Re: [ccp4bb] Structure Determination combining X-ray Data and NMR

[Boaz Shaanan]

Hi, You are touching upon several issues here. The first question to ask is how good and complete are your data to 3.2 A resolution. This should be your first concern. Are they the best you can get at this stage? Second, you're absolutely correct in that there is a lot more to do to improve your model. Although it sounds as if you're on the right track, having the Rw/Rf values so close sounds alarming. Also, with low resolution data you can expect high B's but of course you should try your best to fit residues/atoms to the e.d. Setting high weights to the geometry/stereochemistry restraints in whichever refinement program you're using, can help here too, at least in the initial stages of the refinement if not throughout. If you're using the automatic weights offered by the program you may want to examine them carefully and see whether they can be modified to improve your convergence. So you have a lot to do before considering throwing in your NMR data. As for the latter, I think that there have been a few papers recently from David Baker's lab and Guy Montelione's lab where they've shown how to use rudimentary NMR data (i.e. before converting them to NOE's) in refinement of crystal structures. Which brings up the next question: are you not going to calculate NOE's from your data ? Are the NMR data that you have not sufficient to derive a good solution structure? NOE distance restraints on their own can be used to improve crystallographic structures (I can send you some old and recent references off list, if you're interested).    Cheers,                 Boaz     Boaz Shaanan, Ph.D.                                         Dept. of Life Sciences                                      Ben-Gurion University of the Negev                          Beer-Sheva 84105                                            Israel                                                                                                                  E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220  Skype: boaz.shaanan                  Fax:   972-8-647-2992 or 972-8-646-1710                          From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of 商元 [shangyuan5...@gmail.com] Sent: Friday, January 06, 2012 10:23 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Structure Determination combining X-ray Data and NMR Also, there is one more information I forgot to mention---I also have the NMR assignment(HNCACB spectrum) of the protein, is it possible to combine the NMR data in my refinement? Regards, On Fri, Jan 6, 2012 at 4:14 PM, 商元 <shangyuan5...@gmail.com> wrote: Dear All,    I have a set of 3.2A data containing only 3000 reflections. From the SAD phasing and iterative modeling and density modification, I get a preliminary structure with bad geometric conformations(~8/160 ramachandran outliers in Coot). After Phenix MLHL refinement, the geometry is still bad with (10% ramachandran outliers and 25% Rotamer outliers), and the B-factors are all too high(all between 80 to 170, average ~120), and R-factor/R-free have a value of 0.328/0.326.   The poor geometry of my model and the unusual B-factors indicates there are still a lot improvement in my model. The question is, as I only have ~3000 reflections, and the atoms in the sequence is around 1000, and each atom there are 4 parameters to be refined(X,Y,Z,B-factor, assuming occupancy is 1), so how to refine my model to avoid over-refinement? Should I trust the electron-density map of the refined mtz data, or should I adjust the local geometries using Coot rotamers tools? How to set a reasonable B-factor values in the refinement? Best Regards, Yuan