Hi, My favourite first-stop tool: use your cell parameters at
http://www.rcsb.org/pdb/search/advSearch.do and search for similar Cell Dimensions and Space Group. Or use ftp://ftp.wwpdb.org/pub/pdb/derived_data/index/crystal.idx and a short awk script to list similar PDB entries (it's what I do) ... as Mark says: things happen ;-) Clemens On Thu, Jun 21, 2012 at 06:01:33PM +0800, sonali dhindwal wrote: > Dear Raaij, > > We have not done mass-spec on the band from SDS-PAGE > to confirm if it is our desired protein or any other contaminant. So, > cant say for sure. > > Regards > > -- > Sonali Dhindwal > > “Live as if you were to die tomorrow. Learn as if you were to live forever.” > > --- On Thu, 21/6/12, Mark J van Raaij <[email protected]> wrote: > > From: Mark J van Raaij <[email protected]> > Subject: Re: [ccp4bb] help regarding structure solution > To: "sonali dhindwal" <[email protected]> > Date: Thursday, 21 June, 2012, 11:33 AM > > you didn't answer the most important question - are you 100% sure the protein > in the crystal is not a contaminant? > Unfortunately, these things happen... > Mark J van Raaij > Laboratorio M-4 > Dpto de Estructura de Macromoleculas > Centro Nacional de Biotecnologia - CSIC > c/Darwin 3 > E-28049 Madrid, Spain > tel. (+34) 91 585 4616 > http://www.cnb.csic.es/~mjvanraaij > > > > On 21 Jun 2012, at 06:47, sonali dhindwal wrote: > > > Dear All, > > > > Thanks a lot for your replies. Glad to found so much help. > > Clemens, > > cell parameters are, > > 38.0020 78.0240 56.3800 90.0000 102.2770 90.0000, in P21 spacegroup. > > > > Raaij, Savvas, > > we have checked for the twining and no twining was detected. > > > > Nat, > > DEN is a good suggestion, i will definitely try it today. > > > > Roger, > > Molrep didn't fail but as i mentioned in the mail, it do give 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. But none of the solution fits in the electron density. > > And phaser didn't give any solution. We checked in pointless also, it was > > suggesting the same spacegroup. > > > > Matthew, > > Yes, we don't have the same crystal now. > > > > Garib, > > I will run the balbes server, and will let you know then. > > > > Robert, > > I tried using your server, and found few hits. Will run those templates for > > molecular replacement. > > > > Peter, > > We didnt had MBP tag in the protein. But your idea of doing limited > > proteolysis sounds good, and will definitely try that. > > > > Thanks again to all, for their kind and valuable help. I will write after > > trying all the things as suggested. > > > > Regards > > > > > > -- > > Sonali Dhindwal > > > > “Live as if you were to die tomorrow. Learn as if you were to live forever.” > > > > > > --- On Thu, 21/6/12, Peter Hsu <[email protected]> wrote: > > > > From: Peter Hsu <[email protected]> > > Subject: Re: [ccp4bb] help regarding structure solution > > To: [email protected] > > Date: Thursday, 21 June, 2012, 5:08 AM > > > > Hi Sonali, > > > > Did you use MBP as your purification tag? That's around 45-50kDa if I > > remember right. > > > > If not, I've had a decent amount of luck using in situ proteolysis to get > > crystals of degraded fragments. Try a limited proteolysis first overnight > > at 4C at varying concentrations of trypsin, see which one gives a nice > > stable band after overnight. Use that same concentration to add to your > > protein stock before setting up drops and then try another screen. I always > > use freshly prepared trypsin stock instead of frozen solutions to make sure > > that the freeze thaw doesn't reduce activity of the trypsin and that batch > > to batch is reproducible. > > > > Best of luck, > > Peter > -- *************************************************************** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-------------------------------------------------------------- * BUSTER Development Group (http://www.globalphasing.com) ***************************************************************
