Hi, We’ve had good luck with Phaser in placing many copies of proteins or domains, if the model is reasonably good. Because of the variable elbow angle, you probably want to place the domains separately. However, as soon as you find one convincing assembly you can switch to searching for the whole Fab. Alternatively, you can try a bunch of different models and hope that one is right.
Something that might be useful is a new “MR_GYRE" feature in Phaser. This is a likelihood-based version of the PC refinement that can be done in CNS, to optimise the relative orientations of domains between the rotation and translation searches. If you get stuck, we might be able to give a hand — we haven’t had much experience ourselves with using this on unsolved structures. Of course, the advice you’ve had from others to be certain first of the space group is very good! Best wishes, Randy Read ----- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: +44 1223 336500 Wellcome Trust/MRC Building Fax: +44 1223 336827 Hills Road E-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk On 9 Dec 2014, at 18:31, Mo Wong <mowon...@gmail.com> wrote: > Hi all, > > I’m stuck on a rather complex molecular replacement problem. The crystals are > of an antigen-Fab complex totaling ~67 kDa (waiting to confirm using PAGE > gel). They diffract to ~3.5A at the synchrotron and process in C2 with > dimensions 220x130x230 and beta at 103 so it looks like there are round > 9to12-ish copies in the ASU. The overall Rmerge is high at ~25% with I/sig > cutoff ~2 and redundancy of 5; however, at 4.5A this drops to ~15%. > Furthermore, processing in P1 gives similar Rmerge values too. > > Self-Patterson doesn’t suggest translational symmetry, but the self-rotation > function (SRF) suggests high NCS (see below/attached). > > I’m hoping the SRF might be helpful in trying to confirm/dismiss C2 is the > likely space group, and perhaps suggest a logical assembly with the ASU (I > see strong 2-fold and 3-fold NCS operators suggesting to me dimeric trimers > or vice versa - however, I’ve never had to really analyze SRFs in the absence > of a mol. rep. solution so my interpretation could be wrong). > > Anyway, any help to bringing a molecular replacement solution closer to > reality would be appreciated. > > Thanks! > > <SELF_RF.gif>
