On Wed, Jun 20, 2012 at 11:13 AM, sonali dhindwal < [email protected]> wrote:
> > I am working on a protein for last so many years and for which i have got > crystal now in a tray which i kept 1 years ago. It diffracts well and > resolution is 2.2A, which is good. > > I indexed in HKL2000, mosflm and automar and it shows P21 space group in > all data reduction packages. But when I tried using molrep or phaser then I > do not get any solution. The sequence of my protein is having 46% identity > with other available crystal structure. > Also when I tried to get matthews coffecient, it calculates its molecular > mass less ( about 35 kDa) than which should be (original 54kDa) with > solvent content 47%. > > I have also run the silver staining gel of the protein which contained > crystal that shows about 45 kD protein band which is 10 less than the > original. Also I tried to run gel on crystal but it did not give anything > as it was a small crystal. > > I have tried all combinations of the search model and tried to break > available pdb many ways to make different search models but have not got > any good solution. Molrep gives contrast even 10 or more but no good > electron density map yet. Free R and figure of merit becomes 52% and 42% > respectively in Refmac with all the solutions. > Have you tried using an automated building program on the best solutions you have so far? Refinement programs will often get stuck quickly if the MR solution is poor, but rebuilding from scratch can sometimes do a much better job. Other things to try in cases like this are DEN refinement or MR-Rosetta - both require significant computational resources but also have a wider radius of convergence. The other question to ask yourself in situations like this is "did I really crystallize the protein I'm interested in, or something else?" It's surprisingly easy to crystallize minor contaminants; at last count, I've met at least four different people who've done this. (One of them actually ended up with a decent paper describing a structure he'd never intended to solve.) I suspect there are dozens if not hundreds of datasets lying abandoned because they couldn't be phased or reproduced because they weren't what the researcher thought they were. If there is any way to obtain the mass spec of the crystallized protein, this will be the most useful confirmation either way. The next thing to try is searching for similar unit cells in the PDB, although this doesn't take into account changes in space group that result in a different unit cell without actually changing the lattice. (There are probably multiple tools that can account for this; I can point to one if you're interested.) As a last resort, I would recommend a brute-force approach: https://portal.sbgrid.org/d/apps/wsmr/ -Nat
