[phenixbb] Help needs: SAD-phasing for Cobalt ion

YoungJin yjcho at brandeis.edu
Fri Dec 10 09:47:15 PST 2010

Dear helpers in phenix,

I have anomalous data (Co; wavelength: 1.609A) that I want to use its 
Phase information from SAD-phasing method (autosol).
As I have a native data (from nomal wavelength ~1A) with higher 
resolution (~2A) than SAD data (~2.5A), I wanted to apply phase 
information to this native data for further refinement: My goals were 1. 
I want to confirm where is my heavy metal is; 2. I want to refine 
further using native data as has higher resolution.
At first, when I did autosol, it didn't give me perfect result. So far 
what I tired is as following:
'SAD peak' from imosflm generated mtz file (scaled), 'Phases' from first 
refmac generated mtz file from the native data, and 'MIR native' from 
the native data mtz file (imosflm and scaled).
FOM: 0.186; overall score was 10.75 +/- 12.33.
FYI. I input 2 sites and result suggested 3 sites. Each molecule has one 
binding site of metal ion. I was using old Mac version:1.6.4-486

Anyway, I simply passed the result over Autobuild. It ran and gave me 
better statistics as following:
R/Rfree=0.2734/0.3312; CC 0.76.  However (I have two molecules/ASU) one 
of models was not great. But this time, anomalous density map well 
phased(?!) to 2Fo-Fc map and I could see - it was before only one heavy 
metal I clearly could see- 2 heavy metal signal from 2 molecules and 
those positions are well matched with native density maps.

So I continued phenix.refine using Autobuild made 
'expt_fobs_phase_freeR_flags_1.mtz'. The question is the statistics from 
anomalous data itself (it has slight dispcrepancy between anomalous map 
and 2Fo-Fc/Fo-Fc map due to phase problem) or even those from native 
data is better than my current model which came from native data set 
(SAD-phased as described above) with higher resolution. I think one of 
main reasons is one of molecule has a lid that does not perfectly fit 
into current density map. However, in terms of heavy metal position and 
the active site is better from current map (Autobuild generated model). 
FYI: current model has R/Rfree=0.21/0.27(best was 0.21/0.26); 0.19/0.24 
(for native data only used model) .... Am I too concerning for 
statistics or is there something I am missing?

Now my questions are as following plus any suggestions are welcome:
1. For SAD-phasing, could you tell me which is best way to do so with my 
case (one data from the edge of heavy metal (lower resolution) and 
native data (higher resolution) or where is the point I can convince. 
Also I tried to use DANO/SIGDANO column (from hkl2000 and ccp4) but 
seems like it (Autosol) doesn't take them into account.  Could you tell 
me how can I improve phase by using anomalous data?

2. In case of Co, does anyone have experience about coordination 
numbers? I thought it  should have 6 (or 4) coordination numbers. The 
question what I have now is about the numbers and their lengths ... I 
though it should be within 2A, but not true for my current models (And 
somehow distorted octahedron or else with 2.07-3.28A). I also searched 
PDB files, and found that many deviations from 1.98 to 2.82A (depending 
on corresponding atoms and resolutions: 3KOI, 3KON, 2DD5, and 2XJM). I 
would appreciate if you can give me any tips regarding this issue.

Many thanks in advance,


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