Figure 6 -- analysis of the CRISPR Cas9-sgRNA-DNA ternary complex (4oo8)

[xiangjun]

Figure 6: DSSR applies to RNA-DNA hybrid structures, such as the CRISPR Cas9-sgRNA-DNA ternary complex (chains B and C, PDB id: 4oo8 (47)). (A) The software identifies five helices (depicted by gray lines) and six stems (annotated) in the structure. The longest helix includes the RNA-DNA hybrid duplex (S1, depicted by intertwined gold-red backbone tubes) and the repeat:anti-repeat RNA stem (S2). (B) The secondary structure diagram, derived using DSSR, shows that the hybrid structure does not form a ‘closed’ junction loop. DSSR classifies the CUAG hairpin loop as a diloop (instead of a tetraloop) because the C and G form a Watson-Crick pair that closes the loop, leaving only a two-nucleotide (UA) loop segment. (C) Comparison of the CUAG diloop (center) with the UUGA diloop from a yeast Vts1p-RNA hairpin complex (referred to as part of a pentaloop(59), left) shows the remarkable similarity between the two loops despite the large difference in their base sequences. The CUAG diloop also shares common features with the NMR solution structure of the classic CUUG diloop(60) (often called a tetraloop, right), including the flipped out second position U and the stacking of the closing C–G pair over a neighboring G–C pair. The diloops differ, however, in terms of the inter-pair twist angle at the GpC dinucleotide step. These three images are oriented in the frames of the purines stacked above the terminal nucleotides (A9-left; G58-middle; G8-right) with the minor-groove edges facing the viewer.

Here is the tarball (fig6-CRISPR-Cas9-4oo8.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 CRISPR-Cas9 RNA-DNA hybrid into the most extended view
2. pdb_frag B 1:97 C 1:20 4oo8.pdb 4oo8-nts.pdb
3. rotate_mol 4oo8-nts.pdb temp
4. rotate_mol -r=4oo8.rot temp 4oo8-ok.pdb
5. x3dna-dssr -i=4oo8-ok.pdb --prefix=4oo8-ok -o=4oo8-ok.out
6.  
7. # To get the result illustrated in panel B, load '4oo8-ok-2ndstrs.ct'
8. # or '4oo8-ok-2ndstrs.dbn' into VARNA to draw the planar secondary
9. # structure diagram, exported as .svg for annotation in Inkscape.
10.  
11. # Step #2 -- get the cartoon-block representation, with fitted helices
12. x3dna-dssr -i=4oo8-ok.pdb --helical-axis -o=temp
13. \mv dssr-helicalAxes.pdb 4oo8-ok-helices.pdb
14. x3dna-dssr -i=4oo8-ok.pdb --block-file -o=4oo8-ok-blocks.r3d
15. # see 4oo8-ok.pml -- panel A
16. pymol -qkc 4oo8-ok.pml
17. convert -trim +repage -border 10 -bordercolor white 4oo8-ok-pymol.png 4oo8-ok.png
18.  
19. # Step #3 -- comparison of diloops (panel C)
20. pdb_frag B 5:11 2f8k.pdb temp
21. x3dna-dssr -i=temp --frame=B.9:edge -o=temp2
22. rotate_mol -r=rotxy-180 temp2 2f8k-uuga.pdb
23. x3dna-dssr -i=2f8k-uuga.pdb --block-file -o=2f8k-uuga-blocks.r3d
24. # see file: 2f8k-uuga.pml
25. pymol -qkc 2f8k-uuga.pml
26. convert -trim +repage -border 10 -bordercolor white 2f8k-uuga-pymol.png 2f8k-uuga.png
27.  
28. pdb_frag B 54:60 4oo8-ok.pdb temp
29. x3dna-dssr -i=temp --frame=B.58:edge -o=temp2
30. rotate_mol -r=rotxy-180 temp2 4oo8-cuag.pdb
31. x3dna-dssr -i=4oo8-cuag.pdb --block-file -o=4oo8-cuag-blocks.r3d
32. # see file: 4oo8-cuag.pml
33. pymol -qkc 4oo8-cuag.pml
34. convert -trim +repage -border 10 -bordercolor white 4oo8-cuag-pymol.png 4oo8-cuag.png
35.  
36. ex_str -1 1rng.pdb model1.pdb
37. pdb_frag A 4:9 model1.pdb temp
38. x3dna-dssr -i=temp --frame=A.8:edge -o=temp2
39. rotate_mol -r=rotxy-180 temp2 1rng-cuug.pdb
40. x3dna-dssr -i=1rng-cuug.pdb --block-file -o=1rng-cuug-blocks.r3d
41. # see file: 1rng-cuug.pml
42. pymol -qkc 1rng-cuug.pml
43. convert -trim +repage -border 10 -bordercolor white 1rng-cuug-pymol.png 1rng-cuug.png

Here are the images generated from the above script: