[phenixbb] following on the anomalous map

CPMAS Chen cpmasmit at gmail.com
Thu Aug 28 06:59:30 PDT 2014

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?


On Wed, Aug 27, 2014 at 6:28 AM, Gabor Bunkoczi <gb360 at cam.ac.uk> 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 <nechols at lbl.gov>
>> wrote:
>>  On Sun, Aug 24, 2014 at 6:46 AM, CPMAS Chen <cpmasmit at 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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>> phenixbb at phenix-online.org
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> --
> ##################################################
>      Dr Gabor Bunkoczi
>      Cambridge Institute for Medical Research
>      Wellcome Trust/MRC Building
>      Addenbrooke's Hospital
>      Hills Road
>      Cambridge CB2 0XY
> ##################################################



Charles Chen

Research Associate

University of Pittsburgh School of Medicine

Department of Anesthesiology

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