Re: [ccp4bb] Off-topic Thrombin cleavage

[Seema Nath]

I have used both Novagen thrombin & lyophilized thrombin of 
BDBioscience(dissolved in 10X thrombin dilution buffer provided by Novagen) in 
both 'thrombin cleavge buffer' & others (HEPES pH 7.0, Bicine pH 9.0, MOPS pH 
7.8) without adding calcium chloride - all the conditions permitted the cleavge 
process, but addition of DTT/ BMe slowed the process

Re: [ccp4bb] DNA/RNA modeling

[jens j birktoft]

and if everything else fails including kicking the trash can one could
indeed read the manual/FAQ - thanks  Paul


On Wed, May 23, 2012 at 4:27 PM, Paul Emsley wrote:

>
> On Wed, May 23, 2012 at 12:54 PM, Mark J van Raaij  > wrote:
> even more flexible than COOT?
> - the only thing I can think of that I haven't worked out how to do in
> COOT is grabbing and moving one atom at a time.
>
>
> On 23/05/12 19:50, jens j birktoft wrote:
>
> Thanks Mark.
>
>  As a matter of fact that is what I am really looking for.  The ability
> to move one or a few selected atoms/fragments at a time.
>
>
> And you thought that Coot was not flexible enough to do that?
>
> :-(
>
> Coot FAQ 4.23
>
>
> http://www.biop.ox.ac.uk/coot/doc/coot-faq.html#How-do-I-_0022grab_0022-just-one-atom-in-Real-Space-Refinement_002fRegularization_003f
>
> Also:
>
> If you don't want regularization/refinement, use the Rotate/Translate tool
> and Ctrl-click the atom(s) to move them one at a time.
>
> If you want "on the fly" regularization/refinement of fragments that
> include atoms that don't move when you "drop" them you need to fix them
> with the Fix Atom tool before regularization/refinement.
>
> HTH,
>
> Paul.
>
>


-- 
Jens J. Birktoft
e-mail: jens.kn...@gmail.com
slow-mail: 350 Central Park West, Suite 9F, New York, NY 10025
Phone: 212-749-5057

[ccp4bb] Off-topic: probing hydrophobic surface with ANS

[Huiming Li]

Hi,
 
   Could someone refer me to some papers on using ANS to estimate amount of 
hydrophobic surface in a protein?  I tried the classic way by STEP 1:  
titrating my protein into 1uM ANS till saturation to get the molar fluorescence 
activity of ANS, then STEP 2: titrate ANS into protein to get amount of 
fluorescene at plateau so that I can estimate how much ANS is bound per protein 
molecule.  However, for this protein I am dealing with, I can never get the 
STEP 1 done because the fluorescence signal never saturates. It keeps going up. 
Are there other ways of getting around this?
 
Thanks,
 
Huiming Li

Re: [ccp4bb] DNA/RNA modeling

[Paul Emsley]

On Wed, May 23, 2012 at 12:54 PM, Mark J van Raaij 
mailto:mjvanra...@cnb.csic.es>> wrote:

even more flexible than COOT?
- the only thing I can think of that I haven't worked out how to do in 
COOT is grabbing and moving one atom at a time.



On 23/05/12 19:50, jens j birktoft wrote:

Thanks Mark.

As a matter of fact that is what I am really looking for.  The 
ability to move one or a few selected atoms/fragments at a time.




And you thought that Coot was not flexible enough to do that?

:-(

Coot FAQ 4.23

http://www.biop.ox.ac.uk/coot/doc/coot-faq.html#How-do-I-_0022grab_0022-just-one-atom-in-Real-Space-Refinement_002fRegularization_003f

Also:

If you don't want regularization/refinement, use the Rotate/Translate 
tool and Ctrl-click the atom(s) to move them one at a time.


If you want "on the fly" regularization/refinement of fragments that 
include atoms that don't move when you "drop" them you need to fix them 
with the Fix Atom tool before regularization/refinement.


HTH,

Paul.

[ccp4bb] Off topic: Complexes cloning

[Theresa Hsu]

Dear all

A off-topic question - is there a practical limit for overexpression of 
membrane protein complexes (about 200 kDa in total)? This includes chaperones 
and my target proteins. Can I amplify the gene cluster and simply clone into a 
plasmid?

Thank you.

Re: [ccp4bb] Off-topic Thrombin cleavage

[Debasish Chattopadhyay]

Thrombin works pretty good without any added calcium.  We routinely added 
thrombin to whatever buffer the protein is in provided it doesn't have a lot of 
DTT.  Some beta-mercaptoethanol is alright.  What is the source of your 
thrombin?  

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Yuri 
Pompeu
Sent: Wednesday, May 23, 2012 12:23 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Off-topic Thrombin cleavage

Dear community,
I am trying to cleave a hexaHis tag from my protein prior to crystallization.
As I was setting up my digestion, my protein started to precipitate as soon as 
I added the recommended thrombin buffer.
My question is, if anyone has encountered this, how well does it cleave without 
thrombin buffer?
or even, without the CaCl2? Any other buffer/conditions known to work?
thanks in advance

Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma

[Nikolaos Glykos]

Hi Pete,


I'm curious - which programs have you used for maximum-entropy for
map calculation?


Thanks, I thought no-one would ask :-)

http://utopia.duth.gr/~glykos/graphent.html

Don't download the program today. Or tomorrow. This coming weekend 
there

will be a new release which will contain MacOSX executables.


Nicholas


--
Nicholas M. Glykos, Department of Molecular Biology
 and Genetics, Democritus University of Thrace, University Campus,
  Dragana, 68100 Alexandroupolis, Greece, Tel/Fax (office) 
+302551030620,

Ext.77620, Tel (lab) +302551030615, http://utopia.duth.gr/~glykos/

Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma

[George Sheldrick]

In SHELXL. a refinement program sometimes used by small molecule 
crystallographers, all Fourier map for at least the last 20 years were 
weighted by Ic^2/(Ic^2+sigma^2(I)), where Ic is the calculated squared 
structure factor and sigma(I) is the square root of 1/w. w is the weight 
assigned to a reflection in the refinement (e.g. w=1/(sig(I)^2+(gI)^2), 
where sig(I) is the esd of the measured intensity I and g is a small 
constant. This purely empirical scheme appears to result in a 
significant reduction in the noise level of the map, at least for 
typical small molecule structures. Such schemes have been called 
'maximum likelihood by intuition', a proper maximum likelihood treatment 
taking the esds of the intensities into account would of course do much 
better.


George

On 05/23/2012 06:59 PM, Dale Tronrud wrote:

On 05/23/12 08:06, Nicholas M Glykos wrote:

Hi Ed,



I may be wrong here (and please by all means correct me), but I think
it's not entirely true that experimental errors are not used in modern
map calculation algorithm.  At the very least, the 2mFo-DFc maps are
calibrated to the model error (which can be ideologically seen as the
"error of experiment" if you include model inaccuracies into that).

This is an amplitude modification. It does not change the fact that the
sigmas are not being used in the inversion procedure [and also does not
change the (non) treatment of missing data]. A more direct and relevant
example to discuss (with respect to Francisco's question) would be the
calculation of a Patterson synthesis (where the phases are known and
fixed).



I have not done extensive (or any for that matter) testing, but my
evidence-devoid gut feeling is that maps not using experimental errors
(which in REFAMC can be done either via gui button or by excluding SIGFP
from LABIN in a script) will for a practicing crystallographer be
essentially indistinguishable.

It seems that although you are not doubting the importance of maximum
likelihood for refinement, you do seem to doubt the importance of closely
related probabilistic methods (such as maximum entropy methods) for map
calculation. I think you can't have it both ways ... :-)




The reason for this is that "model errors" as estimated by various
maximum likelihood algorithms tend to exceed experimental errors.  It
may be that these estimates are inflated (heretical thought but when you
think about it uniform inflation of the SIGMA_wc may have only
proportional impact on the log-likelihood or even less so when they
correlate with experimental errors).  Or it may be that the experimental
errors are underestimated (another heretical thought).

My experience from comparing conventional (FFT-based) and maximum-entropy-
related maps is that the main source of differences between the two maps
has more to do with missing data (especially low resolution overloaded
reflections) and putative outliers (for difference Patterson maps), but in
certain cases (with very accurate or inaccurate data) standard deviations
do matter.

In a continuation of this torturous diversion from the original question...

Since your concern is not how the sigma(Fo) plays out in refinement but
how uncertainties are dealt with in the map calculation itself (where an
FFT calculates the most probable density values and maximum entropy would
calculate the "best", or centroid, density values) I believe the most
relevant measure of the uncertainty of the Fourier coefficients would be
sigma(2mFo-DFc).  This would be estimated from a complex calculation of
sigma(sigmaA), sigma(Fo), sigma(Fc) and sigma(Phic).  I expect that the
contribution of sigma(Fo) would be one of the smallest contributors to this
calculation, as long as Fo is "observed".  I wouldn't expect the loss of
sigma(Fo) to be catastrophic.

Wouldn't sigma(sigmaA) be the largest component since sigmaA is a function
of resolution and based only on the test set?

Dale Tronrud




All the best,
Nicholas





--
Prof. George M. Sheldrick FRS
Dept. Structural Chemistry,
University of Goettingen,
Tammannstr. 4,
D37077 Goettingen, Germany
Tel. +49-551-39-3021 or -3068
Fax. +49-551-39-22582

Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma

[Nikolaos Glykos]

Hi Ed,


I am not sure I understand this - perhaps we are talking about 
different
things.  Even if by inversion procedure you mean simple calculation 
of

(2fo-fc)*exp(i*phi), the fc is still technically a product of the
refinement, which unless based on trivial least square target (i.e. 
no
weights) does factor in experimental errors.  The (2mFo-DFc) map is 
even

more obviously dependent on the errors.  Again, I believe that the
differences will be minor, but if one calculates a map with refmac
either with or without factoring in experimental errors, there will 
be

*some difference*.  Thus, the experimental errors will affect the
resulting map.  Could you please clarify?


Yes, we are talking about different things. I refer to the case that we
have an amplitude term with its uncertainty (no matter whether it is Fo 
or
Fo^2 or Fo-Fc or 2mFo-DFc or ...) plus a phase with its uncertainty. In 
normal

everyday applications we use FFT which ignores (i) the uncertainties of
both terms, (ii) the missing data. By doing an FFT we produce a map 
which
exactly reproduces the input data (even if they are missing data which 
are
reproduced with an amplitude of zero). What I have been saying is that 
in

the presence of uncertainties and missing information the data do not
define a single map, but a whole set of maps which are statistically
consistent with the data and the question then arises : 'which map 
should

I be looking at ?'. I happen to mention the maximum entropy method as a
possible solution to this problem.



I think that we are not comparing ML to no-ML (or maximum entropy), 
but
rather ML inflated by experimental errors vs pure ML that ignores 
them.

I may be crazy or stupid (or both), but certainly not crazy/stupid
enough to "doubt the importance of maximum likelihood for 
refinement".
(On the other hand, one who promises to never doubt maximum 
likelihood

shall never use SHELX :)


We definitely talk about different things. My arguments had nothing to 
do
with treatment of errors in refinement. The question I was tackling was 
how you
go from |F|,sig(|F|),phase to a map in the presence of errors and 
missing

data.


Nicholas


--
Nicholas M. Glykos, Department of Molecular Biology
 and Genetics, Democritus University of Thrace, University Campus,
  Dragana, 68100 Alexandroupolis, Greece, Tel/Fax (office) 
+302551030620,

Ext.77620, Tel (lab) +302551030615, http://utopia.duth.gr/~glykos/

Re: [ccp4bb] crystallization analysis software

[Prince, D Bryan]

Ed,

I looked into this a number of years ago, and remember the following papers. I 
did not actually use any of the databases described due to IT issues at the 
time. I hope this helps.

Kind regards,
Bryan

LISA: an intranet-based flexible database for protein crystallography project 
management
http://scripts.iucr.org/cgi-bin/paper?S0907444901009295


HalX: an open-source LIMS (Laboratory Information Management System) for small- 
to large-scale laboratories
http://scripts.iucr.org/cgi-bin/paper?S0907444905001290

Xtrack - a web-based crystallographic notebook
http://scripts.iucr.org/cgi-bin/paper?S0907444902012696

CLIMS: Crystallography Laboratory Information Management System
http://scripts.iucr.org/cgi-bin/paper?za5055

These I found while looking up the others.

MOLE: A data management application based on a protein production data model
http://onlinelibrary.wiley.com/doi/10.1002/prot.20291/full

Design of a data model for developing laboratory information management and 
analysis systems for protein production
http://onlinelibrary.wiley.com/doi/10.1002/prot.20303/full

The Protein Information Management System (PiMS): a generic tool for any 
structural biology research laboratory
http://scripts.iucr.org/cgi-bin/paper?S0907444911007943




--
Confidentiality Notice: This message is private and may contain confidential 
and proprietary information. If you have received this message in error, please 
notify us and remove it from your system and note that you must not copy, 
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disclosure of the contents of this message is not permitted and may be unlawful.
 
-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ed 
Pozharski
Sent: Wednesday, May 23, 2012 12:24 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] crystallization analysis software

Does anyone know of a (non-commercial) software that can analyze results
of a crystallization screen?  What I am looking for is some way to tell
what components/factors favor protein solubility/precipitation based on
binary input (clear drop/precipitate).

I did some googling, but please feel free to use lmgtfy on me :)

Cheers,

Ed.


--
After much deep and profound brain things inside my head,
I have decided to thank you for bringing peace to our home.
Julian, King of Lemurs

Re: [ccp4bb] DNA/RNA modeling

[Francis E Reyes]

For the RNA I recommend Eric Westhof's Assemble. 

F

On May 23, 2012, at 9:32 AM, jens j birktoft wrote:

> Hi everybody,
> 
> Does anyone know of a (non-commercial) software  that are suitable for 
> modeling DNA/RNA structures.  Coot is great, however I am looking something 
> that allows more flexibility
> 
> Thanks
> 
> 
> -- 
> Jens J. Birktoft
> e-mail: jens.birkt...@nyu.edu
> 
> slow-mail: 350 Central Park West, Suite 9F, New York, NY 10025
> Phone: 212-749-5057

[ccp4bb] Off-topic Thrombin cleavage

[Yuri Pompeu]

Dear community,
I am trying to cleave a hexaHis tag from my protein prior to crystallization.
As I was setting up my digestion, my protein started to precipitate as soon as 
I added the recommended thrombin buffer.
My question is, if anyone has encountered this, how well does it cleave without 
thrombin buffer?
or even, without the CaCl2? Any other buffer/conditions known to work?
thanks in advance

Re: [ccp4bb] DNA/RNA modeling

[Tuhin Bhowmick]

Hi Jens,
You can try NAMOT (Nucleic Acid MOdelling Tool), from the links below.

http://namot.sourceforge.net/
http://sourceforge.net/projects/namot/

Best,

Tuhin.

Tuhin Bhowmick
Prof. S. Ramakumar’s Lab
Biocrystallography and Computation
Department of Physics
Indian Institute of Science
Bangalore, 560012
India





On Wed, May 23, 2012 at 10:18 PM, Francisco Hernandez-Guzman <
francisco.hernan...@accelrys.com> wrote:

> Hi Jens,
>
> ** **
>
> You can try the free DS Visualizer:
> http://accelrys.com/products/discovery-studio/visualization-download.php**
> **
>
> ** **
>
> It comes with a free molecular builder for proteins, dna/rna and ligands.
> Please note that if you do need to make anything more interesting (energy
> calculations, minimizations, md, etc) will then need a license.
>
> ** **
>
> I hope this helps.
>
> ** **
>
> Francisco
>
> ** **
>
> Full Disclaimer: I do work for Accelrys. 
>
> ** **
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *jens
> j birktoft
> *Sent:* Wednesday, May 23, 2012 9:32 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] DNA/RNA modeling
>
> ** **
>
> Hi everybody,
>
> ** **
>
> Does anyone know of a (non-commercial) software  that are suitable for
> modeling DNA/RNA structures.  Coot is great, however I am looking something
> that allows more flexibility
>
> ** **
>
> Thanks
>
> ** **
>
> ** **
>
> --
> Jens J. Birktoft
> e-mail: jens.birkt...@nyu.edu 
>
>
> slow-mail: 350 Central Park West, Suite 9F, New York, NY 10025
> Phone: 212-749-5057
>

Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma

[Dale Tronrud]

On 05/23/12 08:06, Nicholas M Glykos wrote:
> Hi Ed,
> 
> 
>> I may be wrong here (and please by all means correct me), but I think
>> it's not entirely true that experimental errors are not used in modern
>> map calculation algorithm.  At the very least, the 2mFo-DFc maps are
>> calibrated to the model error (which can be ideologically seen as the
>> "error of experiment" if you include model inaccuracies into that).
> 
> This is an amplitude modification. It does not change the fact that the 
> sigmas are not being used in the inversion procedure [and also does not 
> change the (non) treatment of missing data]. A more direct and relevant 
> example to discuss (with respect to Francisco's question) would be the 
> calculation of a Patterson synthesis (where the phases are known and 
> fixed).
> 
> 
>> I have not done extensive (or any for that matter) testing, but my 
>> evidence-devoid gut feeling is that maps not using experimental errors 
>> (which in REFAMC can be done either via gui button or by excluding SIGFP 
>> from LABIN in a script) will for a practicing crystallographer be 
>> essentially indistinguishable.
> 
> It seems that although you are not doubting the importance of maximum 
> likelihood for refinement, you do seem to doubt the importance of closely 
> related probabilistic methods (such as maximum entropy methods) for map 
> calculation. I think you can't have it both ways ... :-)
> 
> 
> 
>> The reason for this is that "model errors" as estimated by various
>> maximum likelihood algorithms tend to exceed experimental errors.  It
>> may be that these estimates are inflated (heretical thought but when you
>> think about it uniform inflation of the SIGMA_wc may have only
>> proportional impact on the log-likelihood or even less so when they
>> correlate with experimental errors).  Or it may be that the experimental
>> errors are underestimated (another heretical thought).
> 
> My experience from comparing conventional (FFT-based) and maximum-entropy- 
> related maps is that the main source of differences between the two maps 
> has more to do with missing data (especially low resolution overloaded 
> reflections) and putative outliers (for difference Patterson maps), but in 
> certain cases (with very accurate or inaccurate data) standard deviations 
> do matter.

   In a continuation of this torturous diversion from the original question...

   Since your concern is not how the sigma(Fo) plays out in refinement but
how uncertainties are dealt with in the map calculation itself (where an
FFT calculates the most probable density values and maximum entropy would
calculate the "best", or centroid, density values) I believe the most
relevant measure of the uncertainty of the Fourier coefficients would be
sigma(2mFo-DFc).  This would be estimated from a complex calculation of
sigma(sigmaA), sigma(Fo), sigma(Fc) and sigma(Phic).  I expect that the
contribution of sigma(Fo) would be one of the smallest contributors to this
calculation, as long as Fo is "observed".  I wouldn't expect the loss of
sigma(Fo) to be catastrophic.

   Wouldn't sigma(sigmaA) be the largest component since sigmaA is a function
of resolution and based only on the test set?

Dale Tronrud


> 
> 
> All the best,
> Nicholas
> 
> 

Re: [ccp4bb] DNA/RNA modeling

[Francisco Hernandez-Guzman]

Hi Jens,

You can try the free DS Visualizer: 
http://accelrys.com/products/discovery-studio/visualization-download.php

It comes with a free molecular builder for proteins, dna/rna and ligands. 
Please note that if you do need to make anything more interesting (energy 
calculations, minimizations, md, etc) will then need a license.

I hope this helps.

Francisco

Full Disclaimer: I do work for Accelrys.

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of jens j 
birktoft
Sent: Wednesday, May 23, 2012 9:32 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] DNA/RNA modeling

Hi everybody,

Does anyone know of a (non-commercial) software  that are suitable for modeling 
DNA/RNA structures.  Coot is great, however I am looking something that allows 
more flexibility

Thanks


--
Jens J. Birktoft
e-mail: jens.birkt...@nyu.edu

slow-mail: 350 Central Park West, Suite 9F, New York, NY 10025
Phone: 212-749-5057

[ccp4bb] DNA/RNA modeling

[jens j birktoft]

Hi everybody,

Does anyone know of a (non-commercial) software  that are suitable for
modeling DNA/RNA structures.  Coot is great, however I am looking something
that allows more flexibility

Thanks


-- 
Jens J. Birktoft
e-mail: jens.birkt...@nyu.edu 

slow-mail: 350 Central Park West, Suite 9F, New York, NY 10025
Phone: 212-749-5057

Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma

[Francisco Hernandez-Guzman]

I just wanted to take a moment to thank all of the respondents to the post. 
Indeed, my question was more practical in nature since I wanted to "see" the 
density around the ligand in question. From the first suggestions, I quickly 
did manage to generate the maps and accomplish my goal (special thanks to 
Robbie for actually sending me the converted mtz file from the PDB cif entry).

The additional comments have also been highly educational and helpful to 
further my understanding of some more in-depth crystallography concepts.

Thank you,

Francisco

PS The PDB_REDO (http://www.cmbi.ru.nl/pdb_redo/hf/1hfs/index.html) was indeed 
a great resource and I'm certain to use it again. Thanks!

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Francisco 
Hernandez-Guzman
Sent: Tuesday, May 22, 2012 9:28 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Calculating ED Maps from structure factor files with no sigma

Hello everyone,

My apologies if this comes as basic, but I wanted to get the expert's take on 
whether or not the sigmaF values are required in the calculation of an electron 
density map. If I look at the standard ED equation, sigma's don't appear to be 
a requirement but all the scripts that I've looked at do require sigma values.

I wanted to calculate the electron density for PDB id: 1HFS but the structure 
file only lists the Fo's, Fc's and Phases, but no sigmas. Would such structure 
factor file be considered incomplete?

Thank you for your kind explanation.

Francisco

[ccp4bb] crystallization analysis software

[Ed Pozharski]

Does anyone know of a (non-commercial) software that can analyze results
of a crystallization screen?  What I am looking for is some way to tell
what components/factors favor protein solubility/precipitation based on
binary input (clear drop/precipitate).

I did some googling, but please feel free to use lmgtfy on me :)

Cheers,

Ed.


-- 
After much deep and profound brain things inside my head, 
I have decided to thank you for bringing peace to our home.
Julian, King of Lemurs

Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma

[Ed Pozharski]

On Wed, 2012-05-23 at 18:06 +0300, Nicholas M Glykos wrote:
> It seems that although you are not doubting the importance of maximum 
> likelihood for refinement, you do seem to doubt the importance of
> closely 
> related probabilistic methods (such as maximum entropy methods) for
> map 
> calculation. I think you can't have it both ways ... :-)

Nicholas,

I think that we are not comparing ML to no-ML (or maximum entropy), but
rather ML inflated by experimental errors vs pure ML that ignores them.
I may be crazy or stupid (or both), but certainly not crazy/stupid
enough to "doubt the importance of maximum likelihood for refinement".
(On the other hand, one who promises to never doubt maximum likelihood
shall never use SHELX :)

Cheers,

Ed.


-- 
I don't know why the sacrifice thing didn't work.  
Science behind it seemed so solid.
Julian, King of Lemurs

Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma

[Ed Pozharski]

On Wed, 2012-05-23 at 18:06 +0300, Nicholas M Glykos wrote:
> This is an amplitude modification. It does not change the fact that
> the 
> sigmas are not being used in the inversion procedure 

Nicholas,

I am not sure I understand this - perhaps we are talking about different
things.  Even if by inversion procedure you mean simple calculation of
(2fo-fc)*exp(i*phi), the fc is still technically a product of the
refinement, which unless based on trivial least square target (i.e. no
weights) does factor in experimental errors.  The (2mFo-DFc) map is even
more obviously dependent on the errors.  Again, I believe that the
differences will be minor, but if one calculates a map with refmac
either with or without factoring in experimental errors, there will be
*some difference*.  Thus, the experimental errors will affect the
resulting map.  Could you please clarify?

Cheers, 

Ed

-- 
Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
Julian, King of Lemurs

Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma

[Ed Pozharski]

On Wed, 2012-05-23 at 10:02 -0500, Pete Meyer wrote:
> bviously 
> model and experimental errors do factor into calculation of a
> 2mFo-DFc 
> map - but is weight and structure factor calculation part of map 
> calculation, or a distinct stage of data processing? 

Oh, I see.  Sure, when the map coefficients are already available, the
further calculation does need sigmas, or even Fo's/Fc's.  I think the
practical question Fransisco asked comes to this:

If you have no SIGFP column in the input mtz-file, REFMAC will by
default fail (phenix probably won't - at least I've seen datasets where
all the sigmas were reset to 1.00 and it didn't change the output a
bit).  So one naturally things that sigmas are needed/used in map
calculation.  Again in practical terms, just remove the SIGFP from LABIN
and it works, because refmac can do it both ways.

Cheers,

Ed.

-- 
After much deep and profound brain things inside my head, 
I have decided to thank you for bringing peace to our home.
Julian, King of Lemurs

Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma

[Pete Meyer]

Nicholas,

My experience from comparing conventional (FFT-based) and maximum-entropy- 
related maps is that the main source of differences between the two maps 
has more to do with missing data (especially low resolution overloaded 
reflections) and putative outliers (for difference Patterson maps), but in 
certain cases (with very accurate or inaccurate data) standard deviations 
do matter.


I'm curious - which programs have you used for maximum-entropy for map 
calculation?


Pete

Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma

[Nicholas M Glykos]

Hi Ed,


> I may be wrong here (and please by all means correct me), but I think
> it's not entirely true that experimental errors are not used in modern
> map calculation algorithm.  At the very least, the 2mFo-DFc maps are
> calibrated to the model error (which can be ideologically seen as the
> "error of experiment" if you include model inaccuracies into that).

This is an amplitude modification. It does not change the fact that the 
sigmas are not being used in the inversion procedure [and also does not 
change the (non) treatment of missing data]. A more direct and relevant 
example to discuss (with respect to Francisco's question) would be the 
calculation of a Patterson synthesis (where the phases are known and 
fixed).


> I have not done extensive (or any for that matter) testing, but my 
> evidence-devoid gut feeling is that maps not using experimental errors 
> (which in REFAMC can be done either via gui button or by excluding SIGFP 
> from LABIN in a script) will for a practicing crystallographer be 
> essentially indistinguishable.

It seems that although you are not doubting the importance of maximum 
likelihood for refinement, you do seem to doubt the importance of closely 
related probabilistic methods (such as maximum entropy methods) for map 
calculation. I think you can't have it both ways ... :-)



> The reason for this is that "model errors" as estimated by various
> maximum likelihood algorithms tend to exceed experimental errors.  It
> may be that these estimates are inflated (heretical thought but when you
> think about it uniform inflation of the SIGMA_wc may have only
> proportional impact on the log-likelihood or even less so when they
> correlate with experimental errors).  Or it may be that the experimental
> errors are underestimated (another heretical thought).

My experience from comparing conventional (FFT-based) and maximum-entropy- 
related maps is that the main source of differences between the two maps 
has more to do with missing data (especially low resolution overloaded 
reflections) and putative outliers (for difference Patterson maps), but in 
certain cases (with very accurate or inaccurate data) standard deviations 
do matter.


All the best,
Nicholas


-- 


Nicholas M. Glykos, Department of Molecular Biology
 and Genetics, Democritus University of Thrace, University Campus,
  Dragana, 68100 Alexandroupolis, Greece, Tel/Fax (office) +302551030620,
Ext.77620, Tel (lab) +302551030615, http://utopia.duth.gr/~glykos/

Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma

[Pete Meyer]

Ed,


I may be wrong here (and please by all means correct me), but I think
it's not entirely true that experimental errors are not used in modern
map calculation algorithm.  At the very least, the 2mFo-DFc maps are
calibrated to the model error (which can be ideologically seen as the
"error of experiment" if you include model inaccuracies into that).  And


I supposed my statement may have more precise than helpful.  Obviously 
model and experimental errors do factor into calculation of a 2mFo-DFc 
map - but is weight and structure factor calculation part of map 
calculation, or a distinct stage of data processing?  I tend to think of 
it as separate from map calculation, but this may be up for debate 
(judging by the increasing number of statements along the lines of "I 
looked at my mtz file in coot and saw X").


[snip]

Nevertheless, the perceived situation is that "our models are not as
good as our data", and therefore experimental errors don't matter.  Now
I am playing another devil's advocate and I know how crazy this sounds
to an unbiased experimental scientist (e.g. if they don't matter, why
bother improving data reduction algorithms?).


The errors in our models are almost definitely more extensive than the 
errors in our measurements, but one try at answering this "devil's 
advocate" question would be to point out that the usual likelihood 
equations all require sigF (either as a component of sigma, or for 
bootstrapping sigma).  I've only done limited testing related to this 
(it was actually for something else), but likelihood equations produce 
strange results if you try to get them to ignore sigF.




Pete



I guess maps produced in phenix do not use experimental errors in any
way given that the maximum likelihood formalism implemented there does
not.  Although phenix is not immutable and my understanding may be
outdated.  But this is not the right forum for pondering this specific
question.

Cheers,

Ed.

PS.  I fully realize that Francisco's question was more practical (and
the answer to that is to run REFMAC without SIGFP record in LABIN), but
isn't thread-hijacking fun? :)

On Wed, 2012-05-23 at 10:05 +0300, Nicholas M Glykos wrote:

Hi Francisco,

I'll play devil's advocate, but a measurement without an estimate of its 
error is closer to theology than to science. The fact that the standard 
deviations are not used for calculating an electron density map via FFT is 
only due to the hidden assumption that you have 100% complete, error-free 
data set, extending to sufficient high (infinite) resolution. When these 
assumptions do not apply (as is usually the case with physical reality), 
then the simple-minded FFT is not the correct inversion procedure (and the 
data do not univocally define a single map). Under these conditions other 
inversion mathods are needed (such as maximum entropy) for which the 
standard deviations are actively being used for calculating the map.


My twocents,
Nicholas


On Tue, 22 May 2012, Francisco Hernandez-Guzman wrote:


Hello everyone,

My apologies if this comes as basic, but I wanted to get the expert's 
take on whether or not the sigmaF values are required in the calculation 
of an electron density map. If I look at the standard ED equation, 
sigma's don't appear to be a requirement but all the scripts that I've 
looked at do require sigma values.


I wanted to calculate the electron density for PDB id: 1HFS but the 
structure file only lists the Fo's, Fc's and Phases, but no sigmas. 
Would such structure factor file be considered incomplete?


Thank you for your kind explanation.

Francisco

Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma

[Ed Pozharski]

I may be wrong here (and please by all means correct me), but I think
it's not entirely true that experimental errors are not used in modern
map calculation algorithm.  At the very least, the 2mFo-DFc maps are
calibrated to the model error (which can be ideologically seen as the
"error of experiment" if you include model inaccuracies into that).  And
I quote from Acta D53:240,

"REFMAC also includes the sigma_E0 in the derivation of these terms (m
and D) which usually leads to improved behavior.  In fact in several
cases when this has not been so it has been shown that the sigma_I0 were
wrongly estimated during data processing."

Thus, the experimental errors do affect the maps (albeit indirectly).  I
have not done extensive (or any for that matter) testing, but my
evidence-devoid gut feeling is that maps not using experimental errors
(which in REFAMC can be done either via gui button or by excluding SIGFP
from LABIN in a script) will for a practicing crystallographer be
essentially indistinguishable.

The reason for this is that "model errors" as estimated by various
maximum likelihood algorithms tend to exceed experimental errors.  It
may be that these estimates are inflated (heretical thought but when you
think about it uniform inflation of the SIGMA_wc may have only
proportional impact on the log-likelihood or even less so when they
correlate with experimental errors).  Or it may be that the experimental
errors are underestimated (another heretical thought).

Nevertheless, the perceived situation is that "our models are not as
good as our data", and therefore experimental errors don't matter.  Now
I am playing another devil's advocate and I know how crazy this sounds
to an unbiased experimental scientist (e.g. if they don't matter, why
bother improving data reduction algorithms?).

I guess maps produced in phenix do not use experimental errors in any
way given that the maximum likelihood formalism implemented there does
not.  Although phenix is not immutable and my understanding may be
outdated.  But this is not the right forum for pondering this specific
question.

Cheers,

Ed.

PS.  I fully realize that Francisco's question was more practical (and
the answer to that is to run REFMAC without SIGFP record in LABIN), but
isn't thread-hijacking fun? :)

On Wed, 2012-05-23 at 10:05 +0300, Nicholas M Glykos wrote:
> Hi Francisco,
> 
> I'll play devil's advocate, but a measurement without an estimate of its 
> error is closer to theology than to science. The fact that the standard 
> deviations are not used for calculating an electron density map via FFT is 
> only due to the hidden assumption that you have 100% complete, error-free 
> data set, extending to sufficient high (infinite) resolution. When these 
> assumptions do not apply (as is usually the case with physical reality), 
> then the simple-minded FFT is not the correct inversion procedure (and the 
> data do not univocally define a single map). Under these conditions other 
> inversion mathods are needed (such as maximum entropy) for which the 
> standard deviations are actively being used for calculating the map.
> 
> My twocents,
> Nicholas
> 
> 
> On Tue, 22 May 2012, Francisco Hernandez-Guzman wrote:
> 
> > Hello everyone,
> > 
> > My apologies if this comes as basic, but I wanted to get the expert's 
> > take on whether or not the sigmaF values are required in the calculation 
> > of an electron density map. If I look at the standard ED equation, 
> > sigma's don't appear to be a requirement but all the scripts that I've 
> > looked at do require sigma values.
> > 
> > I wanted to calculate the electron density for PDB id: 1HFS but the 
> > structure file only lists the Fo's, Fc's and Phases, but no sigmas. 
> > Would such structure factor file be considered incomplete?
> > 
> > Thank you for your kind explanation.
> > 
> > Francisco
> > 
> 

-- 
"I'd jump in myself, if I weren't so good at whistling."
   Julian, King of Lemurs

Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma

[Nicholas M Glykos]

Hi Francisco,

I'll play devil's advocate, but a measurement without an estimate of its 
error is closer to theology than to science. The fact that the standard 
deviations are not used for calculating an electron density map via FFT is 
only due to the hidden assumption that you have 100% complete, error-free 
data set, extending to sufficient high (infinite) resolution. When these 
assumptions do not apply (as is usually the case with physical reality), 
then the simple-minded FFT is not the correct inversion procedure (and the 
data do not univocally define a single map). Under these conditions other 
inversion mathods are needed (such as maximum entropy) for which the 
standard deviations are actively being used for calculating the map.

My twocents,
Nicholas


On Tue, 22 May 2012, Francisco Hernandez-Guzman wrote:

> Hello everyone,
> 
> My apologies if this comes as basic, but I wanted to get the expert's 
> take on whether or not the sigmaF values are required in the calculation 
> of an electron density map. If I look at the standard ED equation, 
> sigma's don't appear to be a requirement but all the scripts that I've 
> looked at do require sigma values.
> 
> I wanted to calculate the electron density for PDB id: 1HFS but the 
> structure file only lists the Fo's, Fc's and Phases, but no sigmas. 
> Would such structure factor file be considered incomplete?
> 
> Thank you for your kind explanation.
> 
> Francisco
> 

-- 


Nicholas M. Glykos, Department of Molecular Biology
 and Genetics, Democritus University of Thrace, University Campus,
  Dragana, 68100 Alexandroupolis, Greece, Tel/Fax (office) +302551030620,
Ext.77620, Tel (lab) +302551030615, http://utopia.duth.gr/~glykos/