Hi, I agree with Mark, the R factors suggest that your model is more or less complete. It does not seem like that DNA is bound to your protein. Without knowing more about your project, it’s hard to make specific suggestions, but here goes:
* Have you actually checked that there is DNA in the complex crystals? (There can be other reasons why you have different cell parameters and space groups with and without DNA. It may just be different crystal forms. Were they crystallized in the same condition?) If not, wash the crystals, run them on a gel and stain for DNA. * If you do have phosphate or sulphate in the crystallization condition, try to find a different condition without. It may well compete with DNA binding, since your affinity is low. * Does the protein recognize a specific DNA sequence? If not, it may bind to different parts of the DNA molecule that you give it, and you will have lots of problems getting a complex crystal structure because you don’t have a uniform complex. The affinity you state being rather on the low side, I would suspect that this may be an issue. * If you haven’t already, try different lengths of dsDNA. How much do you know about the footprint of the protein on DNA? How much do you know about the affinity of the protein for different DNA lengths? You need to find the sweet spot between affinity and length, i.e. the DNA should not be so short that it doesn’t bind well to the protein, but not so long that the complex won’t crystallize well because the DNA ends are floppy. You don’t say anything about the size of the protein, but 18 bp seems quite short to me, like that might be exactly the footprint of the protein on the DNA, but nothing more. Adding a few bp might help. * This wouldn’t be my immediate next step since your problem seems to be that there isn’t any DNA in the crystals (at least not bound to the protein) in the first place, and you obviously need to solve that problem first, but using DNA with sticky ends can help to form crystal contacts and generate better crystals. * Finally, are you sure about the space groups? Your R factors certainly suggest that you’re right, but P222 and P2 without any screw axes are very unusual. Also worth mentioning, although that doesn’t seem to be the issue here, is that DNA can cause pseudosymmetry that leads to space group misidentification, i.e. it looks like higher symmetry than it really is because the DNA is almost, but not really perfectly symmetrical. This can happen especially if you have a more or less palindromic DNA sequence, which is often the case with sequence-specific DNA-binding proteins. Best, Julia -- Dr. Julia Griese Assistant Professor Department of Cell and Molecular Biology Uppsala University BMC, Box 596 SE-75124 Uppsala Sweden email: julia.gri...@icm.uu.se phone: +46-(0)18-471 4982 http://www.icm.uu.se/structural-biology/griese-lab/ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Mark Roe <m....@sussex.ac.uk> Reply-To: Mark Roe <m....@sussex.ac.uk> Date: Friday, 17 December 2021 at 12:16 To: "CCP4BB@JISCMAIL.AC.UK" <CCP4BB@JISCMAIL.AC.UK> Subject: Re: [ccp4bb] the complex structure of protein and DNA Hi, With this resolution and R-factors, I would guess you are not missing that much – certainly not large bits of DNA. Do you have Phosphate or Sulphate in the crystallisation buffer? If so, you may just be seeing Phosphate/Sulphate where the DNA would bind. Cheers, Mark Dr S.M. Roe, X-Ray Facility Manager, Tel. (+44) 01273 678863 (Office) School of Life Sciences, Tel. (+44) 01273 872896 (X-Ray Lab) University of Sussex, Tel. (+44) 0782 5501579 (Mobile) Falmer, East Sussex. E-mail m....@sussex.ac.uk<mailto:m....@sussex.ac.uk> BN1 9RQ Web http://www.sussex.ac.uk/lifesci/roelab/ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Meiting Yang <meitingyang1...@163.com> Reply to: Meiting Yang <meitingyang1...@163.com> Date: Friday, 17 December 2021 at 08:04 To: "CCP4BB@JISCMAIL.AC.UK" <CCP4BB@JISCMAIL.AC.UK> Subject: Re: [ccp4bb] the complex structure of protein and DNA Dear Petr, Thank you very much for your reply! The complex structure was refined finally to 2.44 Å resolution with an Rwork of 23.7% and an Rfree of 27.4%. We didn't try automatic DNA building tools, I don't know much about this. Thank you very much for your advice, I'm going to study it. Could you please give some specific suggestions about automatic DNA building tools? Thank you very much. At 2021-12-17 15:01:44, "Petr Kolenko" <petr.kole...@fjfi.cvut.cz> wrote: >Dear Yang Meiting, >There are few things to know better about your structures first: >1) What is the resolution of the complex structure? >2) In what stage of structure refinement you are? Rwork/free would help. >3) Have you tried some automatic DNA building tools? >I am not surprised that you can see only a fraction of DNA.I guess, solvent >flattening may also decrease the visibility of this region. The only thing I >would suggest now, do not expect to see the whole DNA immediately. Just start >with the step-wise building if possible. The rest may appear in the later >stage of model building. >Best regards, >Petr >________________________________________ >From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Meiting Yang ><meitingyang1...@163.com> >Sent: Friday, December 17, 2021 7:29:54 AM >To: CCP4BB@JISCMAIL.AC.UK >Subject: [ccp4bb] the complex structure of protein and DNA > >Dear all， >We have determined two Crystal structures, with one is apo structure and the >other is a complex of the same protein with double-stranded DNA. In the >complex, the protein structure is clearly viible, but the DNA only can be seen >several phosphate groups. We want to know how do we get the complete DNA >structure. >The space group of the apo structure is P222, one asymmetric unit including >two protein molecules. The space group of the complex structure is P2, one >unit containing two protein molecules, 5 phosphate groups just situated near >one protein molecules. The binding ability of the DNA and the protein is about >1 μM. The DNA we used for crystallization is 18 bp double-stranded DNA, but >now only 5 phosphate groups can be observed. The crystal we have identified is >a complex rather than a monomer, the cell parameters of complexes and monomers >are different. >Here, we want to get some suggestions, to get the complexe that contain the >entire DNA structure. >Best regards. > > > > > > >________________________________ > >To unsubscribe from the CCP4BB list, click the following link: >https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 När du har kontakt med oss på Uppsala universitet med e-post så innebär det att vi behandlar dina personuppgifter. 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