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