Dear Niu, It is very unlikely that MBP will be disordered. We use that protein a standard for SAXS calibration and gel filtration it is extremely well behaved. There is an excellent review about MBP enhanced crystallization (by Moon et al) and you can do a quick survey of the PDB to find the cornucopia of structures obtained using this strategy. I would suggest maybe an ensemble MR search in phaser using a sum of all conformations adopted by MBP in these different structures. You have 4 A data and it is difficult sometimes to pull out the solution at low resolution. Trimming the few disordered loops in the MBP model could help you, so you avoid throwing away the good solution because of a few clashes when it packs solutions after rotation and translation searches. We recently solved one structure MBP + target protein of about 10 kDal (2.7A) and it worked extremely well. A linker of 30 aminoacids connected the target to MBP and it ended up tightly packed against the carrier MBP protein even if we were still missing about 25 residues of linker that was actually part of the natural sequence of the protein (we had to generate 5 constructs to get it right). Another approach, may not be smart but it should get the job done, is to selenolabel your fusion and crystallize it. MBP has 5 or 6 methionines , even if you target has none, this will be enough to phase (although you are only at 4 A if I understood well). That should enable you to lock the MBP in position and see what is going on in your crystals.
Good luck Pascal Egea On Fri, May 16, 2014 at 8:03 AM, 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 > -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
