Re: [ccp4bb] Summary for Anisotropic Diffraction In Refinement question

2009-09-16 Thread A Leslie
I would like to thank Justin for his summary of this topic, which I'm  
sure many people found of interest, and is very much in the spirit of  
the bulletin board.


I would just like to correct one factual error, in that it has been  
possible to specify anisotropic resolution limits to MOSFLM for many   
years, the appropriate keywords (described in the MOSFLM Help  
documentation) are:


RESOLUTION ANISO 3.5 2.5 2.5

where the three values are the resolution limits along (or close to)  
a*, b*, c*.


Unfortunately this option is not yet available in imosflm.

I have not personally used this option and so cannot compare its  
efficacy relative to integrating isotropically and then applying an  
anisotropic limit such as Justin describes.



Andrew Leslie

On 15 Sep 2009, at 21:48, Justin Hall wrote:


Dear All;

In response to my Anisotropic Diffraction In Refinement, which  
asked for suggestions for how best to proceed with refinement with  
an anisotropic data set, I received a large number of responses  
which overwhelmingly suggested using the UCLA Anisotropy Server (http://www.doe-mbi.ucla.edu/~sawaya/anisoscale/ 
).


The Anisotripy Server treats scaled/truncated data sets (I used  
Scala and the old Truncate program). Fo and SigFo are analyzed with  
respect to resolution in three dimensions and the data treated in  
three steps:

1) An elliptical resolution boundary is determined and applied.
2) A purely anisotropic B-factor is applied to the Fo and SigFo data  
to cause the data in all directions to fall off equally.
3) A negative isotropic B-factor is then applied to the structure  
factors to force the fall-off in the strongest direction to match  
that of the original data, effectively meaning that the data are not  
scaled to the mean but the weaker data are scaled up to match the  
strongest data.


Application of a elliptical resolution boundary is justified because  
the resolution boundary from common integration programs (Denzo and  
Mosflm for example) is spherical where diffraction for anisotropic  
data is ellipsoidal. A spherical boundary would result in the  
inclusion of numerous poorly measured reflections in the higher  
resolution shells which effectively makes these data more noisy.  
Imposing an ellipsoidal resolution boundary is equivalent to  
removing noise from the higher resolution bins and is simply the  
anisotropic equivalent of the normal resolution limit truncation.


However, I was confused by the second and third steps.  The second  
step of application of anisotropic scale factors is appropriate if  
the refinement program does not include anisotropic scaling in its  
calculation of Fc, however modern refinement programs do this. Pavel  
Afonine touched on this in his CCP4BB general posting in response to  
my original posting where he noted that anisotropic scale factor[s]  
that [are] part of the total structure factor take care of this (https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind0909L=CCP4BBT=0F=S=P=8362 
).


For the third step, applying a negative isotropic B-factor to modify  
the Fo is equivalent to sharpening the peaks in your maps and this  
can be useful.  However, applying the correction to Fo will also  
result in an inappropriate decrease in the average temperature  
factor of the resulting model.  Since B-factors are used as a  
measure of the coordinate error of an atom, modifying your Fo means  
these low B factors will tend to confuse the users of that model
into thinking its quality is better than it really is. If a sharper  
map makes identification of model errors easier, the map can be  
sharpened when it is calculated, without affecting the parameters in  
the PDB file.  The latest versions of Coot, for example, allows you  
to sharpen any map that it calculates.


I brought these points to the attention of the Anisotropy Server  
director (Michael Sawaya), who is now working to provide an option  
to omit steps 2 and 3 for users who do not what their structure  
factors modified.


My thanks to everyone who responded to my original question, and to  
Dale Tronrud and Michael Sawaya in particular for valuable discussion.


Re: [ccp4bb] Summary for Anisotropic Diffraction In Refinement question

2009-09-16 Thread Eleanor Dodson
I would like to have some comments on whether the maps before or after 
truncation are better . (obviously the Rfactors will be lower for the 
truncated data ..)


I suspect it iwill be completely anecdotal - but I confess to a gut 
unhappiness about throwing out measurements..

eleanor

Pavel Afonine wrote:
This is why phenix.refine by default outputs both maps: 2mFo-DFc 
filled and not filled, and it is the best to look at both keeping 
in mind all pros and cons of each of them.


Pavel.


On 9/15/09 5:22 PM, Peter Zwart wrote:
Application of a elliptical resolution boundary is justified because 
the
resolution boundary from common integration programs (Denzo and 
Mosflm for
example) is spherical where diffraction for anisotropic data is 
ellipsoidal.

A spherical boundary would result in the inclusion of numerous poorly
measured reflections in the higher resolution shells which 
effectively makes

these data more noisy. Imposing an ellipsoidal resolution boundary is
equivalent to removing noise from the higher resolution bins and is 
simply

the anisotropic equivalent of the normal resolution limit truncation.



Hi Justin,

Please be careful in interpreting maps from elliptically truncated
maps, there is a potential for introducing some bias. In Refmac (as
well and Phenix) maps are produced that fill in missing amplitudes
with DFcalc. When your mtz file contains only a small fraction of
miller indices in the highest (spherical) shell, all the missing
reflections will be assigned DFcalc. Depending on your anisotropy,
this can be a significant number of reflections.

I'm not sure how serious this issue is, but it might be worthwhile
checking the 'unfilled' maps as well (both phenix.refine and Refmac
allow you to compute these).

HTH

Peter
  




Re: [ccp4bb] Summary for Anisotropic Diffraction In Refinement question

2009-09-16 Thread Pierre Rizkallah
Hi Everyone,

I echo Andrew's thanks for the summary offered by Justin.

I would like to mention another way to trim anisotropic diffraction patterns of 
the weak patches 'at source', as it were, in MOSFLM, by specifying a sigma cut 
off applied to each image.

from the manual:
RESOLUTION [ lowres ]  highres  [CUTOFF sigcut]
The CUTOFF subkeyword allows the resolution limit of reflections written to the 
MTZ file to be different for each image. The resolution limit is set as that 
resolution at which I/sigma(I) drops to below sigcut. The I/sigma(I) for 
fully recorded reflections (if any) is used, otherwise partials. Default sigcut 
0.0

I used this option in the past 2 years on one particular data set, which 
certainly lost all its weak hi-res spots, leaving an ellipsoid of reflections. 
The merging stats improved significantly, but the completness in the outer 
shells went down significantly too. I did not compare maps with and without 
cut-off, in this particular case.

If the drop-off is severe, one can imagine the effect on the maps would be 
streakiness. There is no win-win situation, unfortunately. Some people set the 
resolution limit to be half-way between that of the best and worst directions, 
and take everything, weak and strong. It is a sacrifice of some good data for 
the sake of the overall quality, and produces distortions of its own, maybe 
without the streaks.

Pierre Rizkallah


**
Dr. Pierre Rizkallah, Senior Lecturer in Structural Biology, WHRI, School of 
Medicine, Academic Avenue, Heath Park, Cardiff CF14 4XN
email: rizkall...@cf.ac.uk phone + 44 29 2074 2248
 A Leslie and...@mrc-lmb.cam.ac.uk 16/09/09 9:24 AM 
I would like to thank Justin for his summary of this topic, which I'm  
sure many people found of interest, and is very much in the spirit of  
the bulletin board.

I would just like to correct one factual error, in that it has been  
possible to specify anisotropic resolution limits to MOSFLM for many   
years, the appropriate keywords (described in the MOSFLM Help  
documentation) are:

RESOLUTION ANISO 3.5 2.5 2.5

where the three values are the resolution limits along (or close to)  
a*, b*, c*.

Unfortunately this option is not yet available in imosflm.

I have not personally used this option and so cannot compare its  
efficacy relative to integrating isotropically and then applying an  
anisotropic limit such as Justin describes.


Andrew Leslie

On 15 Sep 2009, at 21:48, Justin Hall wrote:

 Dear All;

 In response to my Anisotropic Diffraction In Refinement, which  
 asked for suggestions for how best to proceed with refinement with  
 an anisotropic data set, I received a large number of responses  
 which overwhelmingly suggested using the UCLA Anisotropy Server 
 (http://www.doe-mbi.ucla.edu/~sawaya/anisoscale/ 
 ).

 The Anisotripy Server treats scaled/truncated data sets (I used  
 Scala and the old Truncate program). Fo and SigFo are analyzed with  
 respect to resolution in three dimensions and the data treated in  
 three steps:
 1) An elliptical resolution boundary is determined and applied.
 2) A purely anisotropic B-factor is applied to the Fo and SigFo data  
 to cause the data in all directions to fall off equally.
 3) A negative isotropic B-factor is then applied to the structure  
 factors to force the fall-off in the strongest direction to match  
 that of the original data, effectively meaning that the data are not  
 scaled to the mean but the weaker data are scaled up to match the  
 strongest data.

 Application of a elliptical resolution boundary is justified because  
 the resolution boundary from common integration programs (Denzo and  
 Mosflm for example) is spherical where diffraction for anisotropic  
 data is ellipsoidal. A spherical boundary would result in the  
 inclusion of numerous poorly measured reflections in the higher  
 resolution shells which effectively makes these data more noisy.  
 Imposing an ellipsoidal resolution boundary is equivalent to  
 removing noise from the higher resolution bins and is simply the  
 anisotropic equivalent of the normal resolution limit truncation.

 However, I was confused by the second and third steps.  The second  
 step of application of anisotropic scale factors is appropriate if  
 the refinement program does not include anisotropic scaling in its  
 calculation of Fc, however modern refinement programs do this. Pavel  
 Afonine touched on this in his CCP4BB general posting in response to  
 my original posting where he noted that anisotropic scale factor[s]  
 that [are] part of the total structure factor take care of this 
 (https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind0909L=CCP4BBT=0F=S=P=8362
  
 ).

 For the third step, applying a negative isotropic B-factor to modify  
 the Fo is equivalent to sharpening the peaks in your maps and this  
 can be useful.  However, applying the correction to Fo will also  
 

Re: [ccp4bb] Summary for Anisotropic Diffraction In Refinement question

2009-09-16 Thread Harry Powell

Just to add my two ha'porth.

I discussed this some years ago with Garib, just after I'd added the  
anisotropic cutoff to the resolution limits in Mosflm (mentioned by  
Andrew below); as I remember (and this is an invitation to Garib to  
contribute and correct me here!), the answer went along the lines that  
the systematically missing volumes of data would affect the maximum  
likelihood model used (which used an isotropic resolution diffraction  
model?), so the refinement of a molecular model produced with  
anisotropic data would be affected badly.


I think I would tend to follow Pierre's route, though, since the model  
I implemented only defines the resolution along a* b* and c*, and the  
sigma cutoff is probably more flexible.


Having said that, there are one or two users out there who have used  
the anisotropic cutoffs and have been very happy with the results (or  
at least that's what they said to me).


On 16 Sep 2009, at 11:13, Pierre Rizkallah wrote:


Hi Everyone,

I echo Andrew's thanks for the summary offered by Justin.

I would like to mention another way to trim anisotropic diffraction  
patterns of the weak patches 'at source', as it were, in MOSFLM, by  
specifying a sigma cut off applied to each image.


from the manual:
RESOLUTION [ lowres ]  highres  [CUTOFF sigcut]
The CUTOFF subkeyword allows the resolution limit of reflections  
written to the MTZ file to be different for each image. The  
resolution limit is set as that resolution at which I/sigma(I) drops  
to below sigcut. The I/sigma(I) for fully recorded reflections (if  
any) is used, otherwise partials. Default sigcut 0.0


I used this option in the past 2 years on one particular data set,  
which certainly lost all its weak hi-res spots, leaving an ellipsoid  
of reflections. The merging stats improved significantly, but the  
completness in the outer shells went down significantly too. I did  
not compare maps with and without cut-off, in this particular case.


If the drop-off is severe, one can imagine the effect on the maps  
would be streakiness. There is no win-win situation, unfortunately.  
Some people set the resolution limit to be half-way between that of  
the best and worst directions, and take everything, weak and strong.  
It is a sacrifice of some good data for the sake of the overall  
quality, and produces distortions of its own, maybe without the  
streaks.


Pierre Rizkallah


**
Dr. Pierre Rizkallah, Senior Lecturer in Structural Biology, WHRI,  
School of Medicine, Academic Avenue, Heath Park, Cardiff CF14 4XN

email: rizkall...@cf.ac.uk phone + 44 29 2074 2248

A Leslie and...@mrc-lmb.cam.ac.uk 16/09/09 9:24 AM 

I would like to thank Justin for his summary of this topic, which I'm
sure many people found of interest, and is very much in the spirit of
the bulletin board.

I would just like to correct one factual error, in that it has been
possible to specify anisotropic resolution limits to MOSFLM for many
years, the appropriate keywords (described in the MOSFLM Help
documentation) are:

RESOLUTION ANISO 3.5 2.5 2.5

where the three values are the resolution limits along (or close to)
a*, b*, c*.

Unfortunately this option is not yet available in imosflm.

I have not personally used this option and so cannot compare its
efficacy relative to integrating isotropically and then applying an
anisotropic limit such as Justin describes.


Andrew Leslie

On 15 Sep 2009, at 21:48, Justin Hall wrote:


Dear All;

In response to my Anisotropic Diffraction In Refinement, which
asked for suggestions for how best to proceed with refinement with
an anisotropic data set, I received a large number of responses
which overwhelmingly suggested using the UCLA Anisotropy Server 
(http://www.doe-mbi.ucla.edu/~sawaya/anisoscale/

).


The Anisotripy Server treats scaled/truncated data sets (I used
Scala and the old Truncate program). Fo and SigFo are analyzed with
respect to resolution in three dimensions and the data treated in
three steps:
1) An elliptical resolution boundary is determined and applied.
2) A purely anisotropic B-factor is applied to the Fo and SigFo data
to cause the data in all directions to fall off equally.
3) A negative isotropic B-factor is then applied to the structure
factors to force the fall-off in the strongest direction to match
that of the original data, effectively meaning that the data are not
scaled to the mean but the weaker data are scaled up to match the
strongest data.

Application of a elliptical resolution boundary is justified because
the resolution boundary from common integration programs (Denzo and
Mosflm for example) is spherical where diffraction for anisotropic
data is ellipsoidal. A spherical boundary would result in the
inclusion of numerous poorly measured reflections in the higher
resolution shells which effectively makes these data more noisy.
Imposing an ellipsoidal resolution boundary is 

[ccp4bb] Summary for Anisotropic Diffraction In Refinement question

2009-09-15 Thread Justin Hall

Dear All;

In response to my Anisotropic Diffraction In Refinement, which asked  
for suggestions for how best to proceed with refinement with an  
anisotropic data set, I received a large number of responses which  
overwhelmingly suggested using the UCLA Anisotropy Server  
(http://www.doe-mbi.ucla.edu/~sawaya/anisoscale/).


The Anisotripy Server treats scaled/truncated data sets (I used Scala  
and the old Truncate program). Fo and SigFo are analyzed with respect  
to resolution in three dimensions and the data treated in three steps:

1) An elliptical resolution boundary is determined and applied.
2) A purely anisotropic B-factor is applied to the Fo and SigFo data  
to cause the data in all directions to fall off equally.
3) A negative isotropic B-factor is then applied to the structure  
factors to force the fall-off in the strongest direction to match that  
of the original data, effectively meaning that the data are not scaled  
to the mean but the weaker data are scaled up to match the strongest  
data.


Application of a elliptical resolution boundary is justified because  
the resolution boundary from common integration programs (Denzo and  
Mosflm for example) is spherical where diffraction for anisotropic  
data is ellipsoidal. A spherical boundary would result in the  
inclusion of numerous poorly measured reflections in the higher  
resolution shells which effectively makes these data more noisy.  
Imposing an ellipsoidal resolution boundary is equivalent to removing  
noise from the higher resolution bins and is simply the anisotropic  
equivalent of the normal resolution limit truncation.


However, I was confused by the second and third steps.  The second  
step of application of anisotropic scale factors is appropriate if the  
refinement program does not include anisotropic scaling in its  
calculation of Fc, however modern refinement programs do this. Pavel  
Afonine touched on this in his CCP4BB general posting in response to  
my original posting where he noted that anisotropic scale factor[s]  
that [are] part of the total structure factor take care of this  
(https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind0909L=CCP4BBT=0F=S=P=8362).


For the third step, applying a negative isotropic B-factor to modify  
the Fo is equivalent to sharpening the peaks in your maps and this can  
be useful.  However, applying the correction to Fo will also result in  
an inappropriate decrease in the average temperature factor of the  
resulting model.  Since B-factors are used as a measure of the  
coordinate error of an atom, modifying your Fo means these low B  
factors will tend to confuse the users of that model
into thinking its quality is better than it really is. If a sharper  
map makes identification of model errors easier, the map can be  
sharpened when it is calculated, without affecting the parameters in  
the PDB file.  The latest versions of Coot, for example, allows you to  
sharpen any map that it calculates.


I brought these points to the attention of the Anisotropy Server  
director (Michael Sawaya), who is now working to provide an option to  
omit steps 2 and 3 for users who do not what their structure factors  
modified.


My thanks to everyone who responded to my original question, and to  
Dale Tronrud and Michael Sawaya in particular for valuable discussion.


Re: [ccp4bb] Summary for Anisotropic Diffraction In Refinement question

2009-09-15 Thread Peter Zwart
 Application of a elliptical resolution boundary is justified because the
 resolution boundary from common integration programs (Denzo and Mosflm for
 example) is spherical where diffraction for anisotropic data is ellipsoidal.
 A spherical boundary would result in the inclusion of numerous poorly
 measured reflections in the higher resolution shells which effectively makes
 these data more noisy. Imposing an ellipsoidal resolution boundary is
 equivalent to removing noise from the higher resolution bins and is simply
 the anisotropic equivalent of the normal resolution limit truncation.

Hi Justin,

Please be careful in interpreting maps from elliptically truncated
maps, there is a potential for introducing some bias. In Refmac (as
well and Phenix) maps are produced that fill in missing amplitudes
with DFcalc. When your mtz file contains only a small fraction of
miller indices in the highest (spherical) shell, all the missing
reflections will be assigned DFcalc. Depending on your anisotropy,
this can be a significant number of reflections.

I'm not sure how serious this issue is, but it might be worthwhile
checking the 'unfilled' maps as well (both phenix.refine and Refmac
allow you to compute these).

HTH

Peter


Re: [ccp4bb] Summary for Anisotropic Diffraction In Refinement question

2009-09-15 Thread Pavel Afonine
This is why phenix.refine by default outputs both maps: 2mFo-DFc 
filled and not filled, and it is the best to look at both keeping in 
mind all pros and cons of each of them.


Pavel.


On 9/15/09 5:22 PM, Peter Zwart wrote:

Application of a elliptical resolution boundary is justified because the
resolution boundary from common integration programs (Denzo and Mosflm for
example) is spherical where diffraction for anisotropic data is ellipsoidal.
A spherical boundary would result in the inclusion of numerous poorly
measured reflections in the higher resolution shells which effectively makes
these data more noisy. Imposing an ellipsoidal resolution boundary is
equivalent to removing noise from the higher resolution bins and is simply
the anisotropic equivalent of the normal resolution limit truncation.



Hi Justin,

Please be careful in interpreting maps from elliptically truncated
maps, there is a potential for introducing some bias. In Refmac (as
well and Phenix) maps are produced that fill in missing amplitudes
with DFcalc. When your mtz file contains only a small fraction of
miller indices in the highest (spherical) shell, all the missing
reflections will be assigned DFcalc. Depending on your anisotropy,
this can be a significant number of reflections.

I'm not sure how serious this issue is, but it might be worthwhile
checking the 'unfilled' maps as well (both phenix.refine and Refmac
allow you to compute these).

HTH

Peter