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 >>> >>> >>> ------------------------------**------------------------------** >>> ------------ >>> >>> >>> >> >
