[ccp4bb] Missing DNA density in Protein DNA complex structure
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
Re: [ccp4bb] Missing DNA density in Protein DNA complex structure
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
Re: [ccp4bb] Missing DNA density in Protein DNA complex structure
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 ashok...@gmail.comwrote: 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
Re: [ccp4bb] Missing DNA density in Protein DNA complex structure
Because your protein binds non-specifically, it could be an interesting case of static disorder where the DNAs are making a pseudo-continuous helix through the crystal regardless of sequence, and 12 bp/turn happens to fit nicely into a generic lattice. How does the density for the individual bases look? You could test that idea by substituting one T with BrdU. I suspect you'll see multiple partially- occupied anomalous peaks in your asymmetric unit for those longer DNAs that pack like 12mers. Here's a reference where an RNA duplex did a similar thing: Crystal structures of two plasmid copy control related RNA duplexes: An 18 base pair duplex at 1.20 A resolution and a 19 base pair duplex at 1.55 A resolution.http://www.ncbi.nlm.nih.gov/pubmed/10555960 Klosterman PS, Shah SA, Steitz TA. Biochemistry. 1999 Nov 9;38(45):14784-92. PMID: 10555960 ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edumailto:pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Raji Edayathumangalam [r...@brandeis.edu] Sent: Sunday, May 05, 2013 7:39 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Missing DNA density in Protein DNA complex structure 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 ashok...@gmail.commailto:ashok...@gmail.com 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
Re: [ccp4bb] Missing DNA density in Protein DNA complex structure
Hi Ashok, You say your DNA is palindromic - have you looked at the possibility that the longer DNAs are actually folded on themselves rather than duplexes of two separete oligos? Depending on the location of the centre of your palindrome and on interactions with adjacent molecules, this could explain why you have shorter duplexes for longer molecules, and possibly some of your other observations. Opher
