Dear Vijay,

 

I managed success on a large, 4.5 Ang SeMet phasing years ago.  I strongly recommend trying to get a heavy atom derivative – even a crappy one -  that allows you to use difference Fouriers to find your Seleniums.  For me this was the only approach that worked, and it required bootstrapping from 5 initial peaks in the difference Fourier maps to eventually the full 70 in a.u.  It was a tedious process, always deleting sites to check if they come back, etc., but it is doable.

 

Good luck-


Steve

 

From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of vijay srivastava
Sent: Tuesday, June 03, 2014 4:38 AM
To: [log in to unmask]
Subject: [ccp4bb] experimental phasing at low resolution

 

Dear All,

Does anyone know of a facility that provides amino acid analysis for determination of SeMet incorporation in a recombinant protein?

We're looking for some advice about how to proceed with a structure we're working on.  Our protein is 350 amino acids (10 methionine are present) and naturally binds magnesium.  We have a SeMet data set at peak that goes down to 5.4 angstroms.  We tried to find the heavy atom position with Shelxcde program. The log file is given as below.

 

Resl. Inf - 8.0 - 6.0 - 5.6 - 5.4 - 5.2 - 5.0 - 4.8 - 4.6 - 4.4 - 4.2 - 3.98

N(data) 529 643 241 29 0 0 0 0 0 0 0

<I/sig> 63.5 19.5 10.6 10.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

%Complete 94.1 98.9 99.2 18.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

<d"/sig> 3.06 1.24 1.07 1.21

 

I am also attaching the .lst and .res file and from that we were considering that only one selenomethionine position.We have one monomer per AU (-unfortunately, MR is not working for this project). The space group is P3 .  We also have a native set down to 3.4 angstroms.  While we understand that we may need more phasing information we're wondering if anyone might have some other suggestions or insights about how we can move forward given the data that we currently have. Can we extend the phases with our native data set. Thanks in advance for any advice.

 

regards

 

Vijay

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