Thanks for the feedback. Twinning is found to be 4.5% with phenix.xtriage. I shall model it, even though I am not so eager to use that option in Refmac. I cut the N-terminal in one and used modeled it for the other monomer in P 32, but were not able to refine it better. The 2-fold symmetry is eventually present, which already let to some group internal discussions. You will see a properly modeled PDB entry at one point!
Have a successful day Harm Otten -- On Mon, Sep 24, 2012 at 5:26 PM, <herman.schreu...@sanofi.com> wrote: > Dear Harm, > > As Edwin pointed out, there might not be any non-crystallographic symmetry to > break, because it is crystallographic. While it is clear that no two > N-terminii can be in the same electron density at the same moment, there is > no reason why this symmetry-breaking should happen in a regular, orderly > fashion throughout the crystal. E.g. that all even molecules in a row would > have conformation A, and all odd molecules would have conformation B. > > What most likely happened, is that randomly, the N-terminus would either > occupy conformation A (in the electron density where it is fitted now) and > randomly in some other orientation outside this electron density. This > electron density A would be strong (100% occupancy; 50% from either > neigboring molecule), while the electron density B would be weak (only 50% > occupancy). I had a similar case and the only reason which convinced myself > to fit alternative conformations, was the physical plausibility argument. > > So for the overlapping N-terminus and the surface lysines, you have to > generate alternative conformations. As I said, the electron density for the > non-overlapping conformation might be quite weak (or even disordered), so you > may have to scroll your contour level quit a bit down. You also have to make > sure that your refinement program recognizes what is going on and does not > push the molecules apart. For buster you have to use the gelly EXCLUDE > keyword, refmac will probably do it automatically. > > Good luck! > Herman > > > > -----Original Message----- > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Harm > Otten > Sent: Monday, September 24, 2012 1:10 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] Space group choice with respect to physical plausability > > For a protein dataset we find an "overlapping" N-terminal stretch of 6 amino > acids in P 32 2 1 (1 monomer/asymmetric unit ASU). We did process the same > case in P1 (6 mon./ASU), C2 (3 mon./ASU) and P3 (2 > mon./ASU) to resolve the physically and chemically hard to explain overlap of > the N-terminal and some surface lysins. We did try to mutate the B-factor > outliers to alanins and also to mutate all of the B-chain to alanins/glycins > (using just the backbone and C-alphas) and keep the A-chain untouched. This > did not break the symmetry, i.e. the strong NCS relationship between the > monomers. > > Some key values are highlighted in this table: > > # Molecules Space Group Cell R-Merge > MR > correlation # Clashes B-factor outliers R-factor R-free > (No.) a b c alpha beta gamma > > 70 Å2 > 6 P1 (1) 95.7 55.4 73.1 90 90 90 > 4.4 0.227 A/B > many: B/C 2 n/a n/a n/a > 3 C2 (5) 95.7 55.4 73.1 90 90 90 > 2.7 0.594 > none 8 /3mol 22 30 > C222 (21) 55.4 95.7 73.1 90 90 90 > 43.9 n/a > n/a n/a n/a n/a > 2 P32 (145) 55.4 55.4 73 90 90 120 > 3.4 n/a > N-term 13 /1mol 29 34 > 1 P32 2 1 (152) 55.4 55.4 73 90 90 120 > n/a > n/a n/a n/a n/a n/a > 1 P3 2 1 (150) 55.4 55.4 73 90 90 120 > 3.7 0.359 N-term > 1 P32 1 2 (151) 55.4 55.4 73 90 90 120 > n/a > n/a n/a n/a n/a n/a > > > Why is it so hard to break the symmetry for two (seemingly) different > monomers? > > Thanks everybody for the multitude and quality of your answers to my question > some time back. One can really find the "authority" on hands-on questions and > standards in this bulletin board and the wwPDB. > > Have a successful day > > Harm > > --- > Harm Otten, PhD > Department of Chemistry > Universitetsparken 5, Office C316 > 2100 Copenhagen, Denmark > # +45 35 32 02 86 > fax +45 35 32 03 22 > email h...@chem.ku.dk > web www.harmotten.com > Please consider the environment before printing this email.
