Xtriage assessment critically depends on the quality of the sigma's. Suboptimal data processing can lead to thinking there is anomalous signal when there isn't. The CC1/2 for the anomalous data could be more informative. The other possibility is of course that your solvent continuum is anomalous: http://www.ncbi.nlm.nih.gov/pubmed/16714785 HTH P On 29 August 2014 02:24, Gabor Bunkoczi wrote:

Hi Charles,

I second Nat's explanation on anomalous signal estimation (although in my experience, xtriage tends to err more often on the pessimistic side than the other way round). However, with an almost perfect model and data collected at the peak, the Br peaks should be enormous. Have you looked at the Phaser-EP logfile? What is the largest peak found in the map? (I can have a look, but please send this logfile to my private email address, and to the list.)

Perhaps it would also help if you re-ran the Phaser-EP job, searching for Br as well as S atoms. With a good model, it is not uncommon that these could be located, even from short wavelength data. This would indicate that the anomalous signal you observe comes from sulfur atoms, and not from bromine, with the obvious conclusion that Br is either not present or disordered.

BW, Gabor

On 2014-08-28 14:59, CPMAS Chen wrote:

...

HI, Gabor,

Thanks for you suggestion.

I tried this method, it did not give out NO sites for Br.

why there is apparent anomalous signal and I cannot identify any Br sites?

As Nat pointed out, other atoms should have very weak anomalous signal diffracted at Br absorption peak (3.8e- for Br, versus 0.2e- for the sulfurs). In the difference map, I can clearly see some peaks at high sigma (> 6), but the anomalous difference map(they are all generated by phenix.refine or phenix.maps) is almost featureless, or the peaks has no overlaps. The anomalous difference gives the possible place of Br while the difference map gives the possible place of the whole ligand, and I assume there should be some overlaps between them. Am I right?

Charles

On Wed, Aug 27, 2014 at 6:28 AM, Gabor Bunkoczi wrote:

...

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 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

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Charles Chen

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University of Pittsburgh School of Medicine

Department of Anesthesiology

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Charles Chen

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Department of Anesthesiology

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