How is the DNA packed in the crystal? Coaxially stacked pseudo-infinite
helices?
--paul
On 05/05/2013 02:21 AM, ASHOK KUMAR Patel wrote:
Hi all,
I am working on a DNA binding protein (mol wt around 30 kDa), which
binds to Duplex DNA in a non-specific sequence manner. The structure
has been published with 12 base pair duplex DNA.
I am trying to understand the DBD protein DNA interaction even more by
choosing different lengths and sequences. In Co-crystallization I used
16, 18, 20 and 22 bases palindromic sequence random DNA bases
(purchased from IDT), annealed and used in crystallization.
I collected some diffraction data on NSLS recently at around 2.1 Å and
2.7 Å. But, when I did data processing, model building and refinement.
I am getting strange results as depicted in the table..
S N
a=
b=
c=
α=
β=
γ=
Space group
No of molecules in asymmetric unit
Length of DNA
Used for crystallization
Duplex DNA found in structure
Resolution
1
38.67
61.43
76.77
90.00
104.17
90.00
P 1 21 1
1
12 base
12 base
2.0 Å
2
86.076
57.099
99.493
90.00
103.90
90.00
P 1 21 1
2
17 base
17 base
3.05 Å
3
37.855
61.668
76.601
90.00
102.24
90.00
P 1 21 1
1
*18base*
*12 base*
2.1 Å
4
37.073
61.864
78.242
90.000
100.810
90.000
P 1 21 1
1
*20 base*
*12 base*
2.7 Å
5
*20 base*
*12 base*
3.1
My question and concerns are as:
1. How I am getting almost identical Cell parameters with different
length of DNA (row 3 and 4) to the first row?
2. Why I am getting only 12 base duplex DNA instead of 18mer or 20 mer
I used in crystallization.
3. Is anything has to do with ODD and EVEN duplex DNA. When odd 17
base duplex was used, it has 17 bases in the structure, while in all
EVEN case of 18, 20 or 20, only 12 bases in the structure.
4. The complex having odd DNA length 17 has 2 molecules in ASU while
all other has 1.
Why only 12 mer DNA density in the complex? Why I am missing 6 or 8
bases in the density? How can we explain the missing DNA in the structure?
I will appreciate any kind of explanation and suggestions.
Thanks
Ashok
--
Ashok kumar patel
Department of Biophysics
Johns Hopkins University
Baltimore, MD 21218
