Somewhat to our surprise, we have found that, even when the halide
signal is too weak to solve the substructure from scratch, when you
find the sites (in our case, by using the protein model as a
"substructure" in Phaser), they can still add significant phase
information. So you can get more out of it than just seeing which
sites are halides, especially since the model can be a rough and
incomplete one.
But if all you're after is seeing which sites are halides, the
procedure in Phaser (which is iterative, in that sites found in the
first round help to find further sites) is more sensitive and
convincingly finds more sites than an anomalous difference Fourier
(which is not iterative).
There's a tutorial on our web page with real (albeit lysozyme) data
illustrating how this works.
Regards,
Randy Read
On 31 Mar 2009, at 18:17, Ethan Merritt wrote:
On Tuesday 31 March 2009 09:57:13 Jose Antonio Cuesta-Seijo wrote:
Hi!
Normally the cell parameters, etc change very very little. You'll
only know if the bromides got in at the synchrotron by looking at the
fluorescence spectrum
That won't help, normally. It only tells you that there is bromide in
the solution, not that it found ordered positions in your crystal.
and at the anomalous signal. Normally some will
make it in and some will be in ordered sites, then it becomes mostly
a question of data quality to detect it.
Right. You have to process the data and look for a real signal.
The mere presence of Br is not enough.
In my experience, the frustrating thing is that even if the Br soak
"works" in the sense that it introduces Br into your lattice, the
signal is often/usually not sufficient to phase the structure
de novo. Nevertheless, when you eventually do solve and refine
the structure, you can go back to your anomalous difference data
and calculate a Bijvoet Difference Fourier that clearly shows the
Br sites.
Ethan
You could also try the equivalent iodide soak. Iodine has a decent
anomalous signal at the copper wavenght and thus the anomalous signal
can be detected at your home source and many times the structure can
be solved by SAD or SIRAS. I would also thing that conditions that
give ordered iodide sites are likely to result in ordered bromide
sites, although the ions are not identical.
Jose.
**************************************
Jose Antonio Cuesta-Seijo
Cancer Genomics and Proteomics
Ontario Cancer Institute, UHN
MaRS TMDT Room 4-902M
101 College Street
M5G 1L7 Toronto, ON, Canada
Phone: (416)581-7544
Fax: (416)581-7562
email: jcue...@uhnres.utoronto.ca
**************************************
On Mar 31, 2009, at 12:19 PM, tat cheung cheng wrote:
Hi all
I am now trying to do bromide soaking, but i am not really sure
does the bromide atom enter my crystal. So is there any signs that
indicate the entry of bromide atom? e.g. does the space group, cell
dimension change? or just nothing change, and the bromide atom just
get in?
Thanks very much.
T.C. Cheng
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--
Ethan A Merritt
Biomolecular Structure Center
University of Washington, Seattle 98195-7742
------
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research Tel: + 44 1223 336500
Wellcome Trust/MRC Building Fax: + 44 1223 336827
Hills Road E-mail: rj...@cam.ac.uk
Cambridge CB2 0XY, U.K. www-
structmed.cimr.cam.ac.uk
