Did you try density modification starting with the sad phases with phase extension to 2.7 A followed by auto-tracing? The model should be better than the one built from the 3.7 A sad map.
Sent from Jack's iPad
On Sep 1, 2011, at 11:24 PM, "Kianoush Sadre-Bazzaz" <[log in to unmask]<mailto:[log in to unmask]>> wrote:
Hi Basu,
You mentioned molecular replacement was not successful for this project. Which model was used for this procedure? Have you tried your partially built structure as a model to obtain preliminary phases for your native (2.7A) data set? If there is any luck with that, you might be able to combine phases from this procedure with the Selenium SAD phases, as already suggested.
Kianoush
--- On Thu, 9/1/11, Basudeb Bhattacharyya <[log in to unmask]<mailto:[log in to unmask]>> wrote:
From: Basudeb Bhattacharyya <[log in to unmask]<mailto:[log in to unmask]>>
Subject: [ccp4bb] Low resolution structure determination advice
To: <mailto:[log in to unmask]> [log in to unmask]<mailto:[log in to unmask]>
Date: Thursday, September 1, 2011, 9:31 AM
Dear all,
We're looking for some advice about how to proceed with a structure we're working on. Our protein is 750 amino acids and naturally binds zinc. We have a SeMet data set that goes down to 3.7 angstroms. 4 of 8 selenium sites are ordered and visible in addition to our zincs and we've modeled about 450 residues of C-alpha backbone off a pure SAD density map
(we've tried other phasing experiments such as Zn SAD, MAD, etc. and the best maps--clear density and visible secondary structures--we get are off Se SAD). We have one monomer per AU (and we have secondary structure coverage over our entire protein based on looking at conserved domains of our protein--unfortunately, MR is not working for this project).
R/Rfree hits a minimum of 37/41 respectively in spacegroup P32 (we've tried P3 and P31, which haven't worked). We also have a native set down to 2.7 angstroms. We are able to place our working model into the native data set, but we are unable to further refine the structure in Refmac (density doesn’t improve and the stats creep up). Addition of side chains only makes our stats worse. The data sets are clean (no twinning, etc.). While we understand that we may need more phasing information (i.e. our initial model may still be quite inaccurate given resolution and size of the protein among other things--we are trying to improve this), 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. Thanks in advance for any advice.
Sincerely,
Basu
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