Hi Charles,

I could be totally misunderstanding what you are trying to achieve, but I think what you need to do is an MR-SAD calculation. First, solve the structure with MR (use the macromolecule from the non-Br structure, or use your current model if you have done refinement on it), and use the Phaser-EP GUI, and select "SAD starting from MR model". This should highlight any Br you may have, and gives you a PDB-file with all the peaks (you can also get a proper anomalous map, but this is only useful for visualization - the peak search will identify any peaks you may have).

Let me know if you need more details!

BW, Gabor

On 2014-08-26 14:49, CPMAS Chen wrote:

Thanks. Nat.

Since I have some MET and CYT in the protein, could I try to supply
their sulfur as the initial anomalous scatters?

As for the anomalous isomorphous difference map, would it be useful I
compare the datasets acquired at 1A wavelength and at the Br
absorption peak (~0.92A)?

Appreciate your help

Charles

On Mon, Aug 25, 2014 at 5:08 PM, Nathaniel Echols <nechols@lbl.gov>
wrote:

On Sun, Aug 24, 2014 at 6:46 AM, CPMAS Chen <cpmasmit@gmail.com>
wrote:

Here is the point I am not clear. If I am using phenix.refine to
generate LLG map, how do I pick the anomalous group since I have
not placed them in the model yet?

You can't. The LLG map only becomes really useful once you have
some anomalous scatterers placed and refined - this is how Phaser
substructure completion works. If your molecules have no other
significant anomalous scatterers other than the expected Br, the LLG
map won't do you much good.

By the way, when I choose ion_placement and specify Br, the result
comes with no Br.

Not too surprising, since the code is tuned to look for ions, not
part of a covalent molecule, and halides also tend to bond
non-specifically and we haven't figured out how to deal with that
yet.

I want to find whether the Br-containing ligand is seen in my
protein which I have a high resolution structure available.

I have data collected at Br wavelength, peak or higher position.
Phenix.xtriage reported that the anomalous signal is present to
about 4A. However, both AutoSol or MR-SAD cannot identify the Br
position. Simply say, AutoSol or MR-SAD can not generate any
solution. Well, of course, the simple answer would be that there
is no such ligand cocrystallized.

The simplest explanation is that you just don't have enough
anomalous signal to determine the substructure, which can be true
even if your ligand is bound. Running experimental phasing to
figure this out is unnecessary and time-consuming.

Anyway, I am trying to see if the anomalous difference map or
LLG(generated by phenix.maps, this would be the initial one I
assume) can tell me anything more useful.

So, my question on this topic would be what is a better way you
guys would recommend to identify these Br-ligands? By the way, I
did have the native datasets for the same protein with ligand.

I think in this case I would start with the simple anomalous
difference map. If you run phenix.find_peaks_holes (it's in the
GUI, of course) and give anomalous data as input, it can pick out
the highest peaks in the anomalous map. If the ligand really is
bound and the Br site is ordered I would expect this to be
detectable. Another alternative is to compute an anomalous
isomorphous difference map between a dataset collected at or above
(in eV) the Br peak, and a dataset collected below the peak. This
will allow you to visualize the wavelength-dependent difference in
anomalous scattering, and it's going to be specific for elements
with absorption edges within that energy range. But I really don't
think this should be necessary to answer your question.

-Nat

--

***************************************************

Charles Chen

Research Associate

University of Pittsburgh School of Medicine

Department of Anesthesiology

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