Dear Sonali, Following Michael's suggestion that you just might have crystallized a contaminant (and all the signs point to that I'm afraid - a small crystal growing after a long time that diffracts surprisingly well), you might expect this crystal form already to have been characterised and deposited in the PDB.
In which case can I suggest you use a tool developed by my student, Varun Ramraj, to check the unit cell against the PDB. Its quick and easy and the URL is http://www.strubi.ox.ac.uk/nearest-cell/nearest-cell.cgi It just might save a lot of wasted effort. Regards, Robert -- Dr. Robert Esnouf, University Research Lecturer and Head of Research Computing, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford OX3 7BN, UK Emails: rob...@strubi.ox.ac.uk Tel: (+44) - 1865 - 287783 and rob...@esnouf.com Fax: (+44) - 1865 - 287547 ---- Original message ---- >Date: Wed, 20 Jun 2012 16:00:06 -0400 >From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> (on behalf of "R. M. Garavito" <rmgarav...@gmail.com>) >Subject: Re: [ccp4bb] help regarding structure solution >To: CCP4BB@JISCMAIL.AC.UK > > Dear Sonali, > I think that first item on your possible to-do list > is to verify that you have indeed crystallized the > protein you purified. We, too, got great crystals > once with protein X (100 kD) and noticed that 1) the > lattice constants, space group symmetry, and > Matthew's coefficient were within expected values > (100 kD monomer in the ASU with moderate solvent > content) and 2) the protein seemed to be cleaved > into 50 kD fragments in the drop and in the crystal > (as expected). However, it was a totally different > protein that crystallized, which was why MR didn't > work at all. After solving the structure by MIR, we > found that a 50 kD minor contaminant (< > 1%) crystallized, while our target protein did not. > While there is still a good chance that the > crystals you looked at contains at least a fragment > of your target protein, make sure first, if you can. > As Matt recommended, analyze your protein by mass > spec and/or N-terminal sequencing to verify that it > is what you think it is. Then, as he recommended, > try cloning and expressing a truncated variant. > Careful limited proteolysis of the full-length > protein would also be worthwhile in getting crystals > faster. > Good luck, > Michael > ************************************************************** ** > R. Michael Garavito, Ph.D. > Professor of Biochemistry & Molecular Biology > 603 Wilson Rd., Rm. 513 > Michigan State University > East Lansing, MI 48824-1319 > Office: (517) 355-9724 Lab: (517) 353-9125 > FAX: (517) 353-9334 > Email: rmgarav...@gmail.com > ************************************************************** ** > On Jun 20, 2012, at 2:13 PM, sonali dhindwal wrote: > > Dear All, > 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. > I will highly appreciate all the suggestions for > this kind of problem. > Thanks and regards > -- > Sonali
