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Questions and answers => RNA structures (DSSR) => DSSR-NAR paper => Topic started by: xiangjun on July 08, 2015, 08:41:15 pm

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Title: Figure 4 -- analysis of the env22 twister ribozyme (4rge)
Post by: xiangjun on July 08, 2015, 08:41:15 pm
(http://x3dna.bio.columbia.edu/dssr-nar/fig4-twister-ribozyme-4rge/fig4-4rge-twister-ribozyme.png)

Quote
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 (http://x3dna.bio.columbia.edu/dssr-nar/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) (http://forum.x3dna.org/dssr-nar-paper/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:
(http://x3dna.bio.columbia.edu/dssr-nar/fig4-twister-ribozyme-4rge/4rge-A-ok-full.png)
(http://x3dna.bio.columbia.edu/dssr-nar/fig4-twister-ribozyme-4rge/4rge-A-ok-jct.png)
(http://x3dna.bio.columbia.edu/dssr-nar/fig4-twister-ribozyme-4rge/4rge-nested-jct.png)
(http://x3dna.bio.columbia.edu/dssr-nar/fig4-twister-ribozyme-4rge/4rge-bifurcated.png)

Funded by the NIH R24GM153869 grant on X3DNA-DSSR, an NIGMS National Resource for Structural Bioinformatics of Nucleic Acids

Created and maintained by Dr. Xiang-Jun Lu, Department of Biological Sciences, Columbia University