Re: [ccp4bb] How it could be possible?
Hi, If there is a hinge motion between the two domains, then allowing for this will give you a much better starting model. As Klaus suggested, you may just be able to do rigid body refinement of the two domains. However, it is also possible to place the two domains separately in Phaser, by putting the two domains in separate PDB files, defining an ENSEMBLE with each of these PDB files, then searching for both of them in the same job. In the ccp4i GUI, you can specify an additional ensemble by clicking the Add ensemble button, and you can add another component to search for in the same job by clicking the Add another search button. I hope that answers the question you were asking! Best wishes, Randy Read On 2 Dec 2013, at 04:11, Prem Prakash prem...@gmail.com wrote: Dear All, The density obtained after molecular replacement using phaser at 2.5 Angstrom and then used buccneer for autobuilding of the model. I am not getting reasonable R value (it is 38.5 %) but the figure of merit is 0.629. As My protein has two domains. So is it possible to fragment the individual domain and then again perform the molecular replacement. How it will improve my phase more and how R-factor will be reduced ? Please help and direct me in proceeding in a right way, and if possible provide a protocol for doing that. Thank you With kind regards -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 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
Re: [ccp4bb] How it could be possible?
Thank you very much for kind suggestions and comment. With kind regards Prem On Mon, Dec 2, 2013 at 5:41 PM, Randy Read rj...@cam.ac.uk wrote: Hi, If there is a hinge motion between the two domains, then allowing for this will give you a much better starting model. As Klaus suggested, you may just be able to do rigid body refinement of the two domains. However, it is also possible to place the two domains separately in Phaser, by putting the two domains in separate PDB files, defining an ENSEMBLE with each of these PDB files, then searching for both of them in the same job. In the ccp4i GUI, you can specify an additional ensemble by clicking the Add ensemble button, and you can add another component to search for in the same job by clicking the Add another search button. I hope that answers the question you were asking! Best wishes, Randy Read On 2 Dec 2013, at 04:11, Prem Prakash prem...@gmail.com wrote: Dear All, The density obtained after molecular replacement using phaser at 2.5 Angstrom and then used buccneer for autobuilding of the model. I am not getting reasonable R value (it is 38.5 %) but the figure of merit is 0.629. As My protein has two domains. So is it possible to fragment the individual domain and then again perform the molecular replacement. How it will improve my phase more and how R-factor will be reduced ? Please help and direct me in proceeding in a right way, and if possible provide a protocol for doing that. Thank you With kind regards -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 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
Re: [ccp4bb] How it could be possible?
Dear Prem, If your protein has 2 domains, it is possible that they their relative orientation is different in your target compared to the search model. Therefore searching with the two domains separately can improve your Z-score in Phaser and give you a better map. However, you did not tell us what your Z-score was after phaser (= 8.0 after translation search?) and how your map looks like after phaser, but before buccaneer. For instance, if you simply refine your initial MR solution in refmac, do you get density for side chains that are not part of your search model? Or do you see extra bits of backbone density that are not in the search model.?What about searching with just one domain, do you see (recognisable) density for the second? Finally, an R-value (free or cryst?) of 38% after an initial MR solution is not unreasonable. You may have to manually rebuild your model in Coot with subsequent refinement in Refmac to lower your R-values. Of course, rebuilding in this case requires that you see some extra density you can build into or some parts of the map where your current model does not fit the density. Have you tried to do a rigid body refinement of your initial MR solution, where you allow each domain to move independently? All the best with this structure. Klaus === Dr. Klaus Fütterer Deputy Head of School Undergraduate Admissions Room 717, Biosciences Tower School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham F: +44-(0)-121-414 5925 Edgbaston E: k.futte...@bham.ac.uk Birmingham, B15 2TT, UK W: http://tinyurl.com/futterer-lab === On 2 Dec 2013, at 04:11, Prem Prakash wrote: Dear All, The density obtained after molecular replacement using phaser at 2.5 Angstrom and then used buccneer for autobuilding of the model. I am not getting reasonable R value (it is 38.5 %) but the figure of merit is 0.629. As My protein has two domains. So is it possible to fragment the individual domain and then again perform the molecular replacement. How it will improve my phase more and how R-factor will be reduced ? Please help and direct me in proceeding in a right way, and if possible provide a protocol for doing that. Thank you With kind regards
