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
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
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] 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
Re: [ccp4bb] phasing with se-met at low resolution
On Mon, May 11, 2009 at 05:22:25PM -0500, Pete Meyer wrote: P.S. I would also appreciate the specific query type for searching the PDB on the web for phasing method (MR, MAD, SAD, MIR, etc.). They seem to have everything under the sun searchable, but I cannot find this one. Last time I emailed the RCSB about this (a few years back), it wasn't possible to search by phasing method. You can try using advanced search - keyword search - advanced and doing a full text search, but this is somewhat less than ideal. To be fair though, I suspect relatively few people searching the PDB are concerned about the phasing method used. Funny, I was looking at that just the other day. You basically need a local copy of the PDB and do some ugly grep/awk into the PDB files ... at least thats what I do (there might be better ways). Anyway, as of 28.04.2009 we have a total of 48969 entries with a line REMARK 200 METHOD USED TO DETERMINE THE STRUCTURE: which show (no guarantee!): Molecular replacement = 26436 Undefined (i.e. NULL, N/A etc) = 9349 MAD= 4125 SAD= 3028 Fourier methods= 2929 MIR= 1706 SIR= 512 Direct methods, ab initio = 327 Rigid-body refinement = 186 RIP= 3 UNKNOWN (everything else) = 504 This might add up to more than the total number of entries, since some have several methods listed. I tried to accomodate mis-spellings - lots of them available: MOLECULARE REPLACEMENT MOLECULAR REPLECEMENT MOLECULAR REPLCEMENT MOLECULAR REPLACEMET MOLECULAR REPL. MOLECULAR REPACEMENT MOLECULAR REEMPLACEMENT MOLECULAR PLACEMENT MOLECULAR EEPLACEMENT MOLECULAR PLACEMENT DIFFERENT FOURIER DIFFERECE FOURIER DFIFFERECE FOURIER etc. Cheers Clemens -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
Re: [ccp4bb] phasing with se-met at low resolution
measuring anomalous differences has nothing to do with resolution. measuring anomalous differences has nothing to do with Rmerge. measuring anomalous differences has EVERYTHING to do with signal and noise. (as does measuring anything else) If your average anomalous difference is going to be ~5%, then you need to be able to measure a 5% change in spot intensity, yes? So, if you take your native data, and compare the merged values of I+ and I- (known in Scala as Ranom), and they are already more than 5% different, then ... you are in trouble. But if Ranom for native data is less than 5%, then you stand a chance of measuring a 5% difference. That is, for native data, the true values of I+ and I- should be the same (within the Bijvoet ratio for the sulfurs, which is usually 0.5%), so comparing I+ and I- for native data is actually a very good way to get your expected anomalous error. You can improve this number by increasing redundancy, even if you reduce the exposure time to compensate. In fact, it is a VERY good idea to do this when trying to measure anomalous differences. Redundancy is good for anomalous, but bad for high-res data. Long exposures and fine slicing are good for high-res data, but bad for anomalous. Resolution comes into play because the anomalous error will approach infinity as your spot intensity approaches zero, so you will never be able to measure anomalous differences for your highest resolution bin. The resolution to which you CAN measure anomalous differences (with a signal-to-noise ratio greater than one) will be the resolution where the cumulative Ranom rises to the Bijvoet ratio (5% in your case). That is, look for the resolution limit where the overall native Ranom is 5%, and that is the resolution to which you will probably get experimental phases. If there is no such resolution limit (Ranom 5% in all bins), then MAD/SAD will not work with your current data collection method. Higher redundancy is called for. However, do not get too excited if this resolution limit is 6 A. Although 6 A phases are better than no phases at all, have you ever LOOKED at a 6 A map? It can be very hard to tell if it is protein or not, even with perfect phases and all the right hand choices, etc. Programs and crystallographers alike can get confused by this. I know that there are still many structural biologists out there who just want to get the structure, but I remind you that you can already get the structure to ~50 A resolution with other techniques. Such as gel filtration. The success of phase extension does depend on resolution. Although I do not have a quantitative argument for it, the success of SAD structure determination at worse than 4 A does seem to drop precipitously. This could simply be correlated with the crappiness of the crystals, but it is important to remember that SAD relies heavily on density modification technology, such as solvent flattening and histogram matching, etc, and these methods loose a great deal of power as the resolution of the map decreases (and the protein-solvent contrast becomes less clear). IMHO it is ALWAYS better to collect MAD data, because then the dichotomous phase ambiguity is resolved experimentally. Two wavelengths are twice as good as one, even with the exposure time cut in half. -James Holton MAD Scientist Engin Ozkan wrote: Hi everyone, I thought I start a new thread while it is unusually quiet on the bb. I am pondering over the practical limitations to MAD and SAD phasing with Se-Met at low resolution. What is the lowest resolution at which people have solved structures only using phases from selenium in a realistic case? Let me further qualify my question: My *realistic* *low* resolution case is where 1. Rmerge over all resolution bins is 6-10% (i.e. your crystals are lousy). 2. Resolution limit is worse than 3.5 Angstroms, where I/sigma in the last resolution bin is between 1 and 3 (i.e. your crystals are really lousy). 3. Assuming good selenium occupancy (~85%; I work with eukaryotic expression systems, so 100% is not usually achieavable), 4. The number of selenium atoms are enough many that the Crick-Magdoff equation would give you *at least* an average 5% change in intensities (assuming 6 electrons contributed per selenium, based on both absorptive and dispersive differences being at about 6 e- at the absorption edge). 5. and specifically, no other phases and molecular replacement solutions are available. Obviously, I have a case very similar to what's described above, and three years of failure with heavy atom derivatization (I am still trying). I would be happy to hear about Se-Met cases, and data collection strategies (2wl vs. 3wl MAD vs. SAD, etc.) and phasing methods used in these cases, or references of them. Again, no other partial phases, and no data cut off at 3.6 A with an I/s of 15 in the last resolution bin. Are there any
Re: [ccp4bb] phasing with se-met at low resolution
Dear James, On Tue, May 12, 2009 at 11:26:55AM -0700, James Holton wrote: However, do not get too excited if this resolution limit is 6 A. Although 6 A phases are better than no phases at all, have you ever LOOKED at a 6 A map? It can be very hard to tell if it is protein or not, even with perfect phases and all the right hand choices, etc. Programs and crystallographers alike can get confused by this. I know that there are still many structural biologists out there who just want to get the structure, Completely agree. The big misconception is that the result of the X-ray experiment is anything else than 'just' such an electron density map. What we usually see as 'the structure' is only a model: a PDB file to help us measuring distances, looking at on the display and making nice pictures ... just a useful interpretation of the electron density. So at lower resolution one needs to think more like an EM structural scientist and not an X-Ray one I guess. The success of phase extension does depend on resolution. Although I do not have a quantitative argument for it, the success of SAD structure determination at worse than 4 A does seem to drop precipitously. Not just SAD, but also MAD, MIR, SIR et al (in my experience). Somewhere below 3.5-4A it becomes VERY hard to extend the phases to the full resolution of the dataset. Unless you have NCS (the more the better) - which is just great in those cases. So if one gets crappy crystals at least get them with a huge asymmetric unit ;-) This could simply be correlated with the crappiness of the crystals, but it is important to remember that SAD relies heavily on density modification technology, such as solvent flattening and histogram matching, etc, and these methods loose a great deal of power as the resolution of the map decreases (and the protein-solvent contrast becomes less clear). I always thought it had more to do with the look-and-feel of lower resolution maps (helices are big sausages, sheets blend into flat patches and side-chains are not visible): the methods modifying the density in real-space have probably different assumptions and default parameters (radii for masking, histograms becoming messy, absolute scaling nearly impossible etc). Also: the typical low resolution (20A and below) that is often neglected (beamstop size and masking? Overloads?) becomes more important. In the end 'resolution' comes into play in some way after all I guess - at least when we see 'resolution' as what it is mostly used in that context: a simple concept to describe several actual difficulties (poor crystals which only diffract to low resolution, weak experimental phases, anisotropy, radiation damage etc). Cheers Clemens -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
Re: [ccp4bb] phasing with se-met at low resolution
Thanks, I do understand all of that. I gave some Rmerge and resolution values to give some idea about errors and noise expected in the data, and an idea for up to what resolution phases would be good. And if such low resolution phases ever yield a meaningful model. Both measures are flawed indicators, even though they are the most common measures of data among us. I will definitely check Ranom (which means I should try scala). What I was curious about is practical aspects: especially in cases in which it really worked. And I/we have gotten quite a few responses in MAD vs SAD, inverse beam strategies, radiation damage control, etc. The take home message for me was that noone agrees on the best data collection strategy, although I still have to read upon some of the case references that were sent. Another point is the success rate of software - be it direct methods based or Patterson based - with such data (where anomalous signal would die at even lower resolution) at solving the substructure. I have seen a reference where if the correct substructure could be provided in a test case, SAD was actually successful. In another case, they confirmed the correct selenium sites with a platinum derivative data to further proceed with phasing. To be honest, I can't ever get shelx to find my platinums with 6 A data :) I would also like to hear about phase extension at low resolution (which you have mentioned). Overall, it appears that with such data, there are too many places for failure. Thanks for everyone's interest. Later, I shall come up with a nice summary. Engin On 5/12/09 11:26 AM, James Holton wrote: measuring anomalous differences has nothing to do with resolution. measuring anomalous differences has nothing to do with Rmerge. measuring anomalous differences has EVERYTHING to do with signal and noise. (as does measuring anything else) If your average anomalous difference is going to be ~5%, then you need to be able to measure a 5% change in spot intensity, yes? So, if you take your native data, and compare the merged values of I+ and I- (known in Scala as Ranom), and they are already more than 5% different, then ... you are in trouble. But if Ranom for native data is less than 5%, then you stand a chance of measuring a 5% difference. That is, for native data, the true values of I+ and I- should be the same (within the Bijvoet ratio for the sulfurs, which is usually 0.5%), so comparing I+ and I- for native data is actually a very good way to get your expected anomalous error. You can improve this number by increasing redundancy, even if you reduce the exposure time to compensate. In fact, it is a VERY good idea to do this when trying to measure anomalous differences. Redundancy is good for anomalous, but bad for high-res data. Long exposures and fine slicing are good for high-res data, but bad for anomalous. Resolution comes into play because the anomalous error will approach infinity as your spot intensity approaches zero, so you will never be able to measure anomalous differences for your highest resolution bin. The resolution to which you CAN measure anomalous differences (with a signal-to-noise ratio greater than one) will be the resolution where the cumulative Ranom rises to the Bijvoet ratio (5% in your case). That is, look for the resolution limit where the overall native Ranom is 5%, and that is the resolution to which you will probably get experimental phases. If there is no such resolution limit (Ranom 5% in all bins), then MAD/SAD will not work with your current data collection method. Higher redundancy is called for. However, do not get too excited if this resolution limit is 6 A. Although 6 A phases are better than no phases at all, have you ever LOOKED at a 6 A map? It can be very hard to tell if it is protein or not, even with perfect phases and all the right hand choices, etc. Programs and crystallographers alike can get confused by this. I know that there are still many structural biologists out there who just want to get the structure, but I remind you that you can already get the structure to ~50 A resolution with other techniques. Such as gel filtration. The success of phase extension does depend on resolution. Although I do not have a quantitative argument for it, the success of SAD structure determination at worse than 4 A does seem to drop precipitously. This could simply be correlated with the crappiness of the crystals, but it is important to remember that SAD relies heavily on density modification technology, such as solvent flattening and histogram matching, etc, and these methods loose a great deal of power as the resolution of the map decreases (and the protein-solvent contrast becomes less clear). IMHO it is ALWAYS better to collect MAD data, because then the dichotomous phase ambiguity is resolved experimentally. Two wavelengths are twice as good as one, even with the
Re: [ccp4bb] phasing with se-met at low resolution
Dear Engin, On Tue, May 12, 2009 at 12:20:31PM -0700, Engin Ozkan wrote: The take home message for me was that noone agrees on the best data collection strategy No - since you have to factor in at least half a dozen parameters: unfortunately there is no silver bullet :-( Another point is the success rate of software - be it direct methods based or Patterson based - with such data (where anomalous signal would die at even lower resolution) at solving the substructure. In general you need better data for finding the HA substructure this way then to solve it (where 'solve' can mean a lot of things, e.g. breaking the phase ambiguity and getting some meaningful map). In another case, they confirmed the correct selenium sites with a platinum derivative data to further proceed with phasing. To be honest, I can't ever get shelx to find my platinums with 6 A data :) How do you know SHELXD hasn't found them? In my experience at this kind of resolution you have to be careful to trust the usual criteria for a good solution (CCall 40% etc). Maybe looking at good old fashioned anomalous Patterson Harker sections? Also: I've never had good experiences with Pt derivatives ... low occupancy, VERY high B-values and generally a pain. I've seen people using them very successfully though. As Jim said: it's all about signal and noise. If you have lousy crystals (large noise) you need to go for a large signal: one of those Ta/W clusters maybe - and bootstrapping your way to the other derivatives from there? Depending on the quality of your crystals (and data collection) a weak Pt derivative might not be enough. Overall, it appears that with such data, there are too many places for failure. Yes, such data isn't forgiving - but that makes success even sweater. Remember that what we now see as a straightforward and nearly trivial project was one of those 'really difficult structures' only a few years back (and people did solve those structures). Cheers Clemens -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
Re: [ccp4bb] phasing with se-met at low resolution
However, do not get too excited if this resolution limit is 6 A. Although 6 A phases are better than no phases at all, have you ever LOOKED at a 6 A map? It can be very hard to tell if it is protein or not, even with perfect phases and all the right hand choices, etc. If the map is a 6 Angstrom SeMet map you may well be right, since if the signal goes to 6 Angstrom the data at 7 Angstrom isn't that hot either. However if this was a Ta6Br12 6 Angstrom map then it can look quite pretty for the resolution because the 7 Angstrom SAD data in that case can be pretty good. Case in point it the one we collected for PP2a ABC holoenzyme cleared up all sorts of things about the partial molecular replacement solution, including some reassurance that the desperation WD40 ensemble MR solution was actually correct. At 6A, the WD40 looked somewhat like a Bagel (or a Bundt Cake if one is familiar) but the helices in one of the other subunits (A) were actually nicely resolved. Excitement may be warranted, even at 6 Angstrom. Phil Jeffrey Princeton
Re: [ccp4bb] phasing with se-met at low resolution
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 DATABECOMES IMPORTANT. Raja From: James Holton jmhol...@lbl.gov To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, 12 May, 2009 11:26:55 AM Subject: Re: [ccp4bb] phasing with se-met at low resolution measuring anomalous differences has nothing to do with resolution. measuring anomalous differences has nothing to do with Rmerge. measuring anomalous differences has EVERYTHING to do with signal and noise. (as does measuring anything else) If your average anomalous difference is going to be ~5%, then you need to be able to measure a 5% change in spot intensity, yes? So, if you take your native data, and compare the merged values of I+ and I- (known in Scala as Ranom), and they are already more than 5% different, then ... you are in trouble. But if Ranom for native data is less than 5%, then you stand a chance of measuring a 5% difference. That is, for native data, the true values of I+ and I- should be the same (within the Bijvoet ratio for the sulfurs, which is usually 0.5%), so comparing I+ and I- for native data is actually a very good way to get your expected anomalous error. You can improve this number by increasing redundancy, even if you reduce the exposure time to compensate. In fact, it is a VERY good idea to do this when trying to measure anomalous differences. Redundancy is good for anomalous, but bad for high-res data. Long exposures and fine slicing are good for high-res data, but bad for anomalous. Resolution comes into play because the anomalous error will approach infinity as your spot intensity approaches zero, so you will never be able to measure anomalous differences for your highest resolution bin. The resolution to which you CAN measure anomalous differences (with a signal-to-noise ratio greater than one) will be the resolution where the cumulative Ranom rises to the Bijvoet ratio (5% in your case). That is, look for the resolution limit where the overall native Ranom is 5%, and that is the resolution to which you will probably get experimental phases. If there is no such resolution limit (Ranom 5% in all bins), then MAD/SAD will not work with your current data collection method. Higher redundancy is called for. However, do not get too excited if this resolution limit is 6 A. Although 6 A phases are better than no phases at all, have you ever LOOKED at a 6 A map? It can be very hard to tell if it is protein or not, even with perfect phases and all the right hand choices, etc. Programs and crystallographers alike can get confused by this. I know that there are still many structural biologists out there who just want to get the structure, but I remind you that you can already get the structure to ~50 A resolution with other techniques. Such as gel filtration. The success of phase extension does depend on resolution. Although I do not have a quantitative argument for it, the success of SAD structure determination at worse than 4 A does seem to drop precipitously. This could simply be correlated with the crappiness of the crystals, but it is important to remember that SAD relies heavily on density modification technology, such as solvent flattening and histogram matching, etc, and these methods loose a great deal of power as the resolution of the map decreases (and the protein-solvent contrast becomes less clear). IMHO it is ALWAYS better to collect MAD data, because then the dichotomous phase ambiguity is resolved experimentally. Two wavelengths are twice as good as one, even with the exposure time cut in half. -James Holton MAD Scientist Engin Ozkan wrote: Hi everyone, I thought I start a new thread while it is unusually quiet on the bb. I am pondering over the practical limitations to MAD and SAD phasing with Se-Met at low resolution. What is the lowest resolution at which people have solved structures only using phases from selenium in a realistic case? Let me further qualify my question: My *realistic* *low* resolution case is where 1. Rmerge over all resolution
Re: [ccp4bb] phasing with se-met at low resolution
Dear Raja, FOR HIGH ANGLE REFLECTIONS ANOMALOUS DATA BECOMES IMPORTANT. Raja this is the theoretical point of view. As James pointed out, in real life the intensities of reflections at high resolution becomes comparable to the noise level so that the accuracy of which the reflections are measured increases significantly, rendering the anomalous difference useless - unfortunately. Tim
Re: [ccp4bb] phasing with se-met at low resolution
Dear Engin I would also like to comment. I our recent structure determination of the sodium pump (3.5 A) (see morth JP et al 2007) we did not have experimental phasing to more than 6 A for the Ta6Br12 clusters and 7 A for the Pt sites. Both with extensive multicrystal averaging and phase combination it was possible to trace the structure. The data was what you could call lousy, but in the end I was able to identify the 3 Rubidium ions present in the data set based on the their anomalous scattering power. Not detectable in the reflection statistics of the native data set (see Schack VR et al 2008). So even though your selenium sites will not help the phasing initially, they will still guide the model building extensively (see Hunte C et al 2005 Nature, 3.5 A res structure with Se) with your improved model phases the selenium site positions will improve and at a later stage they will be valuable, when combined as additional phase information. The initial maps always look really bad, your 3 years of heavy atom derivatisation might not have been in vain, you can still use the initial Se phases to try to locate more heavy atoms sites (Fredslund F et al 2006 jmb) good luck Preben On 11/05/2009, at 05.24, Engin Ozkan wrote: Wow, I got quite a number of responses. Thanks everyone. Let's elaborate. Petr, I don't know my anomalous signal, because I haven't yet done the experiment. If I had, I would have definitely talked about chi2 values for I+/I- merged and unmerged, Anomalous Patterson maps, or other measures of anomalous signal (Dauter, Acta Cryst D, 2006 is a great paper to read on that, I suggest every grad student to present it in their Crystallography Journal Club). I was merely pondering today, but I plan to do the experiment very soon (crystals are waiting for synchrotron time). About your example, 2.9 A diffracting crystals (with 4 A anomalous signal) are in the doable range, as you suggested. The question is what would happen if your crystals diffract to 4 A, and anomalous signal dies at 6 A. The interesting bit of course is 1 Met per 200 residue, which should put to death the 1 in 50 or 1 in 100 Methionine myths: it depends on the quality of your data. Engin On 5/10/09 2:50 PM, Leiman Petr wrote: Dear Engin Ozkan, You have told us how bad your crystals are, but you did not mention how good your anomalous signal is: 1. To what resolution does your anomalous signal extend and what statistic is used for this estimate? 2. Do your dispersive and Bijvoet Pattersons look similar and what is the measure of similarity? This structure http://www.pdb.org/pdb/explore/explore.do?structureId=1K28 which contains ~1100 residues in the asymmetric unit (and ~3500 in the entire complex), was solved using a chimerical SeMet derivative, in which one protein was SeMet labeled (17 Se per a.u.) and the other was native. The Semet dataset had a detectable anomalous signal to 4 A resolution (at most). The diffraction extended to 2.9A resolution. Sincerely, Petr --- Petr Leiman Institut de physique des systèmes biologiques École Polytechnique Fédérale de Lausanne (EPFL) Cubotron/BSP-415 CH-1015 Lausanne Switzerland -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Engin Ozkan Sent: Sunday, May 10, 2009 11:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] phasing with se-met at low resolution Hi everyone, I thought I start a new thread while it is unusually quiet on the bb. I am pondering over the practical limitations to MAD and SAD phasing with Se-Met at low resolution. What is the lowest resolution at which people have solved structures only using phases from selenium in a realistic case? Let me further qualify my question: My *realistic* *low* resolution case is where 1. Rmerge over all resolution bins is 6-10% (i.e. your crystals are lousy). 2. Resolution limit is worse than 3.5 Angstroms, whereI/ sigma in the last resolution bin is between 1 and 3 (i.e. your crystals are really lousy). 3. Assuming good selenium occupancy (~85%; I work with eukaryotic expression systems, so 100% is not usually achieavable), 4. The number of selenium atoms are enough many that the Crick- Magdoff equation would give you *at least* an average 5% change in intensities (assuming 6 electrons contributed per selenium, based on both absorptive and dispersive differences being at about 6 e- at the absorption edge). 5. and specifically, no other phases and molecular replacement solutions are available. Obviously, I have a case very similar to what's described above, and three years of failure with heavy atom derivatization (I am still trying). I would be happy to hear about Se-Met cases, and data collection strategies (2wl vs. 3wl MAD vs. SAD, etc.) and phasing methods used in these cases, or references of them
Re: [ccp4bb] phasing with se-met at low resolution
On Sun, May 10, 2009 at 08:24:34PM -0700, Engin Ozkan wrote: The question is what would happen if your crystals diffract to 4 A, and anomalous signal dies at 6 A. The interesting bit of course is 1 Met per 200 residue, which should put to death the 1 in 50 or 1 in 100 Methionine myths: it depends on the quality of your data. Have a look at 2jk4 ... which had the added 'fun' of being a membrane protein with very anisotropic diffraction (best direction to about 4A) and no NCS for averaging ... Cheers Clemens -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
Re: [ccp4bb] phasing with se-met at low resolution
If experience from intrinsic zinc is ok, I'll add my two cents. trying). I would be happy to hear about Se-Met cases, and data collection strategies (2wl vs. 3wl MAD vs. SAD, etc.) and phasing methods used in these cases, or references of them. Again, no other Bert already mentioned collecting in wedges for SAS, so I'll add to the chorus there. For dispersive differences, wedges (20 degrees at inf, 20 at rmt) helps a great deal for some of our crystals as well. P.S. I would also appreciate the specific query type for searching the PDB on the web for phasing method (MR, MAD, SAD, MIR, etc.). They seem to have everything under the sun searchable, but I cannot find this one. Last time I emailed the RCSB about this (a few years back), it wasn't possible to search by phasing method. You can try using advanced search - keyword search - advanced and doing a full text search, but this is somewhat less than ideal. To be fair though, I suspect relatively few people searching the PDB are concerned about the phasing method used. Pete
[ccp4bb] phasing with se-met at low resolution
Hi everyone, I thought I start a new thread while it is unusually quiet on the bb. I am pondering over the practical limitations to MAD and SAD phasing with Se-Met at low resolution. What is the lowest resolution at which people have solved structures only using phases from selenium in a realistic case? Let me further qualify my question: My *realistic* *low* resolution case is where 1. Rmerge over all resolution bins is 6-10% (i.e. your crystals are lousy). 2. Resolution limit is worse than 3.5 Angstroms, where I/sigma in the last resolution bin is between 1 and 3 (i.e. your crystals are really lousy). 3. Assuming good selenium occupancy (~85%; I work with eukaryotic expression systems, so 100% is not usually achieavable), 4. The number of selenium atoms are enough many that the Crick-Magdoff equation would give you *at least* an average 5% change in intensities (assuming 6 electrons contributed per selenium, based on both absorptive and dispersive differences being at about 6 e- at the absorption edge). 5. and specifically, no other phases and molecular replacement solutions are available. Obviously, I have a case very similar to what's described above, and three years of failure with heavy atom derivatization (I am still trying). I would be happy to hear about Se-Met cases, and data collection strategies (2wl vs. 3wl MAD vs. SAD, etc.) and phasing methods used in these cases, or references of them. Again, no other partial phases, and no data cut off at 3.6 A with an I/s of 15 in the last resolution bin. Are there any examples out there? Searching the RCSB and PubMed did not point out to me many successful cases. Thanks, Engin P.S. I would also appreciate the specific query type for searching the PDB on the web for phasing method (MR, MAD, SAD, MIR, etc.). They seem to have everything under the sun searchable, but I cannot find this one.
Re: [ccp4bb] phasing with se-met at low resolution
Dear Engin Ozkan, You have told us how bad your crystals are, but you did not mention how good your anomalous signal is: 1. To what resolution does your anomalous signal extend and what statistic is used for this estimate? 2. Do your dispersive and Bijvoet Pattersons look similar and what is the measure of similarity? This structure http://www.pdb.org/pdb/explore/explore.do?structureId=1K28 which contains ~1100 residues in the asymmetric unit (and ~3500 in the entire complex), was solved using a chimerical SeMet derivative, in which one protein was SeMet labeled (17 Se per a.u.) and the other was native. The Semet dataset had a detectable anomalous signal to 4 A resolution (at most). The diffraction extended to 2.9A resolution. Sincerely, Petr --- Petr Leiman Institut de physique des systèmes biologiques École Polytechnique Fédérale de Lausanne (EPFL) Cubotron/BSP-415 CH-1015 Lausanne Switzerland -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Engin Ozkan Sent: Sunday, May 10, 2009 11:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] phasing with se-met at low resolution Hi everyone, I thought I start a new thread while it is unusually quiet on the bb. I am pondering over the practical limitations to MAD and SAD phasing with Se-Met at low resolution. What is the lowest resolution at which people have solved structures only using phases from selenium in a realistic case? Let me further qualify my question: My *realistic* *low* resolution case is where 1. Rmerge over all resolution bins is 6-10% (i.e. your crystals are lousy). 2. Resolution limit is worse than 3.5 Angstroms, where I/sigma in the last resolution bin is between 1 and 3 (i.e. your crystals are really lousy). 3. Assuming good selenium occupancy (~85%; I work with eukaryotic expression systems, so 100% is not usually achieavable), 4. The number of selenium atoms are enough many that the Crick-Magdoff equation would give you *at least* an average 5% change in intensities (assuming 6 electrons contributed per selenium, based on both absorptive and dispersive differences being at about 6 e- at the absorption edge). 5. and specifically, no other phases and molecular replacement solutions are available. Obviously, I have a case very similar to what's described above, and three years of failure with heavy atom derivatization (I am still trying). I would be happy to hear about Se-Met cases, and data collection strategies (2wl vs. 3wl MAD vs. SAD, etc.) and phasing methods used in these cases, or references of them. Again, no other partial phases, and no data cut off at 3.6 A with an I/s of 15 in the last resolution bin. Are there any examples out there? Searching the RCSB and PubMed did not point out to me many successful cases. Thanks, Engin P.S. I would also appreciate the specific query type for searching the PDB on the web for phasing method (MR, MAD, SAD, MIR, etc.). They seem to have everything under the sun searchable, but I cannot find this one.
Re: [ccp4bb] phasing with se-met at low resolution
Hi Engin, first off, i would not consider an overall Rmerge of 6-10% lousy data, but quite acceptable for most real-life, interesting problems (so no lysozyme, thaumatin etc). Our structure of the protein translocation channel SecY is an example of de novo low-res Se phasing (PDB code 1RHZ). Phases were obtained by carefully collected (friedel flipping with 10-20 deg wedges) peak-wavelength SAD datasets combined with cross-crystal averaging to get interpretable maps. We didn't have NCS. The best resolution was about 3.5 A for the selenium datasets. I would say it is possible (but hard) for anything with at least orthorhombic symmetry. I have a Se SAD data set myself that will not give interpretable maps; the symmetry is P1 however and there are multiple lattices, so this seems pretty hopeless... I would expect there are a bunch of membrane protein structures that fulfill your criteria. Off the top of my head is the small mechano-sensitive channel MscS solved by the Rees group. I think this is 3.9 A data, with a high degree of NCS however. Good luck, Bert Bert van den Berg University of Massachusetts Medical School Program in Molecular Medicine Biotech II, 373 Plantation Street, Suite 115 Worcester MA 01605 Phone: 508 856 1201 (office); 508 856 1211 (lab) e-mail: bert.vandenb...@umassmed.edu http://www.umassmed.edu/pmm/faculty/vandenberg.cfm -Original Message- From: CCP4 bulletin board on behalf of Engin Ozkan Sent: Sun 5/10/2009 5:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] phasing with se-met at low resolution Hi everyone, I thought I start a new thread while it is unusually quiet on the bb. I am pondering over the practical limitations to MAD and SAD phasing with Se-Met at low resolution. What is the lowest resolution at which people have solved structures only using phases from selenium in a realistic case? Let me further qualify my question: My *realistic* *low* resolution case is where 1. Rmerge over all resolution bins is 6-10% (i.e. your crystals are lousy). 2. Resolution limit is worse than 3.5 Angstroms, where I/sigma in the last resolution bin is between 1 and 3 (i.e. your crystals are really lousy). 3. Assuming good selenium occupancy (~85%; I work with eukaryotic expression systems, so 100% is not usually achieavable), 4. The number of selenium atoms are enough many that the Crick-Magdoff equation would give you *at least* an average 5% change in intensities (assuming 6 electrons contributed per selenium, based on both absorptive and dispersive differences being at about 6 e- at the absorption edge). 5. and specifically, no other phases and molecular replacement solutions are available. Obviously, I have a case very similar to what's described above, and three years of failure with heavy atom derivatization (I am still trying). I would be happy to hear about Se-Met cases, and data collection strategies (2wl vs. 3wl MAD vs. SAD, etc.) and phasing methods used in these cases, or references of them. Again, no other partial phases, and no data cut off at 3.6 A with an I/s of 15 in the last resolution bin. Are there any examples out there? Searching the RCSB and PubMed did not point out to me many successful cases. Thanks, Engin P.S. I would also appreciate the specific query type for searching the PDB on the web for phasing method (MR, MAD, SAD, MIR, etc.). They seem to have everything under the sun searchable, but I cannot find this one.
Re: [ccp4bb] phasing with se-met at low resolution
Wow, I got quite a number of responses. Thanks everyone. Let's elaborate. Petr, I don't know my anomalous signal, because I haven't yet done the experiment. If I had, I would have definitely talked about chi2 values for I+/I- merged and unmerged, Anomalous Patterson maps, or other measures of anomalous signal (Dauter, Acta Cryst D, 2006 is a great paper to read on that, I suggest every grad student to present it in their Crystallography Journal Club). I was merely pondering today, but I plan to do the experiment very soon (crystals are waiting for synchrotron time). About your example, 2.9 A diffracting crystals (with 4 A anomalous signal) are in the doable range, as you suggested. The question is what would happen if your crystals diffract to 4 A, and anomalous signal dies at 6 A. The interesting bit of course is 1 Met per 200 residue, which should put to death the 1 in 50 or 1 in 100 Methionine myths: it depends on the quality of your data. Engin On 5/10/09 2:50 PM, Leiman Petr wrote: Dear Engin Ozkan, You have told us how bad your crystals are, but you did not mention how good your anomalous signal is: 1. To what resolution does your anomalous signal extend and what statistic is used for this estimate? 2. Do your dispersive and Bijvoet Pattersons look similar and what is the measure of similarity? This structure http://www.pdb.org/pdb/explore/explore.do?structureId=1K28 which contains ~1100 residues in the asymmetric unit (and ~3500 in the entire complex), was solved using a chimerical SeMet derivative, in which one protein was SeMet labeled (17 Se per a.u.) and the other was native. The Semet dataset had a detectable anomalous signal to 4 A resolution (at most). The diffraction extended to 2.9A resolution. Sincerely, Petr --- Petr Leiman Institut de physique des systèmes biologiques École Polytechnique Fédérale de Lausanne (EPFL) Cubotron/BSP-415 CH-1015 Lausanne Switzerland -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Engin Ozkan Sent: Sunday, May 10, 2009 11:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] phasing with se-met at low resolution Hi everyone, I thought I start a new thread while it is unusually quiet on the bb. I am pondering over the practical limitations to MAD and SAD phasing with Se-Met at low resolution. What is the lowest resolution at which people have solved structures only using phases from selenium in a realistic case? Let me further qualify my question: My *realistic* *low* resolution case is where 1. Rmerge over all resolution bins is 6-10% (i.e. your crystals are lousy). 2. Resolution limit is worse than 3.5 Angstroms, whereI/sigma in the last resolution bin is between 1 and 3 (i.e. your crystals are really lousy). 3. Assuming good selenium occupancy (~85%; I work with eukaryotic expression systems, so 100% is not usually achieavable), 4. The number of selenium atoms are enough many that the Crick-Magdoff equation would give you *at least* an average 5% change in intensities (assuming 6 electrons contributed per selenium, based on both absorptive and dispersive differences being at about 6 e- at the absorption edge). 5. and specifically, no other phases and molecular replacement solutions are available. Obviously, I have a case very similar to what's described above, and three years of failure with heavy atom derivatization (I am still trying). I would be happy to hear about Se-Met cases, and data collection strategies (2wl vs. 3wl MAD vs. SAD, etc.) and phasing methods used in these cases, or references of them. Again, no other partial phases, and no data cut off at 3.6 A with an I/s of 15 in the last resolution bin. Are there any examples out there? Searching the RCSB and PubMed did not point out to me many successful cases. Thanks, Engin P.S. I would also appreciate the specific query type for searching the PDB on the web for phasing method (MR, MAD, SAD, MIR, etc.). They seem to have everything under the sun searchable, but I cannot find this one.
