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

Randy J. Read rjr27 at cam.ac.uk
Thu Mar 13 14:28:00 PDT 2014

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. 

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. 

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. 

Best wishes
Randy Read

Randy J. Read

> On 13 Mar 2014, at 13:29, Guenter Fritz <guenter.fritz at uni-konstanz.de> wrote:
> 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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