Re: [ccp4bb] Reasoning for Rmeas or Rpim as Cutoff
Hi Jacob, I think a lot of people do use I/sigma :o) However sigma is in some cases poorly defined, and the merging residuals you refer to are calculated only from the I values and are related to I/sigma anyhow... Of course the R values are sometimes also poorly defined for low multiplicity data. Best wishes, Graeme On 27 January 2012 17:55, Jacob Keller wrote: > Clarification: I did not mean I/sigma of 2 per se, I just meant > I/sigma is more directly a measure of signal than R values. > > JPK > > On Fri, Jan 27, 2012 at 11:47 AM, Jacob Keller > wrote: >> Dear Crystallographers, >> >> I cannot think why any of the various flavors of Rmerge/meas/pim >> should be used as a data cutoff and not simply I/sigma--can somebody >> make a good argument or point me to a good reference? My thinking is >> that signal:noise of >2 is definitely still signal, no matter what the >> R values are. Am I wrong? I was thinking also possibly the R value >> cutoff was a historical accident/expedient from when one tried to >> limit the amount of data in the face of limited computational >> power--true? So perhaps now, when the computers are so much more >> powerful, we have the luxury of including more weak data? >> >> JPK >> >> >> -- >> *** >> Jacob Pearson Keller >> Northwestern University >> Medical Scientist Training Program >> email: j-kell...@northwestern.edu >> *** > > > > -- > *** > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > email: j-kell...@northwestern.edu > ***
Re: [ccp4bb] MAD
Ian,
If you visit Isaac Newton's old home at Woolsthorpe (near here) you will
see a conflicting claim for location of the classic prism experiment. You
will also find an apple tree in the garden, but that is another story..
Peter
PS this is my special ccp4bb email account, it doesn't always get the
attention it deserves.
On 19 January 2012 17:50, Ian Tickle wrote:
> Perhaps I could chime in with a bit of history as I understand it.
>
> The term 'dispersion' in optics, as everyone who knows their history
> is aware of, refers to the classic experiment by Sir Isaac Newton at
> Trinity College here in Cambridge where he observed white light being
> split up ('dispersed') into its component colours by a prism. This is
> of course due to the variation in refractive index of glass with
> wavelength, so then we arrive at the usual definition of optical
> dispersion as dn/dlambda, i.e. the first derivative of the refractive
> index with respect to the wavelength.
>
> Now the refractive index of an average crystal at around 1 Ang
> wavelength differs by about 1 part in a million from 1, however it can
> be determined by very careful and precise interferometric experiments.
> It's safe to say therefore that the dispersion of X-rays (anomalous
> or otherwise) has no measurable effect whatsoever as far as the
> average X-ray diffraction experiment (SAD, MAD or otherwise) is
> concerned. The question then is how did the term 'anomalous
> dispersion' get to be applied to X-ray diffraction? The answer is
> that it turns out that the equation ('Kramer-Kronig relationship')
> governing X-ray scattering is completely analogous to that governing
> optical dispersion, so it's legitimate to use the term 'dispersive'
> (meaning 'analogous to dispersion') for the real part of the
> wavelength-dependent component of the X-ray scattering factor, because
> the real part of the refractive index is what describes dispersion
> (the imaginary part in both cases describes absorption).
>
> So then from 'dispersive' to 'dispersion' to describe the wavelength
> dependence of X-ray scattering is only a short step, even though it
> only behaves _like_ dispersion in its dependence on wavelength.
> However having two different meanings for the same word can get
> confusing and clearly should be avoided if at all possible.
>
> So what does this have to do with the MAD acronym? I think it stemmed
> from a visit by Wayne Hendrickson to Birkbeck in London some time
> around 1990: he was invited by Tom Blundell to give a lecture on his
> MAD experiments. At that time Wayne called it multi-wavelength
> anomalous dispersion. Tom pointed out that this was really a misnomer
> for the reasons I've elucidated above. Wayne liked the MAD acronym
> and wanted to keep it so he needed a replacement term starting with D
> and diffraction was the obvious choice, and if you look at the
> literature from then on Wayne at least consistently called it
> multi-wavelength anomalous diffraction.
>
> Cheers
>
> -- Ian
>
> On 18 January 2012 18:23, Phil Jeffrey wrote:
> > Can I be dogmatic about this ?
> >
> > Multiwavelength anomalous diffraction from Hendrickson (1991) Science
> Vol.
> > 254 no. 5028 pp. 51-58
> >
> > Multiwavelength anomalous diffraction (MAD) from the CCP4 proceedings
> > http://www.ccp4.ac.uk/courses/proceedings/1997/j_smith/main.html
> >
> > Multi-wavelength anomalous-diffraction (MAD) from Terwilliger Acta Cryst.
> > (1994). D50, 11-16
> >
> > etc.
> >
> >
> > I don't see where the problem lies:
> >
> > a SAD experiment is a single wavelength experiment where you are using
> the
> > anomalous/dispersive signals for phasing
> >
> > a MAD experiment is a multiple wavelength version of SAD. Hopefully one
> > picks an appropriate range of wavelengths for whatever complex case one
> has.
> >
> > One can have SAD and MAD datasets that exploit anomalous/dispersive
> signals
> > from multiple difference sources. This after all is one of the things
> that
> > SHARP is particularly good at accommodating.
> >
> > If you're not using the anomalous/dispersive signals for phasing, you're
> > collecting native data. After all C,N,O,S etc all have a small anomalous
> > signal at all wavelengths, and metalloproteins usually have even larger
> > signals so the mere presence of a theoretical d" difference does not
> make it
> > a SAD dataset. ALL datasets contain some anomalous/dispersive signals,
> most
> > of the time way down in the noise.
> >
> > Phil Jeffrey
> > Princeton
> >
> >
> >
> > On 1/18/12 12:48 PM, Francis E Reyes wrote:
> >>
> >>
> >> Using the terms 'MAD' and 'SAD' have always been confusing to me when
> >> considering more complex phasing cases. What happens if you have
> intrinsic
> >> Zn's, collect a 3wvl experiment and then derivatize it with SeMet or a
> heavy
> >> atom? Or the MAD+native scenario (SHARP) ?
> >>
> >> Instead of using MAD/SAD nomenclature I favor explicitly stating whether
> >> dispersive/anomalous/isomo
[ccp4bb] protein lost on membrane of centricon!!
Hi all, I tried to concentrate my protein using vivaspin 20 10,000 MWCO PES. The protein was in 50mM Hepes pH 7.5, 500mM KCl and 10% glycerol. I lost about 90% of my protein on the membrane of the centricon. Please suggest some way of concentrating this protein. Will concentrating using peg 20K be a good alternative?? regards rashmi
Re: [ccp4bb] Reasoning for Rmeas or Rpim as Cutoff
Dear Jacob, As an editor I am always mindful that an article is finally under the authors' names. That said the reader always deserves to know at what diffraction resolution average intensities (cease to) exist. The usual statistical practice to do that is to use a given quantity's (ie in this case a reflection intensity) sigma. Good effort is made in data processing programs to protect the quality of the estimate of each reflection intensity's sigma notably the chi square test. Thus I request that the diffraction resolution where crosses 2.0 is quoted in the article, if it is not there already. I agree that 2.0 is arbitrary but it is more 'lenient' than the usual '3sigma' statistical test. Sometimes the title or abstract has to be changed to follow this criterion; eg 'The structure of xxx is determined to 2.4 Angstrom resolution' type of title has to be consistent with the above criterion. I do not follow an 'Rmerge must be less than x% rule'. I think the above follows reasonable general statistical practice, whilst permitting authors reasonable freedom, and also protects the (more innocent) readers of articles. I am aware that the 'correlation coefficient' between randomly portioned parts of data sets is being increasingly discussed, this parameter also having general statistical validity. I am monitoring discussion on this carefully. It has long been a good way of assessing the statistical quality of anomalous differences for example; to my knowledge introduced by Michael Rossmann many years ago. Best wishes, John On Fri, Jan 27, 2012 at 5:55 PM, Jacob Keller wrote: > Clarification: I did not mean I/sigma of 2 per se, I just meant > I/sigma is more directly a measure of signal than R values. > > JPK > > On Fri, Jan 27, 2012 at 11:47 AM, Jacob Keller > wrote: >> Dear Crystallographers, >> >> I cannot think why any of the various flavors of Rmerge/meas/pim >> should be used as a data cutoff and not simply I/sigma--can somebody >> make a good argument or point me to a good reference? My thinking is >> that signal:noise of >2 is definitely still signal, no matter what the >> R values are. Am I wrong? I was thinking also possibly the R value >> cutoff was a historical accident/expedient from when one tried to >> limit the amount of data in the face of limited computational >> power--true? So perhaps now, when the computers are so much more >> powerful, we have the luxury of including more weak data? >> >> JPK >> >> >> -- >> *** >> Jacob Pearson Keller >> Northwestern University >> Medical Scientist Training Program >> email: j-kell...@northwestern.edu >> *** > > > > -- > *** > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > email: j-kell...@northwestern.edu > *** -- Professor John R Helliwell DSc
Re: [ccp4bb] protein lost on membrane of centricon!!
Hello Rashmi, How large are your protein monomer units? Are you expecting these units to have formed an oligomer? As a general rule of thumb, you want your protein molecules to be no smaller than 3x the membrane MWCO. Perhaps all you need is to try concentrating with a lower MWCO membrane, e.g. 3,000. Alternatively, you could try: - change membrane manufacturers (this sometimes does make a difference), - pretreating your membrane (e.g. with BSA) to block all binding sites (but then you'll have to worry about BSA contamination of your sample), - concentrating your protein in under nitrogen pressure (I forget right now what the device is called but I used to use it all the time), - concentrating your protein in a speed vac to evaporate off some of the water, - ammonium sulphate precipitation, - or even TCA precipitation. HTH, C On Sat, Jan 28, 2012 at 10:54 AM, rashmi panigrahi < rashmi.panigrah...@gmail.com> wrote: > Hi all, > I tried to concentrate my protein using vivaspin 20 10,000 MWCO PES. > The protein was in 50mM Hepes pH 7.5, 500mM KCl and 10% glycerol. > I lost about 90% of my protein on the membrane of the centricon. > > Please suggest some way of concentrating this protein. > Will concentrating using peg 20K be a good alternative?? > > regards > > rashmi >
Re: [ccp4bb] protein lost on membrane of centricon!!
P.S. I haven't personally tried concentrating with PEG 20K but I suppose it could work. On Sat, Jan 28, 2012 at 12:41 PM, Cale Dakwar wrote: > > Hello Rashmi, > > How large are your protein monomer units? Are you expecting these units > to have formed an oligomer? As a general rule of thumb, you want your > protein molecules to be no smaller than 3x the membrane MWCO. Perhaps all > you need is to try concentrating with a lower MWCO membrane, e.g. 3,000. > > Alternatively, you could try: > > - change membrane manufacturers (this sometimes does make a difference), > > - pretreating your membrane (e.g. with BSA) to block all binding sites > (but then you'll have to worry about BSA contamination of your sample), > > - concentrating your protein in under nitrogen pressure (I forget right > now what the device is called but I used to use it all the time), > > - concentrating your protein in a speed vac to evaporate off some of the > water, > > - ammonium sulphate precipitation, > > - or even TCA precipitation. > > HTH, > C > > > > > On Sat, Jan 28, 2012 at 10:54 AM, rashmi panigrahi < > rashmi.panigrah...@gmail.com> wrote: > >> Hi all, >> I tried to concentrate my protein using vivaspin 20 10,000 MWCO PES. >> The protein was in 50mM Hepes pH 7.5, 500mM KCl and 10% glycerol. >> I lost about 90% of my protein on the membrane of the centricon. >> >> Please suggest some way of concentrating this protein. >> Will concentrating using peg 20K be a good alternative?? >> >> regards >> >> rashmi >> > >
Re: [ccp4bb] protein lost on membrane of centricon!!
Are you close to the theoretical isoelectric point of your protein ? Change pH of buffer Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Jan 28, 2012, at 10:55, "rashmi panigrahi" wrote: > Hi all, > I tried to concentrate my protein using vivaspin 20 10,000 MWCO PES. > The protein was in 50mM Hepes pH 7.5, 500mM KCl and 10% glycerol. > I lost about 90% of my protein on the membrane of the centricon. > > Please suggest some way of concentrating this protein. > Will concentrating using peg 20K be a good alternative?? > > regards > > rashmi
Re: [ccp4bb] Reasoning for Rmeas or Rpim as Cutoff
Dear all Perhaps a bit off of theme, just an example about resolution cut-off mean I/sigma(I) = 2 for dmin = 3.35 A (please have a look at the attached pdf) I would trust in I/s(I) = 2 (in this case it worked), but why not to determine what is information after the model has been refined to some extent using lower I/s(I) and then cutting the resolution by 0.05-0.1 A? Delta R and (from procheck) Avg-G factors did well. Note that Rfree improved by using data from higher resolution. Perhaps if Rmeas or Rpim were bad at that resolution (3.35 A) the story would be different. Best regards, Horacio Quoting John R Helliwell : Dear Jacob, As an editor I am always mindful that an article is finally under the authors' names. That said the reader always deserves to know at what diffraction resolution average intensities (cease to) exist. The usual statistical practice to do that is to use a given quantity's (ie in this case a reflection intensity) sigma. Good effort is made in data processing programs to protect the quality of the estimate of each reflection intensity's sigma notably the chi square test. Thus I request that the diffraction resolution where crosses 2.0 is quoted in the article, if it is not there already. I agree that 2.0 is arbitrary but it is more 'lenient' than the usual '3sigma' statistical test. Sometimes the title or abstract has to be changed to follow this criterion; eg 'The structure of xxx is determined to 2.4 Angstrom resolution' type of title has to be consistent with the above criterion. I do not follow an 'Rmerge must be less than x% rule'. I think the above follows reasonable general statistical practice, whilst permitting authors reasonable freedom, and also protects the (more innocent) readers of articles. I am aware that the 'correlation coefficient' between randomly portioned parts of data sets is being increasingly discussed, this parameter also having general statistical validity. I am monitoring discussion on this carefully. It has long been a good way of assessing the statistical quality of anomalous differences for example; to my knowledge introduced by Michael Rossmann many years ago. Best wishes, John On Fri, Jan 27, 2012 at 5:55 PM, Jacob Keller wrote: Clarification: I did not mean I/sigma of 2 per se, I just meant I/sigma is more directly a measure of signal than R values. JPK On Fri, Jan 27, 2012 at 11:47 AM, Jacob Keller wrote: Dear Crystallographers, I cannot think why any of the various flavors of Rmerge/meas/pim should be used as a data cutoff and not simply I/sigma--can somebody make a good argument or point me to a good reference? My thinking is that signal:noise of >2 is definitely still signal, no matter what the R values are. Am I wrong? I was thinking also possibly the R value cutoff was a historical accident/expedient from when one tried to limit the amount of data in the face of limited computational power--true? So perhaps now, when the computers are so much more powerful, we have the luxury of including more weak data? JPK -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu *** -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu *** -- Professor John R Helliwell DSc resolution-meanI.pdf Description: Adobe PDF document
Re: [ccp4bb] MAD
Good description from Ian complemented by an amusing aside from Peter.
One small point. Ian says
"The answer is that it turns out that the equation ('Kramer-Kronig
relationship')
governing X-ray scattering is completely analogous to that governing
optical dispersion,"
Analogous implies the phenomena are separate. In fact one can derive the
refractive indices from the atomic scattering factors. See for example
http://xdb.lbl.gov/Section1/Sec_1-7.pdf
Particularly equation 1.
Colin
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Peter
Moody
Sent: 28 January 2012 09:35
To: ccp4bb
Subject: Re: [ccp4bb] MAD
Ian,
If you visit Isaac Newton's old home at Woolsthorpe (near here) you will see a
conflicting claim for location of the classic prism experiment. You will also
find an apple tree in the garden, but that is another story..
Peter
PS this is my special ccp4bb email account, it doesn't always get the attention
it deserves.
On 19 January 2012 17:50, Ian Tickle
mailto:ianj...@gmail.com>> wrote:
Perhaps I could chime in with a bit of history as I understand it.
The term 'dispersion' in optics, as everyone who knows their history
is aware of, refers to the classic experiment by Sir Isaac Newton at
Trinity College here in Cambridge where he observed white light being
split up ('dispersed') into its component colours by a prism. This is
of course due to the variation in refractive index of glass with
wavelength, so then we arrive at the usual definition of optical
dispersion as dn/dlambda, i.e. the first derivative of the refractive
index with respect to the wavelength.
Now the refractive index of an average crystal at around 1 Ang
wavelength differs by about 1 part in a million from 1, however it can
be determined by very careful and precise interferometric experiments.
It's safe to say therefore that the dispersion of X-rays (anomalous
or otherwise) has no measurable effect whatsoever as far as the
average X-ray diffraction experiment (SAD, MAD or otherwise) is
concerned. The question then is how did the term 'anomalous
dispersion' get to be applied to X-ray diffraction? The answer is
that it turns out that the equation ('Kramer-Kronig relationship')
governing X-ray scattering is completely analogous to that governing
optical dispersion, so it's legitimate to use the term 'dispersive'
(meaning 'analogous to dispersion') for the real part of the
wavelength-dependent component of the X-ray scattering factor, because
the real part of the refractive index is what describes dispersion
(the imaginary part in both cases describes absorption).
So then from 'dispersive' to 'dispersion' to describe the wavelength
dependence of X-ray scattering is only a short step, even though it
only behaves _like_ dispersion in its dependence on wavelength.
However having two different meanings for the same word can get
confusing and clearly should be avoided if at all possible.
So what does this have to do with the MAD acronym? I think it stemmed
from a visit by Wayne Hendrickson to Birkbeck in London some time
around 1990: he was invited by Tom Blundell to give a lecture on his
MAD experiments. At that time Wayne called it multi-wavelength
anomalous dispersion. Tom pointed out that this was really a misnomer
for the reasons I've elucidated above. Wayne liked the MAD acronym
and wanted to keep it so he needed a replacement term starting with D
and diffraction was the obvious choice, and if you look at the
literature from then on Wayne at least consistently called it
multi-wavelength anomalous diffraction.
Cheers
-- Ian
On 18 January 2012 18:23, Phil Jeffrey
mailto:pjeff...@princeton.edu>> wrote:
> Can I be dogmatic about this ?
>
> Multiwavelength anomalous diffraction from Hendrickson (1991) Science Vol.
> 254 no. 5028 pp. 51-58
>
> Multiwavelength anomalous diffraction (MAD) from the CCP4 proceedings
> http://www.ccp4.ac.uk/courses/proceedings/1997/j_smith/main.html
>
> Multi-wavelength anomalous-diffraction (MAD) from Terwilliger Acta Cryst.
> (1994). D50, 11-16
>
> etc.
>
>
> I don't see where the problem lies:
>
> a SAD experiment is a single wavelength experiment where you are using the
> anomalous/dispersive signals for phasing
>
> a MAD experiment is a multiple wavelength version of SAD. Hopefully one
> picks an appropriate range of wavelengths for whatever complex case one has.
>
> One can have SAD and MAD datasets that exploit anomalous/dispersive signals
> from multiple difference sources. This after all is one of the things that
> SHARP is particularly good at accommodating.
>
> If you're not using the anomalous/dispersive signals for phasing, you're
> collecting native data. After all C,N,O,S etc all have a small anomalous
> signal at all wavelengths, and metalloproteins usually have even larger
> signals so the mere presence of a theoretical d" difference does not make it
> a SAD dataset. ALL datasets contain some anomalous/dispersive signals, most
> of the tim
