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Messages - Randy Bin Lin

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this is a step-by-step recipe about how to use 3dna to build a dna bulge based on a PDB structure and extend its sequence at both terminals. this is a collection of two posts http://forum.x3dna.org/general-discussions/build-dna-bulge-via-3dna/ and http://forum.x3dna.org/general-discussions/extend-dna-duplex-at-both-terminals/ with much help from Xiang-Jun. Kudos to you!

here's the objective: PDB 1AX6 (http://www.rcsb.org/pdb/files/1AX6.pdb.gz) is a dna bulge of -2 deletion with its sequence as CTCGATGCCATC. I wish to build a similar dna bulge with CT at 5' terminal and AC at 3' terminal as extra. two things worth noting: the first is to preserve the backbone conformation (esp. the bulge region as solved by NMR in the PDB file). the second is essentially how to extend dna duplex (B-DNA in my case). I am sure there are many other ways to do the job (let me know if you know what they are). here's the way I used 3dna to achieve my goal.

1. use find_pair to generate ref_frames.dat for each base pair in 1ax6:
find_pair 1ax6.pdb stdout

2. re-orient 1ax6 based on the reference frame of its first 5' base pair, generate canonical B-DNA of CTC, re-orient it based on the reference frame of its last 3' base:
frame_mol -1 ref_frames.dat 1ax6.pdb 1ax6_ref_frames5.pdb
fiber -b -seq=ctc ctc.pdb
find_pair ctc.pdb ctc.bps
frame_mol -3 ref_frames.dat ctc.pdb ctc_ref_frames5.pdb

3. after step 2, 1ax6_ref_frames5.pdb and ctc_ref_frames5.pdb should superimpose based on the common base pair. cut the coordinates of CT in ctc_ref_frames5.pdb into 1ax6_ref_frames5.pdb and name the new pdb file as 1ax6_extend5.pdb. our sequence has two extra base pairs of CT at 5' terminal with canonical B DNA conformation matching that in 1ax6

4. do the same for the 3' terminal
find_pair 1ax6_extend5.pdb stdout
frame_mol -12 ref_frames.dat 1ax6_extend5.pdb 1ax6_extend5_ref_frames3.pdb
fiber -b -seq=cac cac.pdb
find_pair cac.pdb cac.bps
frame_mol -1 ref_frames.dat cac.pdb cac_ref_frames3.pdb

5. the newly extended pdb at both 5' and 3' terminal is 1ax6_extend53.pdb

6. generate bp_step.par by treating the whole structure as a continuous single helix, this way the bulge of 2 bases is preserved.
find_pair -s 1ax6_extend53.pdb stdout | analyze stdin

7. rebuild dna structure with extended terminals and dna bulge of -2 deletion:
x3dna_utils cp_std BDNA
rebuild -atomic bp_step.par bulge-2-full-3dna.pdb

the dna bulge I meant to build has slightly different sequence from 1ax6. just use mutate_bases in 3dna package to do the job. it's omitted here. but should be easy to do. do that before the first step.

hope it helps! feel free to contact me at randybinlin@gmail.com if you have any questions. or post to the forum, Xiang-Jun knows better and he should be able to answer them fairly quickly.

Randy Bin Lin

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General discussions (Q&As) / Re: extend dna duplex at both terminals?
« on: June 03, 2012, 12:30:26 pm »
thanks, Xiang-Jun. I will do that.

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General discussions (Q&As) / Re: extend dna duplex at both terminals?
« on: June 03, 2012, 11:25:31 am »
The point is: one should use the 'ref_frames.dat' file corresponding to the structure to be reoriented.

xiang-jun, it worked like a charm! thanks a lot.

Can you elaborate a little more what it really means in ref_frames.dat for each base pair? My guess is for different ref_frames.dat somehow they share a common coordinate system or something like that. that is why when one 'frame_mol' a duplex based on its own ref_frames.dat, different molecules align very well against each other.

Randy Bin Lin

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General discussions (Q&As) / Re: extend dna duplex at both terminals?
« on: June 02, 2012, 11:37:07 pm »
1ax6 is available from this link: http://www.rcsb.org/pdb/files/1AX6.pdb.gz

with that, everything is reproducible with 3DNA commands:

find_pair 1ax6.pdb stdout
frame_mol -1 ref_frames.dat 1ax6.pdb 1ax6_ref_frames.pdb
fiber -b -seq=ctc ctc.pdb
frame_mol -3 ref_frames.dat ctc.pdb ctc_ref_frames.pdb

load the output pdbs file into visualization package of choice, it's in the attachment. green stick is 1ax6, cyan is ctc. I think the superimposition could be closer.

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General discussions (Q&As) / Re: extend dna duplex at both terminals?
« on: June 02, 2012, 09:16:09 pm »
Xiang-jun, yeah, that's my bad. -3 should be the correct one to use.

now it looks close, but the sugar and base don't superimpose very well. since we use the same reference frame, they are supposed to match well for the base pair, right?


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General discussions (Q&As) / Re: extend dna duplex at both terminals?
« on: June 02, 2012, 08:16:37 pm »
xiang-jun, here's what I did following your suggestions:

find_pair 1ax6.pdb stdout
frame_mol -1 ref_frames.dat 1ax6.pdb 1ax6_ref_frames.pdb
fiber -b -seq=ctc ctc.pdb
frame_mol -1 ref_frames.dat ctc.pdb ctc_ref_frames.pdb

I used the first base pair at 5' end before extension as reference frame.

after visualizing both output molecules, they don't seem to share the common 5' CG base pair. What did I do wrong here?

Randy Bin Lin

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General discussions (Q&As) / Re: extend dna duplex at both terminals?
« on: June 02, 2012, 09:42:26 am »
Xiang-Jun, sorry for my post being too general. for instance, I have a sequence of DNA duplex, with coordinates available:

CTCGATGCCATC

I want to extend the sequence at both ends by 2, turning it into

CTCTCGATGCCATCAC

hope to preserve the backbone of the common sequence and add the two base pairs following alpha helix (say canonical B conformation).

thank you for your advice.


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General discussions (Q&As) / extend dna duplex at both terminals?
« on: June 01, 2012, 11:37:11 pm »
I have a DNA duplex with coordinates. But there are two base pairs missing at both terminals. I hope to use 3DNA to add the missing base pairs while they still maintain the alpha helix of B-DNA. Is it possible to do it via 3DNA?

Randy Bin Lin

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General discussions (Q&As) / Re: build dna bulge via 3dna?
« on: May 31, 2012, 02:00:39 pm »
xiangjun, thank you for the suggestions. I will definitely give it a try. I will let you know how it goes.

Randy Bin Lin

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General discussions (Q&As) / build dna bulge via 3dna?
« on: May 30, 2012, 08:19:06 pm »
I have been trying to build a DNA duplex with bulges with -1, -2 up to -3 deletion, such as in PDB 1AX6 (-2 deletion). I searched online, there is no protocol detailing how to build them in silico with desired DNA sequence. 3DNA and NAB are the two nucleic acid manipulation tool I have been using. but it is not obvious how to do it in both of them. Any advices on that? thanks!

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Created and maintained by Dr. Xiang-Jun Lu [律祥俊], Principal Investigator of the NIH grant R01GM096889
Dr. Lu is currently affiliated with the Bussemaker Laboratory at the Department of Biological Sciences, Columbia University.