Re: [ccp4bb] Izit dye stained crystal
On Fri, 30 Nov 2012 17:27:41 -0500, Sarathy Karunan Partha sarathyus...@gmail.com wrote: Dear all, Here is the XSCALE.LP output after scaling for the izit dye stained crystak. Sorry for attaching the .INP file. Sarathy On Fri, Nov 30, 2012 at 4:19 PM, Sarathy Karunan Partha sarathyus...@gmail.com wrote: Dear all, We did some Izit dye staining to test our crystal (salt or protein) and we observed that the crystal didn�t take up the dye well. But, showed nice protein diffraction (home source KCr 2.2909 A) and we collected a dataset (360 frames, 1o osc, 5 min exposure) on this dye stained crystal. The data collection statistics looks great and most interestingly we saw some anomalous signal for this data (see attached XSCALE.LP). Hi Sarathy, it would be worth investigating which systematic error prevents your data from having a higher ISa - 10 is really low. Have you tried re-running INTEGRATE CORRECT after mv GXPARM.XDS XPARM.XDS ? Based on the Chi^2 values, you might want to try STRICT_ABSORPTION_CORRECTION=TRUE . If that significantly (say more than 10%) increases ISa and also improves the other statistics, then I would go for it. best, Kay This protein was crystallized in 0.2 M Ca acetate, 30 % PEG 400 pH 4.5. Izit dye is basically methylene blue which contains a sulfur atom (phenothiazine ring) and also has some basic dimethylamio groups. Our protein has many acidic residues that could enhance binding of this basic dye.We think the anomalous signal could be from this dye and the heavy atom search with autoSHARP and Phenix autosol is suggestive of 4 sulfur atoms. Did anyone come across similar situation with using this dye and also welcome any suggestions about using this data for S-SAD phasing. Thanks, Sarathy
Re: [ccp4bb] Izit dye stained crystal
Dear Sarathy, I was going to reply along the same lines as Kay Diederichs just did. At the Cr K-alpha wavelength, you would have strong absorption effects which, given the low symmetry of your crystal, would be very hard to get rid of. We have seen numerous cases where a high anomalous correlation was spurious, in the sense that it did not correspond to a solvable substructure but was very likely to be caused by systematic errors, such as absorption effects or correlations in radiation-damage effects, not removed by the scaling procedure. This can be the big drawback of having a single axis of rotation and low symmetry, as you do here. In such cases, collecting images with at least two crystal orientations by means of a multi-axis goniometer (or from several isomorphous crystals, counting on getting a reasonable sampling of possible orientations just through chance) would enable you to check to what resolution you get consistent values for anomalous differences between orientations or between crystals. This would be a much more significant indicator that the CCanom value within a single dataset. Characteristically, your CCanom values are more or less constant as a function of resolution, which is not a feature of a true anomalous signal. Together with the argument put forward by Richard Gillilan on the basis of the high extinction coefficient of methylene blue, I am not optimistic about the prospect of an izit-based S-SAD solution of your structure - but would of course be delighted if you proved this to be incorrect by actually solving you structure from this dataset! With best wishes, Gerard. -- On Fri, Nov 30, 2012 at 05:27:41PM -0500, Sarathy Karunan Partha wrote: Dear all, Here is the XSCALE.LP output after scaling for the izit dye stained crystak. Sorry for attaching the .INP file. Sarathy On Fri, Nov 30, 2012 at 4:19 PM, Sarathy Karunan Partha sarathyus...@gmail.com wrote: Dear all, We did some Izit dye staining to test our crystal (salt or protein) and we observed that the crystal didn’t take up the dye well. But, showed nice protein diffraction (home source KCr 2.2909 A) and we collected a dataset (360 frames, 1o osc, 5 min exposure) on this dye stained crystal. The data collection statistics looks great and most interestingly we saw some anomalous signal for this data (see attached XSCALE.LP). This protein was crystallized in 0.2 M Ca acetate, 30 % PEG 400 pH 4.5. Izit dye is basically methylene blue which contains a sulfur atom (phenothiazine ring) and also has some basic dimethylamio groups. Our protein has many acidic residues that could enhance binding of this basic dye.We think the anomalous signal could be from this dye and the heavy atom search with autoSHARP and Phenix autosol is suggestive of 4 sulfur atoms. Did anyone come across similar situation with using this dye and also welcome any suggestions about using this data for S-SAD phasing. Thanks, Sarathy -- === * * * Gerard Bricogne g...@globalphasing.com * * * * Global Phasing Ltd. * * Sheraton House, Castle Park Tel: +44-(0)1223-353033 * * Cambridge CB3 0AX, UK Fax: +44-(0)1223-366889 * * * ===
[ccp4bb] Izit dye stained crystal
Dear all, We did some Izit dye staining to test our crystal (salt or protein) and we observed that the crystal didn’t take up the dye well. But, showed nice protein diffraction (home source KCr 2.2909 A) and we collected a dataset (360 frames, 1o osc, 5 min exposure) on this dye stained crystal. The data collection statistics looks great and most interestingly we saw some anomalous signal for this data (see attached XSCALE.LP). This protein was crystallized in 0.2 M Ca acetate, 30 % PEG 400 pH 4.5. Izit dye is basically methylene blue which contains a sulfur atom (phenothiazine ring) and also has some basic dimethylamio groups. Our protein has many acidic residues that could enhance binding of this basic dye.We think the anomalous signal could be from this dye and the heavy atom search with autoSHARP and Phenix autosol is suggestive of 4 sulfur atoms. Did anyone come across similar situation with using this dye and also welcome any suggestions about using this data for S-SAD phasing. Thanks, Sarathy XSCALE.INP Description: Binary data
Re: [ccp4bb] Izit dye stained crystal
Hi Sarathy, Are you sure your anomalous scatterers are not the Ca in the crystallisation buffer? These would also bind to the acidic residues in your protein, and Ca has a greater f'' (~2.5e compared to less than 1.5e for S) at the wavelength you used. Just another possibility - unless you already solved the structures and see density for the izit in your density, of course! Cheers, Dave David C. Briggs PhD http://about.me/david_briggs On 30 November 2012 21:19, Sarathy Karunan Partha sarathyus...@gmail.comwrote: Dear all, We did some Izit dye staining to test our crystal (salt or protein) and we observed that the crystal didn’t take up the dye well. But, showed nice protein diffraction (home source KCr 2.2909 A) and we collected a dataset (360 frames, 1o osc, 5 min exposure) on this dye stained crystal. The data collection statistics looks great and most interestingly we saw some anomalous signal for this data (see attached XSCALE.LP). This protein was crystallized in 0.2 M Ca acetate, 30 % PEG 400 pH 4.5. Izit dye is basically methylene blue which contains a sulfur atom (phenothiazine ring) and also has some basic dimethylamio groups. Our protein has many acidic residues that could enhance binding of this basic dye.We think the anomalous signal could be from this dye and the heavy atom search with autoSHARP and Phenix autosol is suggestive of 4 sulfur atoms. Did anyone come across similar situation with using this dye and also welcome any suggestions about using this data for S-SAD phasing. Thanks, Sarathy
Re: [ccp4bb] Izit dye stained crystal
We've found that high PEG concentration seems to compete with dye binding, so I'm not surprised your didn't see good uptake. I doubt the anomalous signal is from the dye ... if you calculate the molar dye concentration you would need to have significant occupancy in the lattice, you'll probably find that the crystals would look almost black ... unless there was a color change. I recall seeing a poster at the annual ACA meeting 3-4 years ago maybe, in which somebody was using polybrominated or polyiodinated aromatics to both dye the crystals and phase the structures at once. In fact, I think we gave the poster an award for that. I don't have my past notes handy to remember, but could look it up if you're interested. Richard Gillilan MacCHESS On Nov 30, 2012, at 4:19 PM, Sarathy Karunan Partha wrote: Dear all, We did some Izit dye staining to test our crystal (salt or protein) and we observed that the crystal didn’t take up the dye well. But, showed nice protein diffraction (home source KCr 2.2909 A) and we collected a dataset (360 frames, 1o osc, 5 min exposure) on this dye stained crystal. The data collection statistics looks great and most interestingly we saw some anomalous signal for this data (see attached XSCALE.LP). This protein was crystallized in 0.2 M Ca acetate, 30 % PEG 400 pH 4.5. Izit dye is basically methylene blue which contains a sulfur atom (phenothiazine ring) and also has some basic dimethylamio groups. Our protein has many acidic residues that could enhance binding of this basic dye.We think the anomalous signal could be from this dye and the heavy atom search with autoSHARP and Phenix autosol is suggestive of 4 sulfur atoms. Did anyone come across similar situation with using this dye and also welcome any suggestions about using this data for S-SAD phasing. Thanks, Sarathy XSCALE.INP
Re: [ccp4bb] Izit dye stained crystal
Dear all, Here is the XSCALE.LP output after scaling for the izit dye stained crystak. Sorry for attaching the .INP file. Sarathy On Fri, Nov 30, 2012 at 4:19 PM, Sarathy Karunan Partha sarathyus...@gmail.com wrote: Dear all, We did some Izit dye staining to test our crystal (salt or protein) and we observed that the crystal didn’t take up the dye well. But, showed nice protein diffraction (home source KCr 2.2909 A) and we collected a dataset (360 frames, 1o osc, 5 min exposure) on this dye stained crystal. The data collection statistics looks great and most interestingly we saw some anomalous signal for this data (see attached XSCALE.LP). This protein was crystallized in 0.2 M Ca acetate, 30 % PEG 400 pH 4.5. Izit dye is basically methylene blue which contains a sulfur atom (phenothiazine ring) and also has some basic dimethylamio groups. Our protein has many acidic residues that could enhance binding of this basic dye.We think the anomalous signal could be from this dye and the heavy atom search with autoSHARP and Phenix autosol is suggestive of 4 sulfur atoms. Did anyone come across similar situation with using this dye and also welcome any suggestions about using this data for S-SAD phasing. Thanks, Sarathy XSCALE.LP Description: Binary data
Re: [ccp4bb] Izit dye stained crystal
Hi Sarathy, What does the density modified electron density map after phasing from either autoSHARP or Phenix? Can the auto building programs build protein chains into this map? Cheers, Scott On Nov 30, 2012, at 5:27 PM, Sarathy Karunan Partha sarathyus...@gmail.commailto:sarathyus...@gmail.com wrote: Dear all, Here is the XSCALE.LP output after scaling for the izit dye stained crystak. Sorry for attaching the .INP file. Sarathy On Fri, Nov 30, 2012 at 4:19 PM, Sarathy Karunan Partha sarathyus...@gmail.commailto:sarathyus...@gmail.com wrote: Dear all, We did some Izit dye staining to test our crystal (salt or protein) and we observed that the crystal didn’t take up the dye well. But, showed nice protein diffraction (home source KCr 2.2909 A) and we collected a dataset (360 frames, 1o osc, 5 min exposure) on this dye stained crystal. The data collection statistics looks great and most interestingly we saw some anomalous signal for this data (see attached XSCALE.LP). This protein was crystallized in 0.2 M Ca acetate, 30 % PEG 400 pH 4.5. Izit dye is basically methylene blue which contains a sulfur atom (phenothiazine ring) and also has some basic dimethylamio groups. Our protein has many acidic residues that could enhance binding of this basic dye.We think the anomalous signal could be from this dye and the heavy atom search with autoSHARP and Phenix autosol is suggestive of 4 sulfur atoms. Did anyone come across similar situation with using this dye and also welcome any suggestions about using this data for S-SAD phasing. Thanks, Sarathy XSCALE.LP Scott T. R. Walsh, Ph.D. Assistant Professor University of Maryland College Park Dept of Cell Biology and Molecular Genetics Institute for Bioscience and Biotechnology Research Rm 3127E SG II 9600 Gudelsky Drive Rockville, MD 20850 email: swals...@umd.edumailto:swals...@umd.edu phone: (240) 314-6478 fax: (240) 314-6255
Re: [ccp4bb] Izit dye stained crystal
Are you referring to the I3C magic phasing triangle by any chance? Beck, et al Acta Cryst D 61(?) (2008) is the reference I think. Good luck! Bryan From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Richard Gillilan Sent: Friday, November 30, 2012 5:21 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Izit dye stained crystal We've found that high PEG concentration seems to compete with dye binding, so I'm not surprised your didn't see good uptake. I doubt the anomalous signal is from the dye ... if you calculate the molar dye concentration you would need to have significant occupancy in the lattice, you'll probably find that the crystals would look almost black ... unless there was a color change. I recall seeing a poster at the annual ACA meeting 3-4 years ago maybe, in which somebody was using polybrominated or polyiodinated aromatics to both dye the crystals and phase the structures at once. In fact, I think we gave the poster an award for that. I don't have my past notes handy to remember, but could look it up if you're interested. Richard Gillilan MacCHESS On Nov 30, 2012, at 4:19 PM, Sarathy Karunan Partha wrote: Dear all, We did some Izit dye staining to test our crystal (salt or protein) and we observed that the crystal didn't take up the dye well. But, showed nice protein diffraction (home source KCr 2.2909 A) and we collected a dataset (360 frames, 1o osc, 5 min exposure) on this dye stained crystal. The data collection statistics looks great and most interestingly we saw some anomalous signal for this data (see attached XSCALE.LP). This protein was crystallized in 0.2 M Ca acetate, 30 % PEG 400 pH 4.5. Izit dye is basically methylene blue which contains a sulfur atom (phenothiazine ring) and also has some basic dimethylamio groups. Our protein has many acidic residues that could enhance binding of this basic dye.We think the anomalous signal could be from this dye and the heavy atom search with autoSHARP and Phenix autosol is suggestive of 4 sulfur atoms. Did anyone come across similar situation with using this dye and also welcome any suggestions about using this data for S-SAD phasing. Thanks, Sarathy XSCALE.INP -- Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] Izit dye stained crystal
Yes, that's the one I remember! Thanks Richard On Nov 30, 2012, at 7:01 PM, Prince, D Bryan wrote: Are you referring to the I3C magic phasing triangle by any chance? Beck, et al Acta Cryst D 61(?) (2008) is the reference I think. Good luck! Bryan Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Richard Gillilan Sent: Friday, November 30, 2012 5:21 PM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Izit dye stained crystal We've found that high PEG concentration seems to compete with dye binding, so I'm not surprised your didn't see good uptake. I doubt the anomalous signal is from the dye ... if you calculate the molar dye concentration you would need to have significant occupancy in the lattice, you'll probably find that the crystals would look almost black ... unless there was a color change. I recall seeing a poster at the annual ACA meeting 3-4 years ago maybe, in which somebody was using polybrominated or polyiodinated aromatics to both dye the crystals and phase the structures at once. In fact, I think we gave the poster an award for that. I don't have my past notes handy to remember, but could look it up if you're interested. Richard Gillilan MacCHESS On Nov 30, 2012, at 4:19 PM, Sarathy Karunan Partha wrote: Dear all, We did some Izit dye staining to test our crystal (salt or protein) and we observed that the crystal didn’t take up the dye well. But, showed nice protein diffraction (home source KCr2.2909 A) and we collected a dataset (360 frames, 1o osc, 5 min exposure) on this dye stained crystal. The data collection statistics looks great and most interestingly we saw some anomalous signal for this data (see attached XSCALE.LP). This protein was crystallized in 0.2 M Ca acetate, 30 % PEG 400 pH 4.5. Izit dye is basically methylene blue which contains a sulfur atom (phenothiazine ring) and also has some basic dimethylamio groups. Our protein has many acidic residues that could enhance binding of this basic dye.We think the anomalous signal could be from this dye and the heavy atom search with autoSHARP and Phenix autosol is suggestive of 4 sulfur atoms. Did anyone come across similar situation with using this dye and also welcome any suggestions about using this data for S-SAD phasing. Thanks, Sarathy XSCALE.INP
