Re: [ccp4bb] Anomalous signal to noise details

[David Waterman]

Hi all,

Thanks for the useful advice. Clemens, I was indeed reminded of the
 vs / discussion when I started this thread. The
reason I brought it up here is because the ratio-of-means form is the way
it is described in "Estimation of anomalous signal in diffraction data
<https://journals.iucr.org/d/issues/2006/08/00/dz5079/#SEC2>"
(Dauter 2006), and similarly in "Can I solve my structure by SAD phasing?
Anomalous signal in SAD phasing
<https://journals.iucr.org/d/issues/2016/03/00/ba5234/index.html#SEC1>"
(Terwilliger et al. 2015), where it appears as <|Δano|>/<σano>. Therefore,
I'm not sure it is true that everyone really means <|ΔF|/σ(ΔF)> when
discussing the anomalous signal to noise, but I am happy to adopt this
version. The problem is, we have different formulations and different
suggested cut offs, and given that we don't really have independent and
identically distributed random variables, these criteria surely produce
(slightly?) different results depending on which form is adopted.

There is a practical motive here: we want to report this metric in DIALS
and we want to provide some indicative guiding lines on a plot to help the
user make an interpretive judgement. It sounds like a plot of <|ΔF|/σ(ΔF)>
with guidelines at 0.8 and 1.2 would be a good start, but it does seem
there is scope for confusion, not immediately resolved by the literature.
At least we will write the formula out on the plot explicitly, rather than
hiding it behind an ambiguous name. Bernhard, unfortunately the copy of BMC
I use for reference is currently locked up at the office. One of the side
effects of working from home is that the group library of hardcopy
textbooks is sat gathering dust.

Cheers

-- David


On Fri, 18 Dec 2020 at 22:41, Petr Kolenko 
wrote:

> Dear David,
>
> Although this is not exactly a topic of your question, an alternative
> approach is to use the resolution screening and compare the results. I have
> implemented this approach to my program SHELIXIR (because it uses SHELX
> C/D/E), which can be found here:
>
> http://kmlinux.fjfi.cvut.cz/~kolenpe1/shelixir/
>
> It also has its GUI here:
>
> http://kmlinux.fjfi.cvut.cz/~kolenpe1/shelixir/gui/
>
> Once you have enough computational power, you can easily perform such
> testing (and no longer need to understand everything that is written in the
> manuscript :-) ).
>
> I hope that the program will soon be published and I would welcome if you
> (or someone else) used it and potentially gave me some feedback or
> suggestion. The program has other functions like parallelized solvent
> content screening, etc. ;-) Feel free to ask for more.
>
> Best regards,
>
> Petr
>
>
>
> PS: Although tested on a number of cases, the command line is more stable
> than the GUI.
>
>
>
> *From:* CCP4 bulletin board  *On Behalf Of *David
> Waterman
> *Sent:* Friday, December 18, 2020 12:53 PM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] Anomalous signal to noise details
>
>
>
> Hi folks
>
>
>
> The paper "Substructure solution with SHELXD
> <https://journals.iucr.org/d/issues/2002/10/02/gr2280/index.html>"
> (Schneider & Sheldrick, 2002) describes how
>
>
>
> data can be truncated at the resolution at which [ΔF to its estimated
> standard deviation as a function of the resolution] drops to below about 1.3
>
>
>
> Is this referring to the quantity <|ΔF|>/<σ(ΔF)> calculated in resolution
> shells, or the quantity <|ΔF|/σ(ΔF)> ?
>
>
>
> This entry
> <https://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php?title=SHELX_C/D/E#Resolution_cutoff_.28SHEL.29>
> on the ccp4wiki gives a cutoff
>
>
>
> where the mean value of |ΔF|/σ(ΔF) falls below about 1.2 (a value of 0.8
> would indicate pure noise)
>
>
>
> this version sounds to me like <|ΔF|/σ(ΔF)>
>
>
>
> which is the "better" metric, and what do people mean when they say
> DANO/SIGDANO? What is the justification for the 1.3 (or 1.2) value?
>
>
>
> Thanks!
>
> -- David
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Anomalous signal to noise details

[Petr Kolenko]

Dear David,
Although this is not exactly a topic of your question, an alternative approach 
is to use the resolution screening and compare the results. I have implemented 
this approach to my program SHELIXIR (because it uses SHELX C/D/E), which can 
be found here:
http://kmlinux.fjfi.cvut.cz/~kolenpe1/shelixir/
It also has its GUI here:
http://kmlinux.fjfi.cvut.cz/~kolenpe1/shelixir/gui/
Once you have enough computational power, you can easily perform such testing 
(and no longer need to understand everything that is written in the manuscript 
:-) ).
I hope that the program will soon be published and I would welcome if you (or 
someone else) used it and potentially gave me some feedback or suggestion. The 
program has other functions like parallelized solvent content screening, etc. 
;-) Feel free to ask for more.
Best regards,
Petr

PS: Although tested on a number of cases, the command line is more stable than 
the GUI.

From: CCP4 bulletin board  On Behalf Of David Waterman
Sent: Friday, December 18, 2020 12:53 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Anomalous signal to noise details

Hi folks

The paper "Substructure solution with 
SHELXD<https://journals.iucr.org/d/issues/2002/10/02/gr2280/index.html>" 
(Schneider & Sheldrick, 2002) describes how

data can be truncated at the resolution at which [ΔF to its estimated standard 
deviation as a function of the resolution] drops to below about 1.3

Is this referring to the quantity <|ΔF|>/<σ(ΔF)> calculated in resolution 
shells, or the quantity <|ΔF|/σ(ΔF)> ?

This 
entry<https://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php?title=SHELX_C/D/E#Resolution_cutoff_.28SHEL.29>
 on the ccp4wiki gives a cutoff

where the mean value of |ΔF|/σ(ΔF) falls below about 1.2 (a value of 0.8 would 
indicate pure noise)

this version sounds to me like <|ΔF|/σ(ΔF)>

which is the "better" metric, and what do people mean when they say 
DANO/SIGDANO? What is the justification for the 1.3 (or 1.2) value?

Thanks!
-- David



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Re: [ccp4bb] Anomalous signal to noise details

[Ian Tickle]

Conventionally (e.g. in cryo-EM) the SNR is taken as a ratio of averages,
either the ratio of the variance of the signal (average of signal squared)
to the variance of the noise, i.e. var(signal) / var(noise), or the square
root of that, i.e. sd(signal) / sd(noise).  See here:
https://en.wikipedia.org/wiki/Signal-to-noise_ratio .

Alternatively, it may be defined as the ratio of the mean value of the
signal to its standard error, i.e. mean(signal) / sd(noise):
https://en.wikipedia.org/wiki/Signal-to-noise_ratio_(imagi
ng)  , so
again a ratio of averages.  Whenever you read about SNR you have to check
carefully which of the three definitions is in use (and often it's not
stated!).

I think the confusion of ratio-of-averages vs. average-of-ratios has arisen
because in crystallography we're not actually talking about the SNR, rather
we're talking about the _average_ SNR, where the average is taken over
samples of a population that have _different_ distributions in reciprocal
space, whereas in the previous case the samples are taken from a population
whose samples are assumed to have the _same_ distribution (and therefore
same s.d.).  Note that the ratio-of-average and average-of-ratio cases
converge if all samples are drawn from the same population with uniform
distribution and s.d..

So then in our situation it is indeed correct to say that average SNR =
average( I / sd(I) ), i.e. an average of the ratios of averages!  When we
measure an _individual_ intensity with its s.d., we are already taking
averages, but they are averages over time where the standard error is
constant.  Then, when we take the spatial average SNR over different
intensities the sampling distribution and its s.d. naturally varies so we
must take the average of ratios.

Cheers

-- Ian


On Fri, 18 Dec 2020 at 20:14, Bernhard Rupp 
wrote:

> > I don't know the justification; maybe just experience? Surely the higher
> the better.  I've seen George Sheldrick deriving the value of ~0.8 when
> there is _no_ anom signal but I forgot the details, sorry ...
>
> It is derived from the mean absolute error (cf. p414 in Chapter 8 of BMC,
> with help of Ian Tickle), and holds for unmerged data. A reasonable good
> indication where to set the cutoff in practice is to look at the site vs
> occupancy plot. A distinct drop after a few good sites is usually a good
> sign, and that tends to cluster around the ~1.3 ratio.
>
>
> http://www.ruppweb.org/Garland/gallery/Ch10/pages/Biomolecular_Crystallography_Fig_10-30_PART2.htm
>
> The noise level is actually observable in data without anomalous signal
>
>
> http://www.ruppweb.org/Garland/gallery/Ch10/pages/Biomolecular_Crystallography_Fig_10-29.htm
>
> Best BR
>
>
>
>
>
> best wishes,
>
> Kay
>
> >
> >Thanks!
> >-- David
> >
> >###
> >#
> >
> >To unsubscribe from the CCP4BB list, click the following link:
> >https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
> >
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> >
>
> 
>
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Re: [ccp4bb] Anomalous signal to noise details

[Gergely Katona]

Hi,

I am not sure about the 1.2-1.3 limit, but 0.8 probably comes from sqrt(2/pi), 
which is the ratio of the mean of a half-normal and its sigma when the mean 
parameter is 0.

Best wishes,

Gergely


From: CCP4 bulletin board  On Behalf Of David Waterman
Sent: 18 December, 2020 12:53
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Anomalous signal to noise details

Hi folks

The paper "Substructure solution with 
SHELXD<https://journals.iucr.org/d/issues/2002/10/02/gr2280/index.html>" 
(Schneider & Sheldrick, 2002) describes how

data can be truncated at the resolution at which [ΔF to its estimated standard 
deviation as a function of the resolution] drops to below about 1.3

Is this referring to the quantity <|ΔF|>/<σ(ΔF)> calculated in resolution 
shells, or the quantity <|ΔF|/σ(ΔF)> ?

This 
entry<https://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php?title=SHELX_C/D/E#Resolution_cutoff_.28SHEL.29>
 on the ccp4wiki gives a cutoff

where the mean value of |ΔF|/σ(ΔF) falls below about 1.2 (a value of 0.8 would 
indicate pure noise)

this version sounds to me like <|ΔF|/σ(ΔF)>

which is the "better" metric, and what do people mean when they say 
DANO/SIGDANO? What is the justification for the 1.3 (or 1.2) value?

Thanks!
-- David



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Re: [ccp4bb] Anomalous signal to noise details

[Bernhard Rupp]

> I don't know the justification; maybe just experience? Surely the higher the 
> better.  I've seen George Sheldrick deriving the value of ~0.8 when there is 
> _no_ anom signal but I forgot the details, sorry ...

It is derived from the mean absolute error (cf. p414 in Chapter 8 of BMC, with 
help of Ian Tickle), and holds for unmerged data. A reasonable good indication 
where to set the cutoff in practice is to look at the site vs occupancy plot. A 
distinct drop after a few good sites is usually a good sign, and that tends to 
cluster around the ~1.3 ratio.

http://www.ruppweb.org/Garland/gallery/Ch10/pages/Biomolecular_Crystallography_Fig_10-30_PART2.htm

The noise level is actually observable in data without anomalous signal

http://www.ruppweb.org/Garland/gallery/Ch10/pages/Biomolecular_Crystallography_Fig_10-29.htm

Best BR





best wishes,

Kay

>
>Thanks!
>-- David
>
>###
>#
>
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>
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Re: [ccp4bb] Anomalous signal to noise details

[Kay Diederichs]

Hi David,

On Fri, 18 Dec 2020 11:53:08 +, David Waterman  wrote:

>Hi folks
>
>The paper "Substructure solution with SHELXD
>"
>(Schneider & Sheldrick, 2002) describes how
>
>data can be truncated at the resolution at which [ΔF to its estimated
>> standard deviation as a function of the resolution] drops to below about 1.3
>
>
>Is this referring to the quantity <|ΔF|>/<σ(ΔF)> calculated in resolution
>shells, or the quantity <|ΔF|/σ(ΔF)> ?

the latter

the only scaling program that I know of that calculates ratios of averages is 
SCALEPACK; 
the others calculate averages of ratios.

>
>This entry
>
>on the ccp4wiki gives a cutoff
>
>where the mean value of |ΔF|/σ(ΔF) falls below about 1.2 (a value of 0.8
>> would indicate pure noise)
>
>
>this version sounds to me like <|ΔF|/σ(ΔF)>

yes

>
>which is the "better" metric, and what do people mean when they say
>DANO/SIGDANO? What is the justification for the 1.3 (or 1.2) value?

I don't know the justification; maybe just experience? Surely the higher the 
better.  I've seen George Sheldrick deriving the value of ~0.8
when there is _no_ anom signal but I forgot the details, sorry ...

best wishes,

Kay

>
>Thanks!
>-- David
>
>
>
>To unsubscribe from the CCP4BB list, click the following link:
>https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>
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Re: [ccp4bb] Anomalous signal to noise details

[Clemens Vonrhein]

Dear David,

On Fri, Dec 18, 2020 at 11:53:08AM +, David Waterman wrote:
> The paper "Substructure solution with SHELXD
> "
> (Schneider & Sheldrick, 2002) describes how
> 
> data can be truncated at the resolution at which [ΔF to its estimated
> > standard deviation as a function of the resolution] drops to below about 1.3
> 
> 
> Is this referring to the quantity <|ΔF|>/<σ(ΔF)> calculated in resolution
> shells, or the quantity <|ΔF|/σ(ΔF)> ?

I'm nearly 100% sure this refers to the latter - or at least: the
latter is the only one making sense to me. This sounds very much like
the confusion when it comes to

   (1)

==> PDBx/mmCIF: _reflns.pdbx_netI_over_sigmaI   73.6  % of entries
_reflns_shell.pdbx_netI_over_sigmaI_all  0.001% of entries
_reflns_shell.pdbx_netI_over_sigmaI_obs  2.6  % of entries

versus

  / (2)

==> PDBx/mmCIF: _reflns.pdbx_netI_over_av_sigmaI 2.6  % of entries
_reflns_shell.meanI_over_sigI_all0.2  % of entries
_reflns_shell.meanI_over_sigI_obs   53.0  % of entries

As far as I can remember, we always computed and reported (1) and
never (2) - at least when it comes to the scaling/merging programs I'm
familiar with (SCALE, XDS/XSCALE, AIMLESS, d*TREK). What useful
information would (2) or <|ΔF|>/<σ(ΔF)> convey anyway ... ?

If we were to believe these definitions, then we are storing the
"right/useful" value  in the overall statistics, but a very
different value of / in the per-shell statistics. All those
_reflns_shell.meanI_over_sigI_obs are most like mis-labeled (1)
quantities.

> This entry
> 
> on the ccp4wiki gives a cutoff
> 
> where the mean value of |ΔF|/σ(ΔF) falls below about 1.2 (a value of 0.8
> > would indicate pure noise)
> 
> 
> this version sounds to me like <|ΔF|/σ(ΔF)>
> 
> which is the "better" metric, and what do people mean when they say
> DANO/SIGDANO? What is the justification for the 1.3 (or 1.2) value?

I think everyone always refers to <|ΔF|/σ(ΔF)> no matter what it is
called (sometimes programmers shorten the notation to avoid unwieldly
wide columns).

I tend to look for values above 1 (and the higher, the better) - but
maybe even more importantly: check the trend with resolution (higher
at low resolution), maybe in comparison with expectations (type of
scatterer, fluorescence scan, anomalous signal, number of sites,
potential B-factors of scatteres etc).

Cheers

Clemens



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[ccp4bb] Anomalous signal to noise details

[David Waterman]

Hi folks

The paper "Substructure solution with SHELXD
"
(Schneider & Sheldrick, 2002) describes how

data can be truncated at the resolution at which [ΔF to its estimated
> standard deviation as a function of the resolution] drops to below about 1.3


Is this referring to the quantity <|ΔF|>/<σ(ΔF)> calculated in resolution
shells, or the quantity <|ΔF|/σ(ΔF)> ?

This entry

on the ccp4wiki gives a cutoff

where the mean value of |ΔF|/σ(ΔF) falls below about 1.2 (a value of 0.8
> would indicate pure noise)


this version sounds to me like <|ΔF|/σ(ΔF)>

which is the "better" metric, and what do people mean when they say
DANO/SIGDANO? What is the justification for the 1.3 (or 1.2) value?

Thanks!
-- David



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[ccp4bb] anomalous signal

[Faisal Tarique]

Dear all

I am working on a metalloprotein which probably contains Ca at its active
site..The sulfur containing amino acid constitutes almost 5.4% of the total
amino acid residues of this protein..I have collected the data at home
source (CuKalpha=1.54A)..Since f'' of Sulfur is 0.56 and that of Ca is 1.28
we can always expect some  anomalous signal out of the data..My question is
..how we will know if the anomalous signal is coming out of Sulfur or from
Calcium ?? is there any method through which we can get to know the
identity of the scattering molecule through the data..Can FFT anomalous map
from CCP4 is of any help in this direction, if yes then please tell me how
to interpret the output from this..

-- 
Regards

Faisal
School of Life Sciences
JNU

Re: [ccp4bb] anomalous signal

[Tobias Weinert]

Dear Faisal,

you could use Phenix to do an f’’ refinement against the data like described 
here:

Liu, Q., Liu, Q.  Hendrickson, W. A. (2013). Acta Cryst. D69, 1314-1332.


best regards,

Tobias


On 30 Apr 2014, at 14:01, Faisal Tarique faisaltari...@gmail.com wrote:

 Dear all
 
 I am working on a metalloprotein which probably contains Ca at its active 
 site..The sulfur containing amino acid constitutes almost 5.4% of the total 
 amino acid residues of this protein..I have collected the data at home source 
 (CuKalpha=1.54A)..Since f'' of Sulfur is 0.56 and that of Ca is 1.28 we can 
 always expect some  anomalous signal out of the data..My question is ..how we 
 will know if the anomalous signal is coming out of Sulfur or from Calcium ?? 
 is there any method through which we can get to know the identity of the 
 scattering molecule through the data..Can FFT anomalous map from CCP4 is of 
 any help in this direction, if yes then please tell me how to interpret the 
 output from this..
 
 -- 
 Regards
 
 Faisal
 School of Life Sciences
 JNU
 

Re: [ccp4bb] anomalous signal

[Eleanor Dodson]

Well - this is pretty common, and really doesnt matter.
 I just let SHELXC/D give an occupancy to its anom scatterer solutions
and assume that the stronger one is Ca. But in fact it wont matter at all
for the phasing, and IF the experiment works it is easy to sort out S from
Ca in the final map.





On 30 April 2014 13:21, Tobias Weinert tobias.wein...@psi.ch wrote:

 Dear Faisal,

 you could use Phenix to do an f’’ refinement against the data like
 described here:

 Liu, Q., Liu, Q.  Hendrickson, W. A. (2013). Acta Cryst. D69, 1314-1332.


 best regards,

 Tobias


 On 30 Apr 2014, at 14:01, Faisal Tarique faisaltari...@gmail.com wrote:

  Dear all
 
  I am working on a metalloprotein which probably contains Ca at its
 active site..The sulfur containing amino acid constitutes almost 5.4% of
 the total amino acid residues of this protein..I have collected the data at
 home source (CuKalpha=1.54A)..Since f'' of Sulfur is 0.56 and that of Ca is
 1.28 we can always expect some  anomalous signal out of the data..My
 question is ..how we will know if the anomalous signal is coming out of
 Sulfur or from Calcium ?? is there any method through which we can get to
 know the identity of the scattering molecule through the data..Can FFT
 anomalous map from CCP4 is of any help in this direction, if yes then
 please tell me how to interpret the output from this..
 
  --
  Regards
 
  Faisal
  School of Life Sciences
  JNU
 

Re: [ccp4bb] anomalous signal

[Keller, Jacob]

I myself have never seen a separate peak that was a single sulfur atom.

I've seen, in a moderately-radiation-damaged dataset, little shards of 
anomalous scattering density in Phaser-generated LLG maps. They were in the 
interior of the protein, and the closest possible atoms were sulfurs. They were 
not in positions suggestive of Fourier truncation ripples, so I hypothesized 
that these sulfurs were ionized and landed at the nearest convenient location, 
and left it at that.

JPK

Re: [ccp4bb] anomalous signal

[Eleanor Dodson]

Look at the aimless plot of CCanom . That is the best indicator I think and 
very sensitive when you have such high redundancy
Eleanor


On 25 Apr 2014, at 22:13, Jim Pflugrath wrote:

 d/sig should be above 0.80
 
 There seems to be plenty of signal there with all values above 1.02.  We have 
 solved structures with less multiplicity and lower d/sig.
 
 There is a different criteria of signal for when you know the positions of 
 the anomalous substructure atoms and when you need to find the positions of 
 the anomalous substructure atoms.
 
 As for no signal, I think I am on record that there is always an anomalous 
 signal. :)  But can you detect it?
 
 Jim
 
 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Faisal Tarique 
 [faisaltari...@gmail.com]
 Sent: Friday, April 25, 2014 4:06 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: [ccp4bb] anomalous signal
 
 Dear all
 
 sorry about my previous mail where i forgot to mention that the data was 
 collected on home source at Cuk alpha and at 1.54A.
 
 written below is the log file of an anomalous data processed through 
 SHELXC..my question is ..what is the strength of anomalous signal ?? as it is 
 said For zero signal d'/sig and d/sig should be about 0.80. Then in 
 the present case is there really a signal or can be assumed no signal..we are 
 expecting one Ca atom bound to the protein at its active site..the redundancy 
 of the data is 11.6..with this signal strength can we assume Ca to be present 
 there or whatever little anomalous if present is due to something elseor 
 there is no signal at all ??...
 
 Resl.   Inf - 8.0 - 6.0 - 5.0 - 4.0 - 3.8 - 3.6 - 3.4 - 3.2 - 3.0 - 2.8 - 2.60
  N(data) 375   493   580  1319   450   538   679   866  1081  1414  1709
  I/sig58.8  38.6  32.6  38.3  27.7  27.2  21.9  18.4  12.6   9.5   6.1
  %Complete  94.7  99.0  99.3  99.5 100.0  99.6  99.7  99.8  99.6  99.6  90.9
  d/sig   1.65  1.27  1.18  1.25  1.19  1.12  1.11  1.11  0.97  1.02  1.05
 
 -- 
 Regards
 
 Faisal
 School of Life Sciences
 JNU

Re: [ccp4bb] anomalous signal

[Tim Gruene]

Dear Faisal,

the lack of the CCanom line in the shelxc output suggests that your data
are already merged, and my guess is you processed your data with HKL2000
- all other integration programs I am aware of do not merge the data at
such an early stage giving you access to the CCanom Eleanor mentioned.

There might be a switch in HKL2000 to not merge the data. A CCanom 30%
is a good indicator of the presence of an anomalous signal.

Best,
Tim

On 04/26/2014 12:18 PM, Eleanor Dodson wrote:
 Look at the aimless plot of CCanom . That is the best indicator I think and 
 very sensitive when you have such high redundancy
 Eleanor
 
 
 On 25 Apr 2014, at 22:13, Jim Pflugrath wrote:
 
 d/sig should be above 0.80

 There seems to be plenty of signal there with all values above 1.02.  We 
 have solved structures with less multiplicity and lower d/sig.

 There is a different criteria of signal for when you know the positions of 
 the anomalous substructure atoms and when you need to find the positions of 
 the anomalous substructure atoms.

 As for no signal, I think I am on record that there is always an anomalous 
 signal. :)  But can you detect it?

 Jim

 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Faisal 
 Tarique [faisaltari...@gmail.com]
 Sent: Friday, April 25, 2014 4:06 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: [ccp4bb] anomalous signal

 Dear all

 sorry about my previous mail where i forgot to mention that the data was 
 collected on home source at Cuk alpha and at 1.54A.

 written below is the log file of an anomalous data processed through 
 SHELXC..my question is ..what is the strength of anomalous signal ?? as it 
 is said For zero signal d'/sig and d/sig should be about 0.80. Then 
 in the present case is there really a signal or can be assumed no signal..we 
 are expecting one Ca atom bound to the protein at its active site..the 
 redundancy of the data is 11.6..with this signal strength can we assume Ca 
 to be present there or whatever little anomalous if present is due to 
 something elseor there is no signal at all ??...

 Resl.   Inf - 8.0 - 6.0 - 5.0 - 4.0 - 3.8 - 3.6 - 3.4 - 3.2 - 3.0 - 2.8 - 
 2.60
  N(data) 375   493   580  1319   450   538   679   866  1081  1414  1709
  I/sig58.8  38.6  32.6  38.3  27.7  27.2  21.9  18.4  12.6   9.5   6.1
  %Complete  94.7  99.0  99.3  99.5 100.0  99.6  99.7  99.8  99.6  99.6  90.9
  d/sig   1.65  1.27  1.18  1.25  1.19  1.12  1.11  1.11  0.97  1.02  1.05

 -- 
 Regards

 Faisal
 School of Life Sciences
 JNU
 
 

-- 
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A



signature.asc
Description: OpenPGP digital signature

Re: [ccp4bb] anomalous signal

[Phil Evans]

Was there a reason that you turned off the scaling in Aimless (onlymerge)? If 
the data have come from Mosflm, this is definitely wrong - the result is that 
(among other things) you have negative CCanom values which is unusual to say 
the least

Just run it with the default options, that's usually the best thing to do to 
start with

Phil

On 26 Apr 2014, at 14:38, Faisal Tarique faisaltari...@gmail.com wrote:

 Dear Eleanor and Tim.
 
 i have reprocessed the data through imosflm and run the aimless through the 
 unmerged output mtz..i am attaching the output log file of the 
 aimless..please tell me how to interpret the anomalous signal from the log 
 file and where the information is written..
 
 Thanks again for your much needed help.
 
 regards
 
 Faisal
 
 
 On Sat, Apr 26, 2014 at 5:22 PM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote:
 Dear Faisal,
 
 the lack of the CCanom line in the shelxc output suggests that your data
 are already merged, and my guess is you processed your data with HKL2000
 - all other integration programs I am aware of do not merge the data at
 such an early stage giving you access to the CCanom Eleanor mentioned.
 
 There might be a switch in HKL2000 to not merge the data. A CCanom 30%
 is a good indicator of the presence of an anomalous signal.
 
 Best,
 Tim
 
 On 04/26/2014 12:18 PM, Eleanor Dodson wrote:
  Look at the aimless plot of CCanom . That is the best indicator I think and 
  very sensitive when you have such high redundancy
  Eleanor
 
 
  On 25 Apr 2014, at 22:13, Jim Pflugrath wrote:
 
  d/sig should be above 0.80
 
  There seems to be plenty of signal there with all values above 1.02.  We 
  have solved structures with less multiplicity and lower d/sig.
 
  There is a different criteria of signal for when you know the positions 
  of the anomalous substructure atoms and when you need to find the 
  positions of the anomalous substructure atoms.
 
  As for no signal, I think I am on record that there is always an 
  anomalous signal. :)  But can you detect it?
 
  Jim
 
  From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Faisal 
  Tarique [faisaltari...@gmail.com]
  Sent: Friday, April 25, 2014 4:06 PM
  To: CCP4BB@JISCMAIL.AC.UK
  Subject: [ccp4bb] anomalous signal
 
  Dear all
 
  sorry about my previous mail where i forgot to mention that the data was 
  collected on home source at Cuk alpha and at 1.54A.
 
  written below is the log file of an anomalous data processed through 
  SHELXC..my question is ..what is the strength of anomalous signal ?? as it 
  is said For zero signal d'/sig and d/sig should be about 0.80. Then 
  in the present case is there really a signal or can be assumed no 
  signal..we are expecting one Ca atom bound to the protein at its active 
  site..the redundancy of the data is 11.6..with this signal strength can we 
  assume Ca to be present there or whatever little anomalous if present is 
  due to something elseor there is no signal at all ??...
 
  Resl.   Inf - 8.0 - 6.0 - 5.0 - 4.0 - 3.8 - 3.6 - 3.4 - 3.2 - 3.0 - 2.8 - 
  2.60
   N(data) 375   493   580  1319   450   538   679   866  1081  1414  
  1709
   I/sig58.8  38.6  32.6  38.3  27.7  27.2  21.9  18.4  12.6   9.5   
  6.1
   %Complete  94.7  99.0  99.3  99.5 100.0  99.6  99.7  99.8  99.6  99.6  
  90.9
   d/sig   1.65  1.27  1.18  1.25  1.19  1.12  1.11  1.11  0.97  1.02  
  1.05
 
  --
  Regards
 
  Faisal
  School of Life Sciences
  JNU
 
 
 
 --
 Dr Tim Gruene
 Institut fuer anorganische Chemie
 Tammannstr. 4
 D-37077 Goettingen
 
 GPG Key ID = A46BEE1A
 
 
 
 
 -- 
 Regards
 
 Faisal
 School of Life Sciences
 JNU
 
 223_aimless.log

[ccp4bb] anomalous signal

[Faisal Tarique]

Dear all

written below is the log file of an anomalous data processed through
SHELXC..my question is ..what is the strength of anomalous signal ?? as it
is said For zero signal d'/sig and d/sig should be about 0.80. Then
in the present case is there really a signal or can be assumed no
signal..we are expecting one Ca atom bound to the protein at its active
site..the redundancy of the data is 11.6..with this signal strength can we
assume Ca to be present there or whatever little anomalous if present is
due to something elseor there is no signal at all ??...

Resl.   Inf - 8.0 - 6.0 - 5.0 - 4.0 - 3.8 - 3.6 - 3.4 - 3.2 - 3.0 - 2.8 -
2.60
 N(data) 375   493   580  1319   450   538   679   866  1081  1414  1709
 I/sig58.8  38.6  32.6  38.3  27.7  27.2  21.9  18.4  12.6   9.5   6.1
 %Complete  94.7  99.0  99.3  99.5 100.0  99.6  99.7  99.8  99.6  99.6  90.9
 d/sig   1.65  1.27  1.18  1.25  1.19  1.12  1.11  1.11  0.97  1.02  1.05



-- 
Regards

Faisal
School of Life Sciences
JNU

[ccp4bb] anomalous signal

[Faisal Tarique]

Dear all

sorry about my previous mail where i forgot to mention that the data was
collected on home source at Cuk alpha and at 1.54A.

written below is the log file of an anomalous data processed through
SHELXC..my question is ..what is the strength of anomalous signal ?? as it
is said For zero signal d'/sig and d/sig should be about 0.80. Then
in the present case is there really a signal or can be assumed no
signal..we are expecting one Ca atom bound to the protein at its active
site..the redundancy of the data is 11.6..with this signal strength can we
assume Ca to be present there or whatever little anomalous if present is
due to something elseor there is no signal at all ??...

Resl.   Inf - 8.0 - 6.0 - 5.0 - 4.0 - 3.8 - 3.6 - 3.4 - 3.2 - 3.0 - 2.8 -
2.60
 N(data) 375   493   580  1319   450   538   679   866  1081  1414  1709
 I/sig58.8  38.6  32.6  38.3  27.7  27.2  21.9  18.4  12.6   9.5   6.1
 %Complete  94.7  99.0  99.3  99.5 100.0  99.6  99.7  99.8  99.6  99.6  90.9
 d/sig   1.65  1.27  1.18  1.25  1.19  1.12  1.11  1.11  0.97  1.02  1.05

-- 
Regards

Faisal
School of Life Sciences
JNU

Re: [ccp4bb] anomalous signal

[Jim Pflugrath]

d/sig should be above 0.80

There seems to be plenty of signal there with all values above 1.02.  We have 
solved structures with less multiplicity and lower d/sig.

There is a different criteria of signal for when you know the positions of 
the anomalous substructure atoms and when you need to find the positions of the 
anomalous substructure atoms.

As for no signal, I think I am on record that there is always an anomalous 
signal. :)  But can you detect it?

Jim


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Faisal Tarique 
[faisaltari...@gmail.com]
Sent: Friday, April 25, 2014 4:06 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] anomalous signal

Dear all

sorry about my previous mail where i forgot to mention that the data was 
collected on home source at Cuk alpha and at 1.54A.

written below is the log file of an anomalous data processed through SHELXC..my 
question is ..what is the strength of anomalous signal ?? as it is said For 
zero signal d'/sig and d/sig should be about 0.80. Then in the present 
case is there really a signal or can be assumed no signal..we are expecting one 
Ca atom bound to the protein at its active site..the redundancy of the data is 
11.6..with this signal strength can we assume Ca to be present there or 
whatever little anomalous if present is due to something elseor there is no 
signal at all ??...

Resl.   Inf - 8.0 - 6.0 - 5.0 - 4.0 - 3.8 - 3.6 - 3.4 - 3.2 - 3.0 - 2.8 - 2.60
 N(data) 375   493   580  1319   450   538   679   866  1081  1414  1709
 I/sig58.8  38.6  32.6  38.3  27.7  27.2  21.9  18.4  12.6   9.5   6.1
 %Complete  94.7  99.0  99.3  99.5 100.0  99.6  99.7  99.8  99.6  99.6  90.9
 d/sig   1.65  1.27  1.18  1.25  1.19  1.12  1.11  1.11  0.97  1.02  1.05

--
Regards

Faisal
School of Life Sciences
JNU

[ccp4bb] anomalous signal for Mg and Calcium

[Faisal Tarique]

Dear all

Just in the continuation of my previous mail i again want to ask few
question on the metalloprotiens..Apart from factors like occupancy, B
factor, coordination sphere and metal ion-ligand distances to distinguish
Mg or calcium, can anomalous signal  tell the identity and the type of
metal ion bound to the protein,  specifically in the case of Mg and
Calcium..An anomalous data analyzed through Xtriage (phenix) gives a signal
of 0.097 with Magnesium while the same gives a signal of 0.1062 with
Caclium ( both data sets showing Anomalous flag as true )..can anybody shed
some light on which is more true ?? the data has maximum resolution of 2.6A
and i had kept Mg atom at the active site (  protein was incubated with 5mM
MgCl2)..just because it is not matching a typical octahedral geometry and
exact metal ion-oxygen distance as represented by Cambridge structural
database (CSD) my reviewer has asked me to check anomalous signal for both
Mg and Ca and ( he is expecting that scattering metal ion it to be Ca )
give appropriate reason for putting Mg there..please give suggestions..

your help would be greatly appreciated

-- 
Regards

Faisal
School of Life Sciences
JNU

Re: [ccp4bb] anomalous signal for Mg and Calcium

[Nat Echols]

On Mon, Apr 21, 2014 at 3:36 PM, Faisal Tarique faisaltari...@gmail.comwrote:

 Just in the continuation of my previous mail i again want to ask few
 question on the metalloprotiens..Apart from factors like occupancy, B
 factor, coordination sphere and metal ion-ligand distances to distinguish
 Mg or calcium, can anomalous signal  tell the identity and the type of
 metal ion bound to the protein,  specifically in the case of Mg and Calcium.


Short answer: if you see a peak in the anomalous difference map, it's
almost certainly calcium, but if you don't see a peak, you still can't rule
out calcium.

Longer answer: magnesium almost never has observable anomalous signal at
the wavelengths we normally use for data collection.  The exception is if
you collect extremely redundant data; Wayne Hendrickson has a very
convincing example of this (I saw it in a talk, but I'll see if I can find
a reference).  Calcium anomalous signal depends on the data quality, but
with good data and full occupancy it can show up in the anomalous
difference map even at the SeMet K edge (~0.9794Å).  However, this is not
guaranteed, especially if it's not very tightly bound.  At 2.6Å resolution
it may be more difficult to distinguish, especially if you have other
stronger anomalous scatterers.  Collecting very redundant data will help a
lot.

.An anomalous data analyzed through Xtriage (phenix) gives a signal of
 0.097 with Magnesium while the same gives a signal of 0.1062 with Caclium (
 both data sets showing Anomalous flag as true )..can anybody shed some
 light on which is more true ??


I don't understand this - what exactly is the difference between the
datasets?  Anyway, that number is really not intended to be interpreted
this way.


 the data has maximum resolution of 2.6A and i had kept Mg atom at the
 active site (  protein was incubated with 5mM MgCl2)..just because it is
 not matching a typical octahedral geometry and exact metal ion-oxygen
 distance as represented by Cambridge structural database (CSD) my reviewer
 has asked me to check anomalous signal for both Mg and Ca and ( he is
 expecting that scattering metal ion it to be Ca ) give appropriate reason
 for putting Mg there..please give suggestions.


In addition to the anomalous maps, check the difference map (Fo-Fc) and
B-factors after refinement with either element at full occupancy.  If it is
correctly identified, the difference map should be relatively flat and the
B-factor should be similar to the coordinating atoms.  Negative difference
map peaks and/or a high B-factor suggest that the element is too heavy;
positive peaks and/or low B-factors indicate the opposite.

-Nat

Re: [ccp4bb] anomalous signal for Mg and Calcium

[Jim Pflugrath]

Further to what Nat wrote which I completely agree with, you should tell us the 
following:

1. Expecting signal of a Calcium atom and expected signal of a Magnesium atom.

2. Are there any intrinsic anomalous scatterers in the structure that you trust 
such as sulfurs from methionines and cysteines or even selenium at 
selenomethionines?  What are their expected signals and do you see those 
signals?  If not, why not?  Basically, these should give one a positive control 
which they can check their experiment with.


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Faisal Tarique 
[faisaltari...@gmail.com]
Sent: Monday, April 21, 2014 5:36 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] anomalous signal for Mg and Calcium

Dear all

Just in the continuation ...

Re: [ccp4bb] Anomalous signal for chlorides

[Eleanor Dodson]

There are many ways of course to do this..
But if you are using the CCP4 GUI, refmac refinement it is a 2 step process.

CAD to add the DANO SIGDANO columns from the input data back into the 
refmac output


fft
 Select anomalous map

Use DANO SIGDANO PHIC FOM asinput

Switch on the peak search

Read that pdb into coot and check that the highest peaks are on 
anomalous scatterers - you usually find the S are visible too..


 If the data is reasonable I do it routinely to verify results - are 
the SO4s really sulfates etc etc..


Eleanor



 On 11/26/2011 11:18 AM, George Sheldrick wrote:

Dear Yuri,

The new program ANODE (J.Appl.Cryst. (2011) 44, 1285-7, Open Access) is
designed for this and is very simple to use. It may be downloaded from
the SHELX beta-test server (please email me if you require downloading
intructions).

George

On 11/26/2011 06:05 AM, Yuri Pompeu wrote:

Hi Boaz,
Yes indeed, the phosphate group of the molecule looks quite beautiful
at 1.17A and it has a really big peak 18sigma!
Is there a utility for calculating anomalous maps, or is it simply an
option in the refinement program?

Re: [ccp4bb] Anomalous signal for chlorides

[George Sheldrick]

Dear Yuri,

The new program ANODE (J.Appl.Cryst. (2011) 44, 1285-7, Open Access) is 
designed for this and is very simple to use. It may be downloaded from 
the SHELX beta-test server (please email me if you require downloading 
intructions).


George

On 11/26/2011 06:05 AM, Yuri Pompeu wrote:

Hi Boaz,
Yes indeed, the phosphate group of the molecule looks quite beautiful at 1.17A 
and it has a really big peak 18sigma!
Is there a utility for calculating anomalous maps, or is it simply an option in 
the refinement program?

Re: [ccp4bb] Anomalous signal for chlorides

[Randy J. Read]

Dear Yuri,

In addition to the other good options that have been presented, you can use 
the log-likelihood gradient maps in Phaser to find anomalous scatterers. We 
find this to be very sensitive, and it has the advantage of being iterative 
(i.e. when you find some anomalous scatterers, this improves your model and 
thus the sensitivity for finding additional sites).


When you're starting from a refined model, we think it is best to look for 
purely imaginary scatterers to add to the real scattering in your model. In 
the ccp4i GUI, choose the SAD with molecular replacement partial 
structure mode, provide the data (with F+ and F-), the wavelength, and the 
current model, then turn on LLG-map completion and select the Complete 
with purely anomalous scatterer option.


If you want to see the initial LLG map, set the number of completion cycles 
to 0 and turn on the option to output log-likelihood-gradient map 
coefficients. Open these in coot as a difference map, choosing the columns 
FLLG_AX and PHLLG_AX. If you let Phaser complete the sites, then the final 
LLG map should be nearly flat and you have to look at the PDB file 
containing the sites that it found.


Best wishes,

Randy Read
On Nov 26 2011, Yuri Pompeu wrote:

Hi Boaz, Yes indeed, the phosphate group of the molecule looks quite 
beautiful at 1.17A and it has a really big peak 18sigma! Is there a 
utility for calculating anomalous maps, or is it simply an option in the 
refinement program?




--
Randy J. Read

Re: [ccp4bb] Anomalous signal for chlorides

[Jacob Keller]

Not that Phaser needs my approval, but I recently did exactly what
Randy recommended and it really found basically all of the S and Cl
sites, with data at resolution 2.2 Ang and wavelength 0.979 Ang, too.
I also played a bit with the sigma cutoff for adding new sites so that
the stronger sites (Se in my case) are found but the weaker ones not.
Also, don't forget to click the output LLG map coefficients option
to get the right columns in your mtz.

Jacob

On Sat, Nov 26, 2011 at 6:13 AM, Randy J. Read rj...@cam.ac.uk wrote:
 Dear Yuri,

 In addition to the other good options that have been presented, you can use
 the log-likelihood gradient maps in Phaser to find anomalous scatterers. We
 find this to be very sensitive, and it has the advantage of being iterative
 (i.e. when you find some anomalous scatterers, this improves your model and
 thus the sensitivity for finding additional sites).

 When you're starting from a refined model, we think it is best to look for
 purely imaginary scatterers to add to the real scattering in your model. In
 the ccp4i GUI, choose the SAD with molecular replacement partial structure
 mode, provide the data (with F+ and F-), the wavelength, and the current
 model, then turn on LLG-map completion and select the Complete with purely
 anomalous scatterer option.

 If you want to see the initial LLG map, set the number of completion cycles
 to 0 and turn on the option to output log-likelihood-gradient map
 coefficients. Open these in coot as a difference map, choosing the columns
 FLLG_AX and PHLLG_AX. If you let Phaser complete the sites, then the final
 LLG map should be nearly flat and you have to look at the PDB file
 containing the sites that it found.

 Best wishes,

 Randy Read
 On Nov 26 2011, Yuri Pompeu wrote:

 Hi Boaz, Yes indeed, the phosphate group of the molecule looks quite
 beautiful at 1.17A and it has a really big peak 18sigma! Is there a utility
 for calculating anomalous maps, or is it simply an option in the refinement
 program?


 --
 Randy J. Read




-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] Anomalous signal for chlorides

[Boaz Shaanan]

Hi,

You could calculate an anomalous difference map using your current phases and 
see whether you see any peaks around the atoms you suspect are Cl, S or P 
(although the latter should have a clear tetrahedral geometry, certainly at 
your resolution). It all depends of course on the wavelength at which you 
collected your data (check Ethan Merritt's site), but again at your resolution 
you may very well hit lucky.

   Cheers,

  Boaz


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

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






From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Yuri Pompeu 
[yuri.pom...@ufl.edu]
Sent: Saturday, November 26, 2011 1:31 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Anomalous signal for chlorides

Hello everyone,
I have a good data set to 1.17A that I solved by MR.
I come accross some sites that appear to be chlorides. I was
wondering if they could have some anomalous signal.
I also have a well ordered phosphate  and a couple of S from Met´s.
How do I go about probing the signal from these?
Thank you. Best,

Re: [ccp4bb] Anomalous signal for chlorides

[Yuri Pompeu]

Hi Boaz, 
Yes indeed, the phosphate group of the molecule looks quite beautiful at 1.17A 
and it has a really big peak 18sigma!
Is there a utility for calculating anomalous maps, or is it simply an option in 
the refinement program?

Re: [ccp4bb] anomalous signal of Mn and Ca ions

[Nave, C (Colin)]

Dear all
If you simply want to see if Ca and Mn are in your sample, you could
also use x-ray fluorescence excited by the x-rays on the beamline used
to collect the diffraction data. It doesn't need the energy set to be at
each absorption edge, it just has to be above the edges. One then sets
the fluorescence detector (used for wavelength setting in MAD
experiments) to MCA mode, giving a spectrum showing the characteristic
lines for each element present. 

I believe the PIXIE technique has certain advantages. However it is not
available on the beamlines used to collect the data.

Of course, I think the question related to whether specific peaks in a
map were Mn or Ca. Then, as stated before, diffraction data collection
on either side of the Mn edge should reveal all, with good data of
sufficient resolution.

The x-ray fluorescence technique was used at the SSRL to reveal details
of the Archimedes Palimsest. This showed there were in fact two geniuses
coming from Greece -  not just Tassos.

Cheers
   Colin



-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Anastassis Perrakis
Sent: 29 February 2008 10:06
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] anomalous signal of Mn and Ca ions

On Feb 29, 2008, at 10:29, Johan Turkenburg wrote:

 Hi,

 You need to firstly check that you did the map calculation correctly, 
 see comments below:

 Sun Tang wrote:
 Dear All,
  In my structures, I want to assign Mn or Ca ions for some densities.

 But when I did not have  anomalous density in CCP4i. I am not sure 
 whether I was correct. The following was what I did:
  I processed the data with HKL2000 and select anomalous signal in 
 scaling. In CCP4i, I selected Run FFT-Creat Map in the Map Mask 
 Utilities.

 What labels did you assign, and did you select 'anomalous map'?  
 This is crucial, as the protein (model) phase needs to be shifted by 
 90 degrees, and this is done 'automatically' by FFT when you ask for 
 an anomalous map. Certainly with data of sufficient quality collected 
 on a home source with Cu radiation anomalous maps may well show up 
 such ions.


Exactly! Thats the most important bit to get right.

Another comment is that if you want to be sure about the nature of the
ions, you might want to try PIXE.

http://www.ee.surrey.ac.uk/IBC/index.php?target=13:68

I think all other suggestions are of course valid, but, without being
sure, the X-ray suggestions I see, need high resolution data ? PIXE is a
more direct method if it works well for your sample.

A.


 I select O format to cover asymmetric unit and Plot
 section on Z axis from 0 to 1 in steps on 10. All others were by  
 default values. I display in ono10.
  I collected the data at the wavelength of 1 A. Do I need to  
 adjust the wavelength to maximize the anomalous signal from Mn or Ca?
  Any ideas and suggestions are greatly appreciated!
  Sun Tang
 -

 ---
 Never miss a thing. Make Yahoo your homepage. http:// 
 us.rd.yahoo.com/evt=51438/*http://www.yahoo.com/r/hs

 -- 
 +
 Dr. Johan P. Turkenburg X-ray facilities manager
 York Structural Biology Laboratory
 University of York  Phone (+) 44 1904 328251
 York YO10 5DD   UK  Fax   (+) 44 1904 328266
 +
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Re: [ccp4bb] anomalous signal of Mn and Ca ions

[Andrzej Olczak]

You can also calculate expected anomalous scattering  signal 
|Delta F| / F at low resolution range
for your structure for different wavelenghts using a webpage: 

http://alfa.p.lodz.pl/assc/

The webpage is not in its final form and still under development, but should 
work OK.
(abreviation SOF used on this webpage stands for site occupancy factor).

You can then compare the calculated value of |Delta F| / F with the 
experimental one (which should be  greater because of the  experimental 
errors) to get some notion about how large  the signal is in comparison to 
the experimental errors. 

Regards,
Andrzej Olczak



 Dear all
 If you simply want to see if Ca and Mn are in your sample, you could
 also use x-ray fluorescence excited by the x-rays on the beamline used
 to collect the diffraction data. It doesn't need the energy set to be at
 each absorption edge, it just has to be above the edges. One then sets
 the fluorescence detector (used for wavelength setting in MAD
 experiments) to MCA mode, giving a spectrum showing the characteristic
 lines for each element present.

 I believe the PIXIE technique has certain advantages. However it is not
 available on the beamlines used to collect the data.

 Of course, I think the question related to whether specific peaks in a
 map were Mn or Ca. Then, as stated before, diffraction data collection
 on either side of the Mn edge should reveal all, with good data of
 sufficient resolution.

 The x-ray fluorescence technique was used at the SSRL to reveal details
 of the Archimedes Palimsest. This showed there were in fact two geniuses
 coming from Greece -  not just Tassos.

 Cheers
Colin



 -Original Message-
 From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
 Anastassis Perrakis
 Sent: 29 February 2008 10:06
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] anomalous signal of Mn and Ca ions

 On Feb 29, 2008, at 10:29, Johan Turkenburg wrote:
  Hi,
 
  You need to firstly check that you did the map calculation correctly,
  see comments below:
 
  Sun Tang wrote:
  Dear All,
   In my structures, I want to assign Mn or Ca ions for some densities.
 
  But when I did not have  anomalous density in CCP4i. I am not sure
  whether I was correct. The following was what I did:
   I processed the data with HKL2000 and select anomalous signal in
  scaling. In CCP4i, I selected Run FFT-Creat Map in the Map Mask
  Utilities.
 
  What labels did you assign, and did you select 'anomalous map'?
  This is crucial, as the protein (model) phase needs to be shifted by
  90 degrees, and this is done 'automatically' by FFT when you ask for
  an anomalous map. Certainly with data of sufficient quality collected
  on a home source with Cu radiation anomalous maps may well show up
  such ions.

 Exactly! Thats the most important bit to get right.

 Another comment is that if you want to be sure about the nature of the
 ions, you might want to try PIXE.

 http://www.ee.surrey.ac.uk/IBC/index.php?target=13:68

 I think all other suggestions are of course valid, but, without being
 sure, the X-ray suggestions I see, need high resolution data ? PIXE is a
 more direct method if it works well for your sample.

   A.

  I select O format to cover asymmetric unit and Plot
 
  section on Z axis from 0 to 1 in steps on 10. All others were by
  default values. I display in ono10.
   I collected the data at the wavelength of 1 A. Do I need to
  adjust the wavelength to maximize the anomalous signal from Mn or Ca?
   Any ideas and suggestions are greatly appreciated!
   Sun Tang
  -
 
  ---
  Never miss a thing. Make Yahoo your homepage. http://
  us.rd.yahoo.com/evt=51438/*http://www.yahoo.com/r/hs
 
  --
  +
  Dr. Johan P. Turkenburg X-ray facilities manager
  York Structural Biology Laboratory
  University of York  Phone (+) 44 1904 328251
  York YO10 5DD   UK  Fax   (+) 44 1904 328266
  +

 DIVFONT size=1 color=grayThis e-mail and any attachments may
 contain confidential, copyright and or privileged material, and are for the
 use of the intended addressee only. If you are not the intended addressee
 or an authorised recipient of the addressee please notify us of receipt by
 returning the e-mail and do not use, copy, retain, distribute or disclose
 the information in or attached to the e-mail. Any opinions expressed within
 this e-mail are those of the individual and not necessarily of Diamond
 Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this
 e-mail or any attachments are free from viruses and we cannot accept
 liability for any damage which you may sustain as a result of software
 viruses which may be transmitted in or with the message. Diamond Light
 Source Limited

Re: [ccp4bb] anomalous signal of Mn and Ca ions

[Stephen Graham]

As Ethan mentions, your best bet for an 100% positive ID would be to
collect a dataset above and below the Mn edge and see the anomalous
signal from the Mn atom disappear.

If that is not an option, there is a great paper by George Sheldrick
and friends regards using the Calcium Bond Valence Sum to determine
the nature of a metal based on its coordination geometry.  [P. Müller,
S. Köpke and G. M. Sheldrick (2003) Is the bond-valence method able to
identify metal atoms in protein structures? Acta Cryst. D59, 32-37].

shameless self-plug You could also have a look at one of my papers
where we applied this (and integration of the anomalous difference
density around the position of the scatterer, scaled against the
Sulfur signal of the Met's and Cys'es in the protein) to confirm metal
loading of a metalloenzyme [Graham, S, Bond, C, Freeman, H, Guss, J.
(2005) Structural and functional implications of metal ion selection
in aminopeptidase P, a metalloprotease with a dinuclear metal center.
Biochemistry.44: 13820-36]

Good luck,

Stephen

On 2/29/08, Sun Tang [EMAIL PROTECTED] wrote:
 Dear All,

 In my structures, I want to assign Mn or Ca ions for some densities. But
 when I did not have  anomalous density in CCP4i. I am not sure whether I was
 correct. The following was what I did:

 I processed the data with HKL2000 and select anomalous signal in scaling. In
 CCP4i, I selected Run FFT-Creat Map in the Map Mask Utilities. I select
 O format to cover asymmetric unit and Plot section on Z axis from 0 to 1
 in steps on 10. All others were by default values. I display in ono10.

 I collected the data at the wavelength of 1 A. Do I need to adjust the
 wavelength to maximize the anomalous signal from Mn or Ca?

 Any ideas and suggestions are greatly appreciated!

 Sun Tang

  
 Never miss a thing. Make Yahoo your homepage.




-- 
Dr Stephen Graham
Nuffield Medical Fellow
Division of Structural Biology
Wellcome Trust Centre for Human Genetics
Roosevelt Drive
Oxford OX3 7BN
United Kingdom
Phone: +44 1865 287 549

Re: [ccp4bb] anomalous signal of Mn and Ca ions

[Johan Turkenburg]

Hi,

You need to firstly check that you did the map calculation correctly, 
see comments below:


Sun Tang wrote:

Dear All,
 
In my structures, I want to assign Mn or Ca ions for some densities. But 
when I did not have  anomalous density in CCP4i. I am not sure whether I 
was correct. The following was what I did:
 
I processed the data with HKL2000 and select anomalous signal in 
scaling. In CCP4i, I selected Run FFT-Creat Map in the Map Mask 
Utilities. 


What labels did you assign, and did you select 'anomalous map'? This is 
crucial, as the protein (model) phase needs to be shifted by 90 degrees, 
and this is done 'automatically' by FFT when you ask for an anomalous 
map. Certainly with data of sufficient quality collected on a home 
source with Cu radiation anomalous maps may well show up such ions.


I select O format to cover asymmetric unit and Plot
section on Z axis from 0 to 1 in steps on 10. All others were by 
default values. I display in ono10.
 
I collected the data at the wavelength of 1 A. Do I need to adjust the 
wavelength to maximize the anomalous signal from Mn or Ca?
 
Any ideas and suggestions are greatly appreciated!
 
Sun Tang



Never miss a thing. Make Yahoo your homepage. 
http://us.rd.yahoo.com/evt=51438/*http://www.yahoo.com/r/hs


--
+
Dr. Johan P. Turkenburg X-ray facilities manager
York Structural Biology Laboratory  
University of York  Phone (+) 44 1904 328251
York YO10 5DD   UK  Fax   (+) 44 1904 328266
+

Re: [ccp4bb] anomalous signal of Mn and Ca ions

[Anastassis Perrakis]

On Feb 29, 2008, at 10:29, Johan Turkenburg wrote:


Hi,

You need to firstly check that you did the map calculation  
correctly, see comments below:


Sun Tang wrote:

Dear All,
 In my structures, I want to assign Mn or Ca ions for some  
densities. But when I did not have  anomalous density in CCP4i. I  
am not sure whether I was correct. The following was what I did:
 I processed the data with HKL2000 and select anomalous signal in  
scaling. In CCP4i, I selected Run FFT-Creat Map in the Map  
Mask Utilities.


What labels did you assign, and did you select 'anomalous map'?  
This is crucial, as the protein (model) phase needs to be shifted  
by 90 degrees, and this is done 'automatically' by FFT when you ask  
for an anomalous map. Certainly with data of sufficient quality  
collected on a home source with Cu radiation anomalous maps may  
well show up such ions.




Exactly! Thats the most important bit to get right.

Another comment is that if you want to be sure about the nature of  
the ions, you might want to try PIXE.


http://www.ee.surrey.ac.uk/IBC/index.php?target=13:68

I think all other suggestions are of course valid, but, without being  
sure, the X-ray suggestions I see, need high resolution data ? PIXE  
is a more direct method if it works well for your sample.


A.



I select O format to cover asymmetric unit and Plot
section on Z axis from 0 to 1 in steps on 10. All others were by  
default values. I display in ono10.
 I collected the data at the wavelength of 1 A. Do I need to  
adjust the wavelength to maximize the anomalous signal from Mn or Ca?

 Any ideas and suggestions are greatly appreciated!
 Sun Tang
- 
---
Never miss a thing. Make Yahoo your homepage. http:// 
us.rd.yahoo.com/evt=51438/*http://www.yahoo.com/r/hs


--
+
Dr. Johan P. Turkenburg X-ray facilities manager
York Structural Biology Laboratory  
University of York  Phone (+) 44 1904 328251
York YO10 5DD   UK  Fax   (+) 44 1904 328266
+

[ccp4bb] anomalous signal of Mn and Ca ions

[Sun Tang]

Dear All,
   
  In my structures, I want to assign Mn or Ca ions for some densities. But when 
I did not have  anomalous density in CCP4i. I am not sure whether I was 
correct. The following was what I did:
   
  I processed the data with HKL2000 and select anomalous signal in scaling. In 
CCP4i, I selected Run FFT-Creat Map in the Map Mask Utilities. I select O 
format to cover asymmetric unit and Plot section on Z axis from 0 to 1 in 
steps on 10. All others were by default values. I display in ono10.
   
  I collected the data at the wavelength of 1 A. Do I need to adjust the 
wavelength to maximize the anomalous signal from Mn or Ca?
   
  Any ideas and suggestions are greatly appreciated!
   
  Sun Tang

   
-
Never miss a thing.   Make Yahoo your homepage.

Re: [ccp4bb] anomalous signal of Mn and Ca ions

[Ethan A Merritt]

On Thursday 28 February 2008 19:54, Sun Tang wrote:
 Dear All,

   In my structures, I want to assign Mn or Ca ions for some densities. 
   But when I did not have  anomalous density in CCP4i. 
[snip]
   I collected the data at the wavelength of 1 A. 

Mn has only about 1e of anomalous scattering (f) power at 1A.
Ca has essentially 0.
So you should not expect to see any peaks in your map.

   Do I need to adjust the wavelength to maximize the anomalous signal from Mn 
 or Ca? 

Yes. 
To distinguish between them you would need to select an X-ray energy
between their respective K-absorption edges.
You can use the X-ray Anomalous Scattering server to help you:
http://skuld.bmsc.washington.edu/scatter/AS_form.html

This will tell you that you would need an X-ray energy less than the Mn K-edge
at  6.5390 keV   (1.8961 Angstrom)
http://skuld.bmsc.washington.edu/scatter/AS_periodic.html



   I am not sure whether I was correct. The following was what I did:  

   I processed the data with HKL2000 and select anomalous signal in scaling. 
 In CCP4i, I selected Run FFT-Creat Map in the Map Mask Utilities. I 
 select O format to cover asymmetric unit and Plot section on Z axis from 0 
 to 1 in steps on 10. All others were by default values. I display in ono10.

   I collected the data at the wavelength of 1 A. Do I need to adjust the 
 wavelength to maximize the anomalous signal from Mn or Ca?

   Any ideas and suggestions are greatly appreciated!

   Sun Tang
 

-- 
Ethan A Merritt
Biomolecular Structure Center
University of Washington, Seattle 98195-7742

Re: [ccp4bb] anomalous signal

[Harry Powell]

Hi

In the same way the Toyota MRD-2 doesn't sell in France?

MRD would indeed be a poor name. For French scientists it might be difficult 
to get funding for an MRD experiment

Regards
Yves


--

Prof. Yves Muller   Phone: +49-(0)-9131-8523082, 8523081
Lehrstuhl fuer BiotechnikFAX:   +49-(0)-9131-8523080
www.biologie.uni-erlangen.de/biotechnik Institut fuer Biologie
Friedrich-Alexander-Universität, Erlangen-Nuernberg
Im IZMP, Henkestrasse 91, D-91052 Erlangen 





Harry
--
Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills
Road, Cambridge, CB2 2QH

[ccp4bb] anomalous signal

[Yves Muller]

ps perhaps anomalous is better than resonant, as it produces MAD and SAD and not 
MRD and
SRD...


MRD would indeed be a poor name. For French scientists it might be difficult to 
get funding for an MRD experiment
Regards
Yves


--

Prof. Yves Muller   Phone: +49-(0)-9131-8523082, 8523081
Lehrstuhl fuer BiotechnikFAX:   +49-(0)-9131-8523080
www.biologie.uni-erlangen.de/biotechnik 
Institut fuer Biologie

Friedrich-Alexander-Universität, Erlangen-Nuernberg
Im IZMP, Henkestrasse 91, D-91052 Erlangen