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
