[phenixbb] phasing high(er) resolution anomalous difference maps with low resolution phases

Guenter Fritz guenter.fritz at uni-konstanz.de
Thu Mar 13 12:29:20 PDT 2014

Hi Todd,

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.
Best, Guenter
> Hello all-
> 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?
> 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.
> 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.
> any comments would be appreciated? and thanks in advance.
> -Todd
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