Hi, Phil, Is it right to model the whole ligand molecule to the anomalous difference map? For some sites, the overlap between anomalous difference and difference map is small, while the anomalous difference or difference map itself is rather big. The anomalous difference is way bigger than a single Br can be. Thanks! Charles On Mon, Sep 8, 2014 at 1:10 PM, Phil Jeffrey wrote:

In your first sentence your meaning of "difference map" is: ( F_obs - F_calc ), phases

For an anomalous difference map you're calculating: ( F(+) - F(-) ), phases-90

So you would still expect to see a peak in the anomalous difference map in the presence of the anomalous scatterer since you are not subtracting off its contribution to the scattering.

There probably would be (very) small differences in the phases dependent on whether you explicitly model the Br as an anomalous scatterer or not but I have never experimented to see if that makes a small impact on an anomalous difference map.

Cheers, Phil Jeffrey Princeton

-- *************************************************** Charles Chen Research Associate University of Pittsburgh School of Medicine Department of Anesthesiology ******************************************************

Thanks, all. It did clear my thought. Because I always wondering why the LLG map is getting more different from the anomalous difference map and getting close to the 2FoFc map, while the anomalous difference map is almost the same as the beginning when I have no ligand put in the model. Charles On Mon, Sep 8, 2014 at 1:23 PM, Pavel Afonine wrote:

On Mon, Sep 8, 2014 at 9:58 AM, CPMAS Chen wrote:

...

A difference map is used to identify whether there is anything not-modeled, say some ligands, ions. Then when I generate anomalous difference map, I should not put the ligand(which contains Br) in the model, right? Or in phenix, after I put the Br-ligand in the model, I should not see the anomalous difference density at the site, right?

An "anomalous difference map" is a map of the anomalous differences DANO (Fobs(+) - Fobs(-)). It's independent of Fcalc, so it doesn't matter if you put the ligand in or not.

This is not quite an accurate statement. To calculate a Fourier map one requires amplitudes and phases. (Fobs(+) - Fobs(-)) are the amplitudes for the anomalous difference map. The phases come from Fcalc (actually from Fmodel), one way or another.

Pavel

-- *************************************************** Charles Chen Research Associate University of Pittsburgh School of Medicine Department of Anesthesiology ******************************************************

I have a similar question about this. when the anomalous difference map identify possible sites for Br(in my case), I should also expect to see the difference map peak at this site. If not, could this "Br" be something else? Thanks! Charles On Tue, Sep 9, 2014 at 11:40 AM, CPMAS Chen wrote:

Hi, All

Is it possible that I have anomalous and LLG peak, but I have no difference density peak? In this case, is that because the model I have is not good enough or the diffraction data at this site of the model is missing?

Thanks!

Charles

On Mon, Sep 8, 2014 at 1:23 PM, Pavel Afonine wrote:

...

On Mon, Sep 8, 2014 at 9:58 AM, CPMAS Chen wrote:

...

A difference map is used to identify whether there is anything not-modeled, say some ligands, ions. Then when I generate anomalous difference map, I should not put the ligand(which contains Br) in the model, right? Or in phenix, after I put the Br-ligand in the model, I should not see the anomalous difference density at the site, right?

An "anomalous difference map" is a map of the anomalous differences DANO (Fobs(+) - Fobs(-)). It's independent of Fcalc, so it doesn't matter if you put the ligand in or not.

This is not quite an accurate statement. To calculate a Fourier map one requires amplitudes and phases. (Fobs(+) - Fobs(-)) are the amplitudes for the anomalous difference map. The phases come from Fcalc (actually from Fmodel), one way or another.

Pavel

--

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

Charles Chen

Research Associate

University of Pittsburgh School of Medicine

Department of Anesthesiology

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

-- *************************************************** Charles Chen Research Associate University of Pittsburgh School of Medicine Department of Anesthesiology ******************************************************

Thanks, Nat. If this is noise, why the anomalous or LLG peaks could be as high as 5 ~ 6 sigma? Charles On Tue, Sep 9, 2014 at 5:58 PM, Nathaniel Echols wrote:

On Tue, Sep 9, 2014 at 8:40 AM, CPMAS Chen wrote:

...

Is it possible that I have anomalous and LLG peak, but I have no difference density peak? In this case, is that because the model I have is not good enough or the diffraction data at this site of the model is missing?

You should at a minimum see > 1sigma density in the 2mFo-DFc map, and if the site is unmodeled (or modeled as a water) you should see an mFo-DFc peak as well. If neither of these applies, the anomalous and LLG peaks are probably just noise.

-Nat

-- *************************************************** Charles Chen Research Associate University of Pittsburgh School of Medicine Department of Anesthesiology ******************************************************

So I have a followup question to this comment in the thread: what is the origin of this rule of thumb of 1 and 3 sigma that people quote for non-difference and difference maps that you can find all over the internet at various sites, but nobody references? I can't find any papers on it specifically, but it's quite possible I'm not digging far enough back in time. Can anyone give me a lead? Thanks, Scott On Wed, Sep 10, 2014 at 9:26 AM, Pavel Afonine wrote:

Hi Charles,

I think the key here is what you call "no difference density peak". Sites may be partially occupied so rule of thumb for choosing contouring levels for 2mFo-DFc and mFo-DFc maps (1 and 3 sigma, correspondingly) may not be appropriate, for instance.

Pavel

On 9/10/14 6:10 AM, CPMAS Chen wrote:

Thanks, Nat.

If this is noise, why the anomalous or LLG peaks could be as high as 5 ~ 6 sigma?

Charles

On Tue, Sep 9, 2014 at 5:58 PM, Nathaniel Echols wrote:

...

On Tue, Sep 9, 2014 at 8:40 AM, CPMAS Chen wrote:

...

Is it possible that I have anomalous and LLG peak, but I have no difference density peak? In this case, is that because the model I have is not good enough or the diffraction data at this site of the model is missing?

You should at a minimum see > 1sigma density in the 2mFo-DFc map, and if the site is unmodeled (or modeled as a water) you should see an mFo-DFc peak as well. If neither of these applies, the anomalous and LLG peaks are probably just noise.

-Nat

--

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

Charles Chen

Research Associate

University of Pittsburgh School of Medicine

Department of Anesthesiology

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

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

-- Scott Horowitz, Ph.D. Research Associate Howard Hughes Medical Institute University of Michigan Department of Molecular, Cellular, and Developmental Biology Bardwell lab 830 N. University Ave, Room 4007 Ann Arbor, MI 48109 phone: 734-647-6683 fax: 734-615-4226

-----BEGIN PGP SIGNED MESSAGE----- Hash: SHA1 On 10/10/2014 8:24 AM, Scott Horowitz wrote:

So I have a followup question to this comment in the thread: what is the origin of this rule of thumb of 1 and 3 sigma that people quote for non-difference and difference maps that you can find all over the internet at various sites, but nobody references? I can't find any papers on it specifically, but it's quite possible I'm not digging far enough back in time. Can anyone give me a lead?

Thanks, Scott

These particular contour levels are just "rules of thumb" as you say. The mistake of referring to them as "sigmas" gives the illusion of some sort of statistical basis. They are simply rmsd's and have no mathematical justification for being used to assess the quality, significance, or accuracy of a ligand identification (as Pavel noted). The 3 rmds contour level for Fo-Fc style maps is particularly problematic. The rmsd of the difference map for a model in the early stages of refinement will be large while at the end it will be small. The same piece of density due to missing atoms will appear weak in the early map and strong later. Conversely the features visible in an early map will correspond to striking errors in the model while features in a final difference map that appear at exactly the same strength will reflect tiny changes. This lack of calibration results in a continual hunt for poorer and poorer water molecules because no matter how many you build there are still plenty of "3 sigma" peaks in the difference map. The paper by Lang, Holton, Fraser, & Alber, recommended by Pavel, discusses many of these issues. They show that the real standard deviation of electron density maps is much lower than these rmsd values. The problem isn't that density in a 2Fo-Fc style map less than 1 rmsd isn't statistically significant but that it is hard to distinguish between alternate models that all fit this density. Telling the difference between a partially occupied ligand and superimposed puddles of partially occupied water molecules is pretty tough, particularly when the ligand is, itself, superimposed on partially occupied water. To keep my focus on some sort of an absolute scale I always think of contour levels in terms of electrons/A^3. This calculation is also problematic as is described in Lang et al. Another tool I use is to leave out a fully occupied water molecule that I have confidence in. I can compare mystery density with this known control to see if I am puzzling over a fully occupied atom or something weaker. With a raw Fo-Fc map you can never be sure. This "1 and 3 sigma" business really needs to be stomped out. Dale Tronrud

On Wed, Sep 10, 2014 at 9:26 AM, Pavel Afonine wrote:

...

Hi Charles,

I think the key here is what you call "no difference density peak". Sites may be partially occupied so rule of thumb for choosing contouring levels for 2mFo-DFc and mFo-DFc maps (1 and 3 sigma, correspondingly) may not be appropriate, for instance.

Pavel

On 9/10/14 6:10 AM, CPMAS Chen wrote:

Thanks, Nat.

If this is noise, why the anomalous or LLG peaks could be as high as 5 ~ 6 sigma?

Charles

On Tue, Sep 9, 2014 at 5:58 PM, Nathaniel Echols wrote:

...

On Tue, Sep 9, 2014 at 8:40 AM, CPMAS Chen wrote:

...

Is it possible that I have anomalous and LLG peak, but I have no difference density peak? In this case, is that because the model I have is not good enough or the diffraction data at this site of the model is missing?

You should at a minimum see > 1sigma density in the 2mFo-DFc map, and if the site is unmodeled (or modeled as a water) you should see an mFo-DFc peak as well. If neither of these applies, the anomalous and LLG peaks are probably just noise.

-Nat

--

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

Charles Chen

Research Associate

University of Pittsburgh School of Medicine

Department of Anesthesiology

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

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