• 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 matchpoint

Follow first the simple registration and download steps .

In addition to the executables, the Situs_2.5_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 use here the VMD graphics program, Chimera and Sculptor also support Situs format).

Standard EM formats are supported and are converted to cubic lattices in Situs format. This is done with the map2map utility. Subsequently, the data is inspected and, if necessary, prepared for  vector quantization using a variety of visualization and analysis tools. All Situs docking 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 a 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. Enter the axis order option: 1: X Y Z (order is already correct). Now 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. Finally, enter the origin of the map: 0 0 0.

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

Note that 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).

Now do the same for the second, detached monomer. Similarly, one finds that the voxel at position x=19, y=12, z=13 is located near the center of the density of the detached monomer in file 3_s2.situs at a threshold level of 12. The threshold cutoff values were found by trial and error to prevent "leaking" of the density into neighboring monomers. Here we also shrink the maps about the found extracted volumes.

Now we can perform the vector quantization and fast docking of the attached monomer with the qrange utility. At the shell prompt, enter

The program returns a list of the 3! = 6 fits, ranked by their codebook vector rms deviation in the second column. The correlation coefficient is shown in the third column. The fourth column shows the internal permutation of the vectors that determine the superposition and is irrelevant here. Note that the correlation coefficient is within a very narrow numeric range, despite the significant differences in orientation between these fits, also the vector rmsd of the best two fits is within a close range.

Select the first fit (enter 1) and enter the output file name: 5_s1_1.dock.pdb. The positional and orientational accuracy of the fitting is estimated based on the statistical vector variability. The file 5_s1_1.dock.pdb now contains the atomic coordinates fitted to the low-resolution map. Also appended to the PDB file are three codebook vectors each for the atomic and low-resolution data, as well as their rms variability. Now, select the second fit and write it to the file 5_s1_2.dock.pdb.

Now do the same for the second, detached monomer. Don't forget to use a lower cutoff value of 12 for this lower-density map. Is there a unique best fit? What is the lowest rms deviation and the gap to the subsequent fits? You should find again two degenerate fits for k=3. Extract the best fit to the file 5_s2_1.dock.pdb and the second best to 5_s2_2.dock.pdb.

Note: As described in (Birmanns & Wriggers, 2007), the fast matching by codebook vectors can be improved by a post-processing with the fast correlation-based tool colacor. We have explained this workflow in the separate manual docking tutorial.

The following sequence of commands in the VMD text console (cf. VMD user guide) will load the two docked structures, 5_s1_1.dock.pdb and 5_s2_1.dock.pdb, and render them in cartoon representation, coded by color. The script then instructs VMD to render the density maps 3_mt.situs in green, 4_s1.situs in blue, and 4_s2.situs in purple:

The result should look like this:

The programs qpdb and qvol are specialized tools that perform vector quantization to generate coarse-grained point cloud models of the structural data. The matchpoint tool (new in Situs 2.5) supports the registration of point clouds even in cases where they are of unequal size. The usage of these routines is demonstrated on the above examples in part II of this tutorial.