Re: [ccp4bb] high B factor

[Daniel M. Himmel, Ph. D.]

I also should backtrack a little.  I assumed that Anandhi has good
data reduction with good statistics, no data problems (such as twinning),
a good molecular replacement solution, and refinement stuck, but he
didn't give any of those details.  What are the R and Rfree values, the RMS
bonds & angles?  How does the Ramachandran plot look?  By the way,
what were the data statistics like (such as Rsym, completeness,
multiplicity)?
Is the electron density continuous and make sense?

Everything Dale said is well-said.
You could have a perfectly good crystal structure with highly mobile
surface loops and maybe a few disordered surface regions that have
been over-determined and should be removed from the structure,
or a protein with domains that like to rearrange,
all of which could give rise to regions with high B-factors.  More
information
is needed to know if there's a problem.  If there is a problem, then I would
start with Eleanor's suggestions to make sure the data is good and the
MR solution is correct.

Good luck.

-Daniel

On Sun, Nov 11, 2018 at 6:26 PM Dale Tronrud  wrote:

>Okay, I'm an idiot.  The letter did say molecular replacement.  I
> have no excuse for being so blind.
>
>I do stand by my statement: I don't see that an average B factor of
> 82 A^2, by itself, indicates any serious problem with a model at 2.65 A
> resolution.
>
>I have generally found that the average B factor of a model is
> uninformative.  All refinement programs will make certain that the fall
> off in intensity of the calculated F's match those of the observations,
> even if the model is completely wrong.  The average B factor can vary
> widely between different models of the same data if the distribution of
> individual B's differ.
>
>For example, a model built by a person in the "never truncate side
> chains" camp will have a higher average B factor than the model built by
> a "truncater".  That just means that the high end tail of the B factor
> distribution has been cut off.  Atoms with B factors in the 200's only
> affect the intensities of a very small number of reflections anyway and
> do not have any effect on the Wilson B.
>
>If a structure has a wobbly domain or an unusual amount of surface
> area exposed to solvent, the model will have a lot of atoms with large B
> factors.  The average B factor will be high, but none-the-less the
> majority of the atoms will be well ordered and the Wilson B small.  This
> is not an indication of error.  It is exactly what one would expect from
> such a structure.
>
>We don't know what the distribution of individual B factors is in Dr.
> Anandan's model.  We don't know if Dr. Anandan truncates side chains or
> has a liking for building protein main chain through weak density.  We
> don't if there are multiple copies of the protein in the asymmetric
> unit, nor if some are more ordered than others.
>
>All we know is that the resolution of the data is 2.65 A (whatever
> that means), that the B factor average is 82 A^2 (whatever that means)
> and that there is one atom with a B factor of 34.57 A^2 and another with
> 225.13 A^2.  I just don't see that these few facts indicate a problem.
>
>If Dr. Anandan has some reason to distrust this model, like maybe not
> being able to detect the image of the allegedly bound ligand, a great
> number of details of the protein, data collection and the current model
> would have to be shared.  When asking such a question on this BB you
> should never be concerned that your letter is too long.
>
> Dale Tronrud
>
> On 11/11/2018 1:06 PM, Dale Tronrud wrote:
> >
> >Did I miss a follow-up letter with more information?  All I've seen
> > is that Dr. Anandan said that there was a model based on 2.65 A
> > resolution data with an average B factor of 82 A^2.  Does this fact
> > alone call for weeks of work attempting to remove model bias?  Dr.
> > Anandan didn't even say this structure was solved via molecular
> > replacement.  No indication of high R factor or stuck refinement. No
> > details at all.  Just a model with a fairly reasonable, if somewhat low,
> > average B factor.
> >
> > Dale Tronrud
> >
> > On 11/10/2018 6:31 PM, Daniel M. Himmel, Ph. D. wrote:
> >> Anandhi,
> >>
> >> Assuming the data reduction went well, and you're in the right space
> goup,
> >> there could be a lot of model bias in your structure stemming from the
> >> starting model.
> >>
> >> There are a lot of things to try.  I would
> >> set all the B-factors to an artificially low B-factor to help de-mask
> >> errors.  Then,
> >> you can generate a composite omit map and FEM maps to see if any obvious
> >> model errors show up in the electron density.  After correcting these,
> >> you can try running
> >> Autobuild and PhaseAndBuild in Phenix.  Compare all the models you get
> from
> >> each of these, especially in regions where your original model had high
> >> b-factors.
> >> Use Coot to identify areas of poor agreement with electron density and

Re: [ccp4bb] high B factor

[Dale Tronrud]

   Okay, I'm an idiot.  The letter did say molecular replacement.  I
have no excuse for being so blind.

   I do stand by my statement: I don't see that an average B factor of
82 A^2, by itself, indicates any serious problem with a model at 2.65 A
resolution.

   I have generally found that the average B factor of a model is
uninformative.  All refinement programs will make certain that the fall
off in intensity of the calculated F's match those of the observations,
even if the model is completely wrong.  The average B factor can vary
widely between different models of the same data if the distribution of
individual B's differ.

   For example, a model built by a person in the "never truncate side
chains" camp will have a higher average B factor than the model built by
a "truncater".  That just means that the high end tail of the B factor
distribution has been cut off.  Atoms with B factors in the 200's only
affect the intensities of a very small number of reflections anyway and
do not have any effect on the Wilson B.

   If a structure has a wobbly domain or an unusual amount of surface
area exposed to solvent, the model will have a lot of atoms with large B
factors.  The average B factor will be high, but none-the-less the
majority of the atoms will be well ordered and the Wilson B small.  This
is not an indication of error.  It is exactly what one would expect from
such a structure.

   We don't know what the distribution of individual B factors is in Dr.
Anandan's model.  We don't know if Dr. Anandan truncates side chains or
has a liking for building protein main chain through weak density.  We
don't if there are multiple copies of the protein in the asymmetric
unit, nor if some are more ordered than others.

   All we know is that the resolution of the data is 2.65 A (whatever
that means), that the B factor average is 82 A^2 (whatever that means)
and that there is one atom with a B factor of 34.57 A^2 and another with
225.13 A^2.  I just don't see that these few facts indicate a problem.

   If Dr. Anandan has some reason to distrust this model, like maybe not
being able to detect the image of the allegedly bound ligand, a great
number of details of the protein, data collection and the current model
would have to be shared.  When asking such a question on this BB you
should never be concerned that your letter is too long.

Dale Tronrud

On 11/11/2018 1:06 PM, Dale Tronrud wrote:
> 
>Did I miss a follow-up letter with more information?  All I've seen
> is that Dr. Anandan said that there was a model based on 2.65 A
> resolution data with an average B factor of 82 A^2.  Does this fact
> alone call for weeks of work attempting to remove model bias?  Dr.
> Anandan didn't even say this structure was solved via molecular
> replacement.  No indication of high R factor or stuck refinement. No
> details at all.  Just a model with a fairly reasonable, if somewhat low,
> average B factor.
> 
> Dale Tronrud
> 
> On 11/10/2018 6:31 PM, Daniel M. Himmel, Ph. D. wrote:
>> Anandhi,
>>
>> Assuming the data reduction went well, and you're in the right space goup,
>> there could be a lot of model bias in your structure stemming from the
>> starting model.  
>>
>> There are a lot of things to try.  I would
>> set all the B-factors to an artificially low B-factor to help de-mask
>> errors.  Then,
>> you can generate a composite omit map and FEM maps to see if any obvious
>> model errors show up in the electron density.  After correcting these,
>> you can try running
>> Autobuild and PhaseAndBuild in Phenix.  Compare all the models you get from
>> each of these, especially in regions where your original model had high
>> b-factors.
>> Use Coot to identify areas of poor agreement with electron density and areas
>> of poor geometry.  Once you spent a while correcting the model, put it
>> through
>> one or more cycles of simulated annealing at different temperatures in
>> parallel.
>> Select several sets of coordinates that give the best Rfree convergence,
>> and then
>> subject those models to individual B-factor refinement.  After that,
>> check again in Coot
>> for areas of high B-factors and areas of poor geometry (try especially
>> to improve
>> your Ramachandran Plot).  Use MolProbity to help in identifying errors
>> and clashes.
>> If a few rounds of simulated annealing and model building don't improve
>> things, try some 
>> refinement in CCP4 Refmac.  PDB_REDO, which uses Refmac, can also help 
>> give you alternative models.  While doing all these, don't be afraid to
>> "cut-and-paste"
>> regions from one model into another model and then correct the geometry
>> in Coot.
>> If B-factors don't come down no matter what you do, you could be in the
>> wrong space 
>> group or have problems with the original data that need to be addressed.
>>
>> I hope this helps.
>>
>> Daniel
>>
>> ___
>>
>> Daniel M. Himmel, Ph. D.
>>
>> URL:  http://www.DanielMHimmel.com/index_Xtal.html
>> 

Re: [ccp4bb] high B factor

[Dale Tronrud]

   Did I miss a follow-up letter with more information?  All I've seen
is that Dr. Anandan said that there was a model based on 2.65 A
resolution data with an average B factor of 82 A^2.  Does this fact
alone call for weeks of work attempting to remove model bias?  Dr.
Anandan didn't even say this structure was solved via molecular
replacement.  No indication of high R factor or stuck refinement. No
details at all.  Just a model with a fairly reasonable, if somewhat low,
average B factor.

Dale Tronrud

On 11/10/2018 6:31 PM, Daniel M. Himmel, Ph. D. wrote:
> Anandhi,
> 
> Assuming the data reduction went well, and you're in the right space goup,
> there could be a lot of model bias in your structure stemming from the
> starting model.  
> 
> There are a lot of things to try.  I would
> set all the B-factors to an artificially low B-factor to help de-mask
> errors.  Then,
> you can generate a composite omit map and FEM maps to see if any obvious
> model errors show up in the electron density.  After correcting these,
> you can try running
> Autobuild and PhaseAndBuild in Phenix.  Compare all the models you get from
> each of these, especially in regions where your original model had high
> b-factors.
> Use Coot to identify areas of poor agreement with electron density and areas
> of poor geometry.  Once you spent a while correcting the model, put it
> through
> one or more cycles of simulated annealing at different temperatures in
> parallel.
> Select several sets of coordinates that give the best Rfree convergence,
> and then
> subject those models to individual B-factor refinement.  After that,
> check again in Coot
> for areas of high B-factors and areas of poor geometry (try especially
> to improve
> your Ramachandran Plot).  Use MolProbity to help in identifying errors
> and clashes.
> If a few rounds of simulated annealing and model building don't improve
> things, try some 
> refinement in CCP4 Refmac.  PDB_REDO, which uses Refmac, can also help 
> give you alternative models.  While doing all these, don't be afraid to
> "cut-and-paste"
> regions from one model into another model and then correct the geometry
> in Coot.
> If B-factors don't come down no matter what you do, you could be in the
> wrong space 
> group or have problems with the original data that need to be addressed.
> 
> I hope this helps.
> 
> Daniel
> 
> ___
> 
> Daniel M. Himmel, Ph. D.
> 
> URL:  http://www.DanielMHimmel.com/index_Xtal.html
> 
> 
> 
> 
> 
> On Thu, Nov 8, 2018 at 7:41 PM Anandhi Anandan
> mailto:anandhi.anan...@uwa.edu.au>> wrote:
> 
> Hello everyone,
> 
> 
> I am trying to solve the structure of a protein with a bound ligand
> at 2.65 A resolution. XDS was used for data reduction, phaser-MR for
> molecular replacement  and Phenix for refinement. The refinement was
> done with the default settings ( individual B factors, occupancy and
> TLS parameters). The resultant atomic B factors are quite high.  The
> overall B factor is 82 with a minimum value of 34.57 and maximum of
> 225.13. I would like to know if any of the data reduction parameters
> can affect the B factors and how best to deal with this issue.
> 
> 
> Anandhi
> 
> 
> 
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> 
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> 



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Re: [ccp4bb] high B factor

[Eleanor Dodson]

Hard to comment on PHEIX refne but check these things: such results must
either mean your molecular replacement solution is wrong or there is
something wrong with the data.
Refinement packages will always try to match the Mean B factor to the
Wilson plot B so I think the problem must be before refinement..

Look at the Wilson plot. Is it as a protein should show? ( There is CCP4
documemtation with a picture I think..
What is the etimated B from that?

(You can run your data through the aimless pipeline and it will report all
these things. and I am sure Xtriage does too.)

Then look at a plot of  v   v resolution. (again provided by
REMAC and prob. PHENIX)
Does it look sensible? Are the two curves overlapping?

Good luck Eleanor









On Sun, 11 Nov 2018 at 04:11, Daniel M. Himmel, Ph. D. <
danielmhim...@gmail.com> wrote:

> By the way, I would recommend running simulated annealing with each
> composite omit map you generate to help in overcoming model bias.
>
> -Daniel
>
>
> On Sat, Nov 10, 2018 at 9:31 PM Daniel M. Himmel, Ph. D. <
> danielmhim...@gmail.com> wrote:
>
>> Anandhi,
>>
>> Assuming the data reduction went well, and you're in the right space goup,
>> there could be a lot of model bias in your structure stemming from the
>> starting model.
>>
>> There are a lot of things to try.  I would
>> set all the B-factors to an artificially low B-factor to help de-mask
>> errors.  Then,
>> you can generate a composite omit map and FEM maps to see if any obvious
>> model errors show up in the electron density.  After correcting these,
>> you can try running
>> Autobuild and PhaseAndBuild in Phenix.  Compare all the models you get
>> from
>> each of these, especially in regions where your original model had high
>> b-factors.
>> Use Coot to identify areas of poor agreement with electron density and
>> areas
>> of poor geometry.  Once you spent a while correcting the model, put it
>> through
>> one or more cycles of simulated annealing at different temperatures in
>> parallel.
>> Select several sets of coordinates that give the best Rfree convergence,
>> and then
>> subject those models to individual B-factor refinement.  After that,
>> check again in Coot
>> for areas of high B-factors and areas of poor geometry (try especially to
>> improve
>> your Ramachandran Plot).  Use MolProbity to help in identifying errors
>> and clashes.
>> If a few rounds of simulated annealing and model building don't improve
>> things, try some
>> refinement in CCP4 Refmac.  PDB_REDO, which uses Refmac, can also help
>> give you alternative models.  While doing all these, don't be afraid to
>> "cut-and-paste"
>> regions from one model into another model and then correct the geometry
>> in Coot.
>> If B-factors don't come down no matter what you do, you could be in the
>> wrong space
>> group or have problems with the original data that need to be addressed.
>>
>> I hope this helps.
>>
>> Daniel
>>
>> ___
>>
>> Daniel M. Himmel, Ph. D.
>>
>> URL:  http://www.DanielMHimmel.com/index_Xtal.html
>> 
>>
>>
>>
>>
>> On Thu, Nov 8, 2018 at 7:41 PM Anandhi Anandan <
>> anandhi.anan...@uwa.edu.au> wrote:
>>
>>> Hello everyone,
>>>
>>>
>>> I am trying to solve the structure of a protein with a bound ligand at
>>> 2.65 A resolution. XDS was used for data reduction, phaser-MR for molecular
>>> replacement  and Phenix for refinement. The refinement was done with the
>>> default settings ( individual B factors, occupancy and TLS parameters). The
>>> resultant atomic B factors are quite high.  The overall B factor is 82 with
>>> a minimum value of 34.57 and maximum of 225.13. I would like to know if any
>>> of the data reduction parameters can affect the B factors and how best to
>>> deal with this issue.
>>>
>>>
>>> Anandhi
>>>
>>>
>>>
>>>
>>> --
>>>
>>> To unsubscribe from the CCP4BB list, click the following link:
>>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>>
>>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] high B factor

[Daniel M. Himmel, Ph. D.]

By the way, I would recommend running simulated annealing with each
composite omit map you generate to help in overcoming model bias.

-Daniel


On Sat, Nov 10, 2018 at 9:31 PM Daniel M. Himmel, Ph. D. <
danielmhim...@gmail.com> wrote:

> Anandhi,
>
> Assuming the data reduction went well, and you're in the right space goup,
> there could be a lot of model bias in your structure stemming from the
> starting model.
>
> There are a lot of things to try.  I would
> set all the B-factors to an artificially low B-factor to help de-mask
> errors.  Then,
> you can generate a composite omit map and FEM maps to see if any obvious
> model errors show up in the electron density.  After correcting these, you
> can try running
> Autobuild and PhaseAndBuild in Phenix.  Compare all the models you get from
> each of these, especially in regions where your original model had high
> b-factors.
> Use Coot to identify areas of poor agreement with electron density and
> areas
> of poor geometry.  Once you spent a while correcting the model, put it
> through
> one or more cycles of simulated annealing at different temperatures in
> parallel.
> Select several sets of coordinates that give the best Rfree convergence,
> and then
> subject those models to individual B-factor refinement.  After that, check
> again in Coot
> for areas of high B-factors and areas of poor geometry (try especially to
> improve
> your Ramachandran Plot).  Use MolProbity to help in identifying errors and
> clashes.
> If a few rounds of simulated annealing and model building don't improve
> things, try some
> refinement in CCP4 Refmac.  PDB_REDO, which uses Refmac, can also help
> give you alternative models.  While doing all these, don't be afraid to
> "cut-and-paste"
> regions from one model into another model and then correct the geometry in
> Coot.
> If B-factors don't come down no matter what you do, you could be in the
> wrong space
> group or have problems with the original data that need to be addressed.
>
> I hope this helps.
>
> Daniel
>
> ___
>
> Daniel M. Himmel, Ph. D.
>
> URL:  http://www.DanielMHimmel.com/index_Xtal.html
> 
>
>
>
>
> On Thu, Nov 8, 2018 at 7:41 PM Anandhi Anandan 
> wrote:
>
>> Hello everyone,
>>
>>
>> I am trying to solve the structure of a protein with a bound ligand at
>> 2.65 A resolution. XDS was used for data reduction, phaser-MR for molecular
>> replacement  and Phenix for refinement. The refinement was done with the
>> default settings ( individual B factors, occupancy and TLS parameters). The
>> resultant atomic B factors are quite high.  The overall B factor is 82 with
>> a minimum value of 34.57 and maximum of 225.13. I would like to know if any
>> of the data reduction parameters can affect the B factors and how best to
>> deal with this issue.
>>
>>
>> Anandhi
>>
>>
>>
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>



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Re: [ccp4bb] high B factor

[Daniel M. Himmel, Ph. D.]

Anandhi,

Assuming the data reduction went well, and you're in the right space goup,
there could be a lot of model bias in your structure stemming from the
starting model.

There are a lot of things to try.  I would
set all the B-factors to an artificially low B-factor to help de-mask
errors.  Then,
you can generate a composite omit map and FEM maps to see if any obvious
model errors show up in the electron density.  After correcting these, you
can try running
Autobuild and PhaseAndBuild in Phenix.  Compare all the models you get from
each of these, especially in regions where your original model had high
b-factors.
Use Coot to identify areas of poor agreement with electron density and areas
of poor geometry.  Once you spent a while correcting the model, put it
through
one or more cycles of simulated annealing at different temperatures in
parallel.
Select several sets of coordinates that give the best Rfree convergence,
and then
subject those models to individual B-factor refinement.  After that, check
again in Coot
for areas of high B-factors and areas of poor geometry (try especially to
improve
your Ramachandran Plot).  Use MolProbity to help in identifying errors and
clashes.
If a few rounds of simulated annealing and model building don't improve
things, try some
refinement in CCP4 Refmac.  PDB_REDO, which uses Refmac, can also help
give you alternative models.  While doing all these, don't be afraid to
"cut-and-paste"
regions from one model into another model and then correct the geometry in
Coot.
If B-factors don't come down no matter what you do, you could be in the
wrong space
group or have problems with the original data that need to be addressed.

I hope this helps.

Daniel

___

Daniel M. Himmel, Ph. D.

URL:  http://www.DanielMHimmel.com/index_Xtal.html





On Thu, Nov 8, 2018 at 7:41 PM Anandhi Anandan 
wrote:

> Hello everyone,
>
>
> I am trying to solve the structure of a protein with a bound ligand at
> 2.65 A resolution. XDS was used for data reduction, phaser-MR for molecular
> replacement  and Phenix for refinement. The refinement was done with the
> default settings ( individual B factors, occupancy and TLS parameters). The
> resultant atomic B factors are quite high.  The overall B factor is 82 with
> a minimum value of 34.57 and maximum of 225.13. I would like to know if any
> of the data reduction parameters can affect the B factors and how best to
> deal with this issue.
>
>
> Anandhi
>
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] high B factor

[Oliviero Carugo]

Dear Anandhi,

you might be interested in PMID: 29752562.

BW,

Oliviero


On 09.11.2018 01:30, Anandhi Anandan wrote:

Hello everyone,

I am trying to solve the structure of a protein with a bound ligand at
2.65 A resolution. XDS was used for data reduction, phaser-MR for
molecular replacement  and Phenix for refinement. The refinement was
done with the default settings ( individual B factors, occupancy and
TLS parameters). The resultant atomic B factors are quite high.  The
overall B factor is 82 with a minimum value of 34.57 and maximum of
225.13. I would like to know if any of the data reduction parameters
can affect the B factors and how best to deal with this issue.

Anandhi

-

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[ccp4bb] high B factor

[Dale Tronrud]

Hi,

   The "B factors" reflect the rapidity of the decrease in intensity of
scattering with increasing resolution.  The scattering of a crystal
becomes unmeasurable at a lower resolution if its intensities drop
faster.  This results in a connection between the resolution of a data
set and the average B factor of the atoms in the resulting model.  With
lower resolution come higher B factors.

   It is inevitable.  It cannot be avoided.

   An average B factor of 82 A^2 for a 2.65 A data set actually sounds
low to me.  Are you taking the TLS contributions into account when
calculating this average?  In any case, you certainly shouldn't be
worrying about your "high" B factors.

Dale Tronrud

On 11/8/2018 4:30 PM, Anandhi Anandan wrote:
> Hello everyone,
> 
> 
> I am trying to solve the structure of a protein with a bound ligand at
> 2.65 A resolution. XDS was used for data reduction, phaser-MR for
> molecular replacement  and Phenix for refinement. The refinement was
> done with the default settings ( individual B factors, occupancy and TLS
> parameters). The resultant atomic B factors are quite high.  The overall
> B factor is 82 with a minimum value of 34.57 and maximum of 225.13. I
> would like to know if any of the data reduction parameters can affect
> the B factors and how best to deal with this issue.
> 
> 
> Anandhi
> 
> 
> 
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> 



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[ccp4bb] high B factor

[Anandhi Anandan]

Hello everyone,


I am trying to solve the structure of a protein with a bound ligand at 2.65 A 
resolution. XDS was used for data reduction, phaser-MR for molecular 
replacement  and Phenix for refinement. The refinement was done with the 
default settings ( individual B factors, occupancy and TLS parameters). The 
resultant atomic B factors are quite high.  The overall B factor is 82 with a 
minimum value of 34.57 and maximum of 225.13. I would like to know if any of 
the data reduction parameters can affect the B factors and how best to deal 
with this issue.


Anandhi





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[ccp4bb] Antw: Re: [ccp4bb] Antw: Re: [ccp4bb] High B factor

[Matthias Barone]

In my feeling, lowering the occ explains much more such unordered loops (or 
solvent exposed sidechains..). 
B-factors are a measure of uncertainty of the atom position xyz: Letting 
B-factors fly is like broadening a distribution to an extent where the 
expectation value looses its purpose. That in mind, I have no problem reducing 
the occ of a solvent-exposed lysine to 30% or even 10% if the atoms are not 
even visible in a FEM map...
best, matthias




>>> Tim Gruene <tim.gru...@psi.ch> 17.10.16 10.06 Uhr >>>
Dear Carlos and Phoebe,

the B-value describes the movement of an atom at first order approximation, 
i.e. as a harmonic motion. It's very unlikely that an atom that is not visible 
in density, discribes a harmonic motion. 
It never occurred to me, but modelling disorder by strong B-factor restraints 
and refining the occupancy seems quite a good idea and would be a much better 
model than an explosion of B-factors.
Coot marks atoms with reduced occupancy, hence there is no extra work required 
to visualise reduced occupancy. I am aware that some people use graphics 
rendering machinery to work with PDB files instead of a dedicated program like 
Coot, but you can never suit everyone.

Best,
Tim

On Monday, October 17, 2016 09:35:02 AM Carlos CONTRERAS-MARTEL wrote:
> Even that occupancy refinement seems to be very interesting for
> crystallographers, I complete agree with Phoebe.
> 
> On 10/14/16 17:38, Phoebe A. Rice wrote:
> > Interesting way to look at it.  But those loop residues are really in
> > the crystal with an occupancy of 1, so wouldn't letting the B factor
> > fly give a clearer description of what's in the crystal?  Especially
> > as many people know to color the structure by B factors to get a feel
> > for which bits are wiggly, but they'll never think to color it by
> > occupancy.
> 
> Let them fly ... at least for protein atoms ...
> 
> Carlos
> 
> > 
> > *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of
> > Matthias Barone [bar...@fmp-berlin.de]
> > *Sent:* Friday, October 14, 2016 8:00 AM
> > *To:* CCP4BB@JISCMAIL.AC.UK
> > *Subject:* [ccp4bb] Antw: Re: [ccp4bb] High B factor
> > 
> > Picking up the mail of Pavel, Phenix refines occupancies..
> > If you expect the loops to be disordered, did you try to lower the
> > occupancy of these residues, following Ethan Merritt statement that
> > "general uncertainty [...] is represented better by occupancy <1
> > rather than an arbitrary large B factor" (To B or not to B,
> > doi:10.1107/S0907444911028320).
> > If this attempt does not bring the B-factors down, it will surely make
> > the model more accurate, as the atom coordinates of the loops may not
> > be correct at all, no?
> > 
> > matthias
> > 
> > >>> Pavel Afonine <pafon...@gmail.com> 14.10.16 9.36 Uhr >>>
> > >>> 
> > If you are still worry about your Bfactor, you could try TLS,
> > 
> > Or NCS, but SA with MLHL might be better.
> > 
> > (A joke).
> 
> --
>   Carlos CONTRERAS MARTEL, Ph.D.
>   (CR1 CNRS)
> 
>   carlos.contreras-mar...@ibs.fr
> 
>   "Bacterial Pathogenesis Group"
>  Institut de Biologie Structurale
>   UMR5075 CEA-CNRS-UGA
> 
>IBS
>Campus EPN
>71, avenue des Martyrs
>CS 10090
>38044 Grenoble CEDEX 9
>FRANCE
> 
> 
>   tel : (+33) (0)4 57 42 86 41
> 
> http://www.ibs.fr/groupes/groupe-pathogenie-bacterienne/?lang=fr
> http://www.ibs.fr/groups/bacterial-pathogenesis-group/?lang=en
-- 
--
Paul Scherrer Institut
Dr. Tim Gruene
- persoenlich -
Principal Investigator
Biology and Chemistry
OFLC/102
CH-5232 Villigen PSI

Phone: +41 (0)56 310 5297

GPG Key ID = A46BEE1A

Re: [ccp4bb] Antw: Re: [ccp4bb] High B factor

[Tim Gruene]

Dear Carlos and Phoebe,

the B-value describes the movement of an atom at first order approximation, 
i.e. as a harmonic motion. It's very unlikely that an atom that is not visible 
in density, discribes a harmonic motion. 
It never occurred to me, but modelling disorder by strong B-factor restraints 
and refining the occupancy seems quite a good idea and would be a much better 
model than an explosion of B-factors.
Coot marks atoms with reduced occupancy, hence there is no extra work required 
to visualise reduced occupancy. I am aware that some people use graphics 
rendering machinery to work with PDB files instead of a dedicated program like 
Coot, but you can never suit everyone.

Best,
Tim

On Monday, October 17, 2016 09:35:02 AM Carlos CONTRERAS-MARTEL wrote:
> Even that occupancy refinement seems to be very interesting for
> crystallographers, I complete agree with Phoebe.
> 
> On 10/14/16 17:38, Phoebe A. Rice wrote:
> > Interesting way to look at it.  But those loop residues are really in
> > the crystal with an occupancy of 1, so wouldn't letting the B factor
> > fly give a clearer description of what's in the crystal?  Especially
> > as many people know to color the structure by B factors to get a feel
> > for which bits are wiggly, but they'll never think to color it by
> > occupancy.
> 
> Let them fly ... at least for protein atoms ...
> 
> Carlos
> 
> > 
> > *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of
> > Matthias Barone [bar...@fmp-berlin.de]
> > *Sent:* Friday, October 14, 2016 8:00 AM
> > *To:* CCP4BB@JISCMAIL.AC.UK
> > *Subject:* [ccp4bb] Antw: Re: [ccp4bb] High B factor
> > 
> > Picking up the mail of Pavel, Phenix refines occupancies..
> > If you expect the loops to be disordered, did you try to lower the
> > occupancy of these residues, following Ethan Merritt statement that
> > "general uncertainty [...] is represented better by occupancy <1
> > rather than an arbitrary large B factor" (To B or not to B,
> > doi:10.1107/S0907444911028320).
> > If this attempt does not bring the B-factors down, it will surely make
> > the model more accurate, as the atom coordinates of the loops may not
> > be correct at all, no?
> > 
> > matthias
> > 
> > >>> Pavel Afonine <pafon...@gmail.com> 14.10.16 9.36 Uhr >>>
> > >>> 
> > If you are still worry about your Bfactor, you could try TLS,
> > 
> > Or NCS, but SA with MLHL might be better.
> > 
> > (A joke).
> 
> --
>   Carlos CONTRERAS MARTEL, Ph.D.
>   (CR1 CNRS)
> 
>   carlos.contreras-mar...@ibs.fr
> 
>   "Bacterial Pathogenesis Group"
>  Institut de Biologie Structurale
>   UMR5075 CEA-CNRS-UGA
> 
>IBS
>Campus EPN
>71, avenue des Martyrs
>CS 10090
>38044 Grenoble CEDEX 9
>FRANCE
> 
> 
>   tel : (+33) (0)4 57 42 86 41
> 
> http://www.ibs.fr/groupes/groupe-pathogenie-bacterienne/?lang=fr
> http://www.ibs.fr/groups/bacterial-pathogenesis-group/?lang=en
-- 
--
Paul Scherrer Institut
Dr. Tim Gruene
- persoenlich -
Principal Investigator
Biology and Chemistry
OFLC/102
CH-5232 Villigen PSI

Phone: +41 (0)56 310 5297

GPG Key ID = A46BEE1A



signature.asc
Description: This is a digitally signed message part.

Re: [ccp4bb] Antw: Re: [ccp4bb] High B factor

[Carlos CONTRERAS-MARTEL]

Even that occupancy refinement seems to be very interesting for 
crystallographers, I complete agree with Phoebe.



On 10/14/16 17:38, Phoebe A. Rice wrote:
Interesting way to look at it.  But those loop residues are really in 
the crystal with an occupancy of 1, so wouldn't letting the B factor 
fly give a clearer description of what's in the crystal?  Especially 
as many people know to color the structure by B factors to get a feel 
for which bits are wiggly, but they'll never think to color it by 
occupancy.

Let them fly ... at least for protein atoms ...

Carlos




*From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of 
Matthias Barone [bar...@fmp-berlin.de]

*Sent:* Friday, October 14, 2016 8:00 AM
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* [ccp4bb] Antw: Re: [ccp4bb] High B factor

Picking up the mail of Pavel, Phenix refines occupancies..
If you expect the loops to be disordered, did you try to lower the 
occupancy of these residues, following Ethan Merritt statement that 
"general uncertainty [...] is represented better by occupancy <1 
rather than an arbitrary large B factor" (To B or not to B, 
doi:10.1107/S0907444911028320).
If this attempt does not bring the B-factors down, it will surely make 
the model more accurate, as the atom coordinates of the loops may not 
be correct at all, no?


matthias


>>> Pavel Afonine <pafon...@gmail.com> 14.10.16 9.36 Uhr >>>

If you are still worry about your Bfactor, you could try TLS,


Or NCS, but SA with MLHL might be better.

(A joke).




--
 Carlos CONTRERAS MARTEL, Ph.D.
 (CR1 CNRS)

 carlos.contreras-mar...@ibs.fr

 "Bacterial Pathogenesis Group"
Institut de Biologie Structurale
 UMR5075 CEA-CNRS-UGA

  IBS
  Campus EPN
  71, avenue des Martyrs
  CS 10090
  38044 Grenoble CEDEX 9
  FRANCE


 tel : (+33) (0)4 57 42 86 41

http://www.ibs.fr/groupes/groupe-pathogenie-bacterienne/?lang=fr
http://www.ibs.fr/groups/bacterial-pathogenesis-group/?lang=en

Re: [ccp4bb] Antw: Re: [ccp4bb] High B factor

[sunanda williams]

Quoting Dale from an older post
"It's also true that the Wilson B calculation assumes that all the B
factors in the crystal are the same - which is also far from the true in
most macromolecular crystals. A person who holds to the practice of
aggressively building water molecules and loops will create a model with a
higher average B than one who uses the same data but is more restrained.
The Wilson B will, of course, be unchanged.
If you have a protein that is equally ordered throughout and you do not
build in weak water molecules and the crystal diffracts high enough to
allow a reasonably accurate calculation of the Wilson B, your average B and
and the Wilson B should be close to each other. If your protein has mobile
loops, which most lower resolution crystals do (and most higher resolution
crystals for that matter) then your average B will be larger than your
Wilson B."

This kinda agrees with my scenario I think... Wilson B is 83 A*2 and Av B
98 A*2, the discrepancy contributed by mobile loops? (3.0 A resolution).
Refinement with group B factors (two per residue) brought the av B down to
80 A*2. TLS, PDB_REDO etc did not help much.
I got the model by Mol Rep, have not tried PROSMART...
And I agree with Phoebe...The aa's are fully there! Lowering the occupancy
may work with Hetatms, but doesn't make sense to do it with residues!




On Fri, Oct 14, 2016 at 9:08 PM, Phoebe A. Rice <pr...@uchicago.edu> wrote:

> Interesting way to look at it.  But those loop residues are really in the
> crystal with an occupancy of 1, so wouldn't letting the B factor fly give a
> clearer description of what's in the crystal?  Especially as many people
> know to color the structure by B factors to get a feel for which bits are
> wiggly, but they'll never think to color it by occupancy.
>
> --
> *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Matthias
> Barone [bar...@fmp-berlin.de]
> *Sent:* Friday, October 14, 2016 8:00 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] Antw: Re: [ccp4bb] High B factor
>
> Picking up the mail of Pavel, Phenix refines occupancies..
> If you expect the loops to be disordered, did you try to lower the
> occupancy of these residues, following Ethan Merritt statement that
> "general uncertainty [...] is represented better by occupancy <1 rather
> than an arbitrary large B factor" (To B or not to B,
> doi:10.1107/S0907444911028320).
> If this attempt does not bring the B-factors down, it will surely make the
> model more accurate, as the atom coordinates of the loops may not be
> correct at all, no?
>
> matthias
>
>
> >>> Pavel Afonine <pafon...@gmail.com> 14.10.16 9.36 Uhr >>>
>
> If you are still worry about your Bfactor, you could try TLS,
>>
>
> Or NCS, but SA with MLHL might be better.
>
> (A joke).
>
>

Re: [ccp4bb] Antw: Re: [ccp4bb] High B factor

[Phoebe A. Rice]

Interesting way to look at it.  But those loop residues are really in the 
crystal with an occupancy of 1, so wouldn't letting the B factor fly give a 
clearer description of what's in the crystal?  Especially as many people know 
to color the structure by B factors to get a feel for which bits are wiggly, 
but they'll never think to color it by occupancy.


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Matthias Barone 
[bar...@fmp-berlin.de]
Sent: Friday, October 14, 2016 8:00 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Antw: Re: [ccp4bb] High B factor

Picking up the mail of Pavel, Phenix refines occupancies..
If you expect the loops to be disordered, did you try to lower the occupancy of 
these residues, following Ethan Merritt statement that "general uncertainty 
[...] is represented better by occupancy <1 rather than an arbitrary large B 
factor" (To B or not to B, doi:10.1107/S0907444911028320).
If this attempt does not bring the B-factors down, it will surely make the 
model more accurate, as the atom coordinates of the loops may not be correct at 
all, no?

matthias


>>> Pavel Afonine <pafon...@gmail.com> 14.10.16 9.36 Uhr >>>


If you are still worry about your Bfactor, you could try TLS,

Or NCS, but SA with MLHL might be better.

(A joke).

Re: [ccp4bb] High B factor

[Dale Tronrud]

   I agree that it would certainly be bad if your model was inconsistent
with the Wilson B.  When I first ran into people who were worried about
this I recommended that they calculate the Wilson B of their model, not
look at the average B as a proxy.  You can calculate structure factors
from your model and feed those into a Wilson B calculator and get the
proper value.

   When you do this you find pretty quickly that a refinement program
has to be screwed up pretty badly for it to create a model with even a
small difference between the observed and calculated Wilson B.  If your
model is off only a little the R value shoots up!  Other than when
debugging refinement code, I have never seen a refined model where there
was any significant difference between the calculated and observed
Wilson B.  This problem simply doesn't come up.

   Try it for yourself and see.

   When you see a difference between the Wilson B and the average of the
B's in your model, all you are seeing is that you have a variety of B
factors and that is not a surprise.  I would be worried if all my B
factors were equal!

   High B factors are not a problem to be fixed!  Some atoms do move
more than others.  Why would you expect otherwise?

Dale Tronrud

On 10/14/2016 12:28 AM, Carlos CONTRERAS MARTEL wrote:
> Sunanda,
> 
> As "common people", ... "agreement" don't means for me "equals" ...
> 
> So I hope I'm not so "incorrect" if I keep one mind the Wilson B as the
> model
> 
> refinement goes and the average B factor changes with different strategies.
> 
> If you are still worry about your Bfactor, you could try TLS, using the
> right approach could
> 
> improve your results.
> 
> All the best
> 
> Carlos
> 
> 
> 
> On 10/13/16 18:42, Dale Tronrud wrote:
>>It seems to be common for people to make the incorrect assumption
>> that the average of the atomic B factors in the PDB file should equal
>> the Wilson B.  The Wilson B is actually a weighted average of the
>> individual B factors where the weighting is rather mysterious, but the
>> small B factors have much higher weight than the larger ones. While the
>> Wilson B is simply a property of your data set the average of the atomic
>> B factors will also depend on choices you have made in building your
>> model.
>>
>>In your case the core of your protein does have B factors that match
>> your Wilson B.  The fact that you have loops that have larger B's
>> doesn't really mean there is a problem, because those atoms don't
>> contribute much to the higher resolution reflections and don't come into
>> the calculation of the Wilson B.
>>
>>The average of your B factors will increase if you choose to build
>> model for more disordered regions, while the Wilson B will, of course,
>> remain the same.  The average B factor will be larger, for example, if
>> you are more aggressive in building water molecules.  This does not
>> indicate a problem but is an unavoidable consequence of your choice to
>> take more risks.
>>
>> Dale
>>
>>
>>
>> On 10/13/2016 12:16 AM, sunanda williams wrote:
>>> Sorry for not making the problem clear!
>>> The overall B factor for the 3.0 A structure is around 98.00 A*2. Most
>>> of the deposited structures in the PDB site around this resolution
>>> has an av B around 70 A*2
>>> All other statistics looks fine. I got a warning message while trying to
>>> upload the structure in PDB that 98 was higher than the norm!
>>> The reason why the structure has such a high B factor could be due to
>>> disordered loops... All the same I was wondering how acceptable this
>>> structure would be to picky reviewers..
>>> And 'better the B factors' means bring them down...sorry couldn't phrase
>>> it 'better'! I am going to try doing refinement with group B factors!
>>> Would that help?
>>> Thanks Robbie, I think this I need to make the best of the model I have.
>>> Prof. Sekar will try PDB_redo..Thanks!
>>>
>>>
>>>
>>> On Thu, Oct 13, 2016 at 11:41 AM, Pavel Afonine >> > wrote:
>>>
>>> I fully agree with Dale in not understanding what the problem is.
>>> Perhaps I have a better chance if you clearly explain what exactly
>>> you mean by "is there any way to better the B factors".
>>> Pavel
>>>
>>>
>>> On Thu, Oct 13, 2016 at 12:57 PM, Dale Tronrud
>>> > wrote:
>>>
>>>I'm sorry but I don't understand what your problem is.  Do
>>> you think
>>> the B factors are too small for a 3A data set?  A range of 70 to
>>> 75 is a
>>> little smaller than usual but probably not out of bounds.
>>>
>>> Dale Tronrud
>>>
>>> On 10/12/2016 7:59 PM, sunanda williams wrote:
>>> > Hi all,
>>> > I have a structure at 3.0 A and R/Rfree of 24/28. The mean B
>>> value is
>>> > around 98. The B value is especially high at the N terminus
>>> and two loop
>>> > 

[ccp4bb] Antw: Re: [ccp4bb] High B factor

[Matthias Barone]

Picking up the mail of Pavel, Phenix refines occupancies..
If you expect the loops to be disordered, did you try to lower the 
occupancy of these residues, following Ethan Merritt statement that 
"general uncertainty [...] is represented better by occupancy <1 
rather than an arbitrary large B factor" 
(To B or not to B, doi:10.1107/S0907444911028320). 
If this attempt does not bring the 
B-factors down, it will surely make the model more accurate, as the atom
 coordinates of the loops may not be correct at all, no?

matthias



>>> Pavel Afonine  14.10.16 9.36 Uhr >>>

  If you are still worry about your Bfactor, you could try 
TLS,


Or NCS, but SA with MLHL might be better. 

(A joke). 




 

Re: [ccp4bb] High B factor

[Pavel Afonine]

> If you are still worry about your Bfactor, you could try TLS,
>

Or NCS, but SA with MLHL might be better.

(A joke).

Re: [ccp4bb] High B factor

[Carlos CONTRERAS MARTEL]

Sunanda,

As "common people", ... "agreement" don't means for me "equals" ...

So I hope I'm not so "incorrect" if I keep one mind the Wilson B as the 
model


refinement goes and the average B factor changes with different strategies.

If you are still worry about your Bfactor, you could try TLS, using the 
right approach could


improve your results.

All the best

Carlos



On 10/13/16 18:42, Dale Tronrud wrote:

It seems to be common for people to make the incorrect assumption
that the average of the atomic B factors in the PDB file should equal
the Wilson B.  The Wilson B is actually a weighted average of the
individual B factors where the weighting is rather mysterious, but the
small B factors have much higher weight than the larger ones. While the
Wilson B is simply a property of your data set the average of the atomic
B factors will also depend on choices you have made in building your
model.

In your case the core of your protein does have B factors that match
your Wilson B.  The fact that you have loops that have larger B's
doesn't really mean there is a problem, because those atoms don't
contribute much to the higher resolution reflections and don't come into
the calculation of the Wilson B.

The average of your B factors will increase if you choose to build
model for more disordered regions, while the Wilson B will, of course,
remain the same.  The average B factor will be larger, for example, if
you are more aggressive in building water molecules.  This does not
indicate a problem but is an unavoidable consequence of your choice to
take more risks.

Dale



On 10/13/2016 12:16 AM, sunanda williams wrote:

Sorry for not making the problem clear!
The overall B factor for the 3.0 A structure is around 98.00 A*2. Most
of the deposited structures in the PDB site around this resolution
has an av B around 70 A*2
All other statistics looks fine. I got a warning message while trying to
upload the structure in PDB that 98 was higher than the norm!
The reason why the structure has such a high B factor could be due to
disordered loops... All the same I was wondering how acceptable this
structure would be to picky reviewers..
And 'better the B factors' means bring them down...sorry couldn't phrase
it 'better'! I am going to try doing refinement with group B factors!
Would that help?
Thanks Robbie, I think this I need to make the best of the model I have.
Prof. Sekar will try PDB_redo..Thanks!



On Thu, Oct 13, 2016 at 11:41 AM, Pavel Afonine > wrote:

 I fully agree with Dale in not understanding what the problem is.
 Perhaps I have a better chance if you clearly explain what exactly
 you mean by "is there any way to better the B factors".
 Pavel


 On Thu, Oct 13, 2016 at 12:57 PM, Dale Tronrud
 > wrote:

I'm sorry but I don't understand what your problem is.  Do
 you think
 the B factors are too small for a 3A data set?  A range of 70 to
 75 is a
 little smaller than usual but probably not out of bounds.

 Dale Tronrud

 On 10/12/2016 7:59 PM, sunanda williams wrote:
 > Hi all,
 > I have a structure at 3.0 A and R/Rfree of 24/28. The mean B
 value is
 > around 98. The B value is especially high at the N terminus
 and two loop
 > regions (around 120-150 AA).
 > The rest of the structure averaged around 70-75. Has anyone
 faced such a
 > scenario? How reliable is the structure and is there any way
 to better
 > the B factors.
 > Any help is appreciated.
 > Thank you!!

Re: [ccp4bb] High B factor

[Dale Tronrud]

   It seems to be common for people to make the incorrect assumption
that the average of the atomic B factors in the PDB file should equal
the Wilson B.  The Wilson B is actually a weighted average of the
individual B factors where the weighting is rather mysterious, but the
small B factors have much higher weight than the larger ones. While the
Wilson B is simply a property of your data set the average of the atomic
B factors will also depend on choices you have made in building your
model.

   In your case the core of your protein does have B factors that match
your Wilson B.  The fact that you have loops that have larger B's
doesn't really mean there is a problem, because those atoms don't
contribute much to the higher resolution reflections and don't come into
the calculation of the Wilson B.

   The average of your B factors will increase if you choose to build
model for more disordered regions, while the Wilson B will, of course,
remain the same.  The average B factor will be larger, for example, if
you are more aggressive in building water molecules.  This does not
indicate a problem but is an unavoidable consequence of your choice to
take more risks.

Dale



On 10/13/2016 12:16 AM, sunanda williams wrote:
> Sorry for not making the problem clear!
> The overall B factor for the 3.0 A structure is around 98.00 A*2. Most
> of the deposited structures in the PDB site around this resolution
> has an av B around 70 A*2
> All other statistics looks fine. I got a warning message while trying to
> upload the structure in PDB that 98 was higher than the norm!
> The reason why the structure has such a high B factor could be due to
> disordered loops... All the same I was wondering how acceptable this
> structure would be to picky reviewers..
> And 'better the B factors' means bring them down...sorry couldn't phrase
> it 'better'! I am going to try doing refinement with group B factors!
> Would that help?
> Thanks Robbie, I think this I need to make the best of the model I have.
> Prof. Sekar will try PDB_redo..Thanks!
> 
> 
> 
> On Thu, Oct 13, 2016 at 11:41 AM, Pavel Afonine  > wrote:
> 
> I fully agree with Dale in not understanding what the problem is.
> Perhaps I have a better chance if you clearly explain what exactly
> you mean by "is there any way to better the B factors".
> Pavel
> 
> 
> On Thu, Oct 13, 2016 at 12:57 PM, Dale Tronrud
> > wrote:
> 
>I'm sorry but I don't understand what your problem is.  Do
> you think
> the B factors are too small for a 3A data set?  A range of 70 to
> 75 is a
> little smaller than usual but probably not out of bounds.
> 
> Dale Tronrud
> 
> On 10/12/2016 7:59 PM, sunanda williams wrote:
> > Hi all,
> > I have a structure at 3.0 A and R/Rfree of 24/28. The mean B
> value is
> > around 98. The B value is especially high at the N terminus
> and two loop
> > regions (around 120-150 AA).
> > The rest of the structure averaged around 70-75. Has anyone
> faced such a
> > scenario? How reliable is the structure and is there any way
> to better
> > the B factors.
> > Any help is appreciated.
> > Thank you!!
> 
> 
> 



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Description: OpenPGP digital signature

Re: [ccp4bb] High B factor

[sunanda williams]

Sorry for not making the problem clear!
The overall B factor for the 3.0 A structure is around 98.00 A*2. Most of
the deposited structures in the PDB site around this resolution
has an av B around 70 A*2
All other statistics looks fine. I got a warning message while trying to
upload the structure in PDB that 98 was higher than the norm!
The reason why the structure has such a high B factor could be due to
disordered loops... All the same I was wondering how acceptable this
structure would be to picky reviewers..
And 'better the B factors' means bring them down...sorry couldn't phrase it
'better'! I am going to try doing refinement with group B factors! Would
that help?
Thanks Robbie, I think this I need to make the best of the model I have.
Prof. Sekar will try PDB_redo..Thanks!



On Thu, Oct 13, 2016 at 11:41 AM, Pavel Afonine  wrote:

> I fully agree with Dale in not understanding what the problem is. Perhaps
> I have a better chance if you clearly explain what exactly you mean by "is
> there any way to better the B factors".
> Pavel
>
>
> On Thu, Oct 13, 2016 at 12:57 PM, Dale Tronrud 
> wrote:
>
>>I'm sorry but I don't understand what your problem is.  Do you think
>> the B factors are too small for a 3A data set?  A range of 70 to 75 is a
>> little smaller than usual but probably not out of bounds.
>>
>> Dale Tronrud
>>
>> On 10/12/2016 7:59 PM, sunanda williams wrote:
>> > Hi all,
>> > I have a structure at 3.0 A and R/Rfree of 24/28. The mean B value is
>> > around 98. The B value is especially high at the N terminus and two loop
>> > regions (around 120-150 AA).
>> > The rest of the structure averaged around 70-75. Has anyone faced such a
>> > scenario? How reliable is the structure and is there any way to better
>> > the B factors.
>> > Any help is appreciated.
>> > Thank you!!
>>
>
>

Re: [ccp4bb] High B factor

[Carlos CONTRERAS-MARTEL]

Hi Sunanda

They don't need to be "better" ... they need to be in agreement with you 
experimental B factor ... Wilson plot?


Best

Carlos

On 10/13/16 04:59, sunanda williams wrote:

Hi all,
I have a structure at 3.0 A and R/Rfree of 24/28. The mean B value is 
around 98. The B value is especially high at the N terminus and two 
loop regions (around 120-150 AA).
The rest of the structure averaged around 70-75. Has anyone faced such 
a scenario? How reliable is the structure and is there any way to 
better the B factors.

Any help is appreciated.
Thank you!!



--
 Carlos CONTRERAS MARTEL, Ph.D.
 (CR1 CNRS)

 carlos.contreras-mar...@ibs.fr

 "Bacterial Pathogenesis Group"
Institut de Biologie Structurale
 UMR5075 CEA-CNRS-UGA

  IBS
  Campus EPN
  71, avenue des Martyrs
  CS 10090
  38044 Grenoble CEDEX 9
  FRANCE


 tel : (+33) (0)4 57 42 86 41

http://www.ibs.fr/groupes/groupe-pathogenie-bacterienne/?lang=fr
http://www.ibs.fr/groups/bacterial-pathogenesis-group/?lang=en

Re: [ccp4bb] High B factor

[Pavel Afonine]

I fully agree with Dale in not understanding what the problem is. Perhaps I
have a better chance if you clearly explain what exactly you mean by "is
there any way to better the B factors".
Pavel

On Thu, Oct 13, 2016 at 12:57 PM, Dale Tronrud 
wrote:

>I'm sorry but I don't understand what your problem is.  Do you think
> the B factors are too small for a 3A data set?  A range of 70 to 75 is a
> little smaller than usual but probably not out of bounds.
>
> Dale Tronrud
>
> On 10/12/2016 7:59 PM, sunanda williams wrote:
> > Hi all,
> > I have a structure at 3.0 A and R/Rfree of 24/28. The mean B value is
> > around 98. The B value is especially high at the N terminus and two loop
> > regions (around 120-150 AA).
> > The rest of the structure averaged around 70-75. Has anyone faced such a
> > scenario? How reliable is the structure and is there any way to better
> > the B factors.
> > Any help is appreciated.
> > Thank you!!
>

Re: [ccp4bb] High B factor

[Robbie Joosten]

Hi Sunanda,

If you model is good, high average B-factors are just caused by the quality of 
your data. You can sometimes improve this a bit by careful reprocessing (e.g. 
by not using the last frames where radiation damage really kicked in). 
Sometimes you can have a better crystal and your data recording strategy can 
also help a bit.
That said, sometimes you just have to work with what you got. If you just make 
the best model you can, it will be reliable enough to describe you protein, 
just not with very high precision on atomic distances and such. The fact that 
you N-terminus and two loops have higher B-factors than the rest is hardly 
shocking. These are the most flexible bits of your protein.

Cheers,
Robbie

Sent from my Windows 10 phone

Van: sunanda williams<mailto:sunanda.willi...@gmail.com>
Verzonden: donderdag 13 oktober 2016 05:10
Aan: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Onderwerp: [ccp4bb] High B factor

Hi all,
I have a structure at 3.0 A and R/Rfree of 24/28. The mean B value is
around 98. The B value is especially high at the N terminus and two loop
regions (around 120-150 AA).
The rest of the structure averaged around 70-75. Has anyone faced such a
scenario? How reliable is the structure and is there any way to better the
B factors.
Any help is appreciated.
Thank you!!

Re: [ccp4bb] High B factor

[Dale Tronrud]

   I'm sorry but I don't understand what your problem is.  Do you think
the B factors are too small for a 3A data set?  A range of 70 to 75 is a
little smaller than usual but probably not out of bounds.

Dale Tronrud

On 10/12/2016 7:59 PM, sunanda williams wrote:
> Hi all,
> I have a structure at 3.0 A and R/Rfree of 24/28. The mean B value is
> around 98. The B value is especially high at the N terminus and two loop
> regions (around 120-150 AA).
> The rest of the structure averaged around 70-75. Has anyone faced such a
> scenario? How reliable is the structure and is there any way to better
> the B factors.
> Any help is appreciated.
> Thank you!!

[ccp4bb] High B factor

[sunanda williams]

Hi all,
I have a structure at 3.0 A and R/Rfree of 24/28. The mean B value is
around 98. The B value is especially high at the N terminus and two loop
regions (around 120-150 AA).
The rest of the structure averaged around 70-75. Has anyone faced such a
scenario? How reliable is the structure and is there any way to better the
B factors.
Any help is appreciated.
Thank you!!

[ccp4bb] High B-factor for metal

[kavya]

Dear users,

   I have refined a structure in R3 with cadmium bound to it, which
was present in the crystallization condition. There are 2 chains
in the asu. The structure is twinned. R and Rfree is around 22% and
28%. One of the cadmium has extremely high B-factor of 127, I tried
replacing it with water, but there were positive peaks appearing after
refinement, no other components in the protein buffer or crystallization
condition fit there. and there are 2 glutamate residues in the interaction
distance of X that come in that position. So kindly suggest me whether
I need to continue with cadmium ion assuming that its occupancy is low, or
any other options are there?

Thanking you
With regards
M. Kavyashree



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This message has been scanned for viruses and
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Re: [ccp4bb] High B-factor for metal

[Herman . Schreuder]

Dear Kavyashree,

I expect that you have a low-occupancy cadmium ion bound. If your resolution is 
not too low, say better than 2.5 Å, I would try to refine the occupancies of 
all cadmium ions. They are so big that it should work. If this does not work, 
you can make a rough estimate of the occupancy by using the scroll button in 
coot. If the electron density of the regular cadmiums disappears at say 3 
sigma, and of your high-Bfactor cadmium disappears at 1 sigma, you could set 
the occupancy of the latter to 0.3 and see how the cadmium behaves during 
refinement. At low resolution occupancies and Bfactors are to a certain extend 
linked, and errors in your estimate of the occupancy will be compensated by the 
Bfactor.

Good luck,
Herman
  

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
ka...@ssl.serc.iisc.ernet.in
Sent: Wednesday, May 11, 2011 11:30 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] High B-factor for metal

Dear users,

   I have refined a structure in R3 with cadmium bound to it, which was present 
in the crystallization condition. There are 2 chains in the asu. The structure 
is twinned. R and Rfree is around 22% and 28%. One of the cadmium has extremely 
high B-factor of 127, I tried replacing it with water, but there were positive 
peaks appearing after refinement, no other components in the protein buffer or 
crystallization condition fit there. and there are 2 glutamate residues in the 
interaction distance of X that come in that position. So kindly suggest me 
whether I need to continue with cadmium ion assuming that its occupancy is low, 
or any other options are there?

Thanking you
With regards
M. Kavyashree



--
This message has been scanned for viruses and dangerous content by MailScanner, 
and is believed to be clean.

Re: [ccp4bb] High B-factor for metal

[Eleanor Dodson]

On 05/11/2011 10:30 AM, ka...@ssl.serc.iisc.ernet.in wrote:

Dear users,

I have refined a structure in R3 with cadmium bound to it, which
was present in the crystallization condition. There are 2 chains
in the asu. The structure is twinned. R and Rfree is around 22% and
28%. One of the cadmium has extremely high B-factor of 127, I tried
replacing it with water, but there were positive peaks appearing after
refinement, no other components in the protein buffer or crystallization
condition fit there. and there are 2 glutamate residues in the interaction
distance of X that come in that position. So kindly suggest me whether
I need to continue with cadmium ion assuming that its occupancy is low, or
any other options are there?

Thanking you
With regards
M. Kavyashree






It isnt on a specialposition is it? If so you need to reduce the occupancy.
Eleanor

Re: [ccp4bb] High B-factor for metal

[Herman . Schreuder]

Dear Kavyashree,

Did you properly label the dual positions as alternate positions (same residue 
number and with A and B added just in front of the residue name)?. Also, did 
the occupancies sum up to less then 1, which makes perfect physical sense, or 
to more than 1? At 2.35 Å and with a partially occupied dual cadmium positions, 
you cannot expect miracles from the refinement programs. If the sum is more 
than 1, I would round off the occupancies to some sensible values with a sum of 
1 and fix them for the rest of the refinement.

Best regards,
Herman

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
ka...@ssl.serc.iisc.ernet.in
Sent: Wednesday, May 11, 2011 12:00 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] High B-factor for metal

Dear Sir,

The resolution is 2.35Ang. I was trying to refine Cadmium occupancy using 
the option of SAD data directly in refmac (as it cadmium gives anomalous 
signal at 1.54179 wavelength) and then use the obtained occupancy of those 
atoms to refine in usual way. It used to to refine occupancies of both cadmium 
and sulphur in (SO4) which is present in the structure, but 2 of the cadmium 
appears to have dual occupancy (which was confirmed as water added as a 
coordinate sphere used to come closer than 2.0 ang after refinement, so I tried 
giving 2 occupancies instead). After occupancy refinement as stated above, they 
were not summing up to 1, so I had doubts whether to continue this method or 
not.


Thank you
With regards
M. Kavyashree


 Dear Kavyashree,

 I expect that you have a low-occupancy cadmium ion bound. If your 
 resolution is not too low, say better than 2.5 Å, I would try to 
 refine the occupancies of all cadmium ions. They are so big that it should 
 work.
 If this does not work, you can make a rough estimate of the occupancy 
 by using the scroll button in coot. If the electron density of the regular
 cadmiums disappears at say 3 sigma, and of your high-Bfactor cadmium 
 disappears at 1 sigma, you could set the occupancy of the latter to 
 0.3 and see how the cadmium behaves during refinement. At low 
 resolution occupancies and Bfactors are to a certain extend linked, 
 and errors in your estimate of the occupancy will be compensated by the 
 Bfactor.

 Good luck,
 Herman


 -Original Message-
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
 ka...@ssl.serc.iisc.ernet.in
 Sent: Wednesday, May 11, 2011 11:30 AM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: [ccp4bb] High B-factor for metal

 Dear users,

I have refined a structure in R3 with cadmium bound to it, which 
 was present in the crystallization condition. There are 2 chains in 
 the asu. The structure is twinned. R and Rfree is around 22% and 28%. 
 One of the cadmium has extremely high B-factor of 127, I tried 
 replacing it with water, but there were positive peaks appearing after 
 refinement, no other components in the protein buffer or 
 crystallization condition fit there. and there are 2 glutamate 
 residues in the interaction distance of X that come in that 
 position. So kindly suggest me whether I need to continue with cadmium 
 ion assuming that its occupancy is low, or any other options are there?

 Thanking you
 With regards
 M. Kavyashree



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Re: [ccp4bb] High B-factor for metal

[Georg Zocher]

Wouldn't it be possible to estimate the occupancy of the Cd-ions from 
the peak heights of a DANO-map. Especially useful if there is doubt 
about the presence of Cd at this low occupied site.


Cheers, Georg



Am 11.05.11 11:33, schrieb herman.schreu...@sanofi-aventis.com:

Dear Kavyashree,

I expect that you have a low-occupancy cadmium ion bound. If your resolution is not too 
low, say better than 2.5 Å, I would try to refine the occupancies of all cadmium ions. 
They are so big that it should work. If this does not work, you can make a rough estimate 
of the occupancy by using the scroll button in coot. If the electron density of the 
regular cadmiums disappears at say 3 sigma, and of your high-Bfactor cadmium 
disappears at 1 sigma, you could set the occupancy of the latter to 0.3 and see how the 
cadmium behaves during refinement. At low resolution occupancies and Bfactors are to a 
certain extend linked, and errors in your estimate of the occupancy will be compensated 
by the Bfactor.

Good luck,
Herman


-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
ka...@ssl.serc.iisc.ernet.in
Sent: Wednesday, May 11, 2011 11:30 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] High B-factor for metal

Dear users,

I have refined a structure in R3 with cadmium bound to it, which was present in the 
crystallization condition. There are 2 chains in the asu. The structure is twinned. R and 
Rfree is around 22% and 28%. One of the cadmium has extremely high B-factor of 127, I 
tried replacing it with water, but there were positive peaks appearing after refinement, 
no other components in the protein buffer or crystallization condition fit there. and 
there are 2 glutamate residues in the interaction distance of X that come in 
that position. So kindly suggest me whether I need to continue with cadmium ion assuming 
that its occupancy is low, or any other options are there?

Thanking you
With regards
M. Kavyashree



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--
Universität Tübingen
Interfakultäres Institut für Biochemie
Dr. Georg Zocher
Hoppe-Seyler-Str. 4
72076 Tuebingen
Germany

Fon: +49(0)-7071-2973374
Mail: georg.zoc...@uni-tuebingen.de
http://www.ifib.uni-tuebingen.de

Re: [ccp4bb] High B-factor for metal

[kavya]

Dear Sir,

I have labeled it rightly. When I was trying to refine
using the method I mentioned it was giving some less than 1
while some more than 1 as occupancy. I am trying to match with
the peaks now.

Thanking you
With regards
M. Kavyashree

 Dear Kavyashree,

 Did you properly label the dual positions as alternate positions (same
 residue number and with A and B added just in front of the residue name)?.
 Also, did the occupancies sum up to less then 1, which makes perfect
 physical sense, or to more than 1? At 2.35 Å and with a partially occupied
 dual cadmium positions, you cannot expect miracles from the refinement
 programs. If the sum is more than 1, I would round off the occupancies to
 some sensible values with a sum of 1 and fix them for the rest of the
 refinement.

 Best regards,
 Herman

 -Original Message-
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
 ka...@ssl.serc.iisc.ernet.in
 Sent: Wednesday, May 11, 2011 12:00 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] High B-factor for metal

 Dear Sir,

 The resolution is 2.35Ang. I was trying to refine Cadmium occupancy
 using the option of SAD data directly in refmac (as it cadmium gives
 anomalous signal at 1.54179 wavelength) and then use the obtained
 occupancy of those atoms to refine in usual way. It used to to refine
 occupancies of both cadmium and sulphur in (SO4) which is present in
 the structure, but 2 of the cadmium appears to have dual occupancy
 (which was confirmed as water added as a coordinate sphere used to
 come closer than 2.0 ang after refinement, so I tried giving 2
 occupancies instead). After occupancy refinement as stated above, they
 were not summing up to 1, so I had doubts whether to continue this
 method or not.


 Thank you
 With regards
 M. Kavyashree


 Dear Kavyashree,

 I expect that you have a low-occupancy cadmium ion bound. If your
 resolution is not too low, say better than 2.5 Å, I would try to
 refine the occupancies of all cadmium ions. They are so big that it
 should work.
 If this does not work, you can make a rough estimate of the occupancy
 by using the scroll button in coot. If the electron density of the
 regular
 cadmiums disappears at say 3 sigma, and of your high-Bfactor cadmium
 disappears at 1 sigma, you could set the occupancy of the latter to
 0.3 and see how the cadmium behaves during refinement. At low
 resolution occupancies and Bfactors are to a certain extend linked,
 and errors in your estimate of the occupancy will be compensated by the
 Bfactor.

 Good luck,
 Herman


 -Original Message-
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
 ka...@ssl.serc.iisc.ernet.in
 Sent: Wednesday, May 11, 2011 11:30 AM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: [ccp4bb] High B-factor for metal

 Dear users,

I have refined a structure in R3 with cadmium bound to it, which
 was present in the crystallization condition. There are 2 chains in
 the asu. The structure is twinned. R and Rfree is around 22% and 28%.
 One of the cadmium has extremely high B-factor of 127, I tried
 replacing it with water, but there were positive peaks appearing after
 refinement, no other components in the protein buffer or
 crystallization condition fit there. and there are 2 glutamate
 residues in the interaction distance of X that come in that
 position. So kindly suggest me whether I need to continue with cadmium
 ion assuming that its occupancy is low, or any other options are there?

 Thanking you
 With regards
 M. Kavyashree



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