following on the anomalous map

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phenix.maps command line arguments

CPMAS Chen

24 Aug 2014 24 Aug '14

9:46 p.m.

Hi, All Phenix users, I am following this topic. http://www.phenix-online.org/pipermail/phenixbb/2014-April/020622.html. 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? By the way, when I choose ion_placement and specify Br, the result comes with no Br. Let me make my situation clear first. 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. 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. Thanks! Charles -- *************************************************** Charles Chen Research Associate University of Pittsburgh School of Medicine Department of Anesthesiology ******************************************************

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Nathaniel Echols

26 Aug 26 Aug

5:08 a.m.

On Sun, Aug 24, 2014 at 6:46 AM, CPMAS Chen 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

CPMAS Chen

9:49 p.m.

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 wrote:

...

On Sun, Aug 24, 2014 at 6:46 AM, CPMAS Chen 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 ******************************************************

Nathaniel Echols

11:48 p.m.

On Tue, Aug 26, 2014 at 6:49 AM, CPMAS Chen wrote:

...

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

Technically this would work, but I don't see much point. At the Br absorption peak you'd expect an f'' of at least 3.8e- for Br, versus 0.2e- for the sulfurs, which means that Br should be by far the largest peak in the map unless it's just not there at all. Refining anomalous scattering the sulfurs won't change this. (What the LLG map is useful for is the reverse - refine the anomalous scattering for Br, and the LLG map will more clearly show the sulfurs.) 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)?

Yes, the anomalous 0.92Å-minus-1.0Å map would be expected to show a positive peak around any Br atoms (it works for SeMet MAD data, at least). But again, I think if it is bound at all the conventional anomalous difference map for the 0.92Å data should be clear enough if the ligand is bound at high enough occupancy to be modeled accurately. -Nat

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CPMAS Chen
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