Extending DNA in protein-DNA complex - issue with final steps

[alysiamandato]

Hello all, and thank you for this easy to use software!

I have extended my DNA duplex using the instructions in the forum post http://forum.x3dna.org/users-contributions/build-dna-bulges-and-extend-dna-duplex-at-both-terminals-via-3dna/msg921. This was very helpful. I can provide my code if necessary.

I am having an issue when I visualize, though. I followed the steps in the post and repeated the steps to ensure I did them correctly, but it seems that there are some breaks in the backbone structure. I am wondering if there is anyway to fix this using 3DNA or if anyone has ran into this problem before. I've attached an image of what I mean.

Please let me know if there is anything I can provide.

[alysiamandato]

I apologize - I was able to figure out a solution. I cannot figure out how to delete this post.

[xiangjun]

Hi,

Glad to know that you have figured out the issues. Instead of deleting your posts, could you please provide step-by-step procedure on how you solved the problem for the benefit of other users? The summary could also serve as a future reference for yourself.

Best regards,

Xiang-Jun

[alysiamandato]

I actually did not solve my problem, so I would appreciate any advice at all!

Introduction: I have a protein-DNA complex with a few base pairs missing at both 5' and 3' terminals. I extended my DNA duplex using the instructions in the forum post: http://forum.x3dna.org/users-contributions/build-dna-bulges-and-extend-dna-duplex-at-both-terminals-via-3dna/msg921. This was very helpful, and my duplex was extended properly, as indicated in stars in the code below.

The Issue: There were breaks in the backbone structure of my duplex. I found this article about the use of the program "Phenix": http://x3dna.org/highlights/restraint-optimization-of-dna-backbone-geometry-using-phenix. Using Phenix did not fix the backbone structure and it removed my protein from the PDB. I tried to simply replace my original DNA coordinates with the new coordinates in the PDB but this did not solve the issue. I am not sure where to go from here.

I am providing my code with all the steps I have taken:

Find_pair 4wlw_AB.pdb stdout
Frame_mol -1 ref_frames.dat 4wlw_AB.pdb 4wlw_ref_frames5.pdb
Fiber -b -seq=ttg ttg.pdb
Find_pair ttg.pdb ttg.bps
Frame_mol -3 ref_frames.dat ttg.pdb ttg_ref_frames5.pdb

Pymol: open 4wlw_ref_frames5.pdb and ttg_ref_frames5.pdb and see if they superimpose onto G of the original DNA
Cut the coordinates of TT and AA in ttg_ref5.pdb into 4wlw_ref_frames5.pdb and save as 4wlw_extend5.pdb

Find_pair 4wlw_extend5.pdb stdout
Frame_mol -25 ref_frames.dat 4wlw_extend5.pdb 4wlw_extend5_ref_frames3.pdb
Fiber -b -seq=ctaacct ctaacct.pdb
Find_pair ctaacct.pdb ctaacct.bps
Frame_mol -1 ref_frames.dat ctaacct.pdb ctaacct_ref_frames3.pdb

Pymol and cut coordinates again

*the DNA is now extended properly, but there are breaks in the backbone structure*

THIS STEP REMOVED MY PROTEIN
x3dna_utils cp_std BDNA
Find_pair 4wlw_extend53.pdb stdout | analyze stdin
Rebuild -atomic bp_step.par 4wlw_3dna.pdb

Phenix.geometry_minimization 4wlw_3dna.pdb min.params
Find_pair 4wlw_3dna.pdb stdout | analyze stdin
Find_pair 4wlw_3dna_minimized.pdb stdout | analyze stdin

*Both 4wlw_3dna.pdb and 4wlw_3dna_minimized.pdb are only the DNA and not the complex. The backbone is still not correct.

[alysiamandato]

I am replying to let others know that I have solved the problem by extending the DNA without the protein. Then, I overlay the protein-DNA complex with the extended DNA and remove the original DNA.

[xiangjun]

Thanks for letting us know that you have now solved your problem on extending DNA at both ends in a DNA-protein complex. Template-based model building of DNA-protein complexes is a persistent issue, as demonstrated in this thread, the one you referred to, and the most recent questions on the Composite module of Web 3DNA 2.0. The traditional 3DNA-based approach 'works', but it is tedious and error prone. The process is technically challenging for non-expert 3DNA users. There are also certain inherent limitations in the approach, as you experienced.

I have created a brand-new program for template-based modeling of nucleic acid structures, including DNA-protein complexes. Using your example, extending 4wlw on 5'-end by fiber B-DNA duplex TTG and 3'-end by CTAACCT can be easily achieved. It literally takes only a couple of minutes. See the attached PDB coordinate file and a schematic image. This novel modeling program is distributed as a module of DSSR Pro.

Best regards,

Xiang-Jun