DNA extending

[joannaziel@o2.pl]

Hi everyone,

I'm a new user of x3DNA, and I'm considering if there is any possibility to extend DNA from both ends ( both in 3' and 5')
Thank for your help.

J.

[xiangjun]

Welcome to join the 3DNA user community!

Yes, it should be possible, at least in principle. Please provide a concrete example of what you want to achieve, and we will start from there.

Xiang-Jun

[joannaziel@o2.pl]

I have DNA from 1DA0 from PDB database. There is a problem, because this DNA is really short: only 6 bp. I would like extend it to 10-12 bp, but from both 5' and 3' ends to keep symetic position of daunomicin. Type of new base pair doesn't matter for me.


Thank for your help

Julianna

[xiangjun]

Hi Julianna,

Thanks for your follow-up. In addition to serving your purpose of extending DNA from both ends, the PDB entry 1da0 is a good example to help illustrate one subtle point in 3DNA.

By default, 3DNA handles one structure at a time. When given an ensemble (as in NMR) with multiple structures in MODEL/ENDMDL delineated PDB format, 3DNA only analyzes the first model. Since 1da0 is symmetrical, with only one chain (and one daunomycin) in the asymmetric unit, you should download the coordinates in biological unit. The file name would be 1da0.pdb1 (the first and only biological assembly), where the two symmetric-related chains are separated by MODEL/ENDMDL pairs:

Code: [Select]

HETATM  158  O5' DM1 A   7     -12.846   8.039   6.403  1.00 23.07           O 
HETATM  159  C6' DM1 A   7     -14.267   6.866   7.856  1.00 18.53           C 
ENDMDL                                                                         
MODEL        2                                                                 
ATOM      1  O5'  DC A   1     -20.437  18.629   3.262  1.00 14.14           O 
ATOM      2  C5'  DC A   1     -21.455  17.960   2.453  1.00  8.69           C 
To proceed with 3DNA, make the ENDMDL record defective by adding a '#' (or a space) in front of it:

Code: [Select]

#ENDMDLLet's call the modified file 1da0-biou.pdb (attached), which is the starting point of following steps.

First, run find_pair to identify base pairs (bps), and generate the reference frame file (ref_frames.dat):

Code: [Select]

1da0-biou.pdb
1da0-biou.out
    2         # duplex
    6         # number of base-pairs
    1    1    # explicit bp numbering/hetero atoms
    1   13  0 #    1 | ...1>A:...1_:[.DC]C-----G[.DG]:...6_:A<...2  0.47  0.02  5.52  8.64 -4.21
    2   12  0 #    2 | ...1>A:...2_:[.DG]G-----C[.DC]:...5_:A<...2  0.45  0.20 11.07  8.54 -1.60
    3   11  0 #    3 | ...1>A:...3_:[.DA]A-----T[.DT]:...4_:A<...2  0.75  0.01  8.35  8.62 -3.81
    4   10  0 #    4 | ...1>A:...4_:[.DT]T-----A[.DA]:...3_:A<...2  0.75  0.01  8.35  8.62 -3.81
    5    9  0 #    5 | ...1>A:...5_:[.DC]C-----G[.DG]:...2_:A<...2  0.45  0.20 11.07  8.54 -1.60
    6    8  0 #    6 | ...1>A:...6_:[.DG]G-----C[.DC]:...1_:A<...2  0.47  0.02  5.52  8.64 -4.21
##### Base-pair criteria used:     4.00     0.00    15.00     2.50    65.00     4.50     7.80 [ O N]
##### 0 non-Watson-Crick base-pairs, and 1 helix (0 isolated bps)
##### Helix #1 (6): 1 - 6To extend the structure at the 3' end, run:

Code: [Select]

frame_mol -6 ref_frames.dat 1da0-biou.pdb 1da0-biou-ref6.pdbto get file 1da0-biou-ref6.pdb where the structure is in the reference frame of bp #6 (i.e., the last bp).

Then, use the fiber command to generate a B-DNA duplex of your preferred sequence:

Code: [Select]

fiber -seq=AATTTCCGGG frag3.pdbHere the sequence is AATTTCCGGG, and the resultant duplex is named frag3.pdb.

Now set frag3.pdb in the reference frame of the first bp, and name the resultant file frag3-ref1.pdb:

Code: [Select]

find_pair frag3.pdb stdout
frame_mol -1 ref_frames.dat frag3.pdb frag3-ref1.pdb
Now you can combine structures 1da0-biou-ref6.pdb and frag3-ref1.pdb, since they share a common reference frame, into a file named 1da0-biou-extend3.pdb (attached):

Code: [Select]

comb_str 1da0-biou-ref6.pdb frag3-ref1.pdb 1da0-biou-extend3.pdb
Display 1da0-biou-extend3.pdb using Jmol/PyMOL, you will see that 1da0 has been extended in its 3'-end (see figure below). Note the overlap of atoms at the joint bp. You need to remove the redundant part to get a sensible DNA structure.

The method outlined here may look complicated, but it is essential to get a clear understand of the underlying principles. Once passing through the hurdle, it won't be that difficult to automate the whole process to achieve what you want efficiently and reproducibly.

You can follow a similar procedure to extend 1da0 in the 5'-end. It would help if you work out the details and share back with the community.

HTH,

Xiang-Jun

[mar_alishahi]

Hi dear Xiangjun,

First of all, thank you for all your guides and hints....

I have a question and I'll be so grateful if you could help me!
I'm quite new  in using x3dna. I wanna extend an ideal dsdna file(attached) from its 3' and 5' ends. My PDB has originally 40 bps, I wanna to extend it to 200 bps. I've just used the method you described below, but when I typed: "frame_mol -40 ref_frames.dat dsdna.pdb dsdna_ref40.pdb", I received an error "error reading parameter file (number of base-pairs): dsdna.pdb"
Could you please help me?

Best,
Marzieh

[xiangjun]

Hi Marzieh,

Welcome to 3DNA! Could you be more specific with a reproducible example? It seems to me that you are missing a step: i.e., you need to have the file "ref_frames.dat" in the first place. This file can be produced by find_pair and analyze. The following steps would not have the problem you experienced:

find_pair dsdna.pdb stdout
#  this will create file "ref_frames.dat"
frame_mol -40 ref_frames.dat dsdna.pdb dsdna_ref40.pdb

Does this make sense to you now?

Xiang-Jun

[mar_alishahi]

Dear Xiang-Jun,

Thank you again. I've also used "find_pair" command at first, but without "stdout" at its end. Seems that it works now!

Best,

Marzieh

[mar_alishahi]

Dear Xiang-Jun,

I can extend my dsdna pdb file. but the problem is all of Hydrogen atoms are missed in my new pdb file. Although there were Hydrogen atoms in my premier pdb file. I just want to know if there is any way to built pdb file containing Hydrogen atoms.

Thanks in advance,

Marzieh

[xiangjun]

There are many software tools for adding hydrogen atoms given the coordinates of heavy atoms, as is commonly the case for x-ray crystal structures in the PDB. Google is your friend.

Xiang-Jun

[akalantzi]

Dear Xiangjun,

I am a new 3DNA user and I would like to ask you a question about DNA extending. I have a DNA with a missing part in the middle (PDB attached) and I would like to reconstruct this part (with AT pairs for example) by extending the other 2 parts. Is it possible? How could I also calculate how many interval pairs with a simple B-DNA conformation I need?

Best regards,
Alex

[xiangjun]

Hi Alex,

Thanks for using 3DNA and for posting your question on the Forum. I've checked your attached PDB file, and understand what you want to achieve. However, 3DNA currently does not provide a straightforward way for your purpose. Your DNA is irregular, and middle missing part is constrained by the two end parts. So a simple B-DNA fragment won't fit (well). Of course, you can have a try using the strategy outline in this thread, starting from one end. As for how many interval base pairs, you can just play with different numbers. Alternatively, the NAB tool from the Case Lab may be of help.

To me, the issue is sort of like 'loop modeling' in protein (and RNA). Conceptually, one could search for similar fragments in the PDB using the two end-base-pairs (O.DT41--Z.DA2 and X.DC19--Y.DG24) as anchor points. If necessary, this may well become an area for the further development of 3DNA.

Xiang-Jun

[akalantzi]

Dear Xiangjun,

Thank you very much for your answer! Using your instructions above, I build something very close to the DNA that I wanted!

Best regards,
Alex