Secondary Structure Element Identification:

SSEHunter and SSEBuilder

SSEHunter, available with AIRS toolkit, is designed for the simultaneous detection of alpha helices and beta sheets in subnanometer resolution cryoEM structures. This algorithm uses three separate scoring metrics; skeletonization, cross correlation and local geometry calculations, to annotate a volumetric density. SSEHunter produces the following result files: a helix correlation volume map, helix skeleton, sheet skeleton, overall skeleton (all MRC volumes), pseudoatom score (PDB file) and a log file (text). SSEHunter is available from the command line or in UCSF’s Chimera.

SSEBuilder, also part of the AIRS toolkit, is tool for the rapid identification and annotation of SSEHunter results. SSEBuilder uses the output of SSEHunter and UCSF’s Chimera for the annotation of subnanometer resolution cryoEM structures. It requires only the output score file from SSEHunter and produces VRML output for sheets, helices and coils, or optionally can produce PDB-style helices.

SSEHunter and SSEBuilder are part of the AIRS (Analysis of Intermediate Resolution Structures) Toolkit and distributed with EMAN 1.7+. In addition to EMAN and its dependencies, AIRS also requires Numeric Python and Scientific Python. The GUI version of SSEHunter and SSEBuilder also require UCSF’s Chimera version 1.2065+ (production release suggested).

For AIRS installation instructions, please visit:

http://ncmi.bcm.tmc.edu/software/AIRS/doc_html.

Using SSEHunter from the command line:

ssehunter3.py <input mrc> <apix> <resolution> <threshold>

input mrc: an even, cubic MRC file (maps 2ⁿ x 2ⁿ x 2ⁿ work best, should have at least 10-20% padding at the edges of the volume)

apix: Angstroms per pixel, should be less than 2

resolution: map resolution, helix identification works at resolutions better than 10Å, while sheet identification may vary

threshold: the isosurface value that most accurately illustrates the features of the map, changing this parameter may effect identification of SSEs

advanced options:

coeff: the helix coefficient file. This takes the most time to compute but only needs to be calculated once for each map.

atoms: the pseudoatoms file. Default pseudatoms are built using the AIRS program pseudoatoms.py, but any generic PDB style atoms file may be used.

coeffwt: weight for helix correlation in scoring (default=1)

skeletonwt: weight for skeleton in scoring (default=1)

pseudoatomwt: weight for local geometry in scoring (default=1)

Using SSEHunter in Chimera:

The following directions will demonstrate how to run SSEHunter in Chimera. The results of SSEHunter will produce a psuedoatoms score file which can be visualized and interpreted using Chimera and SSEBuilder. Note: The following assumes that the necessary dependencies have been installed and that AIRS has been correctly setup in Chimera.

0. Open volume data set. Adjust the isosurface threshold to the desired level.(Fig. 1)

1. Select SSEHunter from the Tools>AIRS> menu. Additionally, you may use the AIRS dialog window, also located in Tools>AIRS>, to access SSEHunter and SSEBuilder.

2. Select the appropriate input files. If you do not have the files or do not know what they mean, select “None” and these will be generated for you. Enter the remaining information about the map. Threshold should be the isosurface threshold value specified in step 0. (Fig. 2)

3. Press Apply. This will take a few minutes depending on parameters and map size. Typical applications of SSEHunter will take ~3 minutes on an 8Å resolution map that is 128x128x128 voxels in size. Chimera will not respond until completion of SSEHunter.

4. After completion, the pseudoatom score file will be loaded by default. The origin of the density map may need to be changed in order to agree with the results. The new origin of the target density map is (0,0,0).

Figure 1	Figure 2

Visualizing SSEHunter results:

This section explains how to render and color the psuedoatoms generated using SSEHunter. This will prepare you for selecting and annotating the individual SSES. Note: The following assumes that SSEHunter was completed and the results were loaded.

5. Select the results file. There are many ways to do this; one way is to use Favorites>Model Panel. Click on the score file in the left window and then click “Select” in the right menu of Model Panel. (Fig. 3)

6. In the Chimera viewer, press the lower right button, which should say “X atoms and Y bonds”. This will open a selection window.

7. Select atoms in the “Inspect” menu and set “atom style” to sphere.

8. Select bonds in the “Inspect” menu and set “displayed” to false. Unselect the SSEHunter results, which can be done by holding down the control key and pushing the left mouse button in an empty area of the Chimera viewer window.

9. In model panel, select “Render by attribute”. A new window should appear. Select the SSEHunter results in the Models menu. (Fig. 4)

10. Under the “Attribute” menu, select B-factor.

11. Adjust the sliders such that the blue bar is all the way to the left (negative, beta sheets) and the red bar is all the way to the right (positive, helix). The middle grey bar should be adjusted so that it is close to zero. Click Apply. This will color the pseudoatoms in the score file so that the helix like atoms are red and the sheet atoms are blue. This is a continuous scoring function so the intensities are directly proportional to the reliability of the prediction. Also, you may need to adjust the transparency factor of the volume to see the results clearly. (Fig. 5)

Figure 3	Figure 4

Annotating the SSEHunter results with SSEBuilder:

This section explains how to annotate individual SSEs and create the visualization/output using SSEBuilder.

12. Select SSEBuilder from the Tools>AIRS menu.

13. Select the score file in the dropdown menu.

14. At this point, individual secondary structure elements may be selected. Select individual pseudoatoms or groups of pseudoatoms using control+left_click in the Chimera viewer window. Holding down while selecting atoms (shift+ control+left_click) will allow add pseudoatoms to the current selection. Selected atoms will be highlighted with a green outline.

15. When a single element is selected, set the type of SSE identified (helix, sheet, coil) and give this element an id (number, letter, name, etc…). Press Add to add it to the list of elements on the left. (Fig. 6)

16. Repeat steps 15 and 16 until completed.

17. By selecting identified SSEs in the left window of SSEBuilder, the corresponding atoms selected may again be highlighted by pressing “Show”. Identified SSEs in the list may also be deleted by selecting the SSE in the left window and pressing “Delete”.

18. Output files in SSEBuilder will be generated as VRML by default. PDB-style helices can be generated by selecting PDB under “Helix style” in SSEBuilder.

19. Press “Build”. The SSEs described in the left window of SSEBuilder will be generated. (Fig. 7)

Figure 5	Figure 6	Figure 7