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 <mjvanra...@cnb.csic.es> wrote: From: Mark J van Raaij <mjvanra...@cnb.csic.es> Subject: Re: [ccp4bb] help regarding structure solution To: "sonali dhindwal" <sonali11dhind...@yahoo.co.in> 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 <hsuu...@u.washington.edu> wrote: > > From: Peter Hsu <hsuu...@u.washington.edu> > Subject: Re: [ccp4bb] help regarding structure solution > To: CCP4BB@JISCMAIL.AC.UK > 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
