It is quite possible that the S- and Cl- signal is being lost under that
from 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<jubo...@jhsph.edu> 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: j-kell...@northwestern.edu
*******************************************
......................
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/
