Hi Randy, The elbow angle you refer to may be different within one asymmetric unit. The 2FJF has 12 Fab molecules per asymmetric unit with elbow angles ranging between 144 and 192. So, I'd rather think that in a specific case of Fab the search is better off with VH-VL and CH1-CL domain pairs used separate. MolRep has a nice option of not using number of molecules to be found. Program will do it for you to start with. Later on upon examination of packing you may add one or two molecules.
Thank you. Vaheh Oganesyan www.medimmune.com -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Randy Read Sent: Tuesday, December 09, 2014 2:52 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Challenging Mol.Rep. Problem 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> To the extent this electronic communication or any of its attachments contain information that is not in the public domain, such information is considered by MedImmune to be confidential and proprietary. This communication is expected to be read and/or used only by the individual(s) for whom it is intended. If you have received this electronic communication in error, please reply to the sender advising of the error in transmission and delete the original message and any accompanying documents from your system immediately, without copying, reviewing or otherwise using them for any purpose. Thank you for your cooperation. To the extent this electronic communication or any of its attachments contain information that is not in the public domain, such information is considered by MedImmune to be confidential and proprietary. This communication is expected to be read and/or used only by the individual(s) for whom it is intended. If you have received this electronic communication in error, please reply to the sender advising of the error in transmission and delete the original message and any accompanying documents from your system immediately, without copying, reviewing or otherwise using them for any purpose. Thank you for your cooperation.
