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 <[log in to unmask]">[log in to unmask]> wrote:
>
> From: Peter Hsu <[log in to unmask]">[log in to unmask]>
> Subject: Re: [ccp4bb] help regarding structure solution
> To: [log in to unmask]">[log in to unmask]
> 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