-----BEGIN PGP SIGNED MESSAGE----- Hash: SHA1 P.S.: running auto-whatever- when you are dealing with critical data is usually not the way to get the best results. Experience with parameter settings is usually more powerful then generalised default settings. Reading the documentation of autoxds, there is an important caveat, where autoxds might corrupt your anomalous signal: The CORRECT step scales your data. It appears that autoxds will call aimless or scala to scale your data again. This is little harmful for the intensities, but corrupts the sigma values because the error model is applied twice and thus it is harmful for phasing using the anomalous signal because this relies on proper sigma values. At the CCP4 workshop in Chicago Garib Murshudov pointed out that when your CC(1/2) is 40% in the outer resolution shell (or CC*(1/2) = 50%) AND I/sigma is approximately 1.0, your sigma values are probably correct (this may be mentioned in the Karplus/Diederichs paper and attributed accordingly). I found this the most useful rule of thumb so far to decide about the resolution cut-off. Cheers, Tim On 07/10/2014 03:13 PM, Tim Gruene wrote:

Dear Charles Chen,

I am not familiar with autoxds, only xds itself. The reported quality of the anomalous signal is quite reliably and consistent with that of other programs, although the resolution shells listed in CORRECT.LP are a little broad, why I prefer to run xscale and take a look at XSCALE.LP (which would also combine your data by the way).

I would recomment NOT to set FRIEDEL'S_LAW=FALSE in XDS, especially if you only have weak anomalous signal. The small differences in the Bijvoet pairs does not really affect the scaling, but the doubled number of reflections gives you much more reliable scaling so that you end up with a stronger anomalous signal with FRIEDEL'S_LAW=TRUE.

Regards, Tim

On 07/10/2014 03:03 PM, CPMAS Chen wrote:

...

Hi, Phenix Users,

...

I recently collected data at SSRL. I used autoxds and multiscale to merge couple of datasets and hoped to see some anomalous signals. Now comes the questions.

...

1. if I use the default sigma(=2), then autoxds will report weak anomalous signals and leave the friedel flag off. If I lower sigma to 1.5, it will report strong anomalous signals. 2. when I used Xtriage under Phenix to check the data quality, it reported weak anomalous signal or at above 10A. 3. I used aimless (in CCP4) to merge data, it also reported weak anomalous signal.

...

Which result should I trust? By the way, how can I view/display the ***.anamplot file, which is apparently xmgr format file, but I can not display in CCP4i.

...

Thanks!

...

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

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- -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -----BEGIN PGP SIGNATURE----- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Icedove - http://www.enigmail.net/ iD8DBQFTvpOxUxlJ7aRr7hoRAm5HAKDoMjGCRgwy4zP77z6bs5dL0sb9LwCcDN1D Kjh1UPy5LVcnQVg4rXlif6c= =WUoO -----END PGP SIGNATURE-----

-----BEGIN PGP SIGNED MESSAGE----- Hash: SHA1 Hi Francis, I couldn't read it properly, but I see there seems a certain similarity between v(I)=a*(v0(I)+b*I^2) and 􏰇0(Ihl) = Sdfac[􏰇2(Ihl) + (Sdadd ghlhIhi)2]1/2. and doing it twice may corrupt the sigmas. It is better to apply an error model only once, no matter what error model. Best, Tim On 07/10/2014 04:38 PM, Francis Reyes wrote:

Hi Tim

So from the XDS docs, the estimates of the sigmas are performed by the CORRECT step in the following way:

"The residual scatter in intensity of symmetry-equivalent reflections is used to estimate their standard deviations. Here, the initial estimate v0(I) (obtained from the INTEGRATE step) for the variance of the reflection intensity I is replaced by v(I)=a*(v0(I)+b*I^2). The two constants a and b are chosen to minimize discrepancies between v(I) and the variance estimated from sample statistics of symmetry related reflections. Based on the more realistic error estimates for the intensities, outliers are recognized by comparison with other symmetry-equivalent reflections. These outliers are included in the main output file XDS_ASCII.HKL in which they are marked by a negative sign attached to the estimated standard deviations of their intensity...."

SCALA/AIMLESS have a completely different way of estimating the sigmas.

"After scaling, the error estimates can be improved by comparing the observed scatter between observations and the estimated standard deviation, making them equal on average. If the standard deviations 􏰇(Ihl) are correct, then the normalized deviations 􏰂hl = (Ihl - hIh0 i)/􏰇(Ihl) (where hIh0 i is averaged over all observations of reflection h excluding the lth observation) should be distributed with a mean 0.0 and stan- dard deviation 1.0. A simple correction to give improved error estimates is 􏰇0(Ihl) = Sdfac[􏰇2(Ihl) + (Sdadd ghlhIhi)2]1/2. "

And are the two models for estimating the sigmas compatible?

F

On Jul 10, 2014, at 9:22 AM, Tim Gruene wrote:

...

The CORRECT step scales your data. It appears that autoxds will call aimless or scala to scale your data again. This is little harmful for the intensities, but corrupts the sigma values because the error model is applied twice and thus it is harmful for phasing using the anomalous signal because this relies on proper sigma values.

- -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -----BEGIN PGP SIGNATURE----- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Icedove - http://www.enigmail.net/ iD8DBQFTvqeWUxlJ7aRr7hoRAhueAKDJCeALNTaLC8uoLQImWRl4KWhukQCeP4ok LT4mM8ggc3iouehqpzxMHPw= =oFme -----END PGP SIGNATURE-----

Hi Phil, I had borderline cases in mind where you are at the limit of solvability. In such a case you want to get the best out of your data as possible and avoid any harm when possible. And since you never know if a case is borderline until it is solved, I think it is good practice to do it right anyway. My comments are based on experience from Kay and George, who have done a few tests on phasing with shelxd. I agree that the changes are small. Best, Tim On 07/10/2014 04:52 PM, Phil Evans wrote:

I don't think the sigma analysis/correction twice should mess things up too much - there will be some differences but the 2nd "correction" probably won't do much if it thinks the 1st correction has done the job (i.e. it shouldn't hurt)

Phil

On 10 Jul 2014, at 14:13, Tim Gruene wrote:

Dear Charles Chen,

I am not familiar with autoxds, only xds itself. The reported quality of the anomalous signal is quite reliably and consistent with that of other programs, although the resolution shells listed in CORRECT.LP are a little broad, why I prefer to run xscale and take a look at XSCALE.LP (which would also combine your data by the way).

I would recomment NOT to set FRIEDEL'S_LAW=FALSE in XDS, especially if you only have weak anomalous signal. The small differences in the Bijvoet pairs does not really affect the scaling, but the doubled number of reflections gives you much more reliable scaling so that you end up with a stronger anomalous signal with FRIEDEL'S_LAW=TRUE.

Regards, Tim

On 07/10/2014 03:03 PM, CPMAS Chen wrote:

...

...

...

Hi, Phenix Users,

I recently collected data at SSRL. I used autoxds and multiscale to merge couple of datasets and hoped to see some anomalous signals. Now comes the questions.

1. if I use the default sigma(=2), then autoxds will report weak anomalous signals and leave the friedel flag off. If I lower sigma to 1.5, it will report strong anomalous signals. 2. when I used Xtriage under Phenix to check the data quality, it reported weak anomalous signal or at above 10A. 3. I used aimless (in CCP4) to merge data, it also reported weak anomalous signal.

Which result should I trust? By the way, how can I view/display the ***.anamplot file, which is apparently xmgr format file, but I can not display in CCP4i.

Thanks!

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

...

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

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

-- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A

Francis, My anomalous plot for Br is more like your Cobalt 1 site less redundancy. Ryan, As my Br anomalous signals are weak, I merged signals from multiple crystals. Meanwhile I am looking into the detail about the autoxds processing as suggested by Tim. Thanks, On Thu, Jul 10, 2014 at 10:02 AM, Francis Reyes wrote:

In addition to Tim's comments above, using loggraph to see the anomalous CC plot from scala is a good qualitative indicator of whether you have anomalous signal..

Some data points for what reasonable plots (and their corresponding XDS SigANO's) look like: https://dl.dropboxusercontent.com/u/19558536/AnomalousCC.pdf

F

On Jul 10, 2014, at 9:03 AM, CPMAS Chen wrote:

...

Which result should I trust? By the way, how can I view/display the

***.anamplot file, which is apparently xmgr format file, but I can not display in CCP4i.

...

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

Well, let me make this clear. 1. I am using Br anomalous signal to identify the potentially bound ligand. 2. I do shoot the crystals at 0.92A. 3. the different crystals have different resolution, but anomalous signal was weak as reported by autoxds. Charles On Thu, Jul 10, 2014 at 12:25 PM, Ryan Spencer wrote:

So I assume you were not able to shoot the crystal at ~0.92 A at SSRL or are the crystals sensitive? If you were limited to the single wavelength beam which is around 1A you’re dealing with a really low f” of 0.5322. There have been crystals solved with Sulfur using an in-house Cu sources (takes a lot of merged sets and anomalous is stronger at certain crystal angles) which gives about the same anomalous scattering as Br. Is the bromine covalent or is it soaked ion?

Alternatives – soak with potassium iodide and get a dataset from an in-house source (iodine f” 6.6 at 1.54), or at least the anomalous locations. You may get a partial model to use for replacement at that point.

Did each processed dataset have the same level of anomalous signal when processed individually?

Ryan

*From:* [email protected] [mailto: [email protected]] *On Behalf Of *CPMAS Chen *Sent:* Thursday, July 10, 2014 8:55 AM *To:* Francis Reyes *Cc:* [email protected] *Subject:* Re: [phenixbb] Anomalous or not?

Francis,

My anomalous plot for Br is more like your Cobalt 1 site less redundancy.

Ryan,

As my Br anomalous signals are weak, I merged signals from multiple crystals.

Meanwhile I am looking into the detail about the autoxds processing as suggested by Tim.

Thanks,

On Thu, Jul 10, 2014 at 10:02 AM, Francis Reyes < [email protected]> wrote:

In addition to Tim's comments above, using loggraph to see the anomalous CC plot from scala is a good qualitative indicator of whether you have anomalous signal..

Some data points for what reasonable plots (and their corresponding XDS SigANO's) look like: https://dl.dropboxusercontent.com/u/19558536/AnomalousCC.pdf

F

On Jul 10, 2014, at 9:03 AM, CPMAS Chen wrote:

...

Which result should I trust? By the way, how can I view/display the

***.anamplot file, which is apparently xmgr format file, but I can not display in CCP4i.

...

--

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

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

Phil, the Br is in ligand, as you suggest, I might just see weak anomalous. Ryan, you are right. I have no evidence to show the Br-ligand I used is in the protein structure. resolution does not degrade much along with frames I think this is just a weak anomalous signal datasets. Thanks. Charles On Thu, Jul 10, 2014 at 1:03 PM, Phil Jeffrey wrote:

Charles,

So if you have bound ligand with a covalently-linked Bromine, and the ligand isn't abundant in your unit cell, don't you *expect* the anomalous signal to be weak ? Have you tried a DANO model-phased difference map or something comparable ?

Phil Jeffrey Princeton

On 7/10/14 12:52 PM, CPMAS Chen wrote:

...

Well, let me make this clear.

1. I am using Br anomalous signal to identify the potentially bound ligand. 2. I do shoot the crystals at 0.92A. 3. the different crystals have different resolution, but anomalous signal was weak as reported by autoxds.

Charles

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

Yeah. Ideally. But when you have multiple blobs in Fo-Fc map, it is hard to decide. of course, it need to be fitted. That is what I am working with. No matter what, the data is there at about 3A for a membrane protein , I should be able to see something. Thanks for all the suggestions! Best, Charles@Pitt On Thu, Jul 10, 2014 at 1:29 PM, Oganesyan, Vaheh wrote:

Shouldn’t phases from MR be a better way to look for bromated ligand in Fo-Fc maps in such a case?

*Regards,*

*Vaheh*

*8-5851*

*From:* [email protected] [mailto: [email protected]] *On Behalf Of *CPMAS Chen *Sent:* Thursday, July 10, 2014 12:53 PM *To:* Ryan Spencer

*Cc:* [email protected] *Subject:* Re: [phenixbb] Anomalous or not?

Well, let me make this clear.

1. I am using Br anomalous signal to identify the potentially bound ligand.

2. I do shoot the crystals at 0.92A.

3. the different crystals have different resolution, but anomalous signal was weak as reported by autoxds.

Charles

On Thu, Jul 10, 2014 at 12:25 PM, Ryan Spencer wrote:

So I assume you were not able to shoot the crystal at ~0.92 A at SSRL or are the crystals sensitive? If you were limited to the single wavelength beam which is around 1A you’re dealing with a really low f” of 0.5322. There have been crystals solved with Sulfur using an in-house Cu sources (takes a lot of merged sets and anomalous is stronger at certain crystal angles) which gives about the same anomalous scattering as Br. Is the bromine covalent or is it soaked ion?

Alternatives – soak with potassium iodide and get a dataset from an in-house source (iodine f” 6.6 at 1.54), or at least the anomalous locations. You may get a partial model to use for replacement at that point.

Did each processed dataset have the same level of anomalous signal when processed individually?

Ryan

*From:* [email protected] [mailto: [email protected]] *On Behalf Of *CPMAS Chen *Sent:* Thursday, July 10, 2014 8:55 AM *To:* Francis Reyes *Cc:* [email protected] *Subject:* Re: [phenixbb] Anomalous or not?

Francis,

My anomalous plot for Br is more like your Cobalt 1 site less redundancy.

Ryan,

As my Br anomalous signals are weak, I merged signals from multiple crystals.

Meanwhile I am looking into the detail about the autoxds processing as suggested by Tim.

Thanks,

On Thu, Jul 10, 2014 at 10:02 AM, Francis Reyes < [email protected]> wrote:

In addition to Tim's comments above, using loggraph to see the anomalous CC plot from scala is a good qualitative indicator of whether you have anomalous signal..

Some data points for what reasonable plots (and their corresponding XDS SigANO's) look like: https://dl.dropboxusercontent.com/u/19558536/AnomalousCC.pdf

F

On Jul 10, 2014, at 9:03 AM, CPMAS Chen wrote:

...

Which result should I trust? By the way, how can I view/display the

***.anamplot file, which is apparently xmgr format file, but I can not display in CCP4i.

...

--

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

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

****************************************************** To the extent this electronic communication or any of its attachments contain information that is not in the public domain, such information is considered by MedImmune to be confidential and proprietary. This communication is expected to be read and/or used only by the individual(s) for whom it is intended. If you have received this electronic communication in error, please reply to the sender advising of the error in transmission and delete the original message and any accompanying documents from your system immediately, without copying, reviewing or otherwise using them for any purpose. Thank you for your cooperation. To the extent this electronic communication or any of its attachments contain information that is not in the public domain, such information is considered by MedImmune to be confidential and proprietary. This communication is expected to be read and/or used only by the individual(s) for whom it is intended. If you have received this electronic communication in error, please reply to the sender advising of the error in transmission and delete the original message and any accompanying documents from your system immediately, without copying, reviewing or otherwise using them for any purpose. Thank you for your cooperation.

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

There are several reasons for not seeing the bromide such as the ligand has poor occupancy, the bromide is disordered. X-ray induced cleavage of a bromide can be significant, even if the crystal overall does not undergo serious damage. The cleavage may be faster depending on the bromide environment. I have a case of a chirality-dependent binding mode. For the same ligand, but for the chirality, one binding mode buries the bromide and the other exposes it to the bulk solvent. Cleavage is "slow" in the first case (~95% occupancy averaged over the dataset) and "fast" in the 2nd case (~50%). One way to look at it is to divide your dataset in several parts (say 3 thirds) and phase each part as suggested below. Look for a peak near the expected position which would decrease as the data collection proceeds. If cleavage did occur then you have both anomalous signal and one very isomorphous "heavy atom derivative" (by treating the end of the data collection as a native) to phase your structure. The idea of using bromide cleavage as source of phasing information was proposed by Gérard Bricogne. Good luck Thierry -----Original Message----- From: [email protected] [mailto:[email protected]] On Behalf Of Francis Reyes Sent: Thursday, July 10, 2014 2:01 PM To: CPMAS Chen Cc: PHENIX user mailing list ([email protected]) Subject: Re: [phenixbb] Anomalous or not? You should see something in an anomalous difference fourier map using the model as a source of phases (make sure your Br is not in the model used to compute it). F On Jul 10, 2014, at 1:48 PM, CPMAS Chen wrote:

Yeah. Ideally. But when you have multiple blobs in Fo-Fc map, it is hard to decide. of course, it need to be fitted. That is what I am working with. No matter what, the data is there at about 3A for a membrane protein , I should be able to see something.

Thanks for all the suggestions!

Best,

Charles@Pitt

On Thu, Jul 10, 2014 at 1:29 PM, Oganesyan, Vaheh wrote: Shouldn't phases from MR be a better way to look for bromated ligand in Fo-Fc maps in such a case?

Regards,

Vaheh

8-5851

From: [email protected] [mailto:[email protected]] On Behalf Of CPMAS Chen Sent: Thursday, July 10, 2014 12:53 PM To: Ryan Spencer

Cc: [email protected] Subject: Re: [phenixbb] Anomalous or not?

Well, let me make this clear.

1. I am using Br anomalous signal to identify the potentially bound ligand.

2. I do shoot the crystals at 0.92A.

3. the different crystals have different resolution, but anomalous signal was weak as reported by autoxds.

Charles

On Thu, Jul 10, 2014 at 12:25 PM, Ryan Spencer wrote:

So I assume you were not able to shoot the crystal at ~0.92 A at SSRL or are the crystals sensitive? If you were limited to the single wavelength beam which is around 1A you're dealing with a really low f" of 0.5322. There have been crystals solved with Sulfur using an in-house Cu sources (takes a lot of merged sets and anomalous is stronger at certain crystal angles) which gives about the same anomalous scattering as Br. Is the bromine covalent or is it soaked ion?

Alternatives - soak with potassium iodide and get a dataset from an in-house source (iodine f" 6.6 at 1.54), or at least the anomalous locations. You may get a partial model to use for replacement at that point.

Did each processed dataset have the same level of anomalous signal when processed individually?

Ryan

From: [email protected] [mailto:[email protected]] On Behalf Of CPMAS Chen Sent: Thursday, July 10, 2014 8:55 AM To: Francis Reyes Cc: [email protected] Subject: Re: [phenixbb] Anomalous or not?

Francis,

My anomalous plot for Br is more like your Cobalt 1 site less redundancy.

Ryan,

As my Br anomalous signals are weak, I merged signals from multiple crystals.

Meanwhile I am looking into the detail about the autoxds processing as suggested by Tim.

Thanks,

On Thu, Jul 10, 2014 at 10:02 AM, Francis Reyes wrote:

In addition to Tim's comments above, using loggraph to see the anomalous CC plot from scala is a good qualitative indicator of whether you have anomalous signal..

Some data points for what reasonable plots (and their corresponding XDS SigANO's) look like: https://dl.dropboxusercontent.com/u/19558536/AnomalousCC.pdf

F

On Jul 10, 2014, at 9:03 AM, CPMAS Chen wrote:

...

Which result should I trust? By the way, how can I view/display the ***.anamplot file, which is apparently xmgr format file, but I can not display in CCP4i.

--

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

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

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

To the extent this electronic communication or any of its attachments contain information that is not in the public domain, such information is considered by MedImmune to be confidential and proprietary. This communication is expected to be read and/or used only by the individual(s) for whom it is intended. If you have received this electronic communication in error, please reply to the sender advising of the error in transmission and delete the original message and any accompanying documents from your system immediately, without copying, reviewing or otherwise using them for any purpose. Thank you for your cooperation. To the extent this electronic communication or any of its attachments contain information that is not in the public domain, such information is considered by MedImmune to be confidential and proprietary. This communication is expected to be read and/or used only by the individual(s) for whom it is intended. If you have received this electronic communication in error, please reply to the sender advising of the error in transmission and delete the original message and any accompanying documents from your system immediately, without copying, reviewing or otherwise using them for any purpose. Thank you for your cooperation.

--

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

Charles Chen

Research Associate

University of Pittsburgh School of Medicine

Department of Anesthesiology

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

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