Dear Niu, When it works, crystallising a fusion protein can be great, with the big advantage that placing a model for the (known) carrier protein gives free phase information. Certainly there are examples of this working, but in the early days of this (20 years or so ago?), I remember hearing of more than one case where only one component was visible in the electron density, because the other component was unconstrained by crystal packing or by the flexible linker. So you might want to consider whether the MBP component might be disordered.
It would be interesting to hear whether anyone has data on how frequently fusion proteins crystallise and how often both components are well-ordered, because I’m just working from a few anecdotes. The warning from Phaser reflects the fact that signal in the MR search will be limited by the low resolution, the incompleteness of the model, and possibly the quality of the model for the target protein. Best wishes, Randy Read On 16 May 2014, at 16:03, Niu Tou <niutou2...@gmail.com> wrote: > Dear All, > > Recently we collected some data of a MBP fusion protein, at around 4A > resolution. The protein itself is about half of the MBP size. However when we > tried to solve it with MR, it failed. We tried to use MBP alone, homology > model of target protein alone, and MBP+model. It is very strange that MBP > alone can not yield any reasonable solution at all, so does searching with > MBP and model together. While searching with model alone could get some > better results, but when fix it to search MBP, it failed. There are 1 > molecule per ASU with solvent content 55%. The spacegroup should be right and > we tried to search all possible alternatives in each run, we also tried to > lower it down, but did not work either. When running Phenix.phaser, there is > a warning at the beginning saying eLLG suggests placing of ensembles will be > very difficult. > > I wonder if anybody has encountered similar situation before. Any suggestions > will be greatly appreciated! > > Regards, > Niu ------ 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
