Dear Ashok, There are many questions underlying your questions. A couple of things to check right off the bat:
(1) Do you actually know that your crystal still contains all of the DNA bp that you started with? Did you analyze the contents of your crystal by native PAGE, mass spec or other methods? (2) Yes, the number of base pairs do matter, especially if you have quasi-helical DNA stacking interactions that facilitate packing along one of the unit cell dimensions. For example, 12-bp is a little over a turn in contrast to 17-bp, which is a little more than 1.5 turns of a DNA B-form helix. (3) Are the crystal packing interactions in cases 1, 3, 4 and 5 similar? And, is there something unique about the packing in case 2, especially DNA-to-DNA packing? Make sure to display symmetry related molecules. That may explain why you can accommodate more molecules in the unit cell. (4) Compare the DNA sequences in cases 1-5 above and see if there is a pattern to the type(s) of nucleotides that are bound by protein in each case. It is hard to say more without knowing what the models look like but if your project is to investigate the DNA-protein interactions in more detail, the above-mentioned sorts of questions may be a place to start. Good luck! Raji On Sun, May 5, 2013 at 2:21 AM, ASHOK KUMAR Patel <[email protected]>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 > -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
