Are thw Se SAd and native data isomorphous - because if so you can just
cad the data sets together - generate phases for the Se set, then use
those as a starting set for the native data to phase extend. Parrot is
the more up to date version of DM which does this in the GUI.
The AutoSHARP will use SOLOMON I believe to do this.
Eleanor
On 09/01/2011 08:28 PM, Bosch, Juergen wrote:
How about phase extension using DM, sure you say you only have one mol per asu
but it might still be worth trying various approaches of solvent
flattening/flipping.
Don't know what you used to detect your sites and refine them, but it also
might be worth sticking them into Sharp with your partial model and see if the
phases improve.
You say you have 450/750 residues what makes you believe that you placed them
correctly ?
Also do you have space from the crystal lattice packing for the additional 300
residues ? In other words are you certain that what you have crystalized is the
full length version of your protein ?
Adding side chains leading to worse Rfactors would suggest that you are most
likely not in frame.
Since you have a partial backbone you could try finding a "homolog" via EBI SSM
to serve as a better starting model, aligning your sequence to it and replacing the side
chains accordingly.
Another suggestion keep changing programs don't stick to Refmac visit Phenix or
the other way round, and as a last resort you could give GraphENT a try.
Jürgen
On Sep 1, 2011, at 3:00 PM, Pete Meyer wrote:
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
......................
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry& Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab: +1-410-614-4894
Fax: +1-410-955-2926
http://web.mac.com/bosch_lab/
