Re: [ccp4bb] @Ian:Death of Rmerge
Wow, it is quite a lecture here! It is very appreciated. I admit some (most?) of my statements were questionable. Thus, I did not know how sigI would be calculated in case of multiple observations, and, indeed, its proper handling should make sigI/I similar to Rmerge. Consequently, I/sigI substitutes Rmerge fairly well. Now, where the metric Rmerge=0.5 came from? If I remember correctly, It was proposed here at ccp4bb. Also, one reviewer suggested to use it. I admit that this is quite an arbitrary value, but when everyone follows it, structures become comparable by this metric. If there is a better approach to estimate the resolution, lets use it, but the common rule should be enforced, otherwise the resolution becomes another venue for cheating. Once again, I was talking about metric for the resolution, it does not need to be equal to metric for the data cutoff. Alex On Jun 3, 2012, at 2:55 PM, Ian Tickle wrote: Hi Alex On 3 June 2012 07:00, aaleshin aales...@burnham.org wrote: I was also taught that under normal conditions this would occur when the data are collected up to the shell, in which Rmerge = 0.5. Do you have a reference for that? I have not seen a demonstration of such an exact relationship between Rmerge and resolution, even for 'normal' data, and I don't think everyone uses 0.5 as the cut-off anyway (e.g. some people use 0.4, some 0.8 etc - though I agree with Phil that we shouldn't get too hung up about the exact number!). Certainly having used the other suggested criteria for resolution cut-off (I/sigma(I) CC(1/2)), the corresponding Rmerge (and Rpim etc) seems to vary a lot (or maybe my data weren't 'normal'). One can collect more data (up to Rmerge=1.0 or even 100) but the resolution of the electron density map will not change significantly. I think we are all at least agreed that beyond some resolution cut-off, adding further higher resolution 'data' will not result in any further improvement in the map (because the weights will become negligible). So it would appear prudent at least to err on the high resolution side! I solved several structures of my own, and this simple rule worked every time. In what sense do you mean it 'worked'? Do you mean you tried different cut-offs in Rmerge (e.g. 0.25, 0.50, 0.75, 1.00 ...) and then used some metric to judge when there was no further significant change in the map and you noted that the optimal value of your chosen metric always occurs around Rmerge 0.5?; and if so how did you judge a 'significant change'? Personally I go along with Dale's suggestion to use the optical resolution of the map to judge when no further improvement occurs. This would need to be done with the completely refined structure because presumably optical resolution will be reduced by phase errors. Note that it wouldn't be necessary to actually quote the optical resolution in place of the X-ray resolution (that would confuse everyone!), you just need to know the value of the X-ray resolution cut-off where the optical resolution no longer changes (it should be clear from a plot of X-ray vs. optical resolution). I is measured as a number of detector counts in the reflection minus background counts. sigI is measured as sq. root of I plus standard deviation (SD) for the background plus various deviations from ideal experiment (like noise from satellite crystals). The most important contribution to the sigma(I)'s, except maybe for the weak reflections, actually comes from differences between the intensities of equivalent reflections, due to variations in absorption and illuminated volume, and other errors in image scale factors (though these are all highly correlated). These are of course exactly the same differences that contribute to Rmerge. E.g. in Scala the SDFAC SDADD parameters are automatically adjusted to fit the observed QQ plot to the expected one, in order to account for such differences. Obviously, sigI cannot be measured accurately. Moreover, the 'resolution' is related to errors in the structural factors, which are average from several measurements. Errors in their scaling would affect the 'resolution', and I/sigI does not detect them, but Rmerge does! Sorry you've lost me here, I don't see why I/sigI should not detect scaling errors: as indicated above if there are errors in the scale factors this will inflate the sigma(I) values via increased SDFAC and/or SDADD, which will increase the sigma(I) values which will in turn reduce the I/sigma(I) values exactly as expected. I see no difference in the behaviour of Rmerge and I/sigma(I) (or indeed in CC(1/2)) in this respect, since they all depend on the differences between equivalents. Rmerge, it means that the symmetry related reflections did not merge well. Under those conditions, Rmerge becomes a much better criterion for estimation of the 'resolution' than sigi/I. As indicated
Re: [ccp4bb] How to know if ADP exists in the ATP-binding site of bacterial expressed proteins
At 280nm absorbs Trp but Tyr and Phe (much less) as well. So your protein sample even without Trp will absorb. A mass spect experiment should show a presence of molecule with MW 507 (ATP) or MW 427 (ADP) George From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Xinghua Qin Sent: Monday, June 04, 2012 7:52 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] How to know if ADP exists in the ATP-binding site of bacterial expressed proteins Deer CCP4ers, How to know if ADP exists in the ATP-binding site of bacterial expressed proteins? Check the UV spectrum at 280 nm is one way, but there is no Trp in my protein. Is there any other conveniet way to find out? Thanks in advance Best wishes Xinghua Qin -- Xinghua Qin State Key Laboratory of Plant Physiology and biochemistry College of Biological Sciences China Agricultural University No.2, Yuan Ming Yuan West Road Haidian District, Beijing, China 100193 Tel: +86-10-62732672 E-mail: mailto:xhqin1...@gmail.com xinghua...@126.com
[ccp4bb] Position for post-doc and graduate student in Amsterdam
Post-doctoral and PhD positions available in the group of Titia K. Sixma, Netherlands Cancer Institute, Amsterdam We are looking for motivated enthousiastic researchers with strong experience in X-ray crystallography and protein biochemistry. Projects are focussing on ubiquitin conjugation processes in DNA regulation, where we study the roles of E3 ligases and deubiquitinating enzymes. Aims are to reconstitute larger complexes in vitro and study structural and functional aspects of the basic enzymatic activities and correlate these to cellular function. The NKI is well-established as a center of excellence for cancer research, with strong genetics, cell biology and structural biology. The institute is highly interactive and is well set up with strong core facilities. For structural biology we share equipment between the Sixma and Perrakis labs and the protein facility . Setup is excellent for biochemistry, biophysics (ITC, Malls, stopped flow, SPR, fluorescence etc) and X-ray crystallography. Amsterdam is a very livable city with many cultural amenities. The institute is located within a 20 minute tram or bicycle ride from the center of Amsterdam and within 20 minutes from Schiphol airport by car, bus or bicycle. Titia K. Sixma Division of Biochemistry Nederlands Kanker Instituut Plesmanlaan 121 1066 CX Amsterdam E-mail: t.si...@nki.nl Tel: +31-20-5121959 Website: http://xtal.nki.nl
Re: [ccp4bb] @Ian:Death of Rmerge
well, actually i recommend having a look at the old but good scalepack manual for why Rmerge is inferior.. (i thought this was clear long ago.. so i am bit amazed that this discussio is still alive and kicking..) question of where to cut, is a different one and thats where the recent papers and developments start to come in. short quote...(scalepack manual): From a statistical point of view, I/σ is a superior criterion, for two reasons. First, it defines a resolution “limit” since by definition I/σ is the signal to noise of your measurements. In contrast, Rmerge is not directly related to signal to noise. Second, the σ assigned to each intensity derives its validity from the χ2’s, which represent the weighted ratio of the difference between the observed and average value of I, 〈I〉, squared, divided by the square of the error model, the whole thing times a factor correcting for the correlation between I and 〈I〉. Since it depends on an explicit declaration of the expected error in the measurement, the user of the program is part of the Bayesian reasoning process behind the error estimation. ..In short, I/σ is the preferred way of assessing the quality of diffraction data because it derives its validity from the χ2 (likelihood) analysis. credits to Otwinowski et al. end of story, i believe. so R-merge died long back. -tommi On Jun 4, 2012, at 9:00 AM, aaleshin wrote: Wow, it is quite a lecture here! It is very appreciated. I admit some (most?) of my statements were questionable. Thus, I did not know how sigI would be calculated in case of multiple observations, and, indeed, its proper handling should make sigI/I similar to Rmerge. Consequently, I/sigI substitutes Rmerge fairly well. Now, where the metric Rmerge=0.5 came from? If I remember correctly, It was proposed here at ccp4bb. Also, one reviewer suggested to use it. I admit that this is quite an arbitrary value, but when everyone follows it, structures become comparable by this metric. If there is a better approach to estimate the resolution, lets use it, but the common rule should be enforced, otherwise the resolution becomes another venue for cheating. Once again, I was talking about metric for the resolution, it does not need to be equal to metric for the data cutoff. Alex On Jun 3, 2012, at 2:55 PM, Ian Tickle wrote: Hi Alex On 3 June 2012 07:00, aaleshin aales...@burnham.org wrote: I was also taught that under normal conditions this would occur when the data are collected up to the shell, in which Rmerge = 0.5. Do you have a reference for that? I have not seen a demonstration of such an exact relationship between Rmerge and resolution, even for 'normal' data, and I don't think everyone uses 0.5 as the cut-off anyway (e.g. some people use 0.4, some 0.8 etc - though I agree with Phil that we shouldn't get too hung up about the exact number!). Certainly having used the other suggested criteria for resolution cut-off (I/sigma(I) CC(1/2)), the corresponding Rmerge (and Rpim etc) seems to vary a lot (or maybe my data weren't 'normal'). One can collect more data (up to Rmerge=1.0 or even 100) but the resolution of the electron density map will not change significantly. I think we are all at least agreed that beyond some resolution cut-off, adding further higher resolution 'data' will not result in any further improvement in the map (because the weights will become negligible). So it would appear prudent at least to err on the high resolution side! I solved several structures of my own, and this simple rule worked every time. In what sense do you mean it 'worked'? Do you mean you tried different cut-offs in Rmerge (e.g. 0.25, 0.50, 0.75, 1.00 ...) and then used some metric to judge when there was no further significant change in the map and you noted that the optimal value of your chosen metric always occurs around Rmerge 0.5?; and if so how did you judge a 'significant change'? Personally I go along with Dale's suggestion to use the optical resolution of the map to judge when no further improvement occurs. This would need to be done with the completely refined structure because presumably optical resolution will be reduced by phase errors. Note that it wouldn't be necessary to actually quote the optical resolution in place of the X-ray resolution (that would confuse everyone!), you just need to know the value of the X-ray resolution cut-off where the optical resolution no longer changes (it should be clear from a plot of X-ray vs. optical resolution). I is measured as a number of detector counts in the reflection minus background counts. sigI is measured as sq. root of I plus standard deviation (SD) for the background plus various deviations from ideal experiment (like noise from satellite crystals). The most important contribution to the sigma(I)'s, except maybe for the weak reflections, actually comes
Re: [ccp4bb] How to know if ADP exists in the ATP-binding site of bacterial expressed proteins
Ion exchange chromatography, this is the published method. Acoot From: Xinghua Qin xinghua...@126.com To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, 4 June 2012 2:51 PM Subject: [ccp4bb] How to know if ADP exists in the ATP-binding site of bacterial expressed proteins Deer CCP4ers, How to know if ADP exists in the ATP-binding site of bacterial expressed proteins? Check the UV spectrum at 280 nm is one way, but there is no Trp in my protein. Is there any other conveniet way to find out? Thanks in advance Best wishes Xinghua Qin -- Xinghua Qin State Key Laboratory of Plant Physiology and biochemistry College of Biological Sciences China Agricultural University No.2, Yuan Ming Yuan West Road Haidian District, Beijing, China 100193 Tel: +86-10-62732672 E-mail: xinghua...@126.com
[ccp4bb]
Is it reasonable to refine occupancy in phenix at 2.2 A resolution? Implementations may differ, but imgo refining occupancy at 2.2A resolution is not very reasonable under most circumstances, as it will correlate strongly with the B-factor. A reasonable approach might be to fix occupancy at different levels and get a series of refined models. Then you look at (i) B-factor behavior and see at what occupancy it matches the surrounding atoms and (ii) difference density (my unsubstantiated theory is that if you plot it against occupancy it should have a central flat region where B-factors are capable of compensating and two linear regions on extreme ends which should allow to extrapolate the true value. Refmac does occupancy refinement. It's quite fast, so you may try randomizing the initial value and get some idea about convergence. Cheers, Ed. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] @Ed: Death of Rmerge
On Sat, 2012-06-02 at 23:32 -0700, aaleshin wrote: Was not Z. Otwinowski first to use it in his scalepack? Maybe I missed something, but given the hoops I have to jump through to get Rpim calculated after scalepack (basically take unmerged data to either the program from Manfred Weiss or SCALA) I think that scalepack still does not report anything but Rmerge. Perhaps this is because HKL team also does not consider Rmerge and its better behaving variants worthy of the role of resolution-determining factor, but I am not a mind reader (unfortunately).
[ccp4bb] REMINDER - BIOCRYS 2012
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Re: [ccp4bb] @Tommi:Death of Rmerge
i thought this was clear long ago.. so i am bit amazed that this discussio is still alive and kicking.. Even though it does not relate to me, but the explanation may come from the fact that new people start using crystallography, and they do not like reading old papers. So, there is nothing wrong in bringing such fundamental discussions up to life periodically. I had some misconception about accuracy of sigI, which I explained earlier. It is obvious that I/σ is the signal to noise of your measurements at a local point of the reciprocal space, but it is not obvious that it would work as well for the merged data. Thanks to Ian and others, I now understand that there is no problem with it. I am also glad to stumble on the referenced papers about the resolution and data quality (Ed Pozharski's post). I missed them because at the time when they published, I was learning molecular biology, enzymology, virology... A modern crystallographer needs to be a good biologist, and this applies some limitations on how much we know about each technique that we use. Alex On Jun 4, 2012, at 1:48 AM, Tommi Kajander wrote: well, actually i recommend having a look at the old but good scalepack manual for why Rmerge is inferior.. (i thought this was clear long ago.. so i am bit amazed that this discussio is still alive and kicking..) question of where to cut, is a different one and thats where the recent papers and developments start to come in. short quote...(scalepack manual): From a statistical point of view, I/σ is a superior criterion, for two reasons. First, it defines a resolution “limit” since by definition I/σ is the signal to noise of your measurements. In contrast, Rmerge is not directly related to signal to noise. Second, the σ assigned to each intensity derives its validity from the χ2’s, which represent the weighted ratio of the difference between the observed and average value of I, 〈I〉, squared, divided by the square of the error model, the whole thing times a factor correcting for the correlation between I and 〈I〉. Since it depends on an explicit declaration of the expected error in the measurement, the user of the program is part of the Bayesian reasoning process behind the error estimation. ..In short, I/σ is the preferred way of assessing the quality of diffraction data because it derives its validity from the χ2 (likelihood) analysis. credits to Otwinowski et al. end of story, i believe. so R-merge died long back. -tommi On Jun 4, 2012, at 9:00 AM, aaleshin wrote: Wow, it is quite a lecture here! It is very appreciated. I admit some (most?) of my statements were questionable. Thus, I did not know how sigI would be calculated in case of multiple observations, and, indeed, its proper handling should make sigI/I similar to Rmerge. Consequently, I/sigI substitutes Rmerge fairly well. Now, where the metric Rmerge=0.5 came from? If I remember correctly, It was proposed here at ccp4bb. Also, one reviewer suggested to use it. I admit that this is quite an arbitrary value, but when everyone follows it, structures become comparable by this metric. If there is a better approach to estimate the resolution, lets use it, but the common rule should be enforced, otherwise the resolution becomes another venue for cheating. Once again, I was talking about metric for the resolution, it does not need to be equal to metric for the data cutoff. Alex On Jun 3, 2012, at 2:55 PM, Ian Tickle wrote: Hi Alex On 3 June 2012 07:00, aaleshin aales...@burnham.org wrote: I was also taught that under normal conditions this would occur when the data are collected up to the shell, in which Rmerge = 0.5. Do you have a reference for that? I have not seen a demonstration of such an exact relationship between Rmerge and resolution, even for 'normal' data, and I don't think everyone uses 0.5 as the cut-off anyway (e.g. some people use 0.4, some 0.8 etc - though I agree with Phil that we shouldn't get too hung up about the exact number!). Certainly having used the other suggested criteria for resolution cut-off (I/sigma(I) CC(1/2)), the corresponding Rmerge (and Rpim etc) seems to vary a lot (or maybe my data weren't 'normal'). One can collect more data (up to Rmerge=1.0 or even 100) but the resolution of the electron density map will not change significantly. I think we are all at least agreed that beyond some resolution cut-off, adding further higher resolution 'data' will not result in any further improvement in the map (because the weights will become negligible). So it would appear prudent at least to err on the high resolution side! I solved several structures of my own, and this simple rule worked every time. In what sense do you mean it 'worked'? Do you mean you tried different cut-offs in Rmerge (e.g. 0.25, 0.50, 0.75, 1.00 ...) and then used some metric to
[ccp4bb]
Hi Ed, I've actually run that exact test in phenix as an exercise to prove to my PI the validity of occupancy refinement. Though as a disclaimer it was a 1.2 angstrom data set and this was an alternate conformation situation. I ran different input occupancies without occupancy refinement and measure the difference density peak values and average B-factors and ended up with the same occupancy ratio that the program's occupancy refinement spit out. Of course this might not hold true if someone is refining the occupancy of a ligand that is partially bound without an alternate option (i.e. total occupancy 1). I haven't tested that one systematically yet though I suspect Pavel has probably already done this at some point. Cheers, Katherine On Mon, Jun 4, 2012 at 7:35 AM, Ed Pozharski epozh...@umaryland.edu wrote: Is it reasonable to refine occupancy in phenix at 2.2 A resolution? Implementations may differ, but imgo refining occupancy at 2.2A resolution is not very reasonable under most circumstances, as it will correlate strongly with the B-factor. A reasonable approach might be to fix occupancy at different levels and get a series of refined models. Then you look at (i) B-factor behavior and see at what occupancy it matches the surrounding atoms and (ii) difference density (my unsubstantiated theory is that if you plot it against occupancy it should have a central flat region where B-factors are capable of compensating and two linear regions on extreme ends which should allow to extrapolate the true value. Refmac does occupancy refinement. It's quite fast, so you may try randomizing the initial value and get some idea about convergence. Cheers, Ed. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
[ccp4bb]
On Mon, 2012-06-04 at 13:11 -0500, Katherine Sippel wrote: Though as a disclaimer it was a 1.2 angstrom data set Which is about 6x more data than 2.2A... Certainly, at atomic resolution the results of occupancy refinement will be more robust. To be fair, even at 2.2A such refinement may succeed sometimes, e.g. for heavy atoms.
