Re: [ccp4bb] Merohedral twining for P212121. -
Dear Frank, DIRAX is very good at finding the twin lattices in case of non-merohedral twinning. In the reticular case you might want to use the LEPAGE-TWIN routine in PLATON to find the correct subcell. My collegue, Martin Lutz, suggests to change the measurement temperature in order to change the cell parameters and thus the amount of overlap. For integration of the data you may want to use our EVAL15 (J. Appl. Cryst. 43 (2010) 70-82). Have a look at http://www.crystal.chem.uu.nl/distr/ EVAL15 can deconvolute reflections with significant overlap, or otherwise outputs the summed intensties of the two reflections. This method is particularly suitable for your reticular case. Scaling can be done with TWINABS and refinement can be done with SHELX (hklf5 format). Loes. P.S. a collection of twinning literature can be found at http://www.cryst.chem.uu.nl/lutz/twin/gen_twin.html On 06/23/10 11:46, Frank von Delft wrote: My experience with pseudo-merohedral twinning (it was actually the reticular case with half the spots overlapped and the other non-overlapped half on a pseudo C-centred lattice) is that the degree of splitting varies widely over the diffraction pattern. In some places there was complete overlap, in others you see elongation of the spots, in others partial separation, and in others complete separation (and of course all shades in-between), with around 50-50 intensity split. In this situation the mosaicity becomes meaningless! I'm not aware of any software that can handle this kind of thing successfully (and certainly the data we did manage to get turned out to be garbage!). Both DIRAX or SAINT should be able to handle it, you'll need SADABS to scale it. (The latter two are in the Bruker software.) phx. -- __ Dr. Loes Kroon-Batenburg Dept. of Crystal and Structural Chemistry Bijvoet Center for Biomolecular Research Utrecht University Padualaan 8, 3584 CH Utrecht The Netherlands E-mail : l.m.j.kroon-batenb...@uu.nl phone : +31-30-2532865 fax: +31-30-2533940 __
Re: [ccp4bb] Lodovico...
Dear all, A tribute to Lodovico (pictures + letters + a song) can be found on http://www.crystalerice.org/Erice2010/Lodovico/index.htm Fred.
Re: [ccp4bb] f2mtz error: problems reading reflection
Can you attach the first 50 lines of your hkl file? Eleanor Yogesh Gupta wrote: Dear Experts, After a new installation on Mac OS 10.6, i am getting this error (related to f2mtz) during the Find SITES step by SHELX in Autosharp. Data line--- LABO H K L FA SIGFA ALPHA Number of columns to be read in: 6 Data line--- CTYP H H H F Q P Data line--- FORM '(3F4.0,2F8.2,F4.0)' $TEXT:Warning: $$ comment $$ WARNING: Format I replaced with F $$ FORMATTED OLD file opened on unit 1 Logical name: HKLIN, Filename: SHELX/1_fa.hkl $TEXT:Warning: $$ comment $$ WARNING: PROJECTNAME not assigned $$ $TEXT:Warning: $$ comment $$ WARNING: CRYSTALNAME not assigned $$ $TEXT:Warning: $$ comment $$ WARNING: DATASETNAME not assigned $$ *** Read error Check FORMAT -- especially the need for REALs f2mtz: problems reading reflection 9 f2mtz: problems reading reflection 9 I am running ccp4-6.1.13. It looks like a FORTRAN error but do not know how to fix this or which file to edit particularly. SHARP otherwise running fine if i exclude SHELX step by feeding known sites. Thanks for your help. Yogesh
Re: [ccp4bb] Fast,medium,slow axis do not match
You could feed *both* maps through mapmask with AXIS X Y Z to convert
them to the same axis order.
You may also have a problem with the maps having different XYZLIM
ranges. In that case, using XYZLIM MATCH on the second run of mapmask to
match the second map to the first should fix it.
Kevin
Hailiang Zhang wrote:
Hi there,
I was generating the atomic mask using ccpr-sfall, and generating real
maps using ccp4-fft, and then ccp4-overlap these maps to calculate the cc
values. However, ccp4-overlapmap frequently complaints that the
Fast,medium,slow axis do not match for these maps. Following are the
script I was using, and thanks for any advice (I can't manually change the
keywords values one by one since everything needs automated):
sfall xyzin ${OUTTMPDIR}${PDB}-Bsmall-0B.pdb \
mapout ${OUTTMPDIR}${PDB}-Bsmall-mask.map\
END-sfall
TITL Toxd Atom map from final coordinates
MODE ATMMAP RESMOD
GRID ${GRID}
SYMM ${spcgrp}
FORM NGAU 5
END
END-sfall
...
fft ${OUTLOGDIR}${PDB}-0B-fft.log\
hklin ${OUTTMPDIR}${PDB}-0B.mtz \
mapout ${OUTTMPDIR}${PDB}-0B.map\
END-fft
TITL data_toxd
GRID ${GRID}
XYZLIM ASU
RESO 100.0 ${resolution}
LABI F1=FOBS PHI=PHIFMODEL
END
END-fft
*
--
EMAIL DISCLAIMER http://www.york.ac.uk/docs/disclaimer/email.htm
[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 ***
[ccp4bb] P-CUBE - Last chance to sign up for the Workshop and 1st Annual Meeting in Grenoble- Registration is closing soon!
Dear All, Don't miss out on the P-CUBE meeting in Grenoble in September! Sign up today underwww.p-cube.eu. The registration deadline is June 30. 2010. See you in Grenoble P-CUBE Management Team *** Dr. Jutta Tatzel Program Manager P-CUBE Department of Biochemistry University of Zurich Winterthurerstr. 190 CH-8057 Zurich Tel +41 44 635 5593 Email:j.tat...@bioc.uzh.ch
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] Fast,medium,slow axis do not match
Alternatively you can force the fft to generate a map with the sfall
axiis order (sfall has a fixed axis order governed by the spacegroup)
From the fft documentation
You can set
AXIS fast medium slow
Eleanor
Kevin Cowtan wrote:
You could feed *both* maps through mapmask with AXIS X Y Z to convert
them to the same axis order.
You may also have a problem with the maps having different XYZLIM
ranges. In that case, using XYZLIM MATCH on the second run of mapmask to
match the second map to the first should fix it.
Kevin
Hailiang Zhang wrote:
Hi there,
I was generating the atomic mask using ccpr-sfall, and generating real
maps using ccp4-fft, and then ccp4-overlap these maps to calculate the cc
values. However, ccp4-overlapmap frequently complaints that the
Fast,medium,slow axis do not match for these maps. Following are the
script I was using, and thanks for any advice (I can't manually change
the
keywords values one by one since everything needs automated):
sfall xyzin ${OUTTMPDIR}${PDB}-Bsmall-0B.pdb \
mapout ${OUTTMPDIR}${PDB}-Bsmall-mask.map\
END-sfall
TITL Toxd Atom map from final coordinates
MODE ATMMAP RESMOD
GRID ${GRID}
SYMM ${spcgrp}
FORM NGAU 5
END
END-sfall
...
fft ${OUTLOGDIR}${PDB}-0B-fft.log\
hklin ${OUTTMPDIR}${PDB}-0B.mtz \
mapout ${OUTTMPDIR}${PDB}-0B.map\
END-fft
TITL data_toxd
GRID ${GRID}
XYZLIM ASU
RESO 100.0 ${resolution}
LABI F1=FOBS PHI=PHIFMODEL
END
END-fft
*
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
[ccp4bb] Postdoc position
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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
Re: [ccp4bb] detection of anomalous signal using HKL2000
Can anyone explain what Zbyszek Otwinowski means by Chi squared? I can't find a definition in any of his papers (though I may have missed it). Is there a reference? It doesn't seem obviously related to the chi squared distribution (In probability theory and statistics, the chi-square distribution (also chi-squared orχ²-distribution) with k degrees of freedom is the distribution of a sum of the squares of k independent standard normal random variables. http://en.wikipedia.org/wiki/Chi-square_distribution) Phil On 29 Jun 2010, at 21:14, Felix Frolow wrote: Graphical information from Scalepack as used in HKL2000 is of unprecedented help to detect anomalous signal. Anomalous detection for S anomalous data using CHI**2 and Rfactor statistics for reflections with averaged and separated Bijvoet pairs is attached. It is very well described in HKL2000 manual. There is nothing special about data collection (strategy was used) and measurement was relatively fast (4 h on MicroMax007 and RaxisIV++). BTW Rfactor is 1.9% with separated Bijvoet reflections and 2.6% with averaged BTW James Holton website calculate for this case 0.078 crystal Dr Felix Frolow Professor of Structural Biology and Biotechnology Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica D, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 Quoting Bernhard Rupp b...@ruppweb.org: 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 Dr Felix Frolow Professor of Structural Biology and Biotechnology Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica D, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-(0)-3640-8723 Cel: ++972-0547-459-608 Fax: ++972-(0)-3640-9407detectionOfAnomalousSignalBy Scalepack-HKL2000.pdf
Re: [ccp4bb] density quiz
Looks atypical for 3-me-Lys, M3L, (usually in natively methylated products) http://www.ruppweb.org/garland/gallery/Ch2/pages/Biomolecular_Crystallograph y_Fig_2-27.htm nor fits dimethyllysine, MYL, (via chem. red) http://www.ruppweb.org/garland/gallery/Ch4/pages/Biomolecular_Crystallograph y_Fig_4-19.htm BR PS: ...and dont forget to remove those waters later ;-) -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of J. Preben Morth Sent: Tuesday, June 29, 2010 1:44 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] density quiz Hi Christian One guess would be one of the possible post translational modifications seen for Lysine. acetylation or a triple methylation. However propionylations and butyrylations have also been observed on lysine. It looks like you could build in waters, you may want to leave this lysine till later and see if more clear density show up. good luck Preben On 29/06/2010, at 21.52, Christian Biertuempfel wrote: Dear all, Does anyone have an idea what this density at a lysine residue could be? The structure is a protein-DNA complex and I suspect that something left-over from the DNA synthesis reacted with the amino group even though the DNA substrate was purified. christian p.s. The protein has seen the following substances during purification and crystallization: Leupeptin, Pepstatin, AEBSF, MES, Tris, HEPES, DTT, EDTA, imidazole, glycerol, PEG 2K-MME, Mg2+, Na+, K+, Ca2+, Cl-. ___ Dr. Christian Biertümpfel Laboratory of Molecular Biology NIDDK/National Institutes of Health phone: +1 301 402 4647 9000 Rockville Pike, Bldg. 5, Rm. B1-03 fax: +1 301 496 0201 Bethesda, MD 20892-0580 USA ___ density_quiz1 .png density_quiz2 .pngdensity_quiz3.pngdensity_quiz4.pngdensity_quiz5.png Jens Preben Morth, Ph.D Aarhus University Department of Molecular Biology Gustav Wieds Vej 10 C DK - 8000 Aarhus C Tel. +45 8942 5257, Fax. +45 8612 3178 j...@mb.au.dk website: http://person.au.dk/en/j...@mb.au.dk
Re: [ccp4bb] detection of anomalous signal using HKL2000
Quoting Phil Evans p...@mrc-lmb.cam.ac.uk: Can anyone explain what Zbyszek Otwinowski means by Chi squared? If I understand properly, CHI**2 value as used in Scalepack is: SUM(I-Ij)**2/SUMsigma(I)**2 (I have to use formula editor to write it properly, but the idea is clear) and is useful in exhibiting and detection of systematic errors. Intuitively this value will be close to 1.0 if only counting statistics contribute to errors in measurements of I. Drawing CHI**2 distribution as a function of various values such as frame number, shell of resolution, intensity of reflection etc. may show very interesting things related to the status of systematic errors during data collection. In the case of absence of systematic errors and radiation decay and in presence of a stable X-ray beam these distributions will be featureless and their value will be dependent on a counting statistics, quality of a detector and absorption of a crystal mainly. Systematic errors of various sources change these distributions in a sensible way. Despite the fact that it is impossible to correct systematic errors, understanding of CHI**2 distributions lead to better understanding of the experiment and in most cases these systematic errors can be eliminated leading to a perfect data shaped mostly by a counting statistics. CHI**2 distributions a la Otwinowsky - Minor (or else, I also do not know if Zbyszek Otwinovsky developed it by himself or adopted from earlier sources),is as used in Scalepack an instrument of a great power. FF Dr Felix Frolow Professor of Structural Biology and Biotechnology Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica D, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 I can't find a definition in any of his papers (though I may have missed it). Is there a reference? It doesn't seem obviously related to the chi squared distribution (In probability theory and statistics, the chi-square distribution (also chi-squared orχ²-distribution) with k degrees of freedom is the distribution of a sum of the squares of k independent standard normal random variables. http://en.wikipedia.org/wiki/Chi-square_distribution) Phil On 29 Jun 2010, at 21:14, Felix Frolow wrote: Graphical information from Scalepack as used in HKL2000 is of unprecedented help to detect anomalous signal. Anomalous detection for S anomalous data using CHI**2 and Rfactor statistics for reflections with averaged and separated Bijvoet pairs is attached. It is very well described in HKL2000 manual. There is nothing special about data collection (strategy was used) and measurement was relatively fast (4 h on MicroMax007 and RaxisIV++). BTW Rfactor is 1.9% with separated Bijvoet reflections and 2.6% with averaged BTW James Holton website calculate for this case 0.078 crystal Dr Felix Frolow Professor of Structural Biology and Biotechnology Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica D, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 Quoting Bernhard Rupp b...@ruppweb.org: 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
Re: [ccp4bb] vacuum adapter for Dry shipper Cryo Dewar
The part you are looking for is known as a valve operator, at least in the parlance of our supplier. Here's a cut paste of the item we purchased for pumping out our shipping dewars with a nominal 30mm O.D.: V1000 Series Operator for 1 tube. Part Number: V1085-3-25 (KF-25 flange) @ $356.00 each From: DLH INDUSTRIES INC. P.O. Box 562 San Luis Obispo, California 93406 (805) 781-3565 FAX (805) 781-3566 Email: sa...@cryocomp.com Web Site: http://www.cryocomp.com ** So far, we have tried to rescue 3-4 different people's dewars by pumping them down with this thing, and only one of them has worked. Most of the time the vacuum fails because of a crack or leak on the inside of the dewar somewhere, and that cannot easily be fixed. Still, the one success pays for the tool. -James Holton MAD Scientist On 6/28/2010 9:34 PM, Frank von Delft wrote: They seem to have a finite life-time: we had two of these, bought at the same time, self-immolate the way you describe within months of another, after ~5 years of good service. We asked TW about it and they said it would cost more to get it fixed than just get a new one -- then again, they would have said that, wouldn't they. If you figure out how to re-suck the vacuum, do tell the list! Cheers phx. P.S. Sorry, we don't have an adaptor -- you probably figured :) On 29/06/2010 00:07, Gunnar Olovsson wrote: Dear ccp4bb, We have a *Taylor-Wharton *CX100 Dry shipper Dewar that has lost it's vacuum insulation. The vacuum sealing plug popped out of the valve (standard 30mm diameter). Has anyone found a vacuum adapter that fits so that one can re-establish vacuum insulation at home? I would ideally need to find a vacuum adapter that fits the valve of the Dewar and that ends in a KF16 Flange (preferably), so that we can re-establish vacuum insulation ourselves. Thank you very much in advance for your help! Sincerely - Gunnar Dr. Gunnar Olovsson Life Sciences Centre (4th floor) University of British Columbia 2350 Health Sciences Mall Vancouver,BC V6T1Z3 Canada gun...@byron.biochem.ubc.ca mailto:gun...@byron.biochem.ubc.ca
[ccp4bb] prediction of anomalous signal
On 6/29/2010 12:55 PM, Felix Frolow wrote: BTW James Holton website calculate for this case 0.078 crystal So far, every single one of these reports has come down to a misinterpretation of the web interface. I am trying to make it clearer, so I am interested in what values were entered into the various fields when someone gets a result that they think is not consistent with a real experiment that they did! http://bl831.als.lbl.gov/xtalsize.html BTW, this is a JavaScript program, so it runs on your web browser, not my server. This means I am not spying on you, but it also means that there is no way for me to tell what people are typing into it. -James Holton MAD Scientist
Re: [ccp4bb] prediction of anomalous signal
Woops! Sorry in case I just confused a lot of people. The number Felix reports: n_xtals = 0.078 means that the structure solution should have been easy (I.E. it could have been done with a crystal having only 8% of the volume used), and indeed it sounds like it was a slam dunk. So, it sounds like Felix got a good prediction from it. However, I am still very interested if anyone can solve a structure from a case where my predictor says that you can't. -James Holton MAD Scientist On 6/29/2010 4:18 PM, James Holton wrote: On 6/29/2010 12:55 PM, Felix Frolow wrote: BTW James Holton website calculate for this case 0.078 crystal So far, every single one of these reports has come down to a misinterpretation of the web interface. I am trying to make it clearer, so I am interested in what values were entered into the various fields when someone gets a result that they think is not consistent with a real experiment that they did! http://bl831.als.lbl.gov/xtalsize.html BTW, this is a JavaScript program, so it runs on your web browser, not my server. This means I am not spying on you, but it also means that there is no way for me to tell what people are typing into it. -James Holton MAD Scientist
Re: [ccp4bb] Density changes from Positive to negative after ligand addition and refinement
Negative (or positive) density tells you what you that what you've modelled is wrong. If you have 1.15A X-ray data and it's telling you what you think is Ca2+ is not Ca2+, then I would think your X-ray data wins and your previous structures lose (a lot can happen from one structure to the next...). I must say, your 2fo-fc looks like a perfectly good Mg2+, since it is the same size (ish) as the surrounding O and C atoms; a Ca2+ would have given a much larger green blob. phx. On 30/06/2010 02:35, xaravich ivan wrote: Dear CCP4BB, I have come across something that might be pretty obvious to experienced people but is making me crazy. I have this great 1.15 angs data and I know that I have a Calcium ion (pics attached) from previous structures of the same protein, that I have solved. Rightly when I add waters with Arp solvent it does not put water at that positive density. Now whenever I have tried to put the calcium, and refine the structure it is giving me a negative density at the metal site. I csn see that the 2fc-fo is clear there, but why negative density. This is just the start of my refinement and I have to refine multiple ligands in the structure and I would like to get past this issue before that. I tried putting atom at the pointer, adding water and renaming it according to the naming convention in the PDB for Calcium, but nothing. Your suggestions would be invaluable, as always. Ivan
Re: [ccp4bb] Density changes from Positive to negative after ligand addition and refinement
Hi Ivan, these Dale Tronrud's slides might help you with understanding the maps: http://www.ccp4.ac.uk/courses/stwk10/talk_files/Dale_The_Wonderful_World_of_Maps.pdf Good luck! Pavel. On 6/29/10 6:35 PM, xaravich ivan wrote: Dear CCP4BB, I have come across something that might be pretty obvious to experienced people but is making me crazy. I have this great 1.15 angs data and I know that I have a Calcium ion (pics attached) from previous structures of the same protein, that I have solved. Rightly when I add waters with Arp solvent it does not put water at that positive density. Now whenever I have tried to put the calcium, and refine the structure it is giving me a negative density at the metal site. I csn see that the 2fc-fo is clear there, but why negative density. This is just the start of my refinement and I have to refine multiple ligands in the structure and I would like to get past this issue before that. I tried putting atom at the pointer, adding water and renaming it according to the naming convention in the PDB for Calcium, but nothing. Your suggestions would be invaluable, as always. Ivan
Re: [ccp4bb] Density changes from Positive to negative after ligand addition and refinement
Could it be that for some reason (like the components of your solutions) you have Mg2+ in that site? Also, Magnesium is a common contaminant, trace amounts are usually present in one chemical or another. Subtle differences in the site might make it a better site for another lighter ion (we still do not fully understand the subtility of protein structures, and of their modifications). Fred. Fred. Message du 30/06/10 03:36 De : xaravich ivan A : CCP4BB@JISCMAIL.AC.UK Copie à : Objet : [ccp4bb] Density changes from Positive to negative after ligand addition and refinement Dear CCP4BB, I have come across something that might be pretty obvious to experienced people but is making me crazy. I have this great 1.15 angs data and I know that I have a Calcium ion (pics attached) from previous structures of the same protein, that I have solved. Rightly when I add waters with Arp solvent it does not put water at that positive density. Now whenever I have tried to put the calcium, and refine the structure it is giving me a negative density at the metal site. I csn see that the 2fc-fo is clear there, but why negative density. This is just the start of my refinement and I have to refine multiple ligands in the structure and I would like to get past this issue before that. I tried putting atom at the pointer, adding water and renaming it according to the naming convention in the PDB for Calcium, but nothing. Your suggestions would be invaluable, as always. Ivan [ Screen shot 2010-06-29 at 6.15.29 PM.png (375.3 Ko) ] [ Screen shot 2010-06-29 at 6.21.22 PM.png (383.5 Ko) ]
