Hi,
Depending on how many zn sites you have, you may be able to do zn-mad
for your native crystals. You don't mention if you've tried combining
your various sources of phase information; if not, it's worth looking into.
You may also want to look into various multi-crystal techniques
(averaging, phasing and/or merging) - I've had decent luck with
multi-crystal phasing off zn at low resolutions.
Good luck,
Pete
Basudeb Bhattacharyya wrote:
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
