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.
