Dear Randy, Over the last ~6 years, we have tried to use the MBP tag to enhance crystallization on >10 proteins. Only one that couldn't crystallize by itself in the end crystallized with the fusion tag. However, unfortunately, the target protein (~10 kDa) turned out to be in the big cavity of the MBP lattice and was completely disordered. Therefore, I am personally not very optimistic about the method.
Best wishes, Gang DONG On Fri, May 16, 2014 17:57, Randy Read wrote: > 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 >
