Re: [ccp4bb] normalization of B-factor values from different crystal structures

[Pavel Afonine]

Hi,

also keep in mind that the total model structure factor used in refinement
and anywhere where model-to-data agreement needs to be evaluated (such as
maps or R factors) is:

Fmodel = ktotal * (Fcalc_atoms + F_bulk_solvent + F_something_else)

where ktotal ~ scale * exp(-h*Uoverall*h_transpose) .

This makes it obvious that B factor is arbitrarily shared between Uoverall
matrix and atomic B factor.
phenix.refine subtracts as much as possible from the trace of Uoverall and
adds that to atomic B; however, sometimes it is only possible to add a part
of what can be removed from Uoverall.

With this in mind and as James pointed out, atomic B factors are likely
defined up to a constant.

Pavel


On Wed, Aug 2, 2017 at 5:54 PM, James Holton 
wrote:

> Woops, sorry.  There was a typo in my response.  here it is again without
> the typo.
>
> B factors are 78.96x the value of the mean square variation in an atom's
> position.  The square is the important part of how you scale them.  Lets
> say you have static disorder in the crystal lattice, and that gives every
> atom an rms variation of 0.5 A relative to their ideal lattice positions,
> then that static disorder imparts a B factor of 78.96*(0.5)^2 = 19.7 to all
> atoms.  If in addition to lattice disorder you have a side chain flapping
> in the breeze by another rms 1.0 A, that is B = 79, but the combination of
> the two things is an rms fluctuation of sqrt(0.5^2 + 1.0^2) = 1.118 rms A,
> and the total B factor resulting from that is 98.7.  It is not a
> coincidence that 98.7 is the sum of 19.7 and 79.  That is, independent
> sources of disorder _add_ when it comes to the B factors they produce.
>
> So, if you want to "normalize" B factors from one structure to another,
> the best thing to do is subtract a constant.  This is mathematically
> equivalent to "deconvoluting" one source of overall variation from the
> site-to-site differences.  What should the constant be?  Well, the
> structure-wide atomic B factor average isn't a bad choice.  The caveat is
> that a B factor change of 5 in the context of an overall B of 15 is
> probably significant, but in a low resolution structure with an overall B
> factor of 100, it might be nothing more than a random fluctuation.  It's
> like looking at the width of bands on a gel.  A small change in a sharp
> band is significant, but that same change in position for a fat band is
> more dubious.
> Now, crystallographically, all a B factor is is the rate of falloff of the
> contribution of an atom to the diffraction pattern with increasing
> resolution.  So, the overall B factor can be quite well known, but the B
> factor of a single atom in the context of tens of thousands of others can
> be harder to determine.  Refinement programs do their best finding the best
> fit, but in the end you are trying to reconcile a lot of different possible
> contributors to the fall-off of data with resolution.  Because of phases, a
> small change in one B factor can cancel a small change in another.  This is
> why B factor refinement at low resolution is dangerous.
>
> If you want to compare B factors I'd recommend putting "error bars" on
> them.  That is, re-refine the structures of interest after jiggling the
> coordinates and setting all the B factors to a constant.  See how
> reproducible the final B factors are.  This will give you an idea of how
> big a change can happen by pure random chance, even with the same data.
>
> Hope that helps!
>
> -James  Holton
> MAD Scientist
>
> On 8/2/2017 12:09 PM, Asmita wrote:
>
> Hi,
>
> This might look as a very fundamental question. I have a dataset of
> crystal structures better than 3.5Ang resolution. For a qualitative
> analysis, I want to compare the residue-wise B-factors in these structures,
> but due to different procedures adopted in refinement and scaling, I
> understand that these values cannot be compared in a raw manner.
>
> Can someone suggest appropriate normalization methods that could be used
> for scaling these B-factors for a relevant and meaningful comparison? All
> the files have isotropic B-factor values and there are no ANISOU entries in
> any of the files.
>
> Thanks
>
> Asmita
>
>
>

Re: [ccp4bb] refmac output

[Pavel Afonine]

Hi Ed,

your suggestion makes perfect sense to me, and it's trivial to add an
option to do what you want. This will be available in next Phenix nightly
build (clearly not tomorrow given today's power outage).

Command line: use "write_map_coefficients_only=True" (by default is is
False).

Refinement GUI: Configure -> Output -> Other options -> tick "Write map
coefficients only" box.

Pavel

On Wed, Aug 2, 2017 at 1:12 PM, Edwin Pozharski 
wrote:

>
> Just to clarify, how do you use the extra columns in this scenario?  My
> suggestion was to have the output file that includes only the map
> coefficient columns, so you still can look at the map.  IIRC, FP/SIGFP
> columns from refmac output are actually modified from the input (scaled
> with Boverall), so it was not recommended to use refmac output as input of
> any kind.
>
> Also, to provide context, my comment resulted from dealing with a large
> chunk of data that included ~200 output mtz files - which is Gb-sized
> tarball that had to be uploaded/downloaded.  Not the end of the world, but
> cutting it in half seemed like a good idea at the time. :)  Not hard to
> script that, of course.
>
> I am not necessarily advocating for skinny files to be the default, but as
> it stands, refmac/buster/phenix do not even provide the option of doing it
> (It's entirely possible that I am wrong here on specifics and will get
> corrected by Garib, Gerard and Pavel).
>
> Cheers,
>
> Ed.
>
> Edwin Pozharski, PhD, Assistant Professor
> University of Maryland, Baltimore
> --
> When the Way is forgotten duty and justice appear;
> Then knowledge and wisdom are born along with hypocrisy.
> When harmonious relationships dissolve then respect and devotion arise;
> When a nation falls to chaos then loyalty and patriotism are born.
> -- / Lao Tse /
>
>

Re: [ccp4bb] normalization of B-factor values from different crystal structures

[James Holton]

Woops, sorry.  There was a typo in my response.  here it is again 
without the typo.


B factors are 78.96x the value of the mean square variation in an atom's 
position.  The square is the important part of how you scale them.  Lets 
say you have static disorder in the crystal lattice, and that gives 
every atom an rms variation of 0.5 A relative to their ideal lattice 
positions, then that static disorder imparts a B factor of 78.96*(0.5)^2 
= 19.7 to all atoms. If in addition to lattice disorder you have a side 
chain flapping in the breeze by another rms 1.0 A, that is B = 79, but 
the combination of the two things is an rms fluctuation of sqrt(0.5^2 + 
1.0^2) = 1.118 rms A, and the total B factor resulting from that is 
98.7.  It is not a coincidence that 98.7 is the sum of 19.7 and 79.  
That is, independent sources of disorder _add_ when it comes to the B 
factors they produce.


So, if you want to "normalize" B factors from one structure to another, 
the best thing to do is subtract a constant.  This is mathematically 
equivalent to "deconvoluting" one source of overall variation from the 
site-to-site differences.  What should the constant be?  Well, the 
structure-wide atomic B factor average isn't a bad choice.  The caveat 
is that a B factor change of 5 in the context of an overall B of 15 is 
probably significant, but in a low resolution structure with an overall 
B factor of 100, it might be nothing more than a random fluctuation.  
It's like looking at the width of bands on a gel.  A small change in a 
sharp band is significant, but that same change in position for a fat 
band is more dubious.


Now, crystallographically, all a B factor is is the rate of falloff of 
the contribution of an atom to the diffraction pattern with increasing 
resolution.  So, the overall B factor can be quite well known, but the B 
factor of a single atom in the context of tens of thousands of others 
can be harder to determine.  Refinement programs do their best finding 
the best fit, but in the end you are trying to reconcile a lot of 
different possible contributors to the fall-off of data with 
resolution.  Because of phases, a small change in one B factor can 
cancel a small change in another.  This is why B factor refinement at 
low resolution is dangerous.


If you want to compare B factors I'd recommend putting "error bars" on 
them.  That is, re-refine the structures of interest after jiggling the 
coordinates and setting all the B factors to a constant.  See how 
reproducible the final B factors are.  This will give you an idea of how 
big a change can happen by pure random chance, even with the same data.


Hope that helps!

-James  Holton
MAD Scientist

On 8/2/2017 12:09 PM, Asmita wrote:

Hi,

This might look as a very fundamental question. I have a dataset of 
crystal structures better than 3.5Ang resolution. For a qualitative 
analysis, I want to compare the residue-wise B-factors in these 
structures, but due to different procedures adopted in refinement and 
scaling, I understand that these values cannot be compared in a raw 
manner.


Can someone suggest appropriate normalization methods that could be 
used for scaling these B-factors for a relevant and meaningful 
comparison? All the files have isotropic B-factor values and there are 
no ANISOU entries in any of the files.


Thanks

Asmita

Re: [ccp4bb] normalization of B-factor values from different crystal structures

[James Holton]

B factors are 78.96x the value of the mean square variation in an atom's 
position.  The square is the important part of how you scale them.  Lets 
say you have static disorder in the crystal lattice, and that gives 
every atom an rms variation of 0.5 A relative to their ideal lattice 
positions, then that static disorder imparts a B factor of 78.96*(0.3)^2 
= 19.7 to all atoms. If in addition to lattice disorder you have a side 
chain flapping in the breeze by another rms 1.0 A, that is B = 79, but 
the combination of the two things is an rms fluctuation of sqrt(0.5^2 + 
1.0^2) = 1.118 rms A, and the total B factor resulting from that is 
98.7.  It is not a coincidence that 98.7 is the sum of 19.7 and 79.  
That is, independent sources of disorder _add_ when it comes to the B 
factors they produce.


So, if you want to "normalize" B factors from one structure to another, 
the best thing to do is subtract a constant.  This is mathematically 
equivalent to "deconvoluting" one source of overall variation from the 
site-to-site differences.  What should the constant be?  Well, the 
structure-wide atomic B factor average isn't a bad choice.  The caveat 
is that a B factor change of 5 in the context of an overall B of 15 is 
probably significant, but in a low resolution structure with an overall 
B factor of 100, it might be nothing more than a random fluctuation.  
It's like looking at the width of bands on a gel.  A small change in a 
sharp band is significant, but that same change in position for a fat 
band is more dubious.


Now, crystallographically, all a B factor is is the rate of falloff of 
the contribution of an atom to the diffraction pattern with increasing 
resolution.  So, the overall B factor can be quite well known, but the B 
factor of a single atom in the context of tens of thousands of others 
can be harder to determine.  Refinement programs do their best finding 
the best fit, but in the end you are trying to reconcile a lot of 
different possible contributors to the fall-off of data with 
resolution.  Because of phases, a small change in one B factor can 
cancel a small change in another.  This is why B factor refinement at 
low resolution is dangerous.


If you want to compare B factors I'd recommend putting "error bars" on 
them.  That is, re-refine the structures of interest after jiggling the 
coordinates and setting all the B factors to a constant.  See how 
reproducible the final B factors are.  This will give you an idea of how 
big a change can happen by pure random chance, even with the same data.


Hope that helps!

-James  Holton
MAD Scientist

On 8/2/2017 12:09 PM, Asmita wrote:

Hi,

This might look as a very fundamental question. I have a dataset of 
crystal structures better than 3.5Ang resolution. For a qualitative 
analysis, I want to compare the residue-wise B-factors in these 
structures, but due to different procedures adopted in refinement and 
scaling, I understand that these values cannot be compared in a raw 
manner.


Can someone suggest appropriate normalization methods that could be 
used for scaling these B-factors for a relevant and meaningful 
comparison? All the files have isotropic B-factor values and there are 
no ANISOU entries in any of the files.


Thanks

Asmita

[ccp4bb] LBNL power outage

[Nicholas Sauter]

Dear software users,

All LBNL crystallographic servers will be off line due to a power shutdown,
taken as a precaution due to a nearby wild land fire in the Berkeley
hills.  This will affect PHENIX, LABELIT, CCTBX and other servers.  We
apologize for the downtime.

Nick

Nicholas K. Sauter, Ph. D.
Senior Scientist, Molecular Biophysics & Integrated Bioimaging Division
Lawrence Berkeley National Laboratory
1 Cyclotron Rd., Bldg. 33R0345
Berkeley, CA 94720
(510) 486-5713

Re: [ccp4bb] normalization of B-factor values from different crystal structures

[Avinash Punekar]

Hi Asmita,

Try running your different crystal structures through PDB_REDO. That should 
normalize the B-factors to some meaningful values for comparison.

Best wishes,
Avinash

Re: [ccp4bb] normalization of B-factor values from different crystal structures

[Ethan A Merritt]

On Wednesday, 02 August, 2017 21:58:07 Asmita Gupta wrote:
> Hi,
> 
> Thanks for the response! 
> 
> What I have are crystal structures of the same protein in multiple 
> conformations, solved by different groups. I wanted to calculate the 
> residue-wise B-factors for each of these structures and compare how the 
> values are changing for corresponding residues in these different structures. 
> e.g. B-factor variation in residue number 200 (Ala) in 10 different 
> conformations of a protein. 
> 
> Hope I have been able to answer the question!

But what is the biological question?  
You haven't learned much if all you can say is
"The B factor of residue 200 is higher in this structure".

Do you not have a larger question in mind - on the order
of "ligand binding a site X correlates with reduced 
mobility of loop A-B"?   

For such questions I suggest (as I always do :-) using TLSMD
analysis rather than examining individual B factors.  
That allows you to say something like "in structure #1 we
can identify a distinct group motion of residues in loop A-B,
whereas in structure #2 loop A-B appears to move in concert
with the entire subdomain XX".  This is not the same thing
as saying individual B factors are higher or lower.

Ethan

-- 
Ethan A Merritt
Biomolecular Structure Center,  K-428 Health Sciences Bldg
MS 357742,   University of Washington, Seattle 98195-7742

Re: [ccp4bb] normalization of B-factor values from different crystal structures

[Asmita Gupta]

Hi,

Thanks for the response! 

What I have are crystal structures of the same protein in multiple 
conformations, solved by different groups. I wanted to calculate the 
residue-wise B-factors for each of these structures and compare how the values 
are changing for corresponding residues in these different structures. e.g. 
B-factor variation in residue number 200 (Ala) in 10 different conformations of 
a protein. 

Hope I have been able to answer the question!

Re: [ccp4bb] Aimless number of uniques

[Phil Evans]

What do you mean by “unique”? In the absence of anomalous signal, I+ and I- 
should be the same, so are not independent, and it doesn’t make sense to count 
them separately. Even if there is some anomalous signal, it is generally small 
on average compared to the whole intensity, so I+ and I- are highly correlated 
- should they be counted separately? What is the count for anyway?

I think it is confusing to count them differently depending on whether there is 
anomalous signal, however small. 

So “Number of unique refections” does depend on how you define it, and I would 
define it as the count of I+ or I-

Phil


> On 2 Aug 2017, at 21:02, Eugene Osipov  wrote:
> 
> Dear Phil, 
> it is much more confusing. Can you please explain me why do you think they 
> are non unique in case of anomalous signal?
> 
> 
> 2 авг. 2017 г. 19:01 пользователь "Phil Evans"  
> написал:
> 
> Anomalous ON or OFF affects some analyses, and outlier rejection: it doesn’t 
> change which reflections are used. It always outputs I+ and I-, even with 
> ANOMALOUS OFF
> 
> Phenix (and maybe XDS) count I+ & I- as two separate unique reflections: 
> Aimless counts them as one, which I think is more reasonable. I+ and I- are 
> strongly correlated
> 
> Phil
> 
> > On 2 Aug 2017, at 16:33, Eugene Osipov  wrote:
> >
> > Dear community,
> >
> > I am confused by a reported number of unique reflections in AIMLESS version 
> > 0.5.32 : 29/03/17 ( CCP4 7.0.043).
> > I am processing dataset with strong anomalous signal but it seems that 
> > "anomalous" keyword does not affect number of unique reflections.
> > I run:
> > $pointless XDS_ASCII.HKL HKLOUT pointless.mtz
> > .
> > $aimless hklin pointless.mtz hklout aimless.mtz
> >
> > After a program run with either "ANOMALOUS ON" or "ANOMALOUS OFF" I see 
> > 6629 reflections. Meanwile XDS and Phenix report  12 200 unique reflections.
> > Is it bug or I miss something?
> >
> >
> > --
> > Eugene Osipov
> > Junior Research Scientist
> > Laboratory of Enzyme Engineering
> > A.N. Bach Institute of Biochemistry
> > Russian Academy of Sciences
> > Leninsky pr. 33, 119071 Moscow, Russia
> > e-mail: e.m.osi...@gmail.com
> 

Re: [ccp4bb] refmac output

[Ethan A Merritt]

On Wednesday, 02 August, 2017 16:12:30 Edwin Pozharski wrote:
> Just to clarify, how do you use the extra columns in this scenario?  My
> suggestion was to have the output file that includes only the map
> coefficient columns, so you still can look at the map.  IIRC, FP/SIGFP
> columns from refmac output are actually modified from the input (scaled
> with Boverall), so it was not recommended to use refmac output as input of
> any kind.

The scaled Fs are useful to recalculate R as a function of whatever, and
to calculate alternative R values (e.g. for Hamilton R test or comparison
to shelxl R1 R2). It's nice to carry though DANO and SIGDANO so that one
can generate anomalous maps. In general I'd rather err on the side of
including everything rather than discarding something that may be useful
later.  In any case it's trivial to run one additional script to filter
unwanted columns if file size really is a controlling factor.

Ethan




> 
> Also, to provide context, my comment resulted from dealing with a large
> chunk of data that included ~200 output mtz files - which is Gb-sized
> tarball that had to be uploaded/downloaded.  Not the end of the world, but
> cutting it in half seemed like a good idea at the time. :)  Not hard to
> script that, of course.
> 
> I am not necessarily advocating for skinny files to be the default, but as
> it stands, refmac/buster/phenix do not even provide the option of doing it
> (It's entirely possible that I am wrong here on specifics and will get
> corrected by Garib, Gerard and Pavel).
> 
> Cheers,
> 
> Ed.
> 
> Edwin Pozharski, PhD, Assistant Professor
> University of Maryland, Baltimore
> --
> When the Way is forgotten duty and justice appear;
> Then knowledge and wisdom are born along with hypocrisy.
> When harmonious relationships dissolve then respect and devotion arise;
> When a nation falls to chaos then loyalty and patriotism are born.
> -- / Lao Tse /

-- 
Ethan A Merritt
Biomolecular Structure Center,  K-428 Health Sciences Bldg
MS 357742,   University of Washington, Seattle 98195-7742

Re: [ccp4bb] refmac output

[Edwin Pozharski]

Just to clarify, how do you use the extra columns in this scenario?  My
suggestion was to have the output file that includes only the map
coefficient columns, so you still can look at the map.  IIRC, FP/SIGFP
columns from refmac output are actually modified from the input (scaled
with Boverall), so it was not recommended to use refmac output as input of
any kind.

Also, to provide context, my comment resulted from dealing with a large
chunk of data that included ~200 output mtz files - which is Gb-sized
tarball that had to be uploaded/downloaded.  Not the end of the world, but
cutting it in half seemed like a good idea at the time. :)  Not hard to
script that, of course.

I am not necessarily advocating for skinny files to be the default, but as
it stands, refmac/buster/phenix do not even provide the option of doing it
(It's entirely possible that I am wrong here on specifics and will get
corrected by Garib, Gerard and Pavel).

Cheers,

Ed.

Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
--
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
-- / Lao Tse /

Re: [ccp4bb] normalization of B-factor values from different crystal structures

[Ethan A Merritt]

On Wednesday, 02 August, 2017 12:09:35 Asmita wrote:
> Hi,
> 
> This might look as a very fundamental question. I have a dataset of crystal
> structures better than 3.5Ang resolution. For a qualitative analysis, I
> want to compare the residue-wise B-factors in these structures, but due to
> different procedures adopted in refinement and scaling, I understand that
> these values cannot be compared in a raw manner.
> 
> Can someone suggest appropriate normalization methods that could be used
> for scaling these B-factors for a relevant and meaningful comparison? All
> the files have isotropic B-factor values and there are no ANISOU entries in
> any of the files.

I may have an answer, but I first I need a better handle on the question.

Can you take a step back and explain what question or hypothesis you
would like to address by making the comparison?  

Do you want to compare B factors for different residues in the same structure,
or B factors for the same residue in different structures,
or something else entirely?

Ethan

-- 
Ethan A Merritt
Biomolecular Structure Center,  K-428 Health Sciences Bldg
MS 357742,   University of Washington, Seattle 98195-7742

[ccp4bb] normalization of B-factor values from different crystal structures

[Asmita]

Hi,

This might look as a very fundamental question. I have a dataset of crystal
structures better than 3.5Ang resolution. For a qualitative analysis, I
want to compare the residue-wise B-factors in these structures, but due to
different procedures adopted in refinement and scaling, I understand that
these values cannot be compared in a raw manner.

Can someone suggest appropriate normalization methods that could be used
for scaling these B-factors for a relevant and meaningful comparison? All
the files have isotropic B-factor values and there are no ANISOU entries in
any of the files.

Thanks

Asmita

[ccp4bb] Experienced Research Technician in Structural Biology

[Vidya Darbari]

Dear Colleagues,

I would really appreciate if you could help share the information regarding 
this available position for an experienced Research technician in Structural 
Biology.

All necessary information and how to apply is in the link 
https://webapps2.is.qmul.ac.uk/jobs/job.action?jobID=2586

Best  Regards
Vidya

Dr. Vidya Chandran Darbari
Lecturer in Structural Biology
Office 4.34 Fogg building
Department of Chemistry and Biochemistry
School of Biological and Chemical Sciences
Queen Mary University of London
Mile End Road
London E1 4NS
Tel: 0207 882 6360

[ccp4bb] Kendrew Symposium, 16-17 Nov 2017, EMBL Heidelberg, Germany

[Christoph Mueller]

Dear Colleagues,

We would like to draw your attention to the 2-day Kendrew Symposium: 
"Revolutions in Structural Biology: Celebrating the 100th Anniversary of 
Sir John Kendrew" which will take place on 16-17 November 2017 at EMBL 
Heidelberg.


The aim of this event is to celebrate Sir John Kendrew 100th anniversary 
with outstanding leaders in the field of structural biology. The talks 
will explore how revolutionary developments in structural biology 
techniques have maintained structural biology at the forefront of biology.


To view the programme and speakers, please visit:
https://www.embl.de/training/events/2017/JKS17-01/index.html

We very much hope that you can join us at this exciting conference!

Best regards,
Christoph

--

Dr. Christoph W. Müller
Head of Structural and Computational Biology Unit

EMBL
Meyerhofstrasse 1
69117 Heidelberg, Germany

email: cmuel...@embl.de
phone: 0049-6221-387-8320
fax: 0049-6221-387-519
http://www.embl.de
http://www.embl.de/research/units/scb/mueller_christoph/index.html
-