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Hello everyone, Maybe of interest/help to others: The solution of my problem is a totally unexpected structural change of 2 out of 4 protein molecules in the asu! The straight forward solution was additionally obscured through the presence of tNCS. What I did: - placed as many molecules as possible in P1 using phaser - deleted clashing regions (pretty much half of half of the proteins in the asu!) - run refmac, corrected the model and build as many residues as possible in the residual density, etc -> here it became apparent that the loop of a beta hairpin structure shifted, (the loop became now the beta-strand!!!) with a cystein forming a disulfide bond with the neighbouring, short protein! And one could see that a two-fold axis has to run directly through that region. - Than I run phaser again, with the orthorombic cell. First I let it place two full proteins and in the second round I gave it the short protein as search model (with the .sol file from the first run) - cycling through building in the residual density, refinement etc... So the symmetry is apparently P2 21 21, with two protein mols folded as expected and two more mols, where almost half of the residues of the protein go somewhere else, with a shift in a beta-hairpin and disulfide bond formation with the now symmetry related molecule! (despite heaps of reducing agent in the buffer...) Sabine (I also gave the initial P1 solution to Phenix autobuild and Buccaneer, but I didn't do much tweaking here, and in the meantime I'd figured it out by hand) >> Hello everybody, >> >> I got a problem with solving and refining a structure of a small protein >> of 20kDa, where we just swapped two residue at the surface by mutation >> (AF vs FA). >> >> For the FA variant we got three crystal forms, where we had no problem >> whatsoever with MR and refinement >> - P6522 cell 63 63 184 ~2.9A >> - P21212 cell: 53 62 109 ~3A >> - P212121 cell 40 43 96 ~1.8A >> >> However with the AF-variant we only got one crystal (out of 50) that >> diffracted reasonable well to ~2.4A at the synchrotron. And now the >> problem starts: initially the data appear to be orthorhombic, but: >> >> - SG18/19: cell 59 91 109 (I noticed that the b-axis is longer by 1/2b >> compared to the other variant?!) >> -> running phaser checking all possible alternative SGs found initally 2 >> mols/AUS in SG18, but since there was still space (and density!) I let >> it search for another one (increased the clash tolerance) >> SOLU SET RFZ=7.7 TFZ=11.8 PAK=0 LLG=141 TFZ==13.9 RFZ=3.0 TFZ=11.2 PAK=0 >> LLG=280 TFZ==17.1 LLG=288 TFZ==17.3 RFZ=5.9 TFZ=16.0 PAK=16 LLG=979 >> TFZ==15.3 LLG=1479 TFZ==17.4 >> >> - However the third molecule than clashes partly >> >> - running refmac: R/Rfree stays in the high 40 >> >> - analysing the data with Xtriage indicates tNCS >> >> - I also tried P212121, P21 and P1, always with the same result: space >> as well as density between the proteins; placing more protein molecules >> results in clashes (i.e. refinement in P1 gives R/Rfree of 38/41) >> >> - data were processed with XDS as well as Dials/Aimless >> >> - the protein is generally well folded, quite stable, without any loose >> ends or flexible regions >> >> - calculation of selfrotation functions in P21 and P21221 gives peak at >> 90dg (kappa=180dg) >> >> Anyone an idea what could be going on or how to solve that? >> >> >> Thanks a lot in advance! >> >> Sabine