<html><head><meta http-equiv="content-type" content="text/html; charset=utf-8"></head><body dir="auto"><div>Guenter's experience agrees with Tom's suggestion that the helical model could give higher resolution phase information, so I'd definitely try that approach. However, there's something even simpler that has worked for us. Give the low resolution map as a density- based partial model to Phaser for LLG completion. </div><div><br></div><div>The Phaser LLG approaches have the advantage that you bootstrap to higher resolution. Once some of the Se sites have been found they effectively give phase information to the higher resolution limit. </div><div><br></div><div>If the crystals are not quite isomorphous you can still do something similar. Cut out the unique density from the experimental map and provide it to Phaser as an MR model. Rigid body refinement from zero rotation and translation will probably work instead of a full search. Then use that map for LLG completion. </div><div><br></div><div>Best wishes</div><div>Randy Read<br><br>----<br><div><div>Randy J. Read</div></div></div><div><br>On 13 Mar 2014, at 13:29, Guenter Fritz <<a href="mailto:guenter.fritz@uni-konstanz.de">guenter.fritz@uni-konstanz.de</a>> wrote:<br><br></div><blockquote type="cite"><div>
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<div class="moz-cite-prefix">Hi Todd,<br>
<br>
we had a similar case but smaller (100 x 150 x 120, 70 Se-met) .
With phases from heavy atom clusters at 5-6 A I did not get the
Se positions. However, when we used a 'crude' alpha helix model as
input for phenix.phaser rigid body refinement with the Se-met data
we got 90% of the Se-met sites and very good phases. My Se-met
data were just 4 A. <br>
Best, Guenter<br>
</div>
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<div style="direction:ltr; font-family:Tahoma; color:#000000;
font-size:10pt">Hello all-<br>
<br>
I am working on a huge protein assembly(monomer is ~3000 amino
acids, large cell ~200, ~200, ~520) that has between 200 and
300 Se-met residues in the asymmetric unit(depending on the
number of molecules I pick in the a.u.). I believe I have low
resolution phasing from another heavy atom with limits to ~
6.2 angstrom. Density modified FOMs dip below 0.6 at that
point. I can see many tubes that have the diameter of model
alpha helices. I ran phenix.find_helices_strands with the
helices_before_trace=True option and got a series of helices
encompassing about 3400 residues. Assuming that the phasing on
the low resolution derivative is true, is it better to use
phasing from the lower resolution derivative or phasing from
the helical models to fish out the Se-Met sites via an
anomalous difference Fourier? I assume I'm stuck at 6.2 for
the derivative phasing though i guess i could extend phasing
from the map a little further. How far is too far? How high of
resolution can I phase from the helices (or from the
derivative map for that matter) and still get accurate enough
phasing to yield reliable difference peaks to find the Se
sites. I have a couple Se-met datasets between 3 and 2.5
angstroms. What sigma levels should I expect for the
difference peaks?
<br>
<br>
also, what is the best way to pick the highest difference
peaks in the anomalous difference fourier? I didn't see a tool
in phenix. "find difference peaks and holes" seems like a
logical choice but it seems to want to structure. I've just
created maps and used the very old and reliable peakmax in
ccp4.<br>
<br>
I have solved structures like this before but with a lot more
info on ncs, envelopes, ncs averaging and a much much smaller
protein. I remember getting sites and plugging them into
resolve with scripts where you could fix them/or not and phase
without looking for more sites, no build etc. Can i just enter
sites and phase followed by density modification with one of
the gui programs.
<br>
<br>
any comments would be appreciated? and thanks in advance.<br>
-Todd<br>
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