Re: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent Count Numbers

[Edward A. Berry]

On 11/30/2016 10:16 PM, Keller, Jacob wrote:

If you fine slice and everything is then a partial, isn't that *more* sensitive 
to lack of synchronization between the shutter and rotation axis than the 
wide-frame method where there's a larger proportion of fulls that don't 
approach the frame edges (in rotation space) ?  Especially if you're 3D profile 
fitting ?


That is how the argument seems to go in Pflugrath 1999, but I would think that 
shutter jitter is a random error, so it would seem better to have several of 
these random errors for a given spot than just one. Perhaps measuring with high 
multiplicity would have the same averaging effect.


Is fine slicing more or less beneficial at high resolutions relative to lower 
ones ?


In terms of I/sigI, it seems to be the same proportional improvement across all 
resolutions. See Fig 4 of the Pflugrath 1999 paper.

JPK


I think the problem there is that, if the shutter jitter is random with a 
constant sigma, it becomes a larger percent of the total exposure for that 
frame. It would be like taking a 1ml pipetor with an error of 2% of full scale, 
i.e. 20 ul. Because you want to average this out, you set it to 200 ul and 
pipet 5 times. The sigma of that measurement would be sqrt(5) * 20 ul, I think, 
so worse than doing it all in one shot. On the other hand if you take a 200 ul 
pipet with sigma 2% of full scale or 4 ul, and take 5 times, the error is 
sqrt(5) * 4 ul which is less than 20 ul.
Of course this would not apply to reflections that are fully recorded on one 
frame since they are not reflecting while the shutter is open/closing. Then it 
would be only variation in background.



Phil Jeffrey

Princeton

--

*From:*CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Keller, Jacob 
[kell...@janelia.hhmi.org]
*Sent:* Wednesday, November 30, 2016 5:44 PM
*To:* CCP4BB@JISCMAIL.AC.UK 
*Subject:* Re: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent 
Count Numbers

If the mosaicity is, say, 0.5 deg, and one is measuring 1 deg frames, about 
half the time is spent measuring non-spot background noise under spots in phi, 
which is all lumped into the intensity measurement. Fine slicing reduces this. 
But I am conjecturing that there is also fine-slicing-mediated improvement due 
to averaging out things like shutter jitter, which would also be averaged out 
through plain ol’ multiplicity.

I guess a third equal-count dataset would be useful as well: one sweep with 
six-fold finer slicing. So it would be:

One sweep, 0.6 deg, 60s

Six sweeps, 0.6 deg, 10s

One sweep, 0.1 deg, 10s

Or something roughly similar. Who will arrange the bets?

JPK

*From:*Boaz Shaanan [mailto:bshaa...@bgu.ac.il]
*Sent:* Wednesday, November 30, 2016 5:19 PM
*To:* Keller, Jacob mailto:kell...@janelia.hhmi.org>>; 
CCP4BB@JISCMAIL.AC.UK 
*Subject:* RE: Effects of Multiplicity and Fine Phi with Equivalent Count 
Numbers

Hi Jacob,

I may have missed completely your point but as far as my memory goes, the main 
argument in favour of fine slicing has always been reduction of the noise 
arising from incoherent scattering, which in the old days arose from the 
capillary, solvent, air, you name it. The noise reduction in fine slicing is 
achieved by shortening the exposure time per frame. This argument still holds 
today although the sources of incoherent scattering could be different. Of 
course, there are other reasons to go for fine slicing such as long axes and 
others. In any case it's the recommended method these days, and for good 
reasons, isn't it?

   Best regards,

Boaz

/Boaz Shaanan, Ph.D. //
/Dept. of Life Sciences /
/Ben-Gurion University of the Negev /
/Beer-Sheva 84105 /
/Israel /
//
/E-mail: bshaa...@bgu.ac.il /
/Phone: 972-8-647-2220  Skype: boaz.shaanan /
/Fax:   972-8-647-2992 or 972-8-646-1710 //

//

---

Re: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent Count Numbers

[Keller, Jacob]

I am wondering whether part of the benefit of fine slicing is really increased 
multiplicity in disguise. I have seen in papers that empirically things do not 
improve much (or can even get slightly worse) past 0.5*SigPhi, which is the 
XDS-defined version of mosaicity; according to MOSFLM’s definition of 
mosaicity, which is ~2-3 larger than that from XDS, this would correspond to 
~0.15-0.25 * mosaic spread (Mueller et al 2011). Accordingly, a crystal with 
MOSFLM mosaic spread of 0.6 would be best measured with ~0.1 deg oscillations, 
meaning, I think, that the spot would be measured at least six times as a 
partial, leading to some averaging of various errors from frame to frame.

JPK




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Edward 
Snell
Sent: Wednesday, November 30, 2016 8:11 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent 
Count Numbers


There is a very nice paper by Colin Nave on Matching X-ray beam and detector 
properties to protein crystals of different perfection​,J Synchrotron Radiat. 
2014  21, 537–546. Due to the spectral and geometric properties of in house 
sources there is probably no advantage with an oscillation below 0.2 degrees no 
matter how good the crystal is. A well conditioned beamline can gain an 
advantage from a very perfect crystal but once you cryocool them all bets are 
off. Make sure you consider the instrument resolution parameters (spectral and 
geometric divergence) and the actual crystal quality. As to resolution 
dependence, it all comes down to the source of the mosaicity, e.g. domain 
misalignment, imperfections, domain boundary effects and domain size. There is 
a rich literature around but speaking from first hand experience, it's not 
trivial to probe this area.



Cheers,



Eddie.


Edward Snell Ph.D.
President and CEO Hauptman-Woodward Medical Research Institute
Assistant Prof. Department of Structural Biology, University at Buffalo
700 Ellicott Street, Buffalo, NY 14203-1102
Phone: (716) 898 8631 Fax: (716) 898 8660
Skype:  eddie.snell Email: 
esn...@hwi.buffalo.edu
Heisenberg was probably here!​

​


From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Jeffrey, Philip D. 
mailto:pjeff...@princeton.edu>>
Sent: Wednesday, November 30, 2016 7:36 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent 
Count Numbers

Jacob,

If you fine slice and everything is then a partial, isn't that *more* sensitive 
to lack of synchronization between the shutter and rotation axis than the 
wide-frame method where there's a larger proportion of fulls that don't 
approach the frame edges (in rotation space) ?  Especially if you're 3D profile 
fitting ?

Is fine slicing more or less beneficial at high resolutions relative to lower 
ones ?

Phil Jeffrey
Princeton

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Keller, Jacob 
[kell...@janelia.hhmi.org]
Sent: Wednesday, November 30, 2016 5:44 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent 
Count Numbers
If the mosaicity is, say, 0.5 deg, and one is measuring 1 deg frames, about 
half the time is spent measuring non-spot background noise under spots in phi, 
which is all lumped into the intensity measurement. Fine slicing reduces this. 
But I am conjecturing that there is also fine-slicing-mediated improvement due 
to averaging out things like shutter jitter, which would also be averaged out 
through plain ol’ multiplicity.

I guess a third equal-count dataset would be useful as well: one sweep with 
six-fold finer slicing. So it would be:

One sweep, 0.6 deg, 60s
Six sweeps, 0.6 deg, 10s
One sweep, 0.1 deg, 10s

Or something roughly similar. Who will arrange the bets?

JPK


From: Boaz Shaanan [mailto:bshaa...@bgu.ac.il]
Sent: Wednesday, November 30, 2016 5:19 PM
To: Keller, Jacob mailto:kell...@janelia.hhmi.org>>; 
CCP4BB@JISCMAIL.AC.UK
Subject: RE: Effects of Multiplicity and Fine Phi with Equivalent Count Numbers

Hi Jacob,

I may have missed completely your point but as far as my memory goes, the main 
argument in favour of fine slicing has always been reduction of the noise 
arising from incoherent scattering, which in the old days arose from the 
capillary, solvent, air, you name it. The noise reduction in fine slicing is 
achieved by shortening the exposure time per frame. This argument still holds 
today although the sources of incoherent scattering could be different. Of 
course, there are other reasons to go for fine slicing such as long axes and 
others. In any case it's the recommended method these days, and for good 
reasons, isn't it?

  Best regards,

   Boaz

B

Re: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent Count Numbers

[Keller, Jacob]

>If you fine slice and everything is then a partial, isn't that *more* 
>sensitive to lack of synchronization between the shutter and rotation axis 
>than the wide-frame method where there's a larger proportion of fulls that 
>don't approach the frame edges (in rotation space) ?  Especially if you're 3D 
>profile fitting ?

That is how the argument seems to go in Pflugrath 1999, but I would think that 
shutter jitter is a random error, so it would seem better to have several of 
these random errors for a given spot than just one. Perhaps measuring with high 
multiplicity would have the same averaging effect.

>Is fine slicing more or less beneficial at high resolutions relative to lower 
>ones ?

In terms of I/sigI, it seems to be the same proportional improvement across all 
resolutions. See Fig 4 of the Pflugrath 1999 paper.

JPK




Phil Jeffrey
Princeton

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Keller, Jacob 
[kell...@janelia.hhmi.org]
Sent: Wednesday, November 30, 2016 5:44 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent 
Count Numbers
If the mosaicity is, say, 0.5 deg, and one is measuring 1 deg frames, about 
half the time is spent measuring non-spot background noise under spots in phi, 
which is all lumped into the intensity measurement. Fine slicing reduces this. 
But I am conjecturing that there is also fine-slicing-mediated improvement due 
to averaging out things like shutter jitter, which would also be averaged out 
through plain ol' multiplicity.

I guess a third equal-count dataset would be useful as well: one sweep with 
six-fold finer slicing. So it would be:

One sweep, 0.6 deg, 60s
Six sweeps, 0.6 deg, 10s
One sweep, 0.1 deg, 10s

Or something roughly similar. Who will arrange the bets?

JPK


From: Boaz Shaanan [mailto:bshaa...@bgu.ac.il]
Sent: Wednesday, November 30, 2016 5:19 PM
To: Keller, Jacob mailto:kell...@janelia.hhmi.org>>; 
CCP4BB@JISCMAIL.AC.UK
Subject: RE: Effects of Multiplicity and Fine Phi with Equivalent Count Numbers

Hi Jacob,

I may have missed completely your point but as far as my memory goes, the main 
argument in favour of fine slicing has always been reduction of the noise 
arising from incoherent scattering, which in the old days arose from the 
capillary, solvent, air, you name it. The noise reduction in fine slicing is 
achieved by shortening the exposure time per frame. This argument still holds 
today although the sources of incoherent scattering could be different. Of 
course, there are other reasons to go for fine slicing such as long axes and 
others. In any case it's the recommended method these days, and for good 
reasons, isn't it?

  Best regards,

   Boaz

Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel

E-mail: bshaa...@bgu.ac.il
Phone: 972-8-647-2220  Skype: boaz.shaanan
Fax:   972-8-647-2992 or 972-8-646-1710




From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Keller, Jacob 
[kell...@janelia.hhmi.org]
Sent: Wednesday, November 30, 2016 11:37 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent Count 
Numbers
Dear Crystallographers,

I am curious whether the observed effects of fine phi slicing might in part or 
in toto be due to simply higher "pseudo-multiplicity." In other words, under 
normal experimental conditions, does simply increasing the number of 
measurements increase the signal and improve precision, even with the same 
number of total counts in the dataset?

As such, I am looking for a paper which, like Pflugrath's 1999 paper, compares 
two data sets with equivalent total counts but, in this case, different 
multiplicities. For example, is a single sweep with 0.5 degree 60s exposures 
empirically, in real practice, equivalent statistically to six passes with 0.5 
degree 10s frames? Better? Worse? Our home source has been donated away to 
Connecticut, so I can't do this experiment myself anymore.

All the best,

Jacob Keller


***
Jacob Pearson Keller, PhD
Research Scientist
HHMI Janelia Research Campus / Looger lab
Phone: (571)209-4000 x3159
Email: kell...@janelia.hhmi.org
***

Re: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent Count Numbers

[Edward Snell]

There is a very nice paper by Colin Nave on Matching X-ray beam and detector 
properties to protein crystals of different perfection​,J Synchrotron Radiat. 
2014  21, 537–546. Due to the spectral and geometric properties of in house 
sources there is probably no advantage with an oscillation below 0.2 degrees no 
matter how good the crystal is. A well conditioned beamline can gain an 
advantage from a very perfect crystal but once you cryocool them all bets are 
off. Make sure you consider the instrument resolution parameters (spectral and 
geometric divergence) and the actual crystal quality. As to resolution 
dependence, it all comes down to the source of the mosaicity, e.g. domain 
misalignment, imperfections, domain boundary effects and domain size. There is 
a rich literature around but speaking from first hand experience, it's not 
trivial to probe this area.


Cheers,


Eddie.


Edward Snell Ph.D.
President and CEO Hauptman-Woodward Medical Research Institute
Assistant Prof. Department of Structural Biology, University at Buffalo
700 Ellicott Street, Buffalo, NY 14203-1102
Phone: (716) 898 8631 Fax: (716) 898 8660
Skype:  eddie.snell Email: esn...@hwi.buffalo.edu
Heisenberg was probably here!​

​


From: CCP4 bulletin board  on behalf of Jeffrey, Philip 
D. 
Sent: Wednesday, November 30, 2016 7:36 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent 
Count Numbers

Jacob,

If you fine slice and everything is then a partial, isn't that *more* sensitive 
to lack of synchronization between the shutter and rotation axis than the 
wide-frame method where there's a larger proportion of fulls that don't 
approach the frame edges (in rotation space) ?  Especially if you're 3D profile 
fitting ?

Is fine slicing more or less beneficial at high resolutions relative to lower 
ones ?

Phil Jeffrey
Princeton

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Keller, Jacob 
[kell...@janelia.hhmi.org]
Sent: Wednesday, November 30, 2016 5:44 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent 
Count Numbers

If the mosaicity is, say, 0.5 deg, and one is measuring 1 deg frames, about 
half the time is spent measuring non-spot background noise under spots in phi, 
which is all lumped into the intensity measurement. Fine slicing reduces this. 
But I am conjecturing that there is also fine-slicing-mediated improvement due 
to averaging out things like shutter jitter, which would also be averaged out 
through plain ol’ multiplicity.

I guess a third equal-count dataset would be useful as well: one sweep with 
six-fold finer slicing. So it would be:

One sweep, 0.6 deg, 60s
Six sweeps, 0.6 deg, 10s
One sweep, 0.1 deg, 10s

Or something roughly similar. Who will arrange the bets?

JPK


From: Boaz Shaanan [mailto:bshaa...@bgu.ac.il]
Sent: Wednesday, November 30, 2016 5:19 PM
To: Keller, Jacob ; CCP4BB@JISCMAIL.AC.UK
Subject: RE: Effects of Multiplicity and Fine Phi with Equivalent Count Numbers

Hi Jacob,

I may have missed completely your point but as far as my memory goes, the main 
argument in favour of fine slicing has always been reduction of the noise 
arising from incoherent scattering, which in the old days arose from the 
capillary, solvent, air, you name it. The noise reduction in fine slicing is 
achieved by shortening the exposure time per frame. This argument still holds 
today although the sources of incoherent scattering could be different. Of 
course, there are other reasons to go for fine slicing such as long axes and 
others. In any case it's the recommended method these days, and for good 
reasons, isn't it?

  Best regards,

   Boaz

Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel

E-mail: bshaa...@bgu.ac.il
Phone: 972-8-647-2220  Skype: boaz.shaanan
Fax:   972-8-647-2992 or 972-8-646-1710





From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Keller, Jacob 
[kell...@janelia.hhmi.org]
Sent: Wednesday, November 30, 2016 11:37 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent Count 
Numbers
Dear Crystallographers,

I am curious whether the observed effects of fine phi slicing might in part or 
in toto be due to simply higher “pseudo-multiplicity.” In other words, under 
normal experimental conditions, does simply increasing the number of 
measurements increase the signal and improve precision, even with the same 
number of total counts in the dataset?

As such, I am looking for a paper which, like Pflugrath’s 1999 paper, compares 
two data sets with equivalent total counts but, in this case, different 
multiplicities. For example, is a single sweep with 0.5 degree 60s exposures 

Re: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent Count Numbers

[Jeffrey, Philip D.]

Jacob,

If you fine slice and everything is then a partial, isn't that *more* sensitive 
to lack of synchronization between the shutter and rotation axis than the 
wide-frame method where there's a larger proportion of fulls that don't 
approach the frame edges (in rotation space) ?  Especially if you're 3D profile 
fitting ?

Is fine slicing more or less beneficial at high resolutions relative to lower 
ones ?

Phil Jeffrey
Princeton

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Keller, Jacob 
[kell...@janelia.hhmi.org]
Sent: Wednesday, November 30, 2016 5:44 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent 
Count Numbers

If the mosaicity is, say, 0.5 deg, and one is measuring 1 deg frames, about 
half the time is spent measuring non-spot background noise under spots in phi, 
which is all lumped into the intensity measurement. Fine slicing reduces this. 
But I am conjecturing that there is also fine-slicing-mediated improvement due 
to averaging out things like shutter jitter, which would also be averaged out 
through plain ol’ multiplicity.

I guess a third equal-count dataset would be useful as well: one sweep with 
six-fold finer slicing. So it would be:

One sweep, 0.6 deg, 60s
Six sweeps, 0.6 deg, 10s
One sweep, 0.1 deg, 10s

Or something roughly similar. Who will arrange the bets?

JPK


From: Boaz Shaanan [mailto:bshaa...@bgu.ac.il]
Sent: Wednesday, November 30, 2016 5:19 PM
To: Keller, Jacob ; CCP4BB@JISCMAIL.AC.UK
Subject: RE: Effects of Multiplicity and Fine Phi with Equivalent Count Numbers

Hi Jacob,

I may have missed completely your point but as far as my memory goes, the main 
argument in favour of fine slicing has always been reduction of the noise 
arising from incoherent scattering, which in the old days arose from the 
capillary, solvent, air, you name it. The noise reduction in fine slicing is 
achieved by shortening the exposure time per frame. This argument still holds 
today although the sources of incoherent scattering could be different. Of 
course, there are other reasons to go for fine slicing such as long axes and 
others. In any case it's the recommended method these days, and for good 
reasons, isn't it?

  Best regards,

   Boaz

Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel

E-mail: bshaa...@bgu.ac.il
Phone: 972-8-647-2220  Skype: boaz.shaanan
Fax:   972-8-647-2992 or 972-8-646-1710




From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Keller, Jacob 
[kell...@janelia.hhmi.org]
Sent: Wednesday, November 30, 2016 11:37 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent Count 
Numbers
Dear Crystallographers,

I am curious whether the observed effects of fine phi slicing might in part or 
in toto be due to simply higher “pseudo-multiplicity.” In other words, under 
normal experimental conditions, does simply increasing the number of 
measurements increase the signal and improve precision, even with the same 
number of total counts in the dataset?

As such, I am looking for a paper which, like Pflugrath’s 1999 paper, compares 
two data sets with equivalent total counts but, in this case, different 
multiplicities. For example, is a single sweep with 0.5 degree 60s exposures 
empirically, in real practice, equivalent statistically to six passes with 0.5 
degree 10s frames? Better? Worse? Our home source has been donated away to 
Connecticut, so I can’t do this experiment myself anymore.

All the best,

Jacob Keller


***
Jacob Pearson Keller, PhD
Research Scientist
HHMI Janelia Research Campus / Looger lab
Phone: (571)209-4000 x3159
Email: kell...@janelia.hhmi.org
***

[ccp4bb] advice on screen dispensing?

[Olga Moroz]

Dear All,
It looks like our old Hydra died of old age, and new Hydras seem quite 
expensive. We are now looking for an optimal way to transfer crystallisation 
screens
from the deep well blocks into crystallisation plates.  We were told 
Liquidator96 from Rainin, 5-200mkl worked well and was very easy to use.
Are there any other suggestions - what do you use in your labs, how much did 
the instruments cost, are you happy with them?
Thanks a lot!
Olga

Re: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent Count Numbers

[Keller, Jacob]

If the mosaicity is, say, 0.5 deg, and one is measuring 1 deg frames, about 
half the time is spent measuring non-spot background noise under spots in phi, 
which is all lumped into the intensity measurement. Fine slicing reduces this. 
But I am conjecturing that there is also fine-slicing-mediated improvement due 
to averaging out things like shutter jitter, which would also be averaged out 
through plain ol' multiplicity.

I guess a third equal-count dataset would be useful as well: one sweep with 
six-fold finer slicing. So it would be:

One sweep, 0.6 deg, 60s
Six sweeps, 0.6 deg, 10s
One sweep, 0.1 deg, 10s

Or something roughly similar. Who will arrange the bets?

JPK


From: Boaz Shaanan [mailto:bshaa...@bgu.ac.il]
Sent: Wednesday, November 30, 2016 5:19 PM
To: Keller, Jacob ; CCP4BB@JISCMAIL.AC.UK
Subject: RE: Effects of Multiplicity and Fine Phi with Equivalent Count Numbers

Hi Jacob,

I may have missed completely your point but as far as my memory goes, the main 
argument in favour of fine slicing has always been reduction of the noise 
arising from incoherent scattering, which in the old days arose from the 
capillary, solvent, air, you name it. The noise reduction in fine slicing is 
achieved by shortening the exposure time per frame. This argument still holds 
today although the sources of incoherent scattering could be different. Of 
course, there are other reasons to go for fine slicing such as long axes and 
others. In any case it's the recommended method these days, and for good 
reasons, isn't it?

  Best regards,

   Boaz

Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel

E-mail: bshaa...@bgu.ac.il
Phone: 972-8-647-2220  Skype: boaz.shaanan
Fax:   972-8-647-2992 or 972-8-646-1710




From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Keller, Jacob 
[kell...@janelia.hhmi.org]
Sent: Wednesday, November 30, 2016 11:37 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent Count 
Numbers
Dear Crystallographers,

I am curious whether the observed effects of fine phi slicing might in part or 
in toto be due to simply higher "pseudo-multiplicity." In other words, under 
normal experimental conditions, does simply increasing the number of 
measurements increase the signal and improve precision, even with the same 
number of total counts in the dataset?

As such, I am looking for a paper which, like Pflugrath's 1999 paper, compares 
two data sets with equivalent total counts but, in this case, different 
multiplicities. For example, is a single sweep with 0.5 degree 60s exposures 
empirically, in real practice, equivalent statistically to six passes with 0.5 
degree 10s frames? Better? Worse? Our home source has been donated away to 
Connecticut, so I can't do this experiment myself anymore.

All the best,

Jacob Keller


***
Jacob Pearson Keller, PhD
Research Scientist
HHMI Janelia Research Campus / Looger lab
Phone: (571)209-4000 x3159
Email: kell...@janelia.hhmi.org
***

Re: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent Count Numbers

[Boaz Shaanan]

Hi Jacob,


I may have missed completely your point but as far as my memory goes, the main argument in favour of fine slicing has always been reduction of the noise arising from incoherent scattering, which in the old days arose from the capillary, solvent, air, you
 name it. The noise reduction in fine slicing is achieved by shortening the exposure time per frame. This argument still holds today although the sources of incoherent scattering could be different. Of course, there are other reasons to go for fine slicing
 such as long axes and others. In any case it's the recommended method these days, and for good reasons, isn't it?


  Best regards,


                   Boaz
 
Boaz Shaanan, Ph.D.

Dept. of Life Sciences  
Ben-Gurion University of the Negev  
Beer-Sheva 84105    
Israel  
    
E-mail: bshaa...@bgu.ac.il
Phone: 972-8-647-2220  Skype: boaz.shaanan  
Fax:   972-8-647-2992 or 972-8-646-1710
 
 








From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Keller, Jacob [kell...@janelia.hhmi.org]
Sent: Wednesday, November 30, 2016 11:37 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent Count Numbers





Dear Crystallographers,
 
I am curious whether the observed effects of fine phi slicing might in part or in toto be due to simply higher “pseudo-multiplicity.” In other words, under normal experimental conditions, does simply increasing the number of measurements
 increase the signal and improve precision, even with the same number of total counts in the dataset?
 
As such, I am looking for a paper which, like Pflugrath’s 1999 paper, compares two data sets with equivalent total counts but, in this case, different multiplicities. For example, is a single sweep with 0.5 degree 60s exposures empirically,
 in real practice, equivalent statistically to six passes with 0.5 degree 10s frames? Better? Worse? Our home source has been donated away to Connecticut, so I can’t do this experiment myself anymore.
 
All the best,
 
Jacob Keller
 
 
***
Jacob Pearson Keller, PhD
Research Scientist
HHMI Janelia Research Campus / Looger lab
Phone: (571)209-4000 x3159
Email:
kell...@janelia.hhmi.org
***
 

[ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent Count Numbers

[Keller, Jacob]

Dear Crystallographers,

I am curious whether the observed effects of fine phi slicing might in part or 
in toto be due to simply higher "pseudo-multiplicity." In other words, under 
normal experimental conditions, does simply increasing the number of 
measurements increase the signal and improve precision, even with the same 
number of total counts in the dataset?

As such, I am looking for a paper which, like Pflugrath's 1999 paper, compares 
two data sets with equivalent total counts but, in this case, different 
multiplicities. For example, is a single sweep with 0.5 degree 60s exposures 
empirically, in real practice, equivalent statistically to six passes with 0.5 
degree 10s frames? Better? Worse? Our home source has been donated away to 
Connecticut, so I can't do this experiment myself anymore.

All the best,

Jacob Keller


***
Jacob Pearson Keller, PhD
Research Scientist
HHMI Janelia Research Campus / Looger lab
Phone: (571)209-4000 x3159
Email: kell...@janelia.hhmi.org
***

Re: [ccp4bb] RapiData at SSRL 2017

[Thomas, Leonard M.]

Please ignore, wrong email thread

Leonard M. Thomas Ph.D.
Macromolecular Crystallography Laboratory
Oklahoma COBRE in Structural Biology
Price Family Foundation Institute of Structural Biology
Department of Chemistry and Biochemistry
University of Oklahoma
Stephenson Life Sciences Research Center
101 Stephenson Parkway
Norman, OK 73019
405-325-1126
lmtho...@ou.edu
http://barlywine.chem.ou.edu
http://structuralbiology.ou.edu


From: CCP4 bulletin board  on behalf of Thomas, Leonard 
M. 
Sent: Wednesday, November 30, 2016 12:21:38 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] RapiData at SSRL 2017

Hi Clyde,

We have a crystal collecting now.  If it does not finish by the time our time 
is up you can remove it.  It is sort of a lets take some frames and see if we 
can do anything with it.
Overall the run was okay, ended up with some useful information, not sure about 
publishable data but we also have a better idea what to work on.

Cheers,
Len




From: CCP4 bulletin board  on behalf of Smith, Clyde 

Sent: Tuesday, November 29, 2016 7:50:25 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] RapiData at SSRL 2017

There are two weeks left to apply for the RapiData 2017 course on Data 
Collection and Structure Solving. The course will take place at SSRL on April 
16-21 2017. It is a great venue for young scientists to learn how to optimize 
macromolecular crystallography experiments at synchrotron beamlines and how to 
analyze the data from experts in methods and software developers.

RapiData comprises two days of lectures and two days of practical tutorials, 
including hands on data collection at the beamlines.
Students are encouraged to bring their own samples. For new SSRL users, it is 
also a great opportunity to become familiar with our beamlines and experimental 
facilities.

Details about the course can be found at:
http://smb.slac.stanford.edu/rapidata/about.html
The link to the application is:
http://smb.slac.stanford.edu/rapidata/registration.html
The deadline for applications is December 12th. Applications submitted after 
the deadline will be placed in a waiting list and considered if a spot opens. 
Late applications are not eligible for financial support.

Ana, Clyde and Silvia (organizers)

Please direct questions to Ana Gonzalez (a...@slac.stanford.edu) or Clyde Smith 
(csm...@slac.stanford.edu).

Re: [ccp4bb] RapiData at SSRL 2017

[Thomas, Leonard M.]

Hi Clyde,

We have a crystal collecting now.  If it does not finish by the time our time 
is up you can remove it.  It is sort of a lets take some frames and see if we 
can do anything with it.
Overall the run was okay, ended up with some useful information, not sure about 
publishable data but we also have a better idea what to work on.

Cheers,
Len




From: CCP4 bulletin board  on behalf of Smith, Clyde 

Sent: Tuesday, November 29, 2016 7:50:25 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] RapiData at SSRL 2017

There are two weeks left to apply for the RapiData 2017 course on Data 
Collection and Structure Solving. The course will take place at SSRL on April 
16-21 2017. It is a great venue for young scientists to learn how to optimize 
macromolecular crystallography experiments at synchrotron beamlines and how to 
analyze the data from experts in methods and software developers.

RapiData comprises two days of lectures and two days of practical tutorials, 
including hands on data collection at the beamlines.
Students are encouraged to bring their own samples. For new SSRL users, it is 
also a great opportunity to become familiar with our beamlines and experimental 
facilities.

Details about the course can be found at:
http://smb.slac.stanford.edu/rapidata/about.html
The link to the application is:
http://smb.slac.stanford.edu/rapidata/registration.html
The deadline for applications is December 12th. Applications submitted after 
the deadline will be placed in a waiting list and considered if a spot opens. 
Late applications are not eligible for financial support.

Ana, Clyde and Silvia (organizers)

Please direct questions to Ana Gonzalez (a...@slac.stanford.edu) or Clyde Smith 
(csm...@slac.stanford.edu).

Re: [ccp4bb] Unfilled electron density for structure with tNCS

[David Schuller]

There are reasons why MR might fail to find additional copies; perhaps a 
surface loop in the search model interferes with packing. You should 
probably take a look at solvent content.




On 11/30/2016 12:20 PM, Matthew Bratkowski wrote:

Hello all,

I am working on a structure in space group P4 at a resolution of about 
4 angstrom.  Xtriage indicates that translational NCS is present.  I 
am able to solve the structure by molecular replacement with four 
copies in the asymmetric unit, and there are two sets of identical 
copies. However, discontinuous, unfilled Fo-Fc density is present that 
appears to correspond to additional copies of the protein in the 
crystal packing.  I tried having phaser search for additional 
components in the ASU, but in each case the molecular replacement fails.


Is it possible that disordered copies of the protein appear in the ASU 
that is accounting for the unfilled density?  Other than the 
additional density, the structure looks ok.  I did not have too many 
problems with refinement as I was able to get a final Rwork/Rfree of 
around 0.24/0.29, which seems pretty decent considering the low 
resolution.


Any suggestions would be helpful.

Thanks,
Matt




--
===
All Things Serve the Beam
===
   David J. Schuller
   modern man in a post-modern world
   MacCHESS, Cornell University
   schul...@cornell.edu

[ccp4bb] Unfilled electron density for structure with tNCS

[Matthew Bratkowski]

Hello all,

I am working on a structure in space group P4 at a resolution of about 4
angstrom.  Xtriage indicates that translational NCS is present.  I am able
to solve the structure by molecular replacement with four copies in the
asymmetric unit, and there are two sets of identical copies.  However,
discontinuous, unfilled Fo-Fc density is present that appears to correspond
to additional copies of the protein in the crystal packing.  I tried having
phaser search for additional components in the ASU, but in each case the
molecular replacement fails.

Is it possible that disordered copies of the protein appear in the ASU that
is accounting for the unfilled density?  Other than the additional density,
the structure looks ok.  I did not have too many problems with refinement
as I was able to get a final Rwork/Rfree of around 0.24/0.29, which seems
pretty decent considering the low resolution.

Any suggestions would be helpful.

Thanks,
Matt

Re: [ccp4bb] To win or not twinning?

[Eleanor Dodson]

First - there is a useful document about possible twin laws in the CCP4
documentation. It references the SHELX description of twinning for small
molecules.
http://www.ccp4.ac.uk/html/twinning.html

It is possible in a trigonal system that you could have two twin operators
but the twinning stats should lok odd in that case

For a trigonal space group there are several options, but your technique
seems to have correctly found SG P3.
As Jacob Keller says - check POINTLESS to see if P322 etc are options?
Or ask Zanudu..

Just to comment - I think you must have the cell dimensions wrong for the
P63 case? I cant see how the c axis can be longer?

 I like the data processing POINTLESS/AIMLESS/ CTRUNCATE..

POINTLESS lists all axial reflections so you can make an educated guess at
screw axes (or let POINTLESS do it for you...)
POINTLESS analyses each symmetry operator separately. So if the -k, -h, -l
was slightly less convincing than others, AND the twin tests suggested
twinning, you might expect that to be a twin operator and not a symmetry
operator..

ctruncate tests each possible twin operator and scores them. (And so does
XTRIAGE of course..)


On 30 November 2016 at 14:00, Keller, Jacob 
wrote:

> What about spacegroups in PG 32, e.g., p3212?
>
>
>
> JPK
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
> *Napoleao
> Fonseca Valadares
> *Sent:* Wednesday, November 30, 2016 2:01 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] To win or not twinning?
>
>
>
> Dear CCP4ers,
>
> I'd like to kindly ask your advice. Sorry for the long e-mail.
>
> I have crystals of a 12.3 KDa protein that grow in hexagon-like patterns,
> link for the crystal image:
> http://fullonline.org/science/cryst01.jpg
>
> XDS, Phenix and Pointless always suggest that the data sets for these
> crystals belong to the space group P622. However, Phenix, Phaser and
> Pointless indicate that twinning is present.
>
> "Bad looking" diffraction images, diffracted to 1.6 A (collected 6 months
> ago):
> http://fullonline.org/science/dataset1_image37.png
> http://fullonline.org/science/dataset1_image7.png
>
> Best data set, diffracted to 2.01 A (collected a month ago):
> http://fullonline.org/science/dataset2_image51.png
>
> The second data set present better looking images, a better XDS ISa value
> (around 24) and diffracted to 2.2 A. The "bad looking" data set diffracted
> to 1.6 A, but I decided to stop working with it (XDS ISa around 10).
>
> There is a template with 60% identity, I used XDS to try to process the
> data in all trigonal/hexagonal space groups from P3 to P6(3)22, and spend a
> lot of time trying molecular replacement procedures in Phaser and Morda,
> and refining the candidate solutions. Used Zanuda too, trying to figure out
> the space group (and read as much as possible in this CCP4 list looking for
> similar cases).
>
> Unit cells and typical MR results:
>
> P1: 56.430   77.718   77.673 119.99  89.99  89.97
> SOLU SET  RFZ=9.5 TFZ=* PAK=0 LLG=91 RF++ TFZ=18.2 PAK=0 LLG=311
> TFZ==18.4 (&
> TFZ==17.0) LLG+=(311 & 726) LLG=5751 TFZ==64.6 PAK=0 LLG=5751 TFZ==64.6
>
> P3: 77.675   77.675   56.409  90.00  90.00 120.00
> RFZ=5.5 TFZ=8.5 PAK=0 LLG=86 TFZ==9.4 LLG=2575 TFZ==46.5
>
> P6: 77.534   77.534   92.986  90.00  90.00 120.00
> RFZ=11.2 TFZ=16.2 PAK=2 LLG=437 TFZ==25.7 LLG=437 TFZ==25.7
>
> P6(3): 77.675   77.675   56.409  90.00  90.00 120.00
>RFZ=8.8 TFZ=11.5 PAK=0 LLG=66 TFZ==7.6 LLG=66 TFZ==7.6
>
> P622: 77.660   77.660   56.400  90.00  90.00 120.00
>   RFZ=4.8 TFZ=5.4 PAK=1 LLG=22 TFZ==4.7 LLG=24 TFZ==4.8
>
>
> In P1 (LLG=5751 TFZ==64.6) there are 12 molecules in the asymmetric unit,
> and in P3 (LLG=2575 TFZ==46.5) 4 molecules. Packing looks good, in P1 the
> ASU looks like two superposed hexagonal donuts formed by 6 molecules each.
> Refining in P1, without adding waters or TLS, yield r_work = 0.2964 and
> r_free = 0.3428, and it is hard to decrease these values.
> Refining in P3, I managed to get r_work = 0.2934 and r_free = 0.3399, but
> looks like it's not getting any better than this.
> Refining in P6 yields horrible r_free values (>0.50).
>
> Trying to refine MR solutions in any other other space groups yield Rfree
> 0.41 or more.
>
> If in the P3 space group I use the twin operator -k,-h,-l (estimated twin
> fraction of 0.490) suggested by Phenix Xtriage, the values miraculously go
> down to r_work = 0.1923 r_free = 0.2202, without waters (r_free without
> using a twin law = 0.3399). Adding 120 water molecules and doing some
> refining yields 0.1711 r_free = 0.1935 (the asymmetric unit contains 420
> residues and the resolution is 2.01 A).
> I've been reading about twinning refinement and how it can drop the
> Rvalues, but from what I understood if I use it improperly I may compromise
> the refinement quality.
>
> I would like advice on:
> 1 - References that can teach how to look at protein diffraction images
> and understand what I am seeing. The basics l

Re: [ccp4bb] structure determination from a hollow crystal

[Chris Fage]

Hi Eike,

I wouldn't necessarily let the morphology deter you. While working on my
PhD, a colleague collected beautiful data from hollow crystals.

If the crystals collapse while looping, is it possible for you to perform
"micro-surgery" on the crystal so that you only have a single face?
Sometimes you can obtain very nice datasets from plates.

Best,
Chris

On Wed, Nov 30, 2016 at 8:20 AM, Schulz, Eike-Christian <
eike.sch...@mpsd.mpg.de> wrote:

> Dear all,
>
>
>
> Is there a documented case of a structure determination from a hollow
> crystal? We have the unfortunate situation that a protein only crystallizes
> as a hollow tube, where the inside is filled with solvent, which ruins the
> diffraction patterns.
>
>
>
> I was often confronted with similar situations, but in the previous cases
> I could always find a suitable crystal (part) or a different
> crystallization condition saved me.
>
>
>
> If anybody had general advise on how to proceed or could point me to
> relevant literature it would be much appreciated.
>
>
>
> With best regards
>
>
>
> Eike
>
>
>
>

Re: [ccp4bb] To win or not twinning?

[Keller, Jacob]

What about spacegroups in PG 32, e.g., p3212?

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Napoleao 
Fonseca Valadares
Sent: Wednesday, November 30, 2016 2:01 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] To win or not twinning?

Dear CCP4ers,

I'd like to kindly ask your advice. Sorry for the long e-mail.

I have crystals of a 12.3 KDa protein that grow in hexagon-like patterns, link 
for the crystal image:
http://fullonline.org/science/cryst01.jpg

XDS, Phenix and Pointless always suggest that the data sets for these crystals 
belong to the space group P622. However, Phenix, Phaser and Pointless indicate 
that twinning is present.

"Bad looking" diffraction images, diffracted to 1.6 A (collected 6 months ago):
http://fullonline.org/science/dataset1_image37.png
http://fullonline.org/science/dataset1_image7.png

Best data set, diffracted to 2.01 A (collected a month ago):
http://fullonline.org/science/dataset2_image51.png

The second data set present better looking images, a better XDS ISa value 
(around 24) and diffracted to 2.2 A. The "bad looking" data set diffracted to 
1.6 A, but I decided to stop working with it (XDS ISa around 10).

There is a template with 60% identity, I used XDS to try to process the data in 
all trigonal/hexagonal space groups from P3 to P6(3)22, and spend a lot of time 
trying molecular replacement procedures in Phaser and Morda, and refining the 
candidate solutions. Used Zanuda too, trying to figure out the space group (and 
read as much as possible in this CCP4 list looking for similar cases).

Unit cells and typical MR results:

P1: 56.430   77.718   77.673 119.99  89.99  89.97
SOLU SET  RFZ=9.5 TFZ=* PAK=0 LLG=91 RF++ TFZ=18.2 PAK=0 LLG=311 TFZ==18.4 
(&
TFZ==17.0) LLG+=(311 & 726) LLG=5751 TFZ==64.6 PAK=0 LLG=5751 TFZ==64.6

P3: 77.675   77.675   56.409  90.00  90.00 120.00
RFZ=5.5 TFZ=8.5 PAK=0 LLG=86 TFZ==9.4 LLG=2575 TFZ==46.5

P6: 77.534   77.534   92.986  90.00  90.00 120.00
RFZ=11.2 TFZ=16.2 PAK=2 LLG=437 TFZ==25.7 LLG=437 TFZ==25.7

P6(3): 77.675   77.675   56.409  90.00  90.00 120.00
   RFZ=8.8 TFZ=11.5 PAK=0 LLG=66 TFZ==7.6 LLG=66 TFZ==7.6

P622: 77.660   77.660   56.400  90.00  90.00 120.00
  RFZ=4.8 TFZ=5.4 PAK=1 LLG=22 TFZ==4.7 LLG=24 TFZ==4.8


In P1 (LLG=5751 TFZ==64.6) there are 12 molecules in the asymmetric unit, and 
in P3 (LLG=2575 TFZ==46.5) 4 molecules. Packing looks good, in P1 the ASU looks 
like two superposed hexagonal donuts formed by 6 molecules each.
Refining in P1, without adding waters or TLS, yield r_work = 0.2964 and r_free 
= 0.3428, and it is hard to decrease these values.
Refining in P3, I managed to get r_work = 0.2934 and r_free = 0.3399, but looks 
like it's not getting any better than this.
Refining in P6 yields horrible r_free values (>0.50).

Trying to refine MR solutions in any other other space groups yield Rfree 0.41 
or more.

If in the P3 space group I use the twin operator -k,-h,-l (estimated twin 
fraction of 0.490) suggested by Phenix Xtriage, the values miraculously go down 
to r_work = 0.1923 r_free = 0.2202, without waters (r_free without using a twin 
law = 0.3399). Adding 120 water molecules and doing some refining yields 0.1711 
r_free = 0.1935 (the asymmetric unit contains 420 residues and the resolution 
is 2.01 A).
I've been reading about twinning refinement and how it can drop the Rvalues, 
but from what I understood if I use it improperly I may compromise the 
refinement quality.

I would like advice on:
1 - References that can teach how to look at protein diffraction images and 
understand what I am seeing. The basics like recognizing bad data and what 
usually leads to deformity in the spots (for example, elliptical or duplicated) 
would be of great help.

2 - Should I look for other space groups? What else could be tried? Is this a 
case where a twin law should be used in the refinement? If yes, what can I do 
to confirm the need for a twin law in the refinement?

Thank you all in advance.
Regards,
 Napo

Re: [ccp4bb] How to merge two cif files of ligands.

[Wouter Touw]

Dear Peng,

The CCP4 interface for this task can be found in the menu Refinement ->
Restraint Preparation -> Merge monomer libraries.

Cheers,
Wouter

2016-11-30 13:33 GMT+01:00 Peng :

> Hello,
> I was wondering how to merge two cif files of ligands for refinement.
> Thanks,
> Peng
>
>
>
>
>
>
>

[ccp4bb] How to merge two cif files of ligands.

[Peng]

Hello,
I was wondering how to merge two cif files of ligands for refinement.
Thanks,
Peng

Re: [ccp4bb] To win or not twinning?

[frazao]

Hi,


The significant difference of refinement R values of the two models, 
single vs twinned crystal, indicates that the later is a far superior 
model of your crystal, corroborated its lower difference in Rfree-Rwork.



However, I am curious about the refined twinning fraction, did it 
decreased from the initial Xtriage estimate of 0.49?



Carlos




On 11/30/2016 07:01 AM, Napoleao Fonseca Valadares wrote:

Dear CCP4ers,

I'd like to kindly ask your advice. Sorry for the long e-mail.

I have crystals of a 12.3 KDa protein that grow in hexagon-like 
patterns, link for the crystal image:

http://fullonline.org/science/cryst01.jpg

XDS, Phenix and Pointless always suggest that the data sets for these 
crystals belong to the space group P622. However, Phenix, Phaser and 
Pointless indicate that twinning is present.


"Bad looking" diffraction images, diffracted to 1.6 A (collected 6 
months ago):

http://fullonline.org/science/dataset1_image37.png
http://fullonline.org/science/dataset1_image7.png

Best data set, diffracted to 2.01 A (collected a month ago):
http://fullonline.org/science/dataset2_image51.png

The second data set present better looking images, a better XDS ISa 
value (around 24) and diffracted to 2.2 A. The "bad looking" data set 
diffracted to 1.6 A, but I decided to stop working with it (XDS ISa 
around 10).


There is a template with 60% identity, I used XDS to try to process 
the data in all trigonal/hexagonal space groups from P3 to P6(3)22, 
and spend a lot of time trying molecular replacement procedures in 
Phaser and Morda, and refining the candidate solutions. Used Zanuda 
too, trying to figure out the space group (and read as much as 
possible in this CCP4 list looking for similar cases).


Unit cells and typical MR results:

P1: 56.430   77.718   77.673 119.99  89.99  89.97
SOLU SET  RFZ=9.5 TFZ=* PAK=0 LLG=91 RF++ TFZ=18.2 PAK=0 LLG=311 
TFZ==18.4 (&
TFZ==17.0) LLG+=(311 & 726) LLG=5751 TFZ==64.6 PAK=0 LLG=5751 
TFZ==64.6


P3: 77.675   77.675   56.409  90.00  90.00 120.00
RFZ=5.5 TFZ=8.5 PAK=0 LLG=86 TFZ==9.4 LLG=2575 TFZ==46.5

P6: 77.534   77.534   92.986  90.00  90.00 120.00
RFZ=11.2 TFZ=16.2 PAK=2 LLG=437 TFZ==25.7 LLG=437 TFZ==25.7

P6(3): 77.675   77.675   56.409  90.00  90.00 120.00
   RFZ=8.8 TFZ=11.5 PAK=0 LLG=66 TFZ==7.6 LLG=66 TFZ==7.6

P622: 77.660   77.660   56.400  90.00  90.00 120.00
  RFZ=4.8 TFZ=5.4 PAK=1 LLG=22 TFZ==4.7 LLG=24 TFZ==4.8


In P1 (LLG=5751 TFZ==64.6) there are 12 molecules in the asymmetric 
unit, and in P3 (LLG=2575 TFZ==46.5) 4 molecules. Packing looks good, 
in P1 the ASU looks like two superposed hexagonal donuts formed by 6 
molecules each.
Refining in P1, without adding waters or TLS, yield r_work = 0.2964 
and r_free = 0.3428, and it is hard to decrease these values.
Refining in P3, I managed to get r_work = 0.2934 and r_free = 0.3399, 
but looks like it's not getting any better than this.

Refining in P6 yields horrible r_free values (>0.50).

Trying to refine MR solutions in any other other space groups yield 
Rfree 0.41 or more.


If in the P3 space group I use the twin operator -k,-h,-l (estimated 
twin fraction of 0.490) suggested by Phenix Xtriage, the values 
miraculously go down to r_work = 0.1923 r_free = 0.2202, without 
waters (r_free without using a twin law = 0.3399). Adding 120 water 
molecules and doing some refining yields 0.1711 r_free = 0.1935 (the 
asymmetric unit contains 420 residues and the resolution is 2.01 A).
I've been reading about twinning refinement and how it can drop the 
Rvalues, but from what I understood if I use it improperly I may 
compromise the refinement quality.


I would like advice on:
1 - References that can teach how to look at protein diffraction 
images and understand what I am seeing. The basics like recognizing 
bad data and what usually leads to deformity in the spots (for 
example, elliptical or duplicated) would be of great help.


2 - Should I look for other space groups? What else could be tried? Is 
this a case where a twin law should be used in the refinement? If yes, 
what can I do to confirm the need for a twin law in the refinement?


Thank you all in advance.
Regards,
 Napo


--
**
Dr. Carlos Frazao
Structural Biology Laboratory -
Macromolecular Crystallography Unit
ITQB NOVA, Av. da República
2780-157 Oeiras, Portugal

Phone:  (351)-214469666/609
FAX:(351)-214433644
e-mail: fra...@itqb.unl.pt
www.itqb.unl.pt

[ccp4bb] AW: structure determination from a hollow crystal

[Herman . Schreuder]

Dear Eike,

The fact that the crystal is hollow, won’t ruin your diffraction, as long as 
the crystal remains rigid. However, when the crystal is flexible and collapses 
during mounting and freezing, you are in trouble. Against the good practice, I 
would in this case try a big loop and scoop the crystal with a lot of mother 
liquor, so the crystal is completely surrounded by mother liquor during 
freezing. You may get more background this way, but it might save your 
diffraction.

A hollow crystal means that protein around the growing seed gets depleted, so 
you may try to slow down the crystal growth, e.g. by using a lower precipitant 
concentration, maybe in combination with seeding to go to a region in the 
phase-diagram where you only have crystal growth, but no nucleation.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Schulz, 
Eike-Christian
Gesendet: Mittwoch, 30. November 2016 08:20
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] structure determination from a hollow crystal

Dear all,

Is there a documented case of a structure determination from a hollow crystal? 
We have the unfortunate situation that a protein only crystallizes as a hollow 
tube, where the inside is filled with solvent, which ruins the diffraction 
patterns.

I was often confronted with similar situations, but in the previous cases I 
could always find a suitable crystal (part) or a different crystallization 
condition saved me.

If anybody had general advise on how to proceed or could point me to relevant 
literature it would be much appreciated.

With best regards

Eike

[ccp4bb] AW: [ccp4bb] To win or not twinning?

[Herman . Schreuder]

Dear Napo,

For me this looks like a correct treatment of a twinned crystal. What more do 
you want/expect?

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Napoleao 
Fonseca Valadares
Gesendet: Mittwoch, 30. November 2016 08:01
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] To win or not twinning?

Dear CCP4ers,

I'd like to kindly ask your advice. Sorry for the long e-mail.

I have crystals of a 12.3 KDa protein that grow in hexagon-like patterns, link 
for the crystal image:
http://fullonline.org/science/cryst01.jpg

XDS, Phenix and Pointless always suggest that the data sets for these crystals 
belong to the space group P622. However, Phenix, Phaser and Pointless indicate 
that twinning is present.

"Bad looking" diffraction images, diffracted to 1.6 A (collected 6 months ago):
http://fullonline.org/science/dataset1_image37.png
http://fullonline.org/science/dataset1_image7.png

Best data set, diffracted to 2.01 A (collected a month ago):
http://fullonline.org/science/dataset2_image51.png

The second data set present better looking images, a better XDS ISa value 
(around 24) and diffracted to 2.2 A. The "bad looking" data set diffracted to 
1.6 A, but I decided to stop working with it (XDS ISa around 10).

There is a template with 60% identity, I used XDS to try to process the data in 
all trigonal/hexagonal space groups from P3 to P6(3)22, and spend a lot of time 
trying molecular replacement procedures in Phaser and Morda, and refining the 
candidate solutions. Used Zanuda too, trying to figure out the space group (and 
read as much as possible in this CCP4 list looking for similar cases).

Unit cells and typical MR results:

P1: 56.430   77.718   77.673 119.99  89.99  89.97
SOLU SET  RFZ=9.5 TFZ=* PAK=0 LLG=91 RF++ TFZ=18.2 PAK=0 LLG=311 TFZ==18.4 
(&
TFZ==17.0) LLG+=(311 & 726) LLG=5751 TFZ==64.6 PAK=0 LLG=5751 TFZ==64.6

P3: 77.675   77.675   56.409  90.00  90.00 120.00
RFZ=5.5 TFZ=8.5 PAK=0 LLG=86 TFZ==9.4 LLG=2575 TFZ==46.5

P6: 77.534   77.534   92.986  90.00  90.00 120.00
RFZ=11.2 TFZ=16.2 PAK=2 LLG=437 TFZ==25.7 LLG=437 TFZ==25.7

P6(3): 77.675   77.675   56.409  90.00  90.00 120.00
   RFZ=8.8 TFZ=11.5 PAK=0 LLG=66 TFZ==7.6 LLG=66 TFZ==7.6

P622: 77.660   77.660   56.400  90.00  90.00 120.00
  RFZ=4.8 TFZ=5.4 PAK=1 LLG=22 TFZ==4.7 LLG=24 TFZ==4.8


In P1 (LLG=5751 TFZ==64.6) there are 12 molecules in the asymmetric unit, and 
in P3 (LLG=2575 TFZ==46.5) 4 molecules. Packing looks good, in P1 the ASU looks 
like two superposed hexagonal donuts formed by 6 molecules each.
Refining in P1, without adding waters or TLS, yield r_work = 0.2964 and r_free 
= 0.3428, and it is hard to decrease these values.
Refining in P3, I managed to get r_work = 0.2934 and r_free = 0.3399, but looks 
like it's not getting any better than this.
Refining in P6 yields horrible r_free values (>0.50).

Trying to refine MR solutions in any other other space groups yield Rfree 0.41 
or more.

If in the P3 space group I use the twin operator -k,-h,-l (estimated twin 
fraction of 0.490) suggested by Phenix Xtriage, the values miraculously go down 
to r_work = 0.1923 r_free = 0.2202, without waters (r_free without using a twin 
law = 0.3399). Adding 120 water molecules and doing some refining yields 0.1711 
r_free = 0.1935 (the asymmetric unit contains 420 residues and the resolution 
is 2.01 A).
I've been reading about twinning refinement and how it can drop the Rvalues, 
but from what I understood if I use it improperly I may compromise the 
refinement quality.

I would like advice on:
1 - References that can teach how to look at protein diffraction images and 
understand what I am seeing. The basics like recognizing bad data and what 
usually leads to deformity in the spots (for example, elliptical or duplicated) 
would be of great help.

2 - Should I look for other space groups? What else could be tried? Is this a 
case where a twin law should be used in the refinement? If yes, what can I do 
to confirm the need for a twin law in the refinement?

Thank you all in advance.
Regards,
 Napo

Re: [ccp4bb] Calculation of RSRZ Score in PDB Validation Reports

[Jan Dohnalek]

Dear all,
I had this experience: going pedantically to the individual points the RSRZ
and other validation statistics in the form were reporting - in a vast
majority of the cases nothing was wrong at all. So it seems to be somewhat
overdoing its job - not that this is bad on its own - but we are losing
contrast quite a bit between the really serious issues on ... all the other.

Jan Dohnalek




On Wed, Nov 30, 2016 at 2:33 AM, dusan turk  wrote:

> Guys,
>
> I have a two issues to add here:
>
> 1. RSZS validation does not tolerate chain IDs longer than 1 character, so
> it kills one of the very essential reasons why mmCIF format was introduced
> (to enable deposition of large structures in a single file).
>
> 2. I have noticed in validation report of my own structure (4PIA) that the
> RSZS does not ALWAYS work right. For example, the "PHE 63" is well resolved
> with a hole in the ring, yet the validation declares it as a density
> outlier.  Besides there are several other residues in this structure that
> "sit" well in the density, but are considered outliers, whereas several,
> for which side chains the density is missing, are not listed.
>
> Has anyone else had a similar experience?
>
> Taken all remarks together they suggest that something needs to be done
> with RSZS software or density validation procedure to resolve these issues.
>
> 
>
> best,
> dusan
>
>
> On 30/11/16 01:00, CCP4BB automatic digest system wrote:
>
>> Date:Mon, 28 Nov 2016 20:35:44 -0800
>> From:Pavel Afonine
>> Subject: Re: Calculation of RSRZ Score in PDB Validation Reports
>>
>> I find Lothar's comments regarding H and RSRZ excellent! I would think of
>> it as a pretty much bug report. I hope developers at that end listen. This
>> goes very well in line with Phoebe's comment earlier today.
>>
>> Pavel
>>
>> On Mon, Nov 28, 2016 at 2:51 PM, Dale Tronrud
>> wrote:
>>
>> On 11/28/2016 12:52 PM,esse...@helix.nih.gov  wrote:
>>>
 I found that one can get RSRZ to go way down by loosening the geometry
> restraints.  The result is a crappy structure and I don't recommend
>
 doing
>>>
 that, but it does get all the atoms crammed into some sort of density.
>
Your observation is quite interesting. I can add this: when we were

>>> working
>>>
 with low to medium resolution structures, deleting the hydrogen atoms

>>> from
>>>
 the model after refinement moved the very bad RSRZ statistic to about
 the
 average in the given resolution range! Note, no re-refinement was done

>>> just
>>>
 a simple deletion of the riding H-atoms. I find this to be odd given the
 fact that, say the phenix developers favor the inclusion of H-atoms on
 riding positions even in cases of low resolution structures. (I assume

>>> the
>>>
 refmac5 and BUSTER-TNT developers have also a favorable opinion about
 including H-atoms in the final model - and during refinement).

 In my mind, it may be tempting to delete H-atoms to improve this

>>> statistic but
>>>
 when you use them in refinement they should be included regardless of
 the
 outcome of the RSRZ analysis.

>>> Of course, if you trick a validation statistic like this you haven't
>>> accomplished anything.  All you are saying is that one should rank RSRZ
>>> scores with and without hydrogen atoms separately.  Perhaps you should
>>> suggest that to the PDB validation people.
>>>
>>> Dale Tronrud
>>>
 RSRZ, in my most humble of opinions, seems like one of those statistics
>
 that
>>>
 is far more useful in theory than reality.   Particularly for
> medium-resolution structures, the fit of each entire side chain to the
>
 density
>>>
 is likely to be imperfect because the density is imperfect, especially
>
 toward
>>>
 the tips of those side chains.
>
> Then again, it can be a good flag for bits of the structure worth a
>
 second
>>>
 look in rebuilding.
>
The latter is certainly true. It may mean that the developers of RSRZ
 analysis need to tune it a bit to make it fully useful.

 L.

 
> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Matthew
> Bratkowski [mab...@cornell.edu]
> Sent: Tuesday, November 22, 2016 10:12 AM
> To:CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] Calculation of RSRZ Score in PDB Validation Reports
>
> Hello all,
>
> I was wondering if anyone knew how the RSRZ score was calculated in the
> protein data bank validation reports and how useful of a metric this
>
 actually
>>>
 is for structure validation?  I am trying to improve this score on a
>
 structure
>>>
 that I am working on, but I'm not really sure where to begin.  From my
> understanding, the score is based on the number of RSRZ outliers with a
>
 score
>>>
 2.  In my case, I have several res