[phenixbb] following on the anomalous map

Gabor Bunkoczi gb360 at cam.ac.uk
Fri Aug 29 02:24:46 PDT 2014

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 <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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>       Dr Gabor Bunkoczi
>       Cambridge Institute for Medical Research
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> --
> ***************************************************
> Charles Chen
> Research Associate
> University of Pittsburgh School of Medicine
> Department of Anesthesiology
> ******************************************************
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