[ccp4bb] Synthetic RNA for Crystallization
Hello All, I am looking for some advice from some experienced RNA crystallographers. I would like to order some relatively short (90 bases) synthetic RNAs for crystallization trials. I was wondering if anyone could comment on the use of synthetic RNAs for crystallization. Specifically, what is the longest synthetic RNA that can be used for crystallization trials? I've seen some structures in the PDB that are up to 88 bases and are reported to have been obtained with synthetic constructs (3OWI - glycine riboswitch), but I don't really know if that's routine or if it's an exceptional case. Also, for those who have experience with the use of synthetic RNAs, I was wondering where people generally order their synthetic constructs from? Our resident expert in RNA crystallography recommended a company called Dharmacon (part of ThermoFisher), but I was hoping that I might get some other opinions as to which companies make the best quality oligonucleotides, provide samples with the highest purity, and have the most reasonable prices. Thanks in advance for the help! Mike -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] Synthetic RNA for Crystallization
Hi Michael, we normally produce synthetic RNAs following this classic paper if the size is more than let say 40-50 nucleotide, otherwise we buy the RNAs from Dharmacon and the quality is totally OK. Hope it helps javascript:AL_get(this,%20'jour',%20'J%20Mol%20Biol.'); J Mol Biol. javascript:AL_get(this,%20'jour',%20'J%20Mol%20Biol.'); 1995 Jun 2;249(2):398-408. Crystallization of RNA-protein complexes. I. Methods for the large-scale preparation of RNA suitable for crystallographic studies. Price SRhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Price%20SR%22%5BAuthor%5D, Ito N http://www.ncbi.nlm.nih.gov/pubmed?term=%22Ito%20N%22%5BAuthor%5D, Oubridge C http://www.ncbi.nlm.nih.gov/pubmed?term=%22Oubridge%20C%22%5BAuthor%5D, Avis JM http://www.ncbi.nlm.nih.gov/pubmed?term=%22Avis%20JM%22%5BAuthor%5D, Nagai K http://www.ncbi.nlm.nih.gov/pubmed?term=%22Nagai%20K%22%5BAuthor%5D. MRC Laboratory of Molecular Biology, Cambridge, UK. Abstract In vitro transcription using bacteriophage RNA polymerases and linearised plasmid or oligodeoxynucleotide templates has been used extensively to produce RNA for biochemical studies. This method is, however, not ideal for generating RNA for crystallisation because efficient synthesis requires the RNA to have a purine rich sequence at the 5' terminus, also the subsequent RNA is heterogenous in length. We have developed two methods for the large scale production of homogeneous RNA of virtually any sequence for crystallization. In the first method RNA is transcribed together with two flanking intramolecularly-, (cis-), acting ribozymes which excise the desired RNA sequence from the primary transcript, eliminating the promoter sequence and heterogeneous 3' end generated by run-off transcription. We use a combination of two hammerhead ribozymes or a hammerhead and a hairpin ribozyme. The RNA-enzyme activity generates few sequence restrictions at the 3' terminus and none at the 5' terminus, a considerable improvement on current methodologies. In the second method the BsmAI restriction endonuclease is used to linearize plasmid template DNA thereby allowing the generation of RNA with any 3' end. In combination with a 5' cis-acting hammerhead ribozyme any sequence of RNA may be generated by in vitro transcription. This has proven to be extremely useful for the synthesis of short RNAs. 2011/3/13 Michael Thompson mi...@chem.ucla.edu Hello All, I am looking for some advice from some experienced RNA crystallographers. I would like to order some relatively short (90 bases) synthetic RNAs for crystallization trials. I was wondering if anyone could comment on the use of synthetic RNAs for crystallization. Specifically, what is the longest synthetic RNA that can be used for crystallization trials? I've seen some structures in the PDB that are up to 88 bases and are reported to have been obtained with synthetic constructs (3OWI - glycine riboswitch), but I don't really know if that's routine or if it's an exceptional case. Also, for those who have experience with the use of synthetic RNAs, I was wondering where people generally order their synthetic constructs from? Our resident expert in RNA crystallography recommended a company called Dharmacon (part of ThermoFisher), but I was hoping that I might get some other opinions as to which companies make the best quality oligonucleotides, provide samples with the highest purity, and have the most reasonable prices. Thanks in advance for the help! Mike -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu -- Israel Sanchez Fernandez PhD Ramakrishnan-lab MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK
Re: [ccp4bb] I/sigmaI of 3.0 rule
Dear Boaz, You are quite correct, 'latter' and 'former' need to be switched in my email. Apologies to CCP4bb for the confusion caused! Best wishes, George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Sun, 13 Mar 2011, Boaz Shaanan wrote: Dear George, While I agree with you I wonder whether in this statement: ...The practice of quoting R-values both for all data and for F4sigma(F) seems to me to be useful. For example if the latter is much larger than the former, maybe you are including a lot of weak data... Shouldn't it be: ...former (i.e. R for all data) is much larger than the latter (i.e. R for F4sigma(F)... ? Just wondering, although it could be my late night misunderstanding. Best regards, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of George M. Sheldrick [gshe...@shelx.uni-ac.gwdg.de] Sent: Sunday, March 13, 2011 12:11 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] I/sigmaI of 3.0 rule Dear James, I'm a bit puzzled by your negative R-values and unstable behavior. In practice, whether we refine against intensity or against |F|, it is traditional to quote an R-factor (called R1 in small molecule crystallography) R = Sum||Fo|-|Fc|| / Sum|Fo|. Reflections that have negative measured intensities are either given F=0 or (e.g. using TRUNCATE) F is set to a small positive value, both of which avoid having to take the square root of a negative number which most computers don't like doing. Then the 'divide by zero' catastropy and negative R-values cannot happen because Sum|Fo| is always significantly greater than zero, and in my experience there is no problem in calculating an R-value even if the data are complete noise. The practice of quoting R-values both for all data and for F4sigma(F) seems to me to be useful. For example if the latter is much larger than the former, maybe you are including a lot of weak data. Similarly in calculating merging R-values, most programs replace negative intensities by zero, again avoiding the problems you describe. Best wishes, George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Sat, 12 Mar 2011, James Holton wrote: The fundamental mathematical problem of using an R statistic on data with I/sigma(I) 3 is that the assumption that the fractional deviates (I-I)/I obey a Gaussian distribution breaks down. And when that happens, the R calculation itself becomes unstable, giving essentially random R values. Therefore, including weak data in R calculations is equivalent to calculating R with a 3-sigma cutoff, and then adding a random number to the R value. Now, random data is one thing, but if the statistic used to evaluate the data quality is itself random, then it is not what I would call useful. Since I am not very good at math, I always find myself approaching statistics by generating long lists of random numbers, manipulating them in some way, and then graphing the results. For graphing Rmerge vs I/sigma(I), one does find that Bernhard's rule of Rmerge = 0.8/( I/sigma(I) ) generally applies, but only for I/sigma(I) that is = 3. It gets better with high multiplicity, but even with m=100, the Rmerge values for the I/sigma(I) 1 points are all over the place. This is true even if you average the value of Rmerge over a million random number seeds. In fact, one must do so much averaging, that I start to worry about the low-order bits of common random number generators. I have attached images of these Rmerge vs I/sigma graphs. The error bars reflect the rms deviation from the average of a large number of Rmerge values (different random number seeds). The missing values are actually points where the average Rmerge in 60 trials (m=3) was still negative. The reason for this noisy R factor problem becomes clear if you consider the limiting case where the true intensity is zero, and make a histogram of ( I - I )/I. It is not a Gaussian. Rather, it is the Gaussian's evil stepsister: the Lorentzian (or Cauchy distribution). This distribution may look a lot like a Gaussian, but it has longer tails, and these tails give it the weird statistical property of having an undefined mean value. This is counterintuitive! Because you can clearly just look at the histogram and see that it has a central peak (at zero), but if you generate a million
Re: [ccp4bb] Automating liquid dispensing
The Liquidator 96 is at my opinion the best in the market. Is a manual system, but fast without problems related to cross contamination. The electronic ones (from other brands) at some point gives you problems with the electroninc system and the tips are not disposable (they take long time between plates due to the tip washes). Regards, Isabel - Dr. Isabel De Moraes, MRSC Membrane Protein Laboratory Facilities Co-ordinator/Group Leader Membrane Protein Laboratory, Diamond Light Source Ltd, Harwell Science and Innovation Campus, Chilton, Didcot, Oxfordshire, OX11 ODE, UK Tel: 01235 778664 email: isabel.de-mor...@diamond.ac.uk http://www.diamond.ac.uk/Science/MPL/default.htm http://www.rc-harwell.ac.uk/ --- From: CCP4 bulletin board on behalf of Jim Fairman Sent: Fri 3/11/2011 10:57 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Automating liquid dispensing We also recently obtained a Liquidator 96 from Rainin. Working well so far and way below the price of any of the competitors. On Fri, Mar 11, 2011 at 5:41 PM, Engin Özkan eoz...@stanford.edu wrote: Dear Stephen, I did start a similar thread about a year ago. If you want to see the posts, check this out and the responses to it: http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg14758.html In my case, the winner was the Liquidator 96 from Rainin. It has proven to be very useful and too popular, and now it is being utilized for everything under the sun. Best, Engin On 3/11/11 2:00 PM, Stephen McMahon wrote: Hi - We are in the market for a new liquid handling robot specifically for dispensing custom 96 well crystallisation screens. What are people using these days? Likes / dislikes? Many thanks, Stephen -- Engin Özkan Post-doctoral Scholar Howard Hughes Medical Institute Dept of Molecular and Cellular Physiology 279 Campus Drive, Beckman Center B173 Stanford School of Medicine Stanford, CA 94305 ph: (650)-498-7111 -- Jim Fairman, Ph D. Post-Doctoral Fellow National Institutes of Health - NIDDK The Buchanan Lab http://www-mslmb.niddk.nih.gov/buchanan/index.html Lab: 1-301-594-9229 E-mail: fairman@gmail.com james.fair...@nih.gov
Re: [ccp4bb] I/sigmaI of 3.0 rule
You do also have always to consider why you are doing this calculation - usually to satisfy a sceptical and possibly ill-informed referee. A major reason for doing this is to justify including an outer resolution shell of data (see this BB passim), and for this I have come to prefer the random half-dataset correlation coefficient in shells. A CC has a more straightforward distribution than an R-factor (though not entirely without problems). It is independent of the SD estimates, and easy to understand. Phil The fundamental mathematical problem of using an R statistic on data with I/sigma(I) 3 is that the assumption that the fractional deviates (I-I)/I obey a Gaussian distribution breaks down. And when that happens, the R calculation itself becomes unstable, giving essentially random R values. Therefore, including weak data in R calculations is equivalent to calculating R with a 3-sigma cutoff, and then adding a random number to the R value. Now, random data is one thing, but if the statistic used to evaluate the data quality is itself random, then it is not what I would call useful. Since I am not very good at math, I always find myself approaching statistics by generating long lists of random numbers, manipulating them in some way, and then graphing the results. For graphing Rmerge vs I/sigma(I), one does find that Bernhard's rule of Rmerge = 0.8/( I/sigma(I) ) generally applies, but only for I/sigma(I) that is = 3. It gets better with high multiplicity, but even with m=100, the Rmerge values for the I/sigma(I) 1 points are all over the place. This is true even if you average the value of Rmerge over a million random number seeds. In fact, one must do so much averaging, that I start to worry about the low-order bits of common random number generators. I have attached images of these Rmerge vs I/sigma graphs. The error bars reflect the rms deviation from the average of a large number of Rmerge values (different random number seeds). The missing values are actually points where the average Rmerge in 60 trials (m=3) was still negative. The reason for this noisy R factor problem becomes clear if you consider the limiting case where the true intensity is zero, and make a histogram of ( I - I )/I. It is not a Gaussian. Rather, it is the Gaussian's evil stepsister: the Lorentzian (or Cauchy distribution). This distribution may look a lot like a Gaussian, but it has longer tails, and these tails give it the weird statistical property of having an undefined mean value. This is counterintuitive! Because you can clearly just look at the histogram and see that it has a central peak (at zero), but if you generate a million Lorentzian-distributed random numbers and take the average value, you will not get anything close to zero. Try it! You can generate a Lorentzian deviate from a uniform deviate like this: tan(pi*(rand()-0.5)), where rand() makes a random number from 0 to 1. Now, it is not too hard to understand how R could blow up when the true spot intensities are all zero. After all, as I approaches zero, the ratio ( I - I ) / I approaches a divide-by-zero problem. But what about when I/sigma(I) = 1? Or 2? If you look at these histograms, you find that they are a cross between a Gaussian and a Lorentzian (the so-called Voigt function), and the histogram does not become truly Gaussian-looking until I/sigma(I) = 3. At this point, the R factor behaves Bernhard's rule quite well, even with multiplicities as low as 2 or 3. This was the moment when I realized that the early crystallographers who first decided to use this 3-sigma cutoff, were smarter than I am. Now, you can make a Voigt function (or even a Lorentzian) look more like a Gaussian by doing something called outlier rejection, but it is hard to rationalize why the outliers are being rejected. Especially in a simulation! Then again, the silly part of all this is all we really want is the middle of the histogram of ( I - I )/I. In fact, if you just pick the most common Rmerge, you would get a much better estimate of the true Rmerge in a given resolution bin than you would by averaging a hundred times more data. Such procedures are called robust estimators in statistics, and the robust estimator equivalents to the average and the rms deviation from the average are the median and the median absolute deviation from the median. If you make a list of Lorentzian-random numbers as above, and compute the median, you will get a value very close to zero, even with modest multiplicity! And the median absolute deviation from the median rapidly converges to 1, which matches the full width at half maximum of the histogram quite nicely. So, what are the practical implications of this? Perhaps instead of the average Rmerge in each bin we should be looking at the median Rmerge? This will be the same as the average for the cases where I/sigma(I) 3, but still be well
Re: [ccp4bb] Synthetic RNA for Crystallization
1) Make your own with in vitro transcription (straight T7 r T7+RDRP like in Finnzymes kit) 2) Buy from IDTDNA - they are very good Long RNA tends to be expensive. Consider RNA ligase if two or more pieces can be stitched together. Artem On Sun, Mar 13, 2011 at 3:39 AM, Michael Thompson mi...@chem.ucla.eduwrote: Hello All, I am looking for some advice from some experienced RNA crystallographers. I would like to order some relatively short (90 bases) synthetic RNAs for crystallization trials. I was wondering if anyone could comment on the use of synthetic RNAs for crystallization. Specifically, what is the longest synthetic RNA that can be used for crystallization trials? I've seen some structures in the PDB that are up to 88 bases and are reported to have been obtained with synthetic constructs (3OWI - glycine riboswitch), but I don't really know if that's routine or if it's an exceptional case. Also, for those who have experience with the use of synthetic RNAs, I was wondering where people generally order their synthetic constructs from? Our resident expert in RNA crystallography recommended a company called Dharmacon (part of ThermoFisher), but I was hoping that I might get some other opinions as to which companies make the best quality oligonucleotides, provide samples with the highest purity, and have the most reasonable prices. Thanks in advance for the help! Mike -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] Synthetic RNA for Crystallization
Hi Mike, For long RNAs ( 40bases), in vitro transcription is the method of choice. You might want to take a look at this introductory page from Ambion: http://www.ambion.com/techlib/basics/transcription/index.html For structural studies you will have to scale up to milliliter scale. For that you might want to produce your own T7 RNA polymerase. Another thing to consider is the method of purification of your RNA. I believe purification from denaturing gel is the most widely used method, but you should consider native chromatographic purification as well. The following references should get you started on the subject: http://www.ncbi.nlm.nih.gov/pubmed/20946782 http://www.ncbi.nlm.nih.gov/pubmed/17272845 If you absolutely want to go with chemical synthesis, I believe you will have to buy shorter RNA pieces (40 bases or less) and ligate them later using a T4 RNA ligase. There are many good references out there that deal with production, purification and crystallization or RNA. Good luck, Mario Sanches On Sun, Mar 13, 2011 at 4:39 AM, Michael Thompson mi...@chem.ucla.eduwrote: Hello All, I am looking for some advice from some experienced RNA crystallographers. I would like to order some relatively short (90 bases) synthetic RNAs for crystallization trials. I was wondering if anyone could comment on the use of synthetic RNAs for crystallization. Specifically, what is the longest synthetic RNA that can be used for crystallization trials? I've seen some structures in the PDB that are up to 88 bases and are reported to have been obtained with synthetic constructs (3OWI - glycine riboswitch), but I don't really know if that's routine or if it's an exceptional case. Also, for those who have experience with the use of synthetic RNAs, I was wondering where people generally order their synthetic constructs from? Our resident expert in RNA crystallography recommended a company called Dharmacon (part of ThermoFisher), but I was hoping that I might get some other opinions as to which companies make the best quality oligonucleotides, provide samples with the highest purity, and have the most reasonable prices. Thanks in advance for the help! Mike -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu -- Mario Sanches Postdoctoral Fellow Samuel Lunenfeld Research Institute Mount Sinai Hospital 600 University Ave Toronto - Ontario Canada M5G 1X5 http://ca.linkedin.com/in/mariosanches
Re: [ccp4bb] Synthetic RNA for Crystallization
Hi, I'll second Israels's comment. Since the yield per coupling in synthesis is lower for RNA than for DNA it gets really expensive over 30-35 nucleotides. However, you can stitch together several 30 nt oligos using either T4 RNA ligase or T4 DNA ligase (with a DNA splint). Regarding suppliers of synthetic RNA, Dharmacon is still very reliable in terms of actual delivered amount and quality. If you are going for very large scale then oligofactory.com is a good choice. For a 90 nt oligo without any modifications I'd definitely go for T7 in-vitro transcription. On top of Nagai's classic that Israel referred to, I can also recommend the more recent method of native purification developed by Robert Batey and Jeffrey Kieft. See: Batey, R.T. Kieft, J.S. Improved native affinity purification of RNA (2007) RNA, 13, 1384-1389. You are going to need milligrams of T7 RNA polymerase in the end so forget about transcription kits, make your own. Cheers, Martin On Mar 13, 2011, at 10:16 AM, Israel Sanchez wrote: Hi Michael, we normally produce synthetic RNAs following this classic paper if the size is more than let say 40-50 nucleotide, otherwise we buy the RNAs from Dharmacon and the quality is totally OK. Hope it helps J Mol Biol. 1995 Jun 2;249(2):398-408. Crystallization of RNA-protein complexes. I. Methods for the large-scale preparation of RNA suitable for crystallographic studies. Price SR, Ito N, Oubridge C, Avis JM, Nagai K. MRC Laboratory of Molecular Biology, Cambridge, UK. Abstract In vitro transcription using bacteriophage RNA polymerases and linearised plasmid or oligodeoxynucleotide templates has been used extensively to produce RNA for biochemical studies. This method is, however, not ideal for generating RNA for crystallisation because efficient synthesis requires the RNA to have a purine rich sequence at the 5' terminus, also the subsequent RNA is heterogenous in length. We have developed two methods for the large scale production of homogeneous RNA of virtually any sequence for crystallization. In the first method RNA is transcribed together with two flanking intramolecularly-, (cis-), acting ribozymes which excise the desired RNA sequence from the primary transcript, eliminating the promoter sequence and heterogeneous 3' end generated by run-off transcription. We use a combination of two hammerhead ribozymes or a hammerhead and a hairpin ribozyme. The RNA-enzyme activity generates few sequence restrictions at the 3' terminus and none at the 5' terminus, a considerable improvement on current methodologies. In the second method the BsmAI restriction endonuclease is used to linearize plasmid template DNA thereby allowing the generation of RNA with any 3' end. In combination with a 5' cis-acting hammerhead ribozyme any sequence of RNA may be generated by in vitro transcription. This has proven to be extremely useful for the synthesis of short RNAs. 2011/3/13 Michael Thompson mi...@chem.ucla.edu Hello All, I am looking for some advice from some experienced RNA crystallographers. I would like to order some relatively short (90 bases) synthetic RNAs for crystallization trials. I was wondering if anyone could comment on the use of synthetic RNAs for crystallization. Specifically, what is the longest synthetic RNA that can be used for crystallization trials? I've seen some structures in the PDB that are up to 88 bases and are reported to have been obtained with synthetic constructs (3OWI - glycine riboswitch), but I don't really know if that's routine or if it's an exceptional case. Also, for those who have experience with the use of synthetic RNAs, I was wondering where people generally order their synthetic constructs from? Our resident expert in RNA crystallography recommended a company called Dharmacon (part of ThermoFisher), but I was hoping that I might get some other opinions as to which companies make the best quality oligonucleotides, provide samples with the highest purity, and have the most reasonable prices. Thanks in advance for the help! Mike -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu -- Israel Sanchez Fernandez PhD Ramakrishnan-lab MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK
[ccp4bb] MOSFILM
Is MOSFILM 6.2.6 the latest version? Rex Palmer Birkbeck College
Re: [ccp4bb] MOSFILM
According to Harry Powell's website: The current version of Mosflm is version 7.0.7 (uploaded onto this site 22nd December 2010). link: http://www.mrc-lmb.cam.ac.uk/harry/mosflm Cheers, Albert 2011/3/13 REX PALMER rex.pal...@btinternet.com Is MOSFILM 6.2.6 the latest version? Rex Palmer Birkbeck College
Re: [ccp4bb] MOSFILM
Dear Rex, As Albert has already pointed out, the latest version is 7.0.7 and can be downloaded from: http://www.mrc-lmb.cam.ac.uk/harry/mosflm/ 6.2.6 dates from about 2006 I think. It will not deal with Pilatus detectors and there have been a very large number of improvements since then. For ease of use, we would also strongly recommend using the new mosflm interface (iMosflm) which can be downloaded from the same site. Best wishes, Andrew Leslie Is MOSFILM 6.2.6 the latest version? Rex Palmer Birkbeck College
[ccp4bb] MOSFLM
Thanks to all for your prompt, detailed and useful answers. I thought you would all be watching the rugby this afternoon! Rex
[ccp4bb] Hideaki Niwa
Does anyone have an email address for Hide Niwa who was at Birkbeck in the 1990's and went to work back in Japan as a protein crystallographer? The earthquake is rather worrying. Rex Palmer Birkbeck College
Re: [ccp4bb] mosflm gain
Dear James,
Many thanks for the detailed explanation. I do find
your results very interesting and (when time allows
!) I will certainly investigate this effect in more
detail and see if I find similar results for data
that shows significant levels of radiation damage (as
mine invariably seem to do). I have to admit that it
is not entirely clear to me why PSF would result in a
correlation between SDFAC and SDADD, although this is
clearly what you see. It would be rewarding to get to
the bottom of this. As I mentioned earlier, Phil
Evans is currently looking at the refinement of the
SD parameters in relation to Aimless (the imminent
replacement for SCALA) so he is also very interested
in figuring out exactly what is going on here (but
does not have any answers as yet).
Best wishes,
Andrew
Andrew! You don't believe me? Well, I suppose it serves me right for
not explaining where the idea came from (see below).
I do, however, agree with Andrew's assessment that the default-chosen
gain in MOSFLM is adequate for all practical purposes. Any error in
GAIN will be almost exactly compensated for by a corresponding change in
Sdfac in SCALA, and the final value of sigma(I) will be essentially the
same. The only possible difference will be in the sigma-based outlier
rejection within MOSFLM, but since the typical errors in the sigma are
only ~30%, I predict it will be hard to find a situation where this
makes or breaks a structure determination.
So, by way of explanation: there are three things that led me to this
conclusion:
1) the control: fake data with all pixels independent.
adjusting the GAIN as MOSFLM recommends from the BGRATIO analysis
does, in fact, reproduce the correct value of the gain used to
generate the fake data. In SCALA, Sdfac refines to ~1.0, SdB refines to
0, and Sdadd refines to the actual magnitude of fractional error
(introduced by beam flicker, shutter jitter, etc.). No surprises here.
2) blur the fake data with the point-spread function (PSF) empirically
derived for my detector
In this case, the MOSFLM-refined gain is too low. In SCALA,
Sdfac refines to ~1.3, SdB refines to 3-5, and Sdadd is a bit low.
These parameters are about what I see processing good real data.
3) use real data, but force MOSFLM to use the GAIN calibrated
independently for the detector
MOSFLM grumbles a lot about the BGRATIO. In SCALA, Sdfac refines to
~1, and SdB refines to ~0. Sdadd is consistent with my
independently-measured fractional error sources.
Now, I have not evaluated this approach on a huge number of data sets,
but in this case the PSF was both necessary and sufficient to explain
the mystery of SdB. That is: the need for SdB arises because using an
incorrect gain creates a correlation between Sdfac and Sdadd. I
imagine there are other ways to get a non-zero SdB as well, but for
good data I suspect this is the dominant mechanism. I never wrote
this up because I am fairly certain the article would do nothing to
improve the impact factor of the journal in which it was published, but
this anecdote might perhaps be useful to Andrew, Phil, and a few other
readers of this list.
-James Holton
MAD Scientist
On 3/7/2011 2:00 AM, A Leslie wrote:
I have to say that I don't fully agree with James' recommendation to
adjust the GAIN in MOSFLM until the calculated SDFAC parameter in
SCALA is 1.0.
(Background information, the sigmas from Mosflm sd(I) are corrected in
SCALA according to
sd(I) corrected = SdFac * sqrt{sd(I)**2 + SdB*Ihl +
(SdAdd*Ihl)**2}
in order to get the best agreement between corrected sigmas and the
observed differences between symmtery/Friedel related intensities)
While I fully agree with his argument that systematic errors such as
absorption, etc give an error proportional to the intensity, and
therefore should be corrected by the SDADD term rather than SDFAC, in
any real world data set that I have come across the situation is not
so simple. Indeed, according to the usual treatment of errors there
should be no need for the SDB term in SCALA, but in practice it is
essential to have this term to be able to match corrected sigmas with
the observed differences between symmetry related reflections. It also
turns out that the three variable parameters SDFAC, SDB and SDADD are
highly correlated, so one can get rather different values for any
individual parameter from very similar datasets. Radiation damage is
certainly one source of error which would not be expected to follow a
simple error model, or non-isomorphism if multiple crystals have been
used.
Phil Evans is not entirely happy with the behaviour of the refinement
of these parameters and is in fact currently looking at this, but
there is a basic problem here that one is trying to use a simple
error model for a situation where (for whatever reason) it does not
really apply.
The sigma estimates from MOSFLM are
Re: [ccp4bb] Hideaki Niwa
Hello Rex, Nice to hear from you. My contact address hasn't changed since we communicated the last time. I'm okay in Yokohama near Tokyo, though we are facing the grim news and our sympathy goes to those who have suffered in the recent quake. Let's talk off the list. Sincerely, Hideaki Niwa RIKEN Systems and Structural Biology Center(SSBC) 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan Does anyone have an email address for Hide Niwa who was at Birkbeck in the 1990's and went to work back in Japan as a protein crystallographer? The earthquake is rather worrying. #160; Rex Palmer Birkbeck College
[ccp4bb] Postdoctoral position in Cryo-EM and structure determination
A postdoctoral position in Cryo-EM and structure analysis is available at the University of Texas Medical Branch at Galveston to study the structure and active conformations of membrane-associated protein and complexes involved in blood coagulation: http://www.utmb.edu/ncb/faculty/Stoilova-McPhieSvetla.asp. Highly motivated candidates with expertise in macromolecular structure analysis, modeling and biophysics are encouraged to apply. Additional experience with Linux computing systems will be an advantage. The Sealy Center for Structural Biology and Molecular Biophysics at UTMB, as well as the PI’s lab are fully equipped for state of the art structure determination of macromolecular complexes: http://www.scsb.utmb.edu/. UTMB is part of the Gulf Coast Consortium: http://cohesion.rice.edu/centersandinst/gcc/ and the Texas Medical Center, which houses the NIH funded National center for Macromolecular Imaging: http://ncmi.bcm.edu/ncmi/. Svetla Stoilova-McPhie, PhD Assistant Professor, Department of Neuroscience and Cell Biology Scientist, Sealy Centre for Structural Biology and Molecular Biophysics University of Texas Medical Branch at Galveston 301 University Boulevard, Galveston, Texas 77555-0620 Lab: (+1) 409-747-2159 Cell: (+1) 979-319-1348 Fax: (1+) 409-747-2200 Email: svmcp...@utmb.edu http://www.utmb.edu/ncb/faculty/Stoilova-McPhieSvetla.asp
