Re: [ccp4bb] Phaser and Molrep gave different solutions
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 --**--**
Re: [ccp4bb] Phaser and Molrep gave different solutions
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
Re: [ccp4bb] Phaser and Molrep gave different solutions
The self rotation isnt the correct input to the translation function - just run the Auto-Molrep option.. Eleanor On Thu, 21 Jul 2011 20:57:02 +0800, Hubing Lou louhub...@gmail.com wrote: I also processed with Imosflm and ran Pointless, it was P21. Also it was indicated by the Intensity systematic absences in HKL2000 scale.log file. Best, Hubing On Thu, Jul 21, 2011 at 8:44 PM, herman.schreu...@sanofi-aventis.comwrote: ** Dear Hubing, One maybe stupid question: Your are sure the space group is P21 and not P2 or even something else? Did you test other possible space groups? Choosing the wrong space group could exactly lead to the results you observe. Best, Herman -- *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Hubing Lou *Sent:* Thursday, July 21, 2011 6:46 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Phaser and Molrep gave different solutions 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
Re: [ccp4bb] Phaser and Molrep gave different solutions
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
Re: [ccp4bb] Phaser and Molrep gave different solutions
Have you tried using the DNA as your search model? - I have had success this way round - certainly more phasing power than your protein model, I guess. Also, refine with your DNA in place, and your phases/ map should improve - hopefully allowing you to place your protein molecules with ease. Tony. Sent from my iPhone On 21 Jul 2011, at 12:40, Hubing Lou louhub...@gmail.commailto:louhub...@gmail.com wrote: 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 mailto:frederic.velli...@ibs.frfrederic.velli...@ibs.frmailto: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 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
Re: [ccp4bb] Phaser and Molrep gave different solutions
Dear Hubing, One maybe stupid question: Your are sure the space group is P21 and not P2 or even something else? Did you test other possible space groups? Choosing the wrong space group could exactly lead to the results you observe. Best, Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hubing Lou Sent: Thursday, July 21, 2011 6:46 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Phaser and Molrep gave different solutions 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
Re: [ccp4bb] Phaser and Molrep gave different solutions
I also processed with Imosflm and ran Pointless, it was P21. Also it was indicated by the Intensity systematic absences in HKL2000 scale.log file. Best, Hubing On Thu, Jul 21, 2011 at 8:44 PM, herman.schreu...@sanofi-aventis.comwrote: ** Dear Hubing, One maybe stupid question: Your are sure the space group is P21 and not P2 or even something else? Did you test other possible space groups? Choosing the wrong space group could exactly lead to the results you observe. Best, Herman -- *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Hubing Lou *Sent:* Thursday, July 21, 2011 6:46 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Phaser and Molrep gave different solutions 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
Re: [ccp4bb] Phaser and Molrep gave different solutions
Hi, Although it's pretty much true that if TFZ8, the solution is correct, there are hard cases where a correct solution has a significantly lower TFZ score. Also, we've been finding that TFZ scores are generally lower for monoclinic space groups like P21, at least in the search for the first molecule. (Among other things, the search for the first molecule in a polar space group only covers a plane, so there are fewer independent trials.) Rob Oeffner has been doing some detailed statistics, which we're analysing so that we can give better rules of thumb. However, a TFZ of 3.8 for the second molecule (where the search is 3-dimensional) is a bad sign. One of the earlier posts suggested looking just for the DNA, which sounds like a good idea. It's possible that the relative orientation of the protein and DNA is different enough that the complex model doesn't work, so it might well be worth trying the DNA and protein components separately. Good luck! - Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical ResearchTel: +44 1223 336500 Wellcome Trust/MRC Building Fax: +44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk On 21 Jul 2011, at 11:39, Hubing Lou wrote: 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
Re: [ccp4bb] Phaser and Molrep gave different solutions
On 07/21/2011 01:45 PM, 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. Hi, Isn't this a bad sign usually when the TFZ score is lower than the RFZ score? Regards, F. 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
