Re: [ccp4bb] suggestions on refinement of a protein-ligand complex
We probably need more detail to help you. Have you looked carefully at the data processing? Is the Rmerge or Rpim reasonable for all batches? Is there any suggestion of twinning? Does the wilson plot look linear? (These hexagonal SGs can be twinned) How many copies of your molecule do you expect? Then the model you have used.. What is the sequence ID to the protein, and do you have a suitable model for the ligand? I would run a search in all possible SGS - and do the results clearly indicate one choice is better than all others> The final check is in the refinement - does the R value fall (FreeR should too, but your resolution is rather low Now you run the molecular replacement in all possible spacegroups for that pointgroup.. Is there a clear indication of one being preferred? Good luck. Eleanor On Tue, 13 Sept 2022 at 18:29, Kay Diederichs < kay.diederi...@uni-konstanz.de> wrote: > Dear Deepak, > > I guess that the spacegroup in the MTZ file that you use for refinement is > wrong. > > I think you should carefully check the output of PHASER, and in particular > the PDB file that it wrote. The correct spacegroup is given there (and it > may also be P4122 or P4222 or P4322, because likely you used the PHASER > option that tests all possibilities), and you will have to create a MTZ > file for refinement that has that particular spacegroup in its header. > > Hope this helps, & best wishes > Kay > > On Tue, 13 Sep 2022 11:20:44 +0200, Deepak Deepak > wrote: > > >Dear all, > > > >Greetings from Munich. I hope everything is well with you. I am writing to > >take input on a problem related to the structure solution of a > >protein-ligand complex. > > > >I have crystallized a protein (7.6kDa) with a ligand (5.2kDa). The crystal > >diffracted to *2.71 Angstron*, and the data were processed using XDS. > >During the XDS processing, Pointless suggested data be either P6222 (180 > >space group) or P6422 (181 space group) with a 98% probability. > >The data were processed as *P6422*. > > > >Afterward, I ran Phaser MR with protein's PDB as a search ensemble, and I > >found a good solution (with a good TFZ score) for protein alone, where I > >could see some weak density for my ligand. > >In the next step, I ran Phaser MR with a *modeled PDB* of the Ligand as > >search ensemble + *using the partial solution from the previous Phaser MR > >run of Protein*. This Phaser MR run gave me a good fit of ligand onto > >protein surface with nice density for both molecules. > > > >Next, I went ahead with the refinement of this complex. The R-free, in the > >beginning, was 0.55. In the first round of refinement, it went down to > >*0.53,* but in the subsequent rounds, it even* increased*. I am stuck at > >this point and unsure how to proceed. I tried different strategies to > >refine it, but nothing worked. > >Could the space group have been wrong? How can I make sure that? I can > >provide a more detailed explanation to help you better understand the > >problem. > >I appreciate your suggestions and input on this matter. > > > >Kind regards, > >Deepak, > >Ph.D. student, > >LMU Munich, Germany. > > > > > > > >To unsubscribe from the CCP4BB list, click the following link: > >https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > > > >This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a > mailing list hosted by www.jiscmail.ac.uk, terms & conditions are > available at https://www.jiscmail.ac.uk/policyandsecurity/ > > > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > > This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a > mailing list hosted by www.jiscmail.ac.uk, terms & conditions are > available at https://www.jiscmail.ac.uk/policyandsecurity/ > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] suggestions on refinement of a protein-ligand complex
Dear Deepak, I guess that the spacegroup in the MTZ file that you use for refinement is wrong. I think you should carefully check the output of PHASER, and in particular the PDB file that it wrote. The correct spacegroup is given there (and it may also be P4122 or P4222 or P4322, because likely you used the PHASER option that tests all possibilities), and you will have to create a MTZ file for refinement that has that particular spacegroup in its header. Hope this helps, & best wishes Kay On Tue, 13 Sep 2022 11:20:44 +0200, Deepak Deepak wrote: >Dear all, > >Greetings from Munich. I hope everything is well with you. I am writing to >take input on a problem related to the structure solution of a >protein-ligand complex. > >I have crystallized a protein (7.6kDa) with a ligand (5.2kDa). The crystal >diffracted to *2.71 Angstron*, and the data were processed using XDS. >During the XDS processing, Pointless suggested data be either P6222 (180 >space group) or P6422 (181 space group) with a 98% probability. >The data were processed as *P6422*. > >Afterward, I ran Phaser MR with protein's PDB as a search ensemble, and I >found a good solution (with a good TFZ score) for protein alone, where I >could see some weak density for my ligand. >In the next step, I ran Phaser MR with a *modeled PDB* of the Ligand as >search ensemble + *using the partial solution from the previous Phaser MR >run of Protein*. This Phaser MR run gave me a good fit of ligand onto >protein surface with nice density for both molecules. > >Next, I went ahead with the refinement of this complex. The R-free, in the >beginning, was 0.55. In the first round of refinement, it went down to >*0.53,* but in the subsequent rounds, it even* increased*. I am stuck at >this point and unsure how to proceed. I tried different strategies to >refine it, but nothing worked. >Could the space group have been wrong? How can I make sure that? I can >provide a more detailed explanation to help you better understand the >problem. >I appreciate your suggestions and input on this matter. > >Kind regards, >Deepak, >Ph.D. student, >LMU Munich, Germany. > > > >To unsubscribe from the CCP4BB list, click the following link: >https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > >This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing >list hosted by www.jiscmail.ac.uk, terms & conditions are available at >https://www.jiscmail.ac.uk/policyandsecurity/ > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] suggestions on refinement of a protein-ligand complex
Dear all, Greetings from Munich. I hope everything is well with you. I am writing to take input on a problem related to the structure solution of a protein-ligand complex. I have crystallized a protein (7.6kDa) with a ligand (5.2kDa). The crystal diffracted to *2.71 Angstron*, and the data were processed using XDS. During the XDS processing, Pointless suggested data be either P6222 (180 space group) or P6422 (181 space group) with a 98% probability. The data were processed as *P6422*. Afterward, I ran Phaser MR with protein's PDB as a search ensemble, and I found a good solution (with a good TFZ score) for protein alone, where I could see some weak density for my ligand. In the next step, I ran Phaser MR with a *modeled PDB* of the Ligand as search ensemble + *using the partial solution from the previous Phaser MR run of Protein*. This Phaser MR run gave me a good fit of ligand onto protein surface with nice density for both molecules. Next, I went ahead with the refinement of this complex. The R-free, in the beginning, was 0.55. In the first round of refinement, it went down to *0.53,* but in the subsequent rounds, it even* increased*. I am stuck at this point and unsure how to proceed. I tried different strategies to refine it, but nothing worked. Could the space group have been wrong? How can I make sure that? I can provide a more detailed explanation to help you better understand the problem. I appreciate your suggestions and input on this matter. Kind regards, Deepak, Ph.D. student, LMU Munich, Germany. To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
