That looks very hopeful. Do you know how the two molecules are related to each other? Do they form a dimer or have some interesting relationship? I would keep on improving the model - possibly use COOT to average the density and build into that for a start. I presume there are still side chains to mutate and fit, and other corrections to make? You can then save molecule 1, and fit the molecule 1 model back over molecule 2 and save that again. You need to edit the two chains into one PDB in some way and refine again. You might begin to see DNA at a low sigma level in those maps..
(By the way - I dont understand "2) mutate the protein sequence back to my protein and refine the solution in Coot". Chainsaw should output the model with the correct sequence but with atoms missing where there are changes. You would need to rebuild that but not change the sequence? ) And you would hope then that PHASER might find your 2 protein chains with a very high LLG then perhaps place the DNA. but in my limited experience DNA is usually more mobile and therefore harder to pick up than protein. good luck Eleanor On Mon, 25 Jul 2011 15:38:02 +0800, Hubing Lou <louhub...@gmail.com> wrote: > Hi, > > Thanks for those who replied to this thread. > I have been trying all the means that people suggested: search protein > alone, DNA alone. However, both not working out. > One thing Ray Brown suggested "MR works if the molecules have identical > sequences". So I just played around with the following way: 1) use the > chainsaw editted model in MR; 2) mutate the protein sequence back to my > protein and refine the solution in Coot; 3) use the partially refined > protein-DNA as the new search model and run Phaser again then I get the > following results for the two copies in ASU: > RFZ=6.5 TFZ=10.7 LLG=903 RFZ=6.2 TFZ=17.4 LLG=1130. > After one round of rigid body and restraint refinement in Refmac5, the Rfac > and Rfree drops from 0.50/0.51 to 0.46/0.51. I did not refine the DNA > sequences yet. > > I am not sure if this way I can further refine the structure, or do I bring > two much bias into the structure. Please correct me if any. Thanks. > > Best, > Hubing > > > > > On Thu, Jul 21, 2011 at 11:31 PM, <ray-br...@att.net> wrote: > >> I have also tried for years to solve a protein-DNA complex without >> sucess. >> If you have a lot more DNA than protein in the AU then MR will not work. >> You >> always get a good RFZ score but you cannot solve the translation if the >> DNA molecules are forming long stacks. With a plausible packing you will >> of >> course get model phases and a nice map but refinement will not work and >> you >> will get stuck at 40-50% Rf. >> >> You may have a chancewith MR if you only have a small DNA and a much >> bigger >> protein molecule or if the search models and the molecules have identical >> sequences. >> >> To solve this structure you probably have to do Se-labeled protein and >> SAD >> etc. or collect anomalous from metal ions if present. >> >> Cheers. >> >> Ray Brown >> >> ------------------------------ >> *From:* Hubing Lou <louhub...@gmail.com> >> >> *To:* CCP4BB@JISCMAIL.AC.UK >> *Sent:* Thu, July 21, 2011 6:39:49 AM >> *Subject:* Re: [ccp4bb] Phaser and Molrep gave different solutions >> >> I was worried as well with the low TFZ score. Usually successful cases >> with >> score >8. I am still puzzled why Phaser and Molrep gave different >> solutions. >> Does this mean molecular replacement do not work out in this case so more >> crystals have to be prepared? >> >> A little more information might be helpful to dissolve the problem here. >> The model I used is a protein-DNA complex. The protein was Chainsaw >> editted >> but the DNA sequence was directly borrowed from the original model. >> >> Best, >> Hubing >> >> On Thu, Jul 21, 2011 at 3:30 PM, Vellieux Frederic < >> frederic.velli...@ibs.fr> wrote: >> >>> Hi, >>> >>> It's not a bad idea to read the Phaser manual for molecular replacement; >>> see http://www.phaser.cimr.cam.ac.uk/index.php/Molecular_Replacement >>> >>> Soon after the start, in a table on the right hand side, there is: TFZ >>> score < 5, have I solved it ? No. >>> >>> Hence with a TFZ score of 3.8 you do not have a solution using Phaser. >>> >>> Fred. >>> >>> Hubing Lou wrote: >>> >>>> Dear all, >>>> >>>> I am stuck in a molecular replacement case and looking for advices. >>>> I have been working on a protein-DNA complex structure. >>>> Data was processed by HKL2000 to 2.6Ang and some of the data statistics >>>> are shown below: >>>> >>>> Space group: P21, >>>> Unit Cell: 54.73, 104.91, 78.40, 90, 100.2, 90 >>>> Redundancy: 2.8 (2.7) >>>> Completeness: 94.8 (93.1) >>>> Linear R-fac: 0.051 (0.442) >>>> >>>> Data quality was checked by Phenix.xtriage and there's no problem. I >>>> then >>>> prepared a model by Chainsaw. Our protein shares only 30% of sequence >>>> similarity with the model, but structurally they are in the same group >>>> and >>>> almost identical in apo form. Matthrews Coeff indaced two monomers in >>>> AU. I >>>> then ran Phaser in "automated search" mode and there's a solution with >>>> RFZ >>>> score 4.8, TFZ score 3.8. The electron density map was not bad with DNA >>>> double helix clearly seen. However Refmac5 couldn't get Rfree lower >>>> than >>>> 50%. >>>> >>>> I then changed to MolRep, ran "self rotation function" first then used >>>> the first 10 peaks for translation search. Again there's a solution >>>> but it >>>> is different from that from Phaser. I attached a picture here. >>>> Checking in >>>> coot, the packing is the same. But, the refinement couldn't get Rfree >>>> lower >>>> than 50%. >>>> >>>> I have tried to include NCS, TLS refinement in Refmac, both not >>>> working. >>>> Hope someone out there can help. >>>> Thanks very much for your time. >>>> >>>> Hubing >>>> >>>> >>>> ------------------------------**------------------------------** >>>> ------------ >>>> >>>> >>>> >>> >>
