Re: [ccp4bb] phasing with se-met at low resolution
This is absolutely correct - in the analysis you present, the non-anomalous scattering drops with resolution, but the anomalous part does not. And since counting noise varies with intensity, we should actually be better off at high resolution, since there is less non-anomalous scattering to contribute to the noise! (This is somewhat masked by the background, however). So why don't we see this in practice? The reason is that you've missed out one important term: the atomic displacement parameters (B-factors), which describe a combination of thermal motion and positional disorder between unit cells. This motion and disorder applies equally to the core and outer electrons, and so causes a drop-off in both the anomalous and non-anomalous scattering, over and above that caused by the atomic scattering factors. But your reasoning was sound as far as it went, and it is a point which many people haven't recognised! Kevin Raja Dey wrote: Dear James, I don't understand why measuring anomalous differences has nothing to do with resolution. Heavy atoms scatter anomalously because the inner shell electrons of the heavy atom cannot be considered to be free anymore as was assumed for normal Thomson scattering. As a result the atomic scattering factor of the heavy atom becomes complex and this compex contribution to the structure factor leads to non-equality of Friedel pairs in non-centro symmetric systems(excluding centric zone). This feature is taken advantage in phase determination. Since the inner shell electrons being relatively more strongly bound in heavy atoms contribute to anomalous scattering and its effect is more discernable for high angle reflections . Here the anomalous component of the scattering do not decrease much because of the effectively small atomic radii (only inner shell being effective). FOR HIGH ANGLE REFLECTIONS ANOMALOUS DATA BECOMES IMPORTANT. Raja
Re: [ccp4bb] phasing with se-met at low resolution
Kevin Cowtan wrote:
This is absolutely correct - in the analysis you present, the
non-anomalous scattering drops with resolution, but the anomalous part
does not. And since counting noise varies with intensity, we should
actually be better off at high resolution, since there is less
non-anomalous scattering to contribute to the noise! (This is somewhat
masked by the background, however).
So why don't we see this in practice?
The reason is that you've missed out one important term: the atomic
displacement parameters (B-factors), which describe a combination of
thermal motion and positional disorder between unit cells. This motion
and disorder applies equally to the core and outer electrons, and so
causes a drop-off in both the anomalous and non-anomalous scattering,
over and above that caused by the atomic scattering factors.
I agree with everything but would like to add the following: if we
assume an overall atomic displacement parameter, the drop-off in both
the anomalous and non-anomalous scattering is the same. Therefore, the
ratio of anomalous differences over mean intensity (which is what comes
closest to R_{ano} - in whichever way this is defined) is essentially
unaffected by atomic displacements and should still go up at high
resolution, irrespective of the values of the atomic displacement
parameter !
Things are more complicated if individual isotropic atomic displacements
are considered, because the anomalously scattering atoms (e.g. the Se
atoms) may have significantly larger or smaller displacement parameters
than the average.
All this is discussed in section 4.4. of Flack Shmueli (2007) Acta
Cryst. A63, 257--265.
Marc
But your reasoning was sound as far as it went, and it is a point which
many people haven't recognised!
Kevin
Raja Dey wrote:
Dear James,
I don't understand why measuring anomalous differences has nothing to do
with resolution.
Heavy atoms
scatter anomalously because the inner shell electrons
of the heavy atom cannot be considered to be free anymore
as was assumed for normal Thomson scattering. As a result
the atomic scattering factor of the heavy atom becomes
complex and this compex contribution to the structure
factor leads to non-equality of Friedel pairs in non-centro
symmetric systems(excluding centric zone). This feature is taken
advantage in
phase determination. Since the inner shell electrons
being relatively more strongly bound in heavy atoms
contribute to anomalous scattering and its effect
is more discernable for high angle reflections . Here
the anomalous component of the scattering do not
decrease much because of the effectively small atomic
radii (only inner shell being effective). FOR HIGH
ANGLE REFLECTIONS ANOMALOUS DATA
BECOMES IMPORTANT.
Raja
--
Marc SCHILTZ http://lcr.epfl.ch
Re: [ccp4bb] phasing with se-met at low resolution
Marc SCHILTZ wrote:
I agree with everything but would like to add the following: if we
assume an overall atomic displacement parameter, the drop-off in both
the anomalous and non-anomalous scattering is the same. Therefore, the
ratio of anomalous differences over mean intensity (which is what comes
closest to R_{ano} - in whichever way this is defined) is essentially
unaffected by atomic displacements and should still go up at high
resolution, irrespective of the values of the atomic displacement
parameter !
OK, that's new to me. My understanding was that f does not drop off
with resolution in the stationary atom case, since the anomalous
scattering arises from the core atoms. Can you elaborate?
Re: [ccp4bb] phasing with se-met at low resolution
Sorry I don't have instant access to Acta A here so can't comment in the
light of the Flack Shmueli paper. But it seems to me that Kevin's
point is still valid, regardless of whether or not the anomalously
scattering atoms have different ADPs from the average or not. I agree
that this would have the complicating effects described, but I don't see
that it's necessary to invoke it as an explanation. The reason is that
the anomalous phasing power doesn't depend on Rano = |delta-ano|/I,
it depends on the anomalous signal/noise ratio =
|delta-ano|/s.u.(delta-ano), or something related to it, and the
standard uncertainty of course depends largely on the background). So
if the fall-off due to overall thermal motion etc as described by Kevin
causes the S/N ratio to dip much below 1 then the anomalous signal won't
help you.
Cheers
-- Ian
-Original Message-
From: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk]
On
Behalf Of Marc SCHILTZ
Sent: 13 May 2009 11:26
To: Kevin Cowtan; CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] phasing with se-met at low resolution
Kevin Cowtan wrote:
This is absolutely correct - in the analysis you present, the
non-anomalous scattering drops with resolution, but the anomalous
part
does not. And since counting noise varies with intensity, we should
actually be better off at high resolution, since there is less
non-anomalous scattering to contribute to the noise! (This is
somewhat
masked by the background, however).
So why don't we see this in practice?
The reason is that you've missed out one important term: the atomic
displacement parameters (B-factors), which describe a combination of
thermal motion and positional disorder between unit cells. This
motion
and disorder applies equally to the core and outer electrons, and so
causes a drop-off in both the anomalous and non-anomalous
scattering,
over and above that caused by the atomic scattering factors.
I agree with everything but would like to add the following: if we
assume an overall atomic displacement parameter, the drop-off in both
the anomalous and non-anomalous scattering is the same. Therefore, the
ratio of anomalous differences over mean intensity (which is what
comes
closest to R_{ano} - in whichever way this is defined) is essentially
unaffected by atomic displacements and should still go up at high
resolution, irrespective of the values of the atomic displacement
parameter !
Things are more complicated if individual isotropic atomic
displacements
are considered, because the anomalously scattering atoms (e.g. the Se
atoms) may have significantly larger or smaller displacement
parameters
than the average.
All this is discussed in section 4.4. of Flack Shmueli (2007) Acta
Cryst. A63, 257--265.
Marc
But your reasoning was sound as far as it went, and it is a point
which
many people haven't recognised!
Kevin
Raja Dey wrote:
Dear James,
I don't understand why measuring anomalous differences has nothing
to
do
with resolution.
Heavy atoms
scatter anomalously because the inner shell electrons
of the heavy atom cannot be considered to be free anymore
as was assumed for normal Thomson scattering. As a result
the atomic scattering factor of the heavy atom becomes
complex and this compex contribution to the structure
factor leads to non-equality of Friedel pairs in non-centro
symmetric systems(excluding centric zone). This feature is taken
advantage in
phase determination. Since the inner shell electrons
being relatively more strongly bound in heavy atoms
contribute to anomalous scattering and its effect
is more discernable for high angle reflections . Here
the anomalous component of the scattering do not
decrease much because of the effectively small atomic
radii (only inner shell being effective). FOR HIGH
ANGLE REFLECTIONS ANOMALOUS DATA
BECOMES IMPORTANT.
Raja
--
Marc SCHILTZ http://lcr.epfl.ch
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Re: [ccp4bb] phasing with se-met at low resolution
Kevin Cowtan wrote:
Marc SCHILTZ wrote:
I agree with everything but would like to add the following: if we
assume an overall atomic displacement parameter, the drop-off in both
the anomalous and non-anomalous scattering is the same. Therefore, the
ratio of anomalous differences over mean intensity (which is what comes
closest to R_{ano} - in whichever way this is defined) is essentially
unaffected by atomic displacements and should still go up at high
resolution, irrespective of the values of the atomic displacement
parameter !
OK, that's new to me. My understanding was that f does not drop off
with resolution in the stationary atom case, since the anomalous
scattering arises from the core atoms. Can you elaborate?
Yes, this is correct. And if there are atomic displacements, we would
have to multiply f by an overall Debye-Waller factor (t) to get an
effective f which then would drop off with resolution. But the
Debye-Waller factor also affects the normal scattering factors in the
same way. So the ratio of rms Friedel differences over mean intensities
remains essentially unaffected by an overall atomic displacement parameter.
Interpreting the Flack Shmueli (2007) paper :
D = F^2(+) - F^2(-) is the Friedel difference of a reflection and A =
0.5 * [F^2(+) + F^2(-)] is its Friedel average
Then D^2 = t^4 D^2(static) and A = t ^2 A(static)
So the ratio SQRT(D^2) / A is independent of t (i.e. the same as for
the static case).
Marc
--
Marc SCHILTZ http://lcr.epfl.ch
[ccp4bb] xquartz alert
The new OS X 10.5.7 update downgrades your X11 to 2.1.6. There is a new X11 update, 2.3.3, only for 10.5.7 users. It might be prudent to update to 10.5.7 and then xquartz 2.3.3, before reporting that coot or something else is suddenly broken. As usual, very annoying... Engin
Re: [ccp4bb] xquartz alert
My X11 is still on 2.3.1 after updating to 10.5.7 (I could never get X11 2.3.2 to work properly) Coot fine Phil On 13 May 2009, at 17:25, Engin Ozkan wrote: The new OS X 10.5.7 update downgrades your X11 to 2.1.6. There is a new X11 update, 2.3.3, only for 10.5.7 users. It might be prudent to update to 10.5.7 and then xquartz 2.3.3, before reporting that coot or something else is suddenly broken. As usual, very annoying... Engin
Re: [ccp4bb] phasing with se-met at low resolution
The reason is that you've missed out one important term: the atomic displacement parameters (B-factors), which describe a combination of thermal motion and positional disorder between unit cells. A somewhat niggling point: isn't it true that the thermal motion is insignificant at 100K? Does anybody know of a paper which systematically measures B-factors as a function of temperature? The asymptote of the resulting curve would represent all of the non-thermal elements, right? JPK
Re: [ccp4bb] phasing with se-met at low resolution
On Wednesday 13 May 2009 09:30:06 Jacob Keller wrote: The reason is that you've missed out one important term: the atomic displacement parameters (B-factors), which describe a combination of thermal motion and positional disorder between unit cells. A somewhat niggling point: isn't it true that the thermal motion is insignificant at 100K? No. True thermal motion doesn't bottom out until 0 Kelvin. But that is kind of irrelevant, since motion in the sense of things moving in the crystal while we measured the data is only one contribution to the overall ADP (B factor). Does anybody know of a paper which systematically measures B-factors as a function of temperature? The asymptote of the resulting curve would represent all of the non-thermal elements, right? The theory for this is well laid out in Bürgi, H.B., and Förtsch, M. (1999). Dynamic processes and disorder in crystal structures as seen by temperature-dependent diffraction experiments. J. Molecular Structure 486, 457-463. But to the best of my knowledge a full analysis based on temperature-dependent diffraction experiments has never been done for a protein structure. I had a preliminary go at it some years back, but collecting comparable data sets over a range of temperatures spanning liquid He to room temperature is technically challenging. The analysis is also non-trivial. -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
Re: [ccp4bb] phasing with se-met at low resolution
Greg Petsko's group did something like this about a billion years ago (yet, strangely, I remember the paper, even though I'd be stumped if you asked me what I had for breakfast...) They covered the range from room temp down to very cold, using different cryoprotectants (importantly, they were not vitrifying their samples). I recall a plot of ADPs vs. temp that showed an essentially linear decrease down to some temp (maybe around 150 K or so?), after it plateaued, with no further reductions being seen at even very low temp. They rationalized this by saying (I think) that the decrease represented the dynamic disorder, which was damped at low temperatures, and the plateau represented the point where static disorder became the predominant contributor. I remember thinking at the time that this made great intuitive sense. I have no idea if people still buy this. I can't put my finger on the reference, but if you start here you can probably find your way: Ringe D, Petsko GA. Study of protein dynamics by X-ray diffraction. Methods Enzymol. 1986;131:389-433. On 13 May 2009, at 12:30 PM, Jacob Keller wrote: The reason is that you've missed out one important term: the atomic displacement parameters (B-factors), which describe a combination of thermal motion and positional disorder between unit cells. A somewhat niggling point: isn't it true that the thermal motion is insignificant at 100K? Does anybody know of a paper which systematically measures B-factors as a function of temperature? The asymptote of the resulting curve would represent all of the non- thermal elements, right? JPK --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu
[ccp4bb] Postdoctoral Research Position available at UT Southwestern Medical Center
This is posted as a favor for a collaborator, please do not respond to me but directly to Neal Alto. --- A postdoctoral research position is available in the laboratory of Dr. Neal M. Alto, in the department of Microbiology, to study the molecular mechanisms of bacterial type III effectors and specifically, their ability to hijack human signal transduction cascades regulated by Ras-family GTPases and actin cytoskeletal dynamics. The postdoc will be responsible to build upon current biochemical and protein interaction data to express, purify, crystallize, and perform structural analyses involving bacterial type III effectors in complex with human substrates. These studies will aim to complement ongoing research using techniques in cell biology and microbial pathogenesis. Additional project information can be found in [Alto NM et al., Cell 2006] and [Alto NM et al., Journal of Cell Biology 2007]. The University of Texas Southwestern (UTSW) Medical Center is a world- renowned research institute that is built upon foundations of Biochemistry, Structural Biology, and Biomedical Research. Structural studies will be performed in collaboration with the UTSW Structural Biology Laboratory (SBL), a state of the art facility equipped with modern X-ray crystallography equipment including a high-brilliance Rigaku FR-E X-ray generator with an ACTOR crystal-mounting robot, an integrated Phoenix crystallization robot and imaging system, and a Fluidigm microfluidics crystallization robot and imaging system. The Structural Biology Group at UTSW also has guaranteed access to 30 days a year of beamtime at the Structural Biology center (SBC) at the Advanced Photo Source (APS). The minimal requirement is a Doctoral degree in protein chemistry or molecular biology as well as prior experience in techniques of molecular cloning, recombinant protein expression, and X-ray crystallography. The successful applicant must be self-motivated, enthusiastic and work well in a collaborative environment. Competitive salary and fringe benefits will be provided based on UTSW pay scale. Interested individuals should submit a CV, a summary of research achievements, and names of three references to: Dr. Neal Alto (neal.a...@utsouthwestern.edu) * * * * * * * * * * * * * * * * * * * * * * * * * * * * Diana R. Tomchick Associate Professor University of Texas Southwestern Medical Center Department of Biochemistry 5323 Harry Hines Blvd. Rm. ND10.214B Dallas, TX 75390-8816, U.S.A. Email: diana.tomch...@utsouthwestern.edu 214-645-6383 (phone) 214-645-6353 (fax)
Re: [ccp4bb] phasing with se-met at low resolution
So what is the approximate percent contribution of the *temperature-dependent* b-factor at 100K, for an average crystal, or how to determine such? In other words, if I have a crystal with an avg B of 20, when I go from 100K to 0K, how much lower will it drop? I recall seeing papers exploring liquid helium temperatures, which I believe concluded that there was not much gain in lowering the temp, implying that the B's did not go down much after 100K. I had thought that the reason for calling it a temperature factor was more because it represented the many states of the atoms caught *in flagrante vibratio* by the liquid nitrogen plunge upon freezing the crystal, but not actual motions in the crystal. Room temperature is of course different. Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** - Original Message - From: Ethan Merritt merr...@u.washington.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, May 13, 2009 12:12 PM Subject: Re: [ccp4bb] phasing with se-met at low resolution On Wednesday 13 May 2009 09:30:06 Jacob Keller wrote: The reason is that you've missed out one important term: the atomic displacement parameters (B-factors), which describe a combination of thermal motion and positional disorder between unit cells. A somewhat niggling point: isn't it true that the thermal motion is insignificant at 100K? No. True thermal motion doesn't bottom out until 0 Kelvin. But that is kind of irrelevant, since motion in the sense of things moving in the crystal while we measured the data is only one contribution to the overall ADP (B factor). Does anybody know of a paper which systematically measures B-factors as a function of temperature? The asymptote of the resulting curve would represent all of the non-thermal elements, right? The theory for this is well laid out in Bürgi, H.B., and Förtsch, M. (1999). Dynamic processes and disorder in crystal structures as seen by temperature-dependent diffraction experiments. J. Molecular Structure 486, 457-463. But to the best of my knowledge a full analysis based on temperature-dependent diffraction experiments has never been done for a protein structure. I had a preliminary go at it some years back, but collecting comparable data sets over a range of temperatures spanning liquid He to room temperature is technically challenging. The analysis is also non-trivial. -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
Re: [ccp4bb] xquartz alert
I was so looking forward to being able to report that Apple's recent 600 MB bugfix had got the jitters out of PyMOL on an external screen, but no. It still flickers like a German disco in the 90s when I ray trace. X11 2.4. should be coming out soon. There's always hope. Andreas Engin Ozkan wrote: The new OS X 10.5.7 update downgrades your X11 to 2.1.6. There is a new X11 update, 2.3.3, only for 10.5.7 users. It might be prudent to update to 10.5.7 and then xquartz 2.3.3, before reporting that coot or something else is suddenly broken. As usual, very annoying... Engin
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A postdoctoral position is available at the Department of Pharmacology, UT Southwestern Medical Center at Dallas, TX. Our lab uses both NMR and X-ray crystallography to characterize the atomic structures of macromolecules involved in cancer-related signaling pathways. We are also interested in validation of structure-based functional hypotheses using biochemical and cell biological approaches. Applicants should be highly motivated individuals and must have a recent PhD degree in biochemistry, biophysics or other related field. Experience in molecular biology, large-scale protein expression and purification, and X-ray crystallography is strongly preferred. Interested applicants should send his/her CV with names of three referees to: Xuelian Sue Luo, Ph.D. Assistant Professor Department of Pharmacology UT Southwestern Medical Center 6001 Forest Park Road, ND6.136CC Dallas, TX 75390-9041 Email: xuelian@utsouthwestern.edu
Re: [ccp4bb] Software for RNA model building
Dear Rafal, I'm developing a graphical tool to construct RNA 3D models. You can find all the details at this address: http://www.bioinformatics.org/assemble/ It is open-source. At now, i'm searching beta-testers before the official 1.0 release. If you're interested, I will send you an email explaining you how to download the tool. Best, Fabrice Jossinet -- Laboratoire de Bioinformatique, modelisation et simulation des acides nucleiques Institut de biologie moleculaire et cellulaire du CNRS UPR9002, Architecture et Reactivite de l'ARN 15 rue Rene Descartes F - 67084 Strasbourg
Re: [ccp4bb] phasing with se-met at low resolution
On Wednesday 13 May 2009 10:22:54 Patrick Loll wrote: Greg Petsko's group did something like this about a billion years ago (yet, strangely, I remember the paper, even though I'd be stumped if you asked me what I had for breakfast...) They covered the range from room temp down to very cold, using different cryoprotectants (importantly, they were not vitrifying their samples). I recall a plot of ADPs vs. temp that showed an essentially linear decrease down to some temp (maybe around 150 K or so?), after it plateaued, with no further reductions being seen at even very low temp. They rationalized this by saying (I think) that the decrease represented the dynamic disorder, which was damped at low temperatures, and the plateau represented the point where static disorder became the predominant contributor. The problem with this and other older protein work is that it predates our current capabilities to handle models of anisotropy in protein structures. The interesting temperature-dependent effect manifests most significantly as an evolution of anisotropy. It is not well captured by looking only at isotropic B factors. Ethan I remember thinking at the time that this made great intuitive sense. I have no idea if people still buy this. I can't put my finger on the reference, but if you start here you can probably find your way: Ringe D, Petsko GA. Study of protein dynamics by X-ray diffraction. Methods Enzymol. 1986;131:389-433. On 13 May 2009, at 12:30 PM, Jacob Keller wrote: The reason is that you've missed out one important term: the atomic displacement parameters (B-factors), which describe a combination of thermal motion and positional disorder between unit cells. A somewhat niggling point: isn't it true that the thermal motion is insignificant at 100K? Does anybody know of a paper which systematically measures B-factors as a function of temperature? The asymptote of the resulting curve would represent all of the non- thermal elements, right? JPK --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
