Re: [ccp4bb] Using multiple crystals for structure solution in P1 using MAD/SAS/SAD
Dear Bram, Thankyou for the plug, but your email could be misunderstood. In fact we integrated and scaled the domains (2 for insulin, 3 for glucose isomerase) SIMULTANEOUSLY, taking the overlap into acount. This produced two data files (SHELX HKLF 4 and 5 formats). The HKLF 4 file that we used for the SAD phasing and initial refinement contained a unique set of data (Friedel opposites not merged) and had been obtained by a restrained least-squares analysis of all the data in which the 'independent parameters' were the intensities of all unique reflections and the twin fractions, the 'observations' were the total intensities of the overlapping reflections in each integration box and the (weak) 'restraints' were the approximate intensity ratios within each integration box for those composite reflections for which partial overlap made it possible to estimate them. The HKLF 5 format file treats the SUM of the intensities of all reflection components within one integration box as the 'experimental observations' for structure refinement. I am happy to be able to report that both phenix_refine and refmac will soon be able to refine against such data. This HKLF 5 format has been used extensively for many years for refining non-merohedral and pseudo-merohedral twins of small molecules (with shelxl) and if there is significant reflection overlap it always gives better results than various ways of attempting to 'detwin' the data. I am trying to write all this up but am getting lazy and senile. 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 Wed, 23 Jul 2008, Bram Schierbeek wrote: > Hi Hari, > > I have seen a poster last year's DGK by Ina Dix and Madhumati Sevanna from > George Sheldricks' group. Not sure it was published yet. > They collected data on non-merohedrally twinned crystals and processed and > scaled the domains separately and merged them afterwards, thus increasing the > redundancy. Indeed they got better results with S-SAD phasing when using the > combined data. This was on Insulin and Glucose isomerase, which do diffract > better then the average crystal and since the data came from the same crystal, > albeit twinned, the were no problems of non-isomorphism. > > On the other hand, to come back to the original question: You could also use a > kappa goniostat for your P1 crystal to get complete and redundant data. > > Best wishes, > > Bram > > > Bram Schierbeek > Application Scientist Structural Biology Solutions > Bruker AXS BV > Oostsingel 209,P.O.Box 811 > 2600 AV Delft, the Netherlands > T: +31 (0)152 152 508 > F: +31 (0)152 152 599 > E: [EMAIL PROTECTED] > W: www.bruker-axs.com > > > Jacob Keller wrote: > > Shouldn't all of the "crystal-to-crystal" differences be taken out > > automatically by scaling, and is there not the same proportional anomalous > > signal in every isomorphous crystal, regardless of the background? I would > > think that using multiple crystals would give a better idea of "the truth," > > as if taking many snapshots of the same object, and putting them together to > > form a three-dimensional object. In Hazes' language, don't all isomorphous > > crystals "draw from the same [underlying] distribution?" > > > > Jacob Keller > > > > ps admittedly if there is radiation damage or other non-isomorphisms, this > > reasoning does not apply. > > > > *** > > Jacob Pearson Keller > > Northwestern University > > Medical Scientist Training Program > > Dallos Laboratory > > F. Searle 1-240 > > 2240 Campus Drive > > Evanston IL 60208 > > lab: 847.491.2438 > > cel: 773.608.9185 > > email: [EMAIL PROTECTED] > > *** > > > > - Original Message - From: "Bart Hazes" <[EMAIL PROTECTED]> > > To: > > Sent: Wednesday, July 23, 2008 10:05 AM > > Subject: Re: [ccp4bb] Using multiple crystals for structure solution in P1 > > using MAD/SAS/SAD > > > > > > > Increasing redundancy only helps if all data draw from the same > > > distribution so you get a more accurate estimate of the mean of the > > > distribution. When dealing with different crystals, crystal-to-crystal > > > variation is likely larger than the anomalous signal you are looking f
Re: [ccp4bb] Using multiple crystals for structure solution in P1 using MAD/SAS/SAD
Hi Hari, I have seen a poster last year's DGK by Ina Dix and Madhumati Sevanna from George Sheldricks' group. Not sure it was published yet. They collected data on non-merohedrally twinned crystals and processed and scaled the domains separately and merged them afterwards, thus increasing the redundancy. Indeed they got better results with S-SAD phasing when using the combined data. This was on Insulin and Glucose isomerase, which do diffract better then the average crystal and since the data came from the same crystal, albeit twinned, the were no problems of non-isomorphism. On the other hand, to come back to the original question: You could also use a kappa goniostat for your P1 crystal to get complete and redundant data. Best wishes, Bram Bram Schierbeek Application Scientist Structural Biology Solutions Bruker AXS BV Oostsingel 209,P.O.Box 811 2600 AV Delft, the Netherlands T: +31 (0)152 152 508 F: +31 (0)152 152 599 E: [EMAIL PROTECTED] W: www.bruker-axs.com Jacob Keller wrote: Shouldn't all of the "crystal-to-crystal" differences be taken out automatically by scaling, and is there not the same proportional anomalous signal in every isomorphous crystal, regardless of the background? I would think that using multiple crystals would give a better idea of "the truth," as if taking many snapshots of the same object, and putting them together to form a three-dimensional object. In Hazes' language, don't all isomorphous crystals "draw from the same [underlying] distribution?" Jacob Keller ps admittedly if there is radiation damage or other non-isomorphisms, this reasoning does not apply. *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] *** - Original Message - From: "Bart Hazes" <[EMAIL PROTECTED]> To: Sent: Wednesday, July 23, 2008 10:05 AM Subject: Re: [ccp4bb] Using multiple crystals for structure solution in P1 using MAD/SAS/SAD Increasing redundancy only helps if all data draw from the same distribution so you get a more accurate estimate of the mean of the distribution. When dealing with different crystals, crystal-to-crystal variation is likely larger than the anomalous signal you are looking for and I'm therefore not convinced that merging of data is a good idea (never hurts to try though). I wonder if it would work better to derive anomalous differences for the individual data sets first and then merge those anomalous differences. This may allow the subtraction between F+ and F- to remove some of the systematic differences there may be between crystal forms. Bart Kay Diederichs wrote: hari jayaram schrieb: ... I was wondering if anyone could comment on combining datasets from multiple P1 crystals to increase the redundancy even further for such heavy atom ( SAS / SAD ) or MAD experiments. Hari, well, my comment would be that it should be possible in principle from what you describe, but the outcome strongly depends on the details (size of expected and observed anomalous and isomorphous signal, internal anomalous correlation coefficients, I/sigma and R-factors, radiation damage, are crystals isomorphous, ...). To increase the quality of the reduced data it would be advisable to rotate around different axes, which is possible at some - but not all - beamlines. This is even more true in P1. For all of the major data reduction programs there exist specific programs for merging data, and it does make a lot of sense to merge your passes (but don't merge radiation-damaged data with undamaged data)!. I would suggest to use at least two different data reduction packages - everything depends on the quality of the data reduction, and the programs have strengths in different areas. HTH, Kay -- == Bart Hazes (Assistant Professor) Dept. of Medical Microbiology & Immunology University of Alberta 1-15 Medical Sciences Building Edmonton, Alberta Canada, T6G 2H7 phone: 1-780-492-0042 fax:1-780-492-7521 ==
Re: [ccp4bb] Using multiple crystals for structure solution in P1 using MAD/SAS/SAD
Scala has an option (ANOMALOUS MATCH INRUN) to select "matching" Bijvoet pairs, which will have a similar effect to average DelAnom, but when I've tried it, it has given worse results than just chucking everything in together Phil On 23 Jul 2008, at 16:53, Bart Hazes wrote: Jacob Keller wrote: Shouldn't all of the "crystal-to-crystal" differences be taken out automatically by scaling, Scaling only takes out differences in overall scale, B-factor and, if you have enough data, it can correct to some extend for absorption or other more local effects. Systematic differences due to slight differences in unit cell, molecular packing etc lead to different relative intensities that are not removed by scaling. and is there not the same proportional anomalous signal in every isomorphous crystal, regardless of the background? I would think that using multiple crystals would give a better idea of "the truth," as if taking many snapshots of the same object, and putting them together to form a three-dimensional object. In Hazes' language, don't all isomorphous crystals "draw from the same [underlying] distribution?" The answer is yes when the crystals are truly isomorphous. In reality they rarely if ever are. The differences tend to be small enough that you normally don't have to worry about it for heavy atom derivatives or native data sets. However, for weak anomalous signals it is a different story. Bart Jacob Keller ps admittedly if there is radiation damage or other non- isomorphisms, this reasoning does not apply. *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] *** - Original Message - From: "Bart Hazes" <[EMAIL PROTECTED] > To: Sent: Wednesday, July 23, 2008 10:05 AM Subject: Re: [ccp4bb] Using multiple crystals for structure solution in P1 using MAD/SAS/SAD Increasing redundancy only helps if all data draw from the same distribution so you get a more accurate estimate of the mean of the distribution. When dealing with different crystals, crystal-to- crystal variation is likely larger than the anomalous signal you are looking for and I'm therefore not convinced that merging of data is a good idea (never hurts to try though). I wonder if it would work better to derive anomalous differences for the individual data sets first and then merge those anomalous differences. This may allow the subtraction between F+ and F- to remove some of the systematic differences there may be between crystal forms. Bart Kay Diederichs wrote: hari jayaram schrieb: ... I was wondering if anyone could comment on combining datasets from multiple P1 crystals to increase the redundancy even further for such heavy atom ( SAS / SAD ) or MAD experiments. Hari, well, my comment would be that it should be possible in principle from what you describe, but the outcome strongly depends on the details (size of expected and observed anomalous and isomorphous signal, internal anomalous correlation coefficients, I/sigma and R-factors, radiation damage, are crystals isomorphous, ...). To increase the quality of the reduced data it would be advisable to rotate around different axes, which is possible at some - but not all - beamlines. This is even more true in P1. For all of the major data reduction programs there exist specific programs for merging data, and it does make a lot of sense to merge your passes (but don't merge radiation-damaged data with undamaged data)!. I would suggest to use at least two different data reduction packages - everything depends on the quality of the data reduction, and the programs have strengths in different areas. HTH, Kay -- = = = = = = = = = = Bart Hazes (Assistant Professor) Dept. of Medical Microbiology & Immunology University of Alberta 1-15 Medical Sciences Building Edmonton, Alberta Canada, T6G 2H7 phone: 1-780-492-0042 fax:1-780-492-7521 = = = = = = = = = = -- = = = = = = = = == Bart Hazes (Assistant Professor) Dept. of Medical Microbiology & Immunology University of Alberta 1-15 Medical Sciences Building Edmonton, Alberta Canada, T6G 2H7 phone: 1-780-492-0042 fax:1-780-492-7521 = = = = = = = = ==
Re: [ccp4bb] Using multiple crystals for structure solution in P1 using MAD/SAS/SAD
Jacob Keller wrote: Shouldn't all of the "crystal-to-crystal" differences be taken out automatically by scaling, Scaling only takes out differences in overall scale, B-factor and, if you have enough data, it can correct to some extend for absorption or other more local effects. Systematic differences due to slight differences in unit cell, molecular packing etc lead to different relative intensities that are not removed by scaling. and is there not the same proportional anomalous signal in every isomorphous crystal, regardless of the background? I would think that using multiple crystals would give a better idea of "the truth," as if taking many snapshots of the same object, and putting them together to form a three-dimensional object. In Hazes' language, don't all isomorphous crystals "draw from the same [underlying] distribution?" The answer is yes when the crystals are truly isomorphous. In reality they rarely if ever are. The differences tend to be small enough that you normally don't have to worry about it for heavy atom derivatives or native data sets. However, for weak anomalous signals it is a different story. Bart Jacob Keller ps admittedly if there is radiation damage or other non-isomorphisms, this reasoning does not apply. *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] *** - Original Message - From: "Bart Hazes" <[EMAIL PROTECTED]> To: Sent: Wednesday, July 23, 2008 10:05 AM Subject: Re: [ccp4bb] Using multiple crystals for structure solution in P1 using MAD/SAS/SAD Increasing redundancy only helps if all data draw from the same distribution so you get a more accurate estimate of the mean of the distribution. When dealing with different crystals, crystal-to-crystal variation is likely larger than the anomalous signal you are looking for and I'm therefore not convinced that merging of data is a good idea (never hurts to try though). I wonder if it would work better to derive anomalous differences for the individual data sets first and then merge those anomalous differences. This may allow the subtraction between F+ and F- to remove some of the systematic differences there may be between crystal forms. Bart Kay Diederichs wrote: hari jayaram schrieb: ... I was wondering if anyone could comment on combining datasets from multiple P1 crystals to increase the redundancy even further for such heavy atom ( SAS / SAD ) or MAD experiments. Hari, well, my comment would be that it should be possible in principle from what you describe, but the outcome strongly depends on the details (size of expected and observed anomalous and isomorphous signal, internal anomalous correlation coefficients, I/sigma and R-factors, radiation damage, are crystals isomorphous, ...). To increase the quality of the reduced data it would be advisable to rotate around different axes, which is possible at some - but not all - beamlines. This is even more true in P1. For all of the major data reduction programs there exist specific programs for merging data, and it does make a lot of sense to merge your passes (but don't merge radiation-damaged data with undamaged data)!. I would suggest to use at least two different data reduction packages - everything depends on the quality of the data reduction, and the programs have strengths in different areas. HTH, Kay -- == Bart Hazes (Assistant Professor) Dept. of Medical Microbiology & Immunology University of Alberta 1-15 Medical Sciences Building Edmonton, Alberta Canada, T6G 2H7 phone: 1-780-492-0042 fax:1-780-492-7521 == -- == Bart Hazes (Assistant Professor) Dept. of Medical Microbiology & Immunology University of Alberta 1-15 Medical Sciences Building Edmonton, Alberta Canada, T6G 2H7 phone: 1-780-492-0042 fax:1-780-492-7521 ==
Re: [ccp4bb] Using multiple crystals for structure solution in P1 using MAD/SAS/SAD
Shouldn't all of the "crystal-to-crystal" differences be taken out automatically by scaling, and is there not the same proportional anomalous signal in every isomorphous crystal, regardless of the background? I would think that using multiple crystals would give a better idea of "the truth," as if taking many snapshots of the same object, and putting them together to form a three-dimensional object. In Hazes' language, don't all isomorphous crystals "draw from the same [underlying] distribution?" Jacob Keller ps admittedly if there is radiation damage or other non-isomorphisms, this reasoning does not apply. *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] *** - Original Message - From: "Bart Hazes" <[EMAIL PROTECTED]> To: Sent: Wednesday, July 23, 2008 10:05 AM Subject: Re: [ccp4bb] Using multiple crystals for structure solution in P1 using MAD/SAS/SAD Increasing redundancy only helps if all data draw from the same distribution so you get a more accurate estimate of the mean of the distribution. When dealing with different crystals, crystal-to-crystal variation is likely larger than the anomalous signal you are looking for and I'm therefore not convinced that merging of data is a good idea (never hurts to try though). I wonder if it would work better to derive anomalous differences for the individual data sets first and then merge those anomalous differences. This may allow the subtraction between F+ and F- to remove some of the systematic differences there may be between crystal forms. Bart Kay Diederichs wrote: hari jayaram schrieb: ... I was wondering if anyone could comment on combining datasets from multiple P1 crystals to increase the redundancy even further for such heavy atom ( SAS / SAD ) or MAD experiments. Hari, well, my comment would be that it should be possible in principle from what you describe, but the outcome strongly depends on the details (size of expected and observed anomalous and isomorphous signal, internal anomalous correlation coefficients, I/sigma and R-factors, radiation damage, are crystals isomorphous, ...). To increase the quality of the reduced data it would be advisable to rotate around different axes, which is possible at some - but not all - beamlines. This is even more true in P1. For all of the major data reduction programs there exist specific programs for merging data, and it does make a lot of sense to merge your passes (but don't merge radiation-damaged data with undamaged data)!. I would suggest to use at least two different data reduction packages - everything depends on the quality of the data reduction, and the programs have strengths in different areas. HTH, Kay -- == Bart Hazes (Assistant Professor) Dept. of Medical Microbiology & Immunology University of Alberta 1-15 Medical Sciences Building Edmonton, Alberta Canada, T6G 2H7 phone: 1-780-492-0042 fax:1-780-492-7521 ==
Re: [ccp4bb] Using multiple crystals for structure solution in P1 using MAD/SAS/SAD
Increasing redundancy only helps if all data draw from the same distribution so you get a more accurate estimate of the mean of the distribution. When dealing with different crystals, crystal-to-crystal variation is likely larger than the anomalous signal you are looking for and I'm therefore not convinced that merging of data is a good idea (never hurts to try though). I wonder if it would work better to derive anomalous differences for the individual data sets first and then merge those anomalous differences. This may allow the subtraction between F+ and F- to remove some of the systematic differences there may be between crystal forms. Bart Kay Diederichs wrote: hari jayaram schrieb: ... I was wondering if anyone could comment on combining datasets from multiple P1 crystals to increase the redundancy even further for such heavy atom ( SAS / SAD ) or MAD experiments. Hari, well, my comment would be that it should be possible in principle from what you describe, but the outcome strongly depends on the details (size of expected and observed anomalous and isomorphous signal, internal anomalous correlation coefficients, I/sigma and R-factors, radiation damage, are crystals isomorphous, ...). To increase the quality of the reduced data it would be advisable to rotate around different axes, which is possible at some - but not all - beamlines. This is even more true in P1. For all of the major data reduction programs there exist specific programs for merging data, and it does make a lot of sense to merge your passes (but don't merge radiation-damaged data with undamaged data)!. I would suggest to use at least two different data reduction packages - everything depends on the quality of the data reduction, and the programs have strengths in different areas. HTH, Kay -- == Bart Hazes (Assistant Professor) Dept. of Medical Microbiology & Immunology University of Alberta 1-15 Medical Sciences Building Edmonton, Alberta Canada, T6G 2H7 phone: 1-780-492-0042 fax:1-780-492-7521 ==
Re: [ccp4bb] Using multiple crystals for structure solution in P1 using MAD/SAS/SAD
hari jayaram schrieb: ... I was wondering if anyone could comment on combining datasets from multiple P1 crystals to increase the redundancy even further for such heavy atom ( SAS / SAD ) or MAD experiments. Hari, well, my comment would be that it should be possible in principle from what you describe, but the outcome strongly depends on the details (size of expected and observed anomalous and isomorphous signal, internal anomalous correlation coefficients, I/sigma and R-factors, radiation damage, are crystals isomorphous, ...). To increase the quality of the reduced data it would be advisable to rotate around different axes, which is possible at some - but not all - beamlines. This is even more true in P1. For all of the major data reduction programs there exist specific programs for merging data, and it does make a lot of sense to merge your passes (but don't merge radiation-damaged data with undamaged data)!. I would suggest to use at least two different data reduction packages - everything depends on the quality of the data reduction, and the programs have strengths in different areas. HTH, Kay smime.p7s Description: S/MIME Cryptographic Signature
[ccp4bb] Using multiple crystals for structure solution in P1 using MAD/SAS/SAD
We are faced with phasing a structure for a protein that refuses to crystallize in any spacegroup but P1. To add to the fun , the resolution for most selenomethionine and heavy atom soak datasets ranges from 3.8 to 4.2 A . In order to increase the redundancy we have been taking many inverse beam datasets from each crystal by making sure the beam is significantly attenuated. We now have 360 times 6, ( i.e 6 passes) and in some case 8 passes, datasets for a few crystals collected at the peak wavelength in the case of Selenomethionine crystals. In some cases we even managed an inflection dataset . Needless to say the anomolous signal seems quite week at these resolutions and low redundancies for any single crystal dataset. I was wondering if anyone could comment on combining datasets from multiple P1 crystals to increase the redundancy even further for such heavy atom ( SAS / SAD ) or MAD experiments. Thanks a lot for your help and suggestions in advance hari
