Figure 4 -- analysis of the env22 twister ribozyme (4rge)

[xiangjun]

Figure 4: DSSR discloses complexity in the folding of the env22 twister ribozyme not apparent in the two-armed tertiary structure (chain A, PDB id: 4rge (43)). (A) The software automatically detects the long helical arm with five coaxially stacked stems and the short single-stemmed arm of the molecule. Failing to account for the pseudoknots within the structure leads to a characterization of the molecule very different from its real organization. When pseudoknots are omitted, the RNA appears to form a simplified [2,1,3] three-way junction as shown in both planar (B) and linear (C) secondary structure diagrams. In reality, the DSSR-derived dot-bracket notation points to a double-pseudoknotted structure (D) with two types of brackets distinguishing the pseudoknotted pairs (matched [] and {}), and uncovers a novel [4,2,2,0,1,3,0,0,1,1] ten-way junction loop (D,E). The junction, which can be traced by following the arrows along the red arcs and bases (starting from U3, marked with *) in D, contains both ends of four of the six stems and follows a supercoiled pathway in 3D (Supplementary Figure S5). In contrast, without consideration of pseudoknots (F), the junction forms a simple relaxed circle (Supplementary Figure S5). DSSR also detects three previously ignored base pairs that help to anchor the consecutive A-minor motifs reported in the literature (43) (G). U41 pairs with A42 and A43 through bifurcated hydrogen bonding, as well as with A26 (Supplementary Figure S4C,D). Moreover, U41 and A42 constitute a UpA dinucleotide platform, and in combination with G25 and A26, create a unique network of eight interacting nucleotides (G). All eight nucleotides are involved in the ten-way junction loop (labeled red in (E)).

Here is the tarball (fig4-twister-ribozyme-4rge.tar.gz) with the script and all related data files.
The content of the full script (named tasks) is shown below. Please see also notes for "Figure 2 -- analysis of the yeast phenylalanine tRNA (1ehz)".

Code: Bash

1. # Step #1 -- reorient the twister ribozyme vertically
2. pdb_frag A 1:56 4rge.pdb 4rge-A.pdb
3. x3dna-dssr -i=4rge-A.pdb -o=4rge-A.out --more --prefix=4rge-A
4.  
5. # To get the result illustrated in panel D, load '4rge-A-2ndstrs.ct'
6. # or '4rge-A-2ndstrs.dbn' into VARNA to draw the linear secondary
7. # structure diagram, exported as .svg for annotation in Inkscape.
8.  
9. # Extract the two helical axes from 4rge-A.out to file: 4rge-A.rot1
10. # then reorient the structure vertically: 4rge-A.rot2
11. rotate_mol -t=4rge-A.rot1 4rge-A.pdb 4rge-A-rot1.pdb
12. rotate_mol -r=4rge-A.rot2 4rge-A-rot1.pdb 4rge-A-ok.pdb
13.  
14. # Step #2 -- get the cartoon-block representation with the two
15. #            ls-fitted helical axes.
16. x3dna-dssr -i=4rge-A-ok.pdb --helical-axis -o=temp
17. \mv dssr-helicalAxes.pdb 4rge-A-ok-helices.pdb
18. x3dna-dssr -i=4rge-A-ok.pdb --block-file -o=4rge-A-ok-blocks.r3d
19.  
20. # Step #3 -- simplified representation of the [4,2,2,0,1,3,0,0,1,1]
21. #            10-way junction in 3D -- panel E
22. #   note the '--raw-xyz' option: it keeps the original coordinates
23. x3dna-dssr -i=4rge-A-ok.pdb --raw-xyz --simple-junction -o=temp
24. \mv dssr-simplifiedJcts.pdb 4rge-A-ok-jct.pdb
25. # see file: 4rge-A-ok-jct.pml
26. pymol -qkc 4rge-A-ok-jct.pml
27. convert -trim +repage -border 10 -bordercolor white 4rge-A-ok-jct-pymol.png 4rge-A-ok-jct.png
28.  
29. # see file: 4rge-A-ok-full.pml (cartoon-block with the schematic
30. # junction overlaid) -- panel A
31. pymol -qkc 4rge-A-ok-full.pml
32. convert -trim +repage -border 10 -bordercolor white 4rge-A-ok-full-pymol.png 4rge-A-ok-full.png
33.  
34. # Step #4 -- remove pseudoknots to get a fully nested structure. It now
35. #            has only a [2,1,3] 3-way junction -- panels B, C, and F
36. \cp 4rge-A-ok.pdb 4rge-nested.pdb
37. x3dna-dssr -i=4rge-nested.pdb --nested --raw-xyz --simple-junction --prefix=4rge-nested -o=4rge-nested.out
38.  
39. # The planar (panel B) and linear (panel C) secondary structure
40. # diagrams are produced by loading '4rge-nested-2ndstrs.ct' or
41. # '4rge-nested-2ndstrs.dbn' into VARNA, exported as .svg, and
42. # annotated with Inkscape.
43.  
44. # see file: 4rge-nested-jct.pml -- panel F
45. pymol -qkc 4rge-nested-jct.pml
46. convert -trim +repage -border 10 -bordercolor white 4rge-nested-jct-pymol.png 4rge-nested-jct.png
47.  
48. # Step #5 -- bifurcated U-A pairs in a network of 8 nucleotides
49. pdb_frag A 13:14 A 25:26 A 36 A 41:43 4rge-A-ok.pdb 4rge-bifurcated.pdb
50. x3dna-dssr -i=4rge-bifurcated.pdb --block-file -o=4rge-bifurcated.r3d
51. # see file: 4rge-bifurcated.pml -- panel G
52. pymol -qkc 4rge-bifurcated.pml
53. convert -trim +repage -border 10 -bordercolor white 4rge-bifurcated-pymol.png 4rge-bifurcated.png

Here are the images generated from the above script: