When something like this happens I always go back to the data. What do the mages look like - are the spots ugly streaky ones, or quite tidy. Then is the spacegroup right ? Did you integrate assuming C/mmm symmetry? If so go back and do it again in P1! Look carefully at the pointless analysis; I have just struggled the a case which is probably P21 but with two molecules related by a two fold parallel to a* , which masqueraded as C/mmm. What is the multiplicity - high enough to be sure?
Check the self rotation function - is there a clear non-crystallographic match (doesnt have to be a two fold..) I like the MOLREP plots to see that. And it is often hard to build at 2.8A - can you squeeze a higher resolution? All the best Eleanor On Tue, 24 Mar 2020 at 08:55, Randy Read <[email protected]> wrote: > Dear Zhu, > > Questions specific to Phenix should really go to the Phenix-BB, so I am > cross-posting my reply there. Here I’ll focus more on generic issues. > There are also CCP4 tools that you could consider and presumably other > people will offer advice on those. > > One point you raise comes up so much that we have an entry in our FAQ ( > https://www.phaser.cimr.cam.ac.uk/index.php/FAQ) about it: “Can I use > Phaser to check the correctness of a model I have already built and > refined?” The answer is no, because once you’ve refined the model it > becomes better than random at predicting the data and therefore achieves a > high LLG score, regardless of whether or not it is correct. > > In this case, you might be able to use Phaser to help complete the model > if one of the two copies of the complex is more completely modelled than > the other. If, say, you had a model for one copy you could fix that and > search for a second copy: this should work because the refinement didn’t > know anything about the second copy. > > Phenix.autosol attempts to determine the NCS operators, so you need to > check whether that has succeeded. If not, you might need to try some more > manual approaches to defining the NCS, which would help a great deal in map > improvement. Tom Terwilliger might answer this in more detail (perhaps on > the Phenix-BB), but I don’t think you can build protein and nucleic acid in > the same job, so you should look at the documentation to see how to do that. > > Good luck! > > Randy Read > ----- > Randy J. Read > Department of Haematology, University of Cambridge > Cambridge Institute for Medical Research Tel: +44 1223 336500 > The Keith Peters Building Fax: +44 1223 > 336827 > Hills Road E-mail: > [email protected] > Cambridge CB2 0XY, U.K. > www-structmed.cimr.cam.ac.uk > > > On 24 Mar 2020, at 04:31, Zhu Qiao <[email protected]> wrote: > > > > Dear All > > > > I am sorry for the long context. > > > > I have one protein (252 AAs, 2 Met) bound to double-stranded DNA (24 bp) > crystalized. I collected the Se-Met data of the crystal in C222 up to 2.8 > angstrom. the space group is confirmed by running the pointless. > > > > I used the Phenix.Autosol to find the heavy atoms and get a quite nice > map after the density modification. It seems there are two proteins and > two DNA duplex are in one ASU. Phenix.Autobuild can only build less than > half of the protein sequence into the map and fill in the potential DNA map > with amino acids. The Rwork/Rfree is 0.40 and 0.46, with the map CC=0.60. > If I do the MR with the initial model built by Autobuild, the result > TFZ=40, LLG=200+, which suggests the partial correction of the initial > model. > > > > Here is the problem. From the map, I can see one of my protein domain > and a clear feature of DNA double helix. But whatever I go further for > manual build using coot, like building the DNA double-strand into the map > and building the resolved domain, the refinement statistics go bad with R > free ~0.50. > > > > I am wondering what's going wrong and how come the refinement can't > improve the R factor. > > > > I have attached the relevant photos. > > > https://drive.google.com/drive/folders/1dJ4kn7CEHkCL3sMcCJtBqa5OsVFQngBx?usp=sharing > > > > > > Sincerely > > Zhu > > > > ######################################################################## > > > > To unsubscribe from the CCP4BB list, click the following link: > > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > > ######################################################################## > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
