[ccp4bb] measure of anamolous signal
Dear all, I have collected a 2.7 angstrom home source data with Cu-Kalpha source for a protein with 6 cysteines, with a multiplicity of around 23. I need to know, is there any significant anamolous signal present in the data set, since there is no good model for my protein. Can any one tell, which program to run, and what parameter to see? Thanks in advance. cheers, Murugan
Re: [ccp4bb] measure of anamolous signal
Vandu Murugan wrote: Dear all, I have collected a 2.7 angstrom home source data with Cu-Kalpha source for a protein with 6 cysteines, with a multiplicity of around 23. I need to know, is there any significant anamolous signal present in the data set, since there is no good model for my protein. Can any one tell, which program to run, and what parameter to see? Thanks in advance. cheers, Murugan When processing the data, ensure that Bijvoet pairs are not merged. The data processing software should provide you with an R-ano value and that is already a start. The values provided should tell you if you have an anomalous signal or not. You may also have to play with the number of frames to integrate in order to obtain an optimal anomalous signal in the resulting data set. There are several publications describing Sulphur SAD, but one of them which is freely available on the Web can be found here: http://www.stoe.com/pages/brochure/labnote_genix_cu.pdf , Schiltz M (pp 4-6). Good luck! Fred.
Re: [ccp4bb] measure of anamolous signal
Dear Murugan, you can use the program hkl2map from Thomas Schneider, available at http://webapps.embl-hamburg.de/hkl2map/ It's a graphical interface to the programs shelx c/d/e which are available from http://shelx.uni-ac.gwdg.de/SHELX/index.html With SAD data you want to look at the d/sigma line at the end of the shelxc output. Where that drops below about 1.3 is approximately where your anomalous signal ends. You might get slightly improved statistics with xprep instead of shelxc, but xprep is not free and you have to get a copy from Bruker-AXS. Tim On Tue, Jun 29, 2010 at 02:35:29PM +0530, Vandu Murugan wrote: Dear all, I have collected a 2.7 angstrom home source data with Cu-Kalpha source for a protein with 6 cysteines, with a multiplicity of around 23. I need to know, is there any significant anamolous signal present in the data set, since there is no good model for my protein. Can any one tell, which program to run, and what parameter to see? Thanks in advance. cheers, Murugan -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] measure of anamolous signal
Hi Murugan, One useful indicator of raw anomalous signal is the ANOMPLOT graph from Scala - this shows the differences between reflections compared with the expected differences. If the gradient of the plot is 1 there's no more differences that you would expect. If the gradient is more than one there is (or may be.) - also check out the merging statistics as a function of batch, if there's significant radiation damage this may mess things up. Scala writes out the gradient (assuming you told it anomalous on) in the summary Another rule-of-thumb is the resolution limit where cc-anom is 0.5. The most practical indicator of the anomalous signal is of course the success or failure of the subsequent phasing :o) Best wishes, Graeme On 29 June 2010 10:05, Vandu Murugan wandumuru...@gmail.com wrote: Dear all, I have collected a 2.7 angstrom home source data with Cu-Kalpha source for a protein with 6 cysteines, with a multiplicity of around 23. I need to know, is there any significant anamolous signal present in the data set, since there is no good model for my protein. Can any one tell, which program to run, and what parameter to see? Thanks in advance. cheers, Murugan
Re: [ccp4bb] measure of anamolous signal
Dear Murugan, Am 29.06.10 11:05, schrieb Vandu Murugan: Dear all, I have collected a 2.7 angstrom home source data with Cu-Kalpha source for a protein with 6 cysteines, with a multiplicity of around 23. I need to know, is there any significant anamolous signal present in the data set, since there is no good model for my protein. Can any one tell, which program to run, and what parameter to see? Thanks in advance. cheers, Murugan estimating the quality of the anomalous signal is not trivial, and several quality indicators have been discussed (see for example Fu, Rose Wang, Acta Cryst D60, 499-506 (2004), or Zwart, Acta Cryst D61, 1437-1444 (2005)). If you process your data with XDS, there are two quality indicators for the anomalous signal, given both in the CORRECT.LP and in the XSCALE.LP file. One is the correlation of anomalous differences between two randomly chosen subsets that should have the same anomalous difference due to crystallographic symmetry, called RANOM. The other describes the absolute anomalous difference divided by their standard deviation, called SIGANO. A typical rule of thumb (and the one that I use) is, that RANOM should be ~ 30%, and SIGANO should be ~ 1.2. However, these indicators might not be realistic (see references above) and therefore should be taken with a grain of salt. Good luck, Dirk. -- *** Dirk Kostrewa Gene Center Munich, A5.07 Department of Biochemistry Ludwig-Maximilians-Universität München Feodor-Lynen-Str. 25 D-81377 Munich Germany Phone: +49-89-2180-76845 Fax:+49-89-2180-76999 E-mail: kostr...@genzentrum.lmu.de WWW:www.genzentrum.lmu.de ***
Re: [ccp4bb] measure of anamolous signal
I've found the scala CC-anom significantly underestimates the anomalous signal, relative to e.g. xprep. I don't know why that is, but the latter seems to agree with what shelxd is happy with. Cheers phx On 29/06/2010 10:35, Graeme Winter wrote: Hi Murugan, One useful indicator of raw anomalous signal is the ANOMPLOT graph from Scala - this shows the differences between reflections compared with the expected differences. If the gradient of the plot is 1 there's no more differences that you would expect. If the gradient is more than one there is (or may be.) - also check out the merging statistics as a function of batch, if there's significant radiation damage this may mess things up. Scala writes out the gradient (assuming you told it anomalous on) in the summary Another rule-of-thumb is the resolution limit where cc-anom is 0.5. The most practical indicator of the anomalous signal is of course the success or failure of the subsequent phasing :o) Best wishes, Graeme On 29 June 2010 10:05, Vandu Muruganwandumuru...@gmail.com wrote: Dear all, I have collected a 2.7 angstrom home source data with Cu-Kalpha source for a protein with 6 cysteines, with a multiplicity of around 23. I need to know, is there any significant anamolous signal present in the data set, since there is no good model for my protein. Can any one tell, which program to run, and what parameter to see? Thanks in advance. cheers, Murugan
Re: [ccp4bb] measure of anamolous signal
I would say CC-anom 0.3 or even 0.2 (note that the scala CC-anom is defined on I not F) Phil On 29 Jun 2010, at 14:37, Frank von Delft wrote: I've found the scala CC-anom significantly underestimates the anomalous signal, relative to e.g. xprep. I don't know why that is, but the latter seems to agree with what shelxd is happy with. Cheers phx On 29/06/2010 10:35, Graeme Winter wrote: Hi Murugan, One useful indicator of raw anomalous signal is the ANOMPLOT graph from Scala - this shows the differences between reflections compared with the expected differences. If the gradient of the plot is 1 there's no more differences that you would expect. If the gradient is more than one there is (or may be.) - also check out the merging statistics as a function of batch, if there's significant radiation damage this may mess things up. Scala writes out the gradient (assuming you told it anomalous on) in the summary Another rule-of-thumb is the resolution limit where cc-anom is 0.5. The most practical indicator of the anomalous signal is of course the success or failure of the subsequent phasing :o) Best wishes, Graeme On 29 June 2010 10:05, Vandu Muruganwandumuru...@gmail.com wrote: Dear all, I have collected a 2.7 angstrom home source data with Cu-Kalpha source for a protein with 6 cysteines, with a multiplicity of around 23. I need to know, is there any significant anamolous signal present in the data set, since there is no good model for my protein. Can any one tell, which program to run, and what parameter to see? Thanks in advance. cheers, Murugan
Re: [ccp4bb] measure of anamolous signal
Answering the question should I even bother trying? can be complicated, but I get asked that a lot at the beamline! I recently incorporated a number of data quality formulas into a little interactive web page here: http://bl831.als.lbl.gov/xtalsize.html which focuses on calculating how many crystals of a given size you will need to average together to attain a specified goal (either weak spots (high res) or subtle differences (anomalous)) given radiation damage limits. For those interested in explanation: A quick-and-dirty way to estimate your Bijvoet ratio is with the formula: 0.75*fpp*sqrt(sites/MW) where sites is the number of sites you expect per MW, where MW is in Daltons (amu). Note that MW can represent the protein monomer, asymmetric unit, or whatever, as long as sites refers to the same thing. fpp is the expected number of anomalous electrons, which you can get from the CCP4 program crossec or several websites. Now, since you didn't mention your molecular weight, I will guess that it is about 30 kDa, which means your Bijvoet ratio at CuKa (where the fpp of sulfur is ~0.56 electron) will be about 0.6%. Now, detecting a 0.6% difference requires that the two things you are subtracting (I+ and I-) be measured to at least a precision of 0.42% (because the error in the difference will then be: sqrt(0.42%^2+0.42%^2) = 0.6%). This is the BARE minimum (where the signal is equal to the noise), but even to reach this goal, your signal-to-noise ratio must be 1/0.42% = 240. This is challenging! Typical data sets have I/sig(I) ~ 30 in their best bins (see Deiderichs 2010 http://dx.doi.org/10.1107/S0907444910014836). A multiplicity of 23 can push I/sig(I) to 30*sqrt(23) = 143, which is good, but still not close to 240. You will probably need a multiplicity of ~65 to measure Bijvoet differences to an accuracy of 0.6% Then again, if your protein is only 10 kDa, then your Bijvoet ratio is ~1% and you will only need I/sig(I) 140. -James Holton MAD Scientist Vandu Murugan wrote: Dear all, I have collected a 2.7 angstrom home source data with Cu-Kalpha source for a protein with 6 cysteines, with a multiplicity of around 23. I need to know, is there any significant anamolous signal present in the data set, since there is no good model for my protein. Can any one tell, which program to run, and what parameter to see? Thanks in advance. cheers, Murugan
Re: [ccp4bb] measure of anamolous signal
On Tue, Jun 29, 2010 at 09:30:30AM -0700, James Holton wrote: Answering the question should I even bother trying? can be complicated, [...] -James Holton MAD Scientist Since 'trying' may only take a semi-experienced user about 30min until they could have a poly-Alanine trace of their protein, I would say the answer is definitely Yes irrespective of what statistics may tell you - unless you don't have a protein in your crystal, but e.g. DNA/ RNA... Tim Vandu Murugan wrote: Dear all, I have collected a 2.7 angstrom home source data with Cu-Kalpha source for a protein with 6 cysteines, with a multiplicity of around 23. I need to know, is there any significant anamolous signal present in the data set, since there is no good model for my protein. Can any one tell, which program to run, and what parameter to see? Thanks in advance. cheers, Murugan -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] measure of anamolous signal
I second the hkl2map/SHELXCDE approach. Two complete examples explaining how to do this for MAD and S-SAD cases are in my book. I wish to emphasize the importance of a) running enough trials b) careful selection of resolution cutoffs c) look at the solution distribution d) play with SHELXE parameters. The hkl2map graphs are enormously helpful for this purpose. BR -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Tim Gruene Sent: Tuesday, June 29, 2010 2:29 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] measure of anamolous signal Dear Murugan, you can use the program hkl2map from Thomas Schneider, available at http://webapps.embl-hamburg.de/hkl2map/ It's a graphical interface to the programs shelx c/d/e which are available from http://shelx.uni-ac.gwdg.de/SHELX/index.html With SAD data you want to look at the d/sigma line at the end of the shelxc output. Where that drops below about 1.3 is approximately where your anomalous signal ends. You might get slightly improved statistics with xprep instead of shelxc, but xprep is not free and you have to get a copy from Bruker-AXS. Tim On Tue, Jun 29, 2010 at 02:35:29PM +0530, Vandu Murugan wrote: Dear all, I have collected a 2.7 angstrom home source data with Cu-Kalpha source for a protein with 6 cysteines, with a multiplicity of around 23. I need to know, is there any significant anamolous signal present in the data set, since there is no good model for my protein. Can any one tell, which program to run, and what parameter to see? Thanks in advance. cheers, Murugan -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
