download and Installation 
• Data Flow and Design 
• Converting the EM Maps 
• Computing the Difference Density Maps 
• Inspecting Data Cross Sections 
• Extracting Regions of Interest 
• Inspecting the Voxel Histogram 
• Extracting Single-Molecule Densities 
• Vector Quantization and Docking 
• Visualization 
• Part II: Rigid-Body Docking with qdock 

Follow first the simple registration and download steps .

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

• 0_ncd.pdb: Atomic coordinates of a ncd monomer as described in Wriggers et al., 1999.
• 0_mt.ascii: ASCII-formatted density map of the undecorated microtubule, from the Milligan lab data site.
• 0_nm.ascii: ASCII-formatted density map of the ncd monomer decorated microtubule, from the Milligan lab data site.
• 0_nd.ascii: ASCII-formatted density map of the ncd dimer decorated microtubule, from the Milligan lab data site.
  

In the following, we will use these four files to dock ncd to the microtubule, following the steps described in Wriggers et al., 1999. The user can compare all generated files to the files in the solutions directory.

Schematic diagram of EM 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 data 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).

Standard EM formats are supported and are converted to cubic lattices in Situs format. Subsequently, the data is inspected and, if necessary, prepared for the vector quantization using a variety of visualization and analysis tools. All Situs tools require one volume and one PDB structure for the fitting. Atomic coordinates in PDB format can be transformed to low-resolution maps, if necessary, and vice versa, to allow docking of maps to maps or structures to structures. After the vector quantization, the high-resolution structure is docked to the low-resolution density by the corresponding codebook vectors. The resulting docked complex can be inspected using the graphics program. 

We convert EM maps to Situs format with the map2map utility. Enter at the shell prompt:
 

Enter the file format option 1: ASCII. At the program prompt enter the number of columns: 89, the number of rows: 89, and the number of sections: 61. Enter the grid (lattice) spacing in Angstrom: 6. Enter the order option: 1: x > y > z (order is already correct).

Repeat this procedure similarly for the other two EM maps to create the files 1_nm.situs and 1_nd.situs.

We need to specify the grid indexing. Also, we do not know the unit cell of the map, so the origin of the coordinate system is set to the first voxel in the map. 

Similarly, to compute the difference density map of the second, detached monomer, subtract the monomer-decorated microtubule data from the dimer-decorated microtubule data:  

Now we inspect the generated Situs maps with the voledit utility. voledit requires a fixed-width font in the shell window. 

Here is an example voledit session for file 1_nd.situs:  

Similarly, we create the files 3_nd.situs, 3_nm.situs, 3_s1.situs, and 3_s2.situs from the corresponding files 1_nd.situs, 1_nm.situs, 2_s1.situs, and 2_s2.situs. 

As an exercise, check out what happens if you specify an output file "test" and an offset density value of 10 and a scaling factor of 2 are entered at the program prompt. 

The inspection shows that the background peak is already at the origin, so we remove the output file "test". Note also that volhist can be run without an output file as argument, in which case the program simply shows the histogram.

The volhist  tool is also useful for discrepancy / difference mapping of simulated maps (created with pdb2vol) that are to be subtracted from experimental maps. In this case a rescaling of the simulated map is in order until the histogram matches that of the experimental map (or until the histogram of the difference map is balanced between positive and negative values if the subtracted map is of same size).