To repeat James's points
1) DANO (or Diso) maps are are equivalent to calculating a projection -
even for perfect data the term is F" sin(PhiP-PhiA) so
it wont be as clear as a LLG map.. from either SHARP or Phaser .
2) the SIGMA level will reflect the Se signal strength
I usually calculate a DANO map and do a peak search to check whether
there is a weak signal. (GUI FFT anomalous - peak search - then read
those "coordinates" into coot to compare to the existing model. The
weak scatterers are usually there but often way down the list..
3) Of course if the anom diff is a bit damaged by radiation or something
else your signal may be buried by the noise.
Eleanor
On 09/02/2011 03:02 PM, James Holton wrote:
> It is quite possible that the S- and Cl- signal is being lost under that
> from) so the Se sites. Could be:
> 1) simply noise that would swamp the S-SAD signal anyway
> 2) you just aren't contouring your map low enough ("sigma" is not on an
> absolute scale)
> 3) trigonometry. Remember, with SAD you are not looking at the heavy
> atom contribution (FH) directly, you are looking at the projection of FH
> that is orthogonal to FP (the protein contribution). Hence the weaker
> atom positions have less and less influence on DANO as the Se signal
> becomes stronger.
>
> One way to test the last hypothesis is to calculate anomalous
> differences from your model and see what that anomalous-difference map
> looks like. You can get DANOcalc for a given PDB file and wavelength
> using the CCP4 Suite and my script:
> http://bl831.als.lbl.gov/~jamesh/mlfsom/ano_sfall.com
>
> or you can do it with phenix.fmodel after you look up the f', f" for all
> your elements at the wavelength of interest.
>
> It is interesting to see what happens if you set f" = 100 electrons or
> more. If f" is too big, then it swamps FP, and you can no longer get
> phases by SAD.
>
> -James Holton
> MAD Scientist
>
> On 9/1/2011 3:29 PM, Jacob Keller wrote:
>> Update:
>>
>> I tried more anomalous maps, this time with the originally-deposited
>> data at 1.8 Ang (mine were similar, substrate-soaked crystals) and
>> phases from the refined model, and the Se sites are now ~40-50 sigma,
>> and there is still totally nothing at the Cl and S sites, even though
>> in 2Fo-Fc the Cl is ~9 sigma, and the S is 8 sigma (the Se is ~15
>> sigma). If it has reasonably-high electron density, shouldn't it have
>> at least some anomalous scattering? I am wondering whether somehow the
>> model phases are biasing the map, but I can't really imagine how that
>> would be...
>>
>> JPK
>>
>>
>> On Thu, Sep 1, 2011 at 3:15 PM, Bosch, Juergen<[log in to unmask]> wrote:
>>> Where in refinement of your model are you ?
>>> At an early stage I wouldn't be surprised to only see SeMets but once
>>> you've
>>> refined your structure and go back to calculate an anomalous map with
>>> the
>>> improved phases you might double your signal for SeMet and start seeing
>>> sulfurs.
>>> An alternative explanation, you've blasted your crystals at the
>>> synchrotron
>>> and the remaining anomalous signal is too weak to show the sulfurs.
>>> Just two thoughts,
>>> Jürgen
>>> On Sep 1, 2011, at 4:03 PM, Jacob Keller wrote:
>>>
>>> Dear Crystallographers,
>>>
>>> I recently have been working with a 2.5 Ang SeMet peak wavelength
>>> dataset which contains 2 cys's and also a couple of bona fide Cl ions
>>> (reasonable b-factor/site is semi-buried/water does not work). In the
>>> FFT anomalous difference map using PhiC from the refined model and
>>> Dano, I can see the MSE's at ~10 sigma, but no Cl ions, even though Cl
>>> should have f" = ~0.3 versus Se's f" = ~4, and no S's in the cys,
>>> despite f" = 0.23e. There is really no anomalous peak at all--is it
>>> just the smallness of the signal, or are the Se's somehow "swamping
>>> out" the other signal? Perhaps the phases are tainted by the presence
>>> of semet in the model?
>>>
>>> Looking for suggestions,
>>>
>>> Jacob Keller
>>>
>>> *******************************************
>>> Jacob Pearson Keller
>>> Northwestern University
>>> Medical Scientist Training Program
>>> cel: 773.608.9185
>>> email: [log in to unmask]
>>> *******************************************
>>>
>>> ......................
>>> Jürgen Bosch
>>> Johns Hopkins University
>>> Bloomberg School of Public Health
>>> Department of Biochemistry& Molecular Biology
>>> Johns Hopkins Malaria Research Institute
>>> 615 North Wolfe Street, W8708
>>> Baltimore, MD 21205
>>> Office: +1-410-614-4742
>>> Lab: +1-410-614-4894
>>> Fax: +1-410-955-2926
>>> http://web.mac.com/bosch_lab/
>>>
>>>
>>>
>>>
>>>
>>
>>
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