Tutorial - Advanced Interpretation of Volumetric Data

Sculptor can detect alpha-helices and other filaments in 3D EM maps (Rusu and Wriggers, 2012, Rusu et al., 2012). Maps can first be denoised using our DPSV filter (Starosolski et al., 2012).

1. Work flow for denoising maps using DPSV

Here is the HIV virion example from our manuscripts (Starosolski et al., 2012, supplemtary data 1 in Rusu et al., 2012):

  • Download the map file 1155_inverted_int16.map (original source: EMDB ID 1155).
  • Load the map using "File"->"Open" menu, you should now see a new document being listed in the document list in the top-left part of the main Sculptor window. Suggested contour level: 21
  • Select the map by right-clicking on the previously opened document.3C92_AI.pdb
  • Chose from menu "Volume" -> "DPSV Fliter" and next set the parameters in popup window as follows: 
  • select "6" for 6-neighborhood model. (3D-neighborhood) 
  • set "Beta" to value "0.0005",  
  • leave the rest of parameters at default: "Mask Size" = 5, and "Path Length" = 2. 
  • Click "OK" for staring the calculation ( it will take a while, depending on CPU speed and number of cores). 
  • When the filtration is done you will notice the asterisk before the selected document, indicating that this document was changed.

2. Work flow for detecting alpha helices in intermediate resolution maps

The following video (shown at half size) descibes the necessary steps in tracing alpha helices. You can download the video here for full size viewing. You can also download the map file emd_1740_AI.situs and the PDB file 3C92_AI.pdb to follow the video along.

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Most of the VolTrac parameters shown in the video are intuitive, but  for sake of clarity we illustrate here the geometry of the template used in the tracing: 

3C92_AI.pdbtemplate dimensions

3. Work flow for visualizing and manually editing the traces

A frequently asked question is how to visualize the VolTrac traces in Sculptor. Here are four figures that show the procedure. Click on the thumbnails to see the full size figures:

1. Load PDB load PDB
2. Show Tubes  show tubes
3. Increase Radius increase radius
4. Output output

Often, a user may wish to manually edit the traces to eliminate false positives (e.g. alpha helices placed in beta sheet regions). The manual editing and merging of the results is described in the following series of screenshots (click on the thumbnails to see the full size figures):

5. Show Tubes show tubes
6. Select Extract  select extract
7. Extract Tubes extract tubes
8. Hide Results make invisible
9. Change Rendering change rendering
10. Merge Highlighted merge results

4. Work flow for tracing filaments in tomograms

actin filaments in filopodium
If you are interested in tracing filamentous density in 3D maps of cellular structures from tomography or other in vivo imaging techniques, we recommend to follow first the tutorials in steps 2 and 3 above to learn the basic functionality of the VolTrac algorithm.  Then, refer to Rusu et al., 2012 which describes in the main text the parameters and steps taken in the tracing of actin filaments in a filopodium protrusion. Supplementary data files 2-6 of this paper comprise snapshots of this workflow.  Supplementary data file 4 lists the steps taken prior to VolTrac, whereas the VolTrac parameters are given in the main text of the paper. The final results shown in Fig. 3B of the paper are provided in supplementary file 6 for comparison purposes. You can visualize these traces as described for alpha-helices under step 3 above.