Re: [ccp4bb] refmac5.7 refine pesudo-translational symmetry
Dear Randy, Thanks very much for your detailed explanation and helpful advice. I have run the phaser job just as you have said. This is the result. The resolution of this dataset is 2.45A. = I added the three commands to phaser job for all alternative space group of P212121: TNCS USE ON TNCS NMOL 4 TNCS PATT PERCENT 80.0 The phaser got a SINGLE solution for space group P22121, and Rval=88.2 SOLU SET RFZ=14.5 TFZ=22.3 PAK=0 +TNCS PAK=0 +TNCS PAK=0 +TNCS PAK=0 LLG=2565LLG=4322 SOLU SPAC P 2 21 21 SOLU 6DIM ENSE ensemble1 EULER 91.7 89.4 90.3 FRAC 0.49 0.51 0.99 BFAC 0.00 SOLU 6DIM ENSE ensemble1 EULER 269.3 89.4 90.2 FRAC -0.01 -0.00 0.74 BFAC 0.00 SOLU 6DIM ENSE ensemble1 EULER 269.0 89.1 90.0 FRAC 0.49 -0.00 1.00 BFAC 0.00 SOLU 6DIM ENSE ensemble1 EULER 91.5 89.3 90.2 FRAC 0.99 0.51 0.74 BFAC 0.00 After 50 cycles rigidbody refinement, R/Rfree=0.36/0.37 After 50 cycles restraint refinement (with jellybody refine and twin refine), R/Rfree=0.31/0.35 After several cycles of coot model building and restraint refinement, R/Rfree=0.27/0.31 After finding waters, R/Rfree=0.2478/0.2984 === If I run phaser job without those three added commands, the phaser got single solution at P21212 space group (Rval=0.3%): SOLU SET RFZ=17.0 TFZ=20.1 PAK=0 LLG=457 TFZ==16.2 RFZ=16.3 TFZ=62.1 PAK=0 LLG=2599 TFZ==19.8 ( TFZ=59.0 PAK=0 LLG=2403) LLG+=(2608 4349) LLG=4200 TFZ==22.2 PAK=0 RFZ=10.8 TFZ=56.4 PAK=0 LLG=5751 TFZ==22.5 LLG=9274 TFZ==27.5 SOLU SPAC P 21 21 2 SOLU 6DIM ENSE ensemble1 EULER 90.0 90.7 270.1 FRAC -0.00 0.26 0.13 BFAC -4.09 SOLU 6DIM ENSE ensemble1 EULER 271.2 89.4 90.3 FRAC 0.01 0.24 -0.38 BFAC -3.00 SOLU 6DIM ENSE ensemble1 EULER 270.3 90.8 270.1 FRAC 0.50 -0.25 -0.12 BFAC -1.25 SOLU 6DIM ENSE ensemble1 EULER 87.5 90.6 270.4 FRAC 0.02 0.26 -0.37 BFAC 11.58 And the electron density is worse than the P22121 solution. == Just as I have said last email, I can also get single solution at P212121 space group, and after 50 cycles rigidbody refinement and 50 cycles restraint refinement with jellybody and twin, I can get R/Rfree=0.30/0.33. But the electron density is worse than the P22121 solution, so I do not carry out the model building in coot. === My questiones: 1) Is the P22121 solution is my correct solution? These three solutions really confused me. 2) Why the R/Rfree is high even after I have good electron density and have found waters? (R/Rfree=0.478/0.2984 for 2.45A data) 3) What's the function of the command TNCS USE ON? Is it necessary? 4) I found if I used twin refinement in refmac5, the R/Rfree would decrease about 0.02 comparing to without twinn refinement. Is it reasonable? Is there any tNCS refinement options in refmac? Thanks for your help. Best wishes, Qixu Cai 2012/7/31 Randy Read rj...@cam.ac.uk Hi, The second Patterson peak is twice the first (considering lattice translations, where 1 is equivalent to 0 modulo 1), and then if you triple the first vector you'll get minus the first vector (again considering lattice translations, i.e. 3/4 is equal to 1 - 1/4 which is equivalent to -1/4), which is equivalent by symmetry to the first vector so wouldn't appear in the peak list. So the Patterson indicates 4 copies separated by 0, 1, 2 and 3 times the top Patterson vector, in approximately the same orientation. We've haven't fully dealt with the complications of multiple tNCS-related copies in Phaser yet, but for this type of case there is a reasonable treatment. You should add two commands to the Phaser job: TNCS NMOL 4 TNCS PATT PERCENT 80 The first says that the Patterson translation is repeated 4 times, and the second will cause the second Patterson peak to be ignored. I'd suggest repeating the Phaser run with these commands and making sure that you end up with the same solution as you got when the tNCS was ignored. When tNCS is ignored, it's possible to end up with a solution that is only partially correct, which would be one explanation for having some molecules that look better in density than others. Best wishes, Randy Read On 31 Jul 2012, at 13:11, Qixu Cai wrote: It's a P212121 dataset. I have used phaser to find four solution in ASU. This is the phaser log file: PEUDO-TRANSLATIONAL NCS VECTOR -- Space Group : P 21 21 21 Patterson Symmetry: P m m m Resolution of All Data (Number):2.45 49.00 (22968) Resolution of Patterson (Number): 5.00 9.99 (2364) There were 2 non-origin distinct peaks (i.e. more than 15 angstroms from the origin)
Re: [ccp4bb] refmac5.7 refine pesudo-translational symmetry
Are your unit cell and SG correct? I think you maybe should reindex to get a cell volume 25% of this one, and maybe SG P21 That patterson peak is enormous.. Eleanor On 1 Aug 2012, at 08:45, Qixu Cai wrote: Dear Randy, Thanks very much for your detailed explanation and helpful advice. I have run the phaser job just as you have said. This is the result. The resolution of this dataset is 2.45A. = I added the three commands to phaser job for all alternative space group of P212121: TNCS USE ON TNCS NMOL 4 TNCS PATT PERCENT 80.0 The phaser got a SINGLE solution for space group P22121, and Rval=88.2 SOLU SET RFZ=14.5 TFZ=22.3 PAK=0 +TNCS PAK=0 +TNCS PAK=0 +TNCS PAK=0 LLG=2565LLG=4322 SOLU SPAC P 2 21 21 SOLU 6DIM ENSE ensemble1 EULER 91.7 89.4 90.3 FRAC 0.49 0.51 0.99 BFAC 0.00 SOLU 6DIM ENSE ensemble1 EULER 269.3 89.4 90.2 FRAC -0.01 -0.00 0.74 BFAC 0.00 SOLU 6DIM ENSE ensemble1 EULER 269.0 89.1 90.0 FRAC 0.49 -0.00 1.00 BFAC 0.00 SOLU 6DIM ENSE ensemble1 EULER 91.5 89.3 90.2 FRAC 0.99 0.51 0.74 BFAC 0.00 After 50 cycles rigidbody refinement, R/Rfree=0.36/0.37 After 50 cycles restraint refinement (with jellybody refine and twin refine), R/Rfree=0.31/0.35 After several cycles of coot model building and restraint refinement, R/Rfree=0.27/0.31 After finding waters, R/Rfree=0.2478/0.2984 === If I run phaser job without those three added commands, the phaser got single solution at P21212 space group (Rval=0.3%): SOLU SET RFZ=17.0 TFZ=20.1 PAK=0 LLG=457 TFZ==16.2 RFZ=16.3 TFZ=62.1 PAK=0 LLG=2599 TFZ==19.8 ( TFZ=59.0 PAK=0 LLG=2403) LLG+=(2608 4349) LLG=4200 TFZ==22.2 PAK=0 RFZ=10.8 TFZ=56.4 PAK=0 LLG=5751 TFZ==22.5 LLG=9274 TFZ==27.5 SOLU SPAC P 21 21 2 SOLU 6DIM ENSE ensemble1 EULER 90.0 90.7 270.1 FRAC -0.00 0.26 0.13 BFAC -4.09 SOLU 6DIM ENSE ensemble1 EULER 271.2 89.4 90.3 FRAC 0.01 0.24 -0.38 BFAC -3.00 SOLU 6DIM ENSE ensemble1 EULER 270.3 90.8 270.1 FRAC 0.50 -0.25 -0.12 BFAC -1.25 SOLU 6DIM ENSE ensemble1 EULER 87.5 90.6 270.4 FRAC 0.02 0.26 -0.37 BFAC 11.58 And the electron density is worse than the P22121 solution. == Just as I have said last email, I can also get single solution at P212121 space group, and after 50 cycles rigidbody refinement and 50 cycles restraint refinement with jellybody and twin, I can get R/Rfree=0.30/0.33. But the electron density is worse than the P22121 solution, so I do not carry out the model building in coot. === My questiones: 1) Is the P22121 solution is my correct solution? These three solutions really confused me. 2) Why the R/Rfree is high even after I have good electron density and have found waters? (R/Rfree=0.478/0.2984 for 2.45A data) 3) What's the function of the command TNCS USE ON? Is it necessary? 4) I found if I used twin refinement in refmac5, the R/Rfree would decrease about 0.02 comparing to without twinn refinement. Is it reasonable? Is there any tNCS refinement options in refmac? Thanks for your help. Best wishes, Qixu Cai 2012/7/31 Randy Read rj...@cam.ac.uk Hi, The second Patterson peak is twice the first (considering lattice translations, where 1 is equivalent to 0 modulo 1), and then if you triple the first vector you'll get minus the first vector (again considering lattice translations, i.e. 3/4 is equal to 1 - 1/4 which is equivalent to -1/4), which is equivalent by symmetry to the first vector so wouldn't appear in the peak list. So the Patterson indicates 4 copies separated by 0, 1, 2 and 3 times the top Patterson vector, in approximately the same orientation. We've haven't fully dealt with the complications of multiple tNCS-related copies in Phaser yet, but for this type of case there is a reasonable treatment. You should add two commands to the Phaser job: TNCS NMOL 4 TNCS PATT PERCENT 80 The first says that the Patterson translation is repeated 4 times, and the second will cause the second Patterson peak to be ignored. I'd suggest repeating the Phaser run with these commands and making sure that you end up with the same solution as you got when the tNCS was ignored. When tNCS is ignored, it's possible to end up with a solution that is only partially correct, which would be one explanation for having some molecules that look better in density than others. Best wishes, Randy Read On 31 Jul 2012, at 13:11, Qixu Cai wrote: It's a P212121 dataset. I have used phaser to find four solution in ASU. This is the phaser log file: PEUDO-TRANSLATIONAL NCS VECTOR
Re: [ccp4bb] refmac5.7 refine pesudo-translational symmetry
Dear Eleanor, The cell dimensions of the dataset with tNCS are 63.995 75.459 128.860 90.00 90.00 90.00 in P 2 21 21 space group. But for the other dataset without tNCS of the same protein, the cell dimensions are 64.203 65.319 76.443 90.00 90.00 90.00 in C 2 2 21. So the cell volume of C2221 is 25% of the P22121. But I cannot index the data to C2221 space group. Thanks a lot. Best wishes, Qixu Cai 2012/8/1 Eleanor Dodson eleanor.dod...@york.ac.uk Are your unit cell and SG correct? I think you maybe should reindex to get a cell volume 25% of this one, and maybe SG P21 That patterson peak is enormous.. Eleanor On 1 Aug 2012, at 08:45, Qixu Cai wrote: Dear Randy, Thanks very much for your detailed explanation and helpful advice. I have run the phaser job just as you have said. This is the result. The resolution of this dataset is 2.45A. = I added the three commands to phaser job for all alternative space groupof P212121: TNCS USE ON TNCS NMOL 4 TNCS PATT PERCENT 80.0 The phaser got a SINGLE solution for space group P22121, and Rval=88.2 SOLU SET RFZ=14.5 TFZ=22.3 PAK=0 +TNCS PAK=0 +TNCS PAK=0 +TNCS PAK=0 LLG=2565LLG=4322 SOLU SPAC P 2 21 21 SOLU 6DIM ENSE ensemble1 EULER 91.7 89.4 90.3 FRAC 0.49 0.51 0.99 BFAC 0.00 SOLU 6DIM ENSE ensemble1 EULER 269.3 89.4 90.2 FRAC -0.01 -0.00 0.74 BFAC 0.00 SOLU 6DIM ENSE ensemble1 EULER 269.0 89.1 90.0 FRAC 0.49 -0.00 1.00 BFAC 0.00 SOLU 6DIM ENSE ensemble1 EULER 91.5 89.3 90.2 FRAC 0.99 0.51 0.74 BFAC 0.00 After 50 cycles rigidbody refinement, R/Rfree=0.36/0.37 After 50 cycles restraint refinement (with jellybody refine and twin refine), R/Rfree=0.31/0.35 After several cycles of coot model building and restraint refinement, R/Rfree=0.27/0.31 After finding waters, R/Rfree=0.2478/0.2984 === If I run phaser job without those three added commands, the phaser got single solution at P21212 space group (Rval=0.3%): SOLU SET RFZ=17.0 TFZ=20.1 PAK=0 LLG=457 TFZ==16.2 RFZ=16.3 TFZ=62.1 PAK=0 LLG=2599 TFZ==19.8 ( TFZ=59.0 PAK=0 LLG=2403) LLG+=(2608 4349) LLG=4200 TFZ==22.2 PAK=0 RFZ=10.8 TFZ=56.4 PAK=0 LLG=5751 TFZ==22.5 LLG=9274 TFZ==27.5 SOLU SPAC P 21 21 2 SOLU 6DIM ENSE ensemble1 EULER 90.0 90.7 270.1 FRAC -0.00 0.26 0.13 BFAC -4.09 SOLU 6DIM ENSE ensemble1 EULER 271.2 89.4 90.3 FRAC 0.01 0.24 -0.38 BFAC -3.00 SOLU 6DIM ENSE ensemble1 EULER 270.3 90.8 270.1 FRAC 0.50 -0.25 -0.12 BFAC -1.25 SOLU 6DIM ENSE ensemble1 EULER 87.5 90.6 270.4 FRAC 0.02 0.26 -0.37 BFAC 11.58 And the electron density is worse than the P22121 solution. == Just as I have said last email, I can also get single solution at P212121 space group, and after 50 cycles rigidbody refinement and 50 cycles restraint refinement with jellybody and twin, I can get R/Rfree=0.30/0.33. But the electron density is worse than the P22121 solution, so I do not carry out the model building in coot. === My questiones: 1) Is the P22121 solution is my correct solution? These three solutions really confused me. 2) Why the R/Rfree is high even after I have good electron density and have found waters? (R/Rfree=0.478/0.2984 for 2.45A data) 3) What's the function of the command TNCS USE ON? Is it necessary? 4) I found if I used twin refinement in refmac5, the R/Rfree would decrease about 0.02 comparing to without twinn refinement. Is it reasonable? Is there any tNCS refinement options in refmac? Thanks for your help. Best wishes, Qixu Cai 2012/7/31 Randy Read rj...@cam.ac.uk Hi, The second Patterson peak is twice the first (considering lattice translations, where 1 is equivalent to 0 modulo 1), and then if you triple the first vector you'll get minus the first vector (again considering lattice translations, i.e. 3/4 is equal to 1 - 1/4 which is equivalent to -1/4), which is equivalent by symmetry to the first vector so wouldn't appear in the peak list. So the Patterson indicates 4 copies separated by 0, 1, 2 and 3 times the top Patterson vector, in approximately the same orientation. We've haven't fully dealt with the complications of multiple tNCS-related copies in Phaser yet, but for this type of case there is a reasonable treatment. You should add two commands to the Phaser job: TNCS NMOL 4 TNCS PATT PERCENT 80 The first says that the Patterson translation is repeated 4 times, and the second will cause the second Patterson peak to be ignored. I'd suggest repeating the Phaser run with these commands and making sure that you end up with the same solution as you got when the tNCS was ignored. When tNCS is ignored, it's possible to end up with a solution that is only partially
[ccp4bb] Process multiple data sets
Dear All: I collected 5 data sets from one crystal and would like to process them together. Here is how I did: In HKL2000, load the all data sets. Index each set. When I try Intergrate, the program automatically go through the whole data sets there, and do not go through. I then process data sets by loading one at each time. Index, intergrate and scale all go through very smoothly. But when I put them together, the program just goes crazy. Thank you for advice Uma
[ccp4bb] Iron induced reduction of crystal
Hi All, I'm currently working of a protein with a ferredoxin protein with anIron-Sulphur cluster. I was harvesting some crystals the other day and a piece of my scalpel blade broke off and ended up in the well solution. I Sealed the well without noticing, the shard of iron oxidised and the crystals lost most of their red colour: Ordinary crystals: http://s1058.photobucket.com/albums/t401/__Rhys__/?action=viewcurrent=MBPR_Rodcluster2edit.png Crystals from Blade containing well: http://s1058.photobucket.com/albums/t401/__Rhys__/?action=viewcurrent=MBPR_Bleached.png My explanation for this (If someone has a different one that'd be great too) Is that the oxidation of the metallic iron the well, created reducing conditions in the chamber and reduced the iron-sulphur cluster (reduced ferredoxin is much less strongly coloured). Which got me to thinking...Could this be applied as a technique to create reducing conditions in protein crystallography, as the use of reducing agents isn't always practical. Cheers, Rhys Grinter PhD Candidate University of Glasgow
Re: [ccp4bb] Process multiple data sets
The data sets were collected from the same crystal by scan collecting 40 frames from each section. The space group of this crystal is P2. My guess that I may have to index and integrate each set indivadually, and then scale them together. Thanks Uma On Wed, Aug 1, 2012 at 9:01 AM, Machius, Mischa Christian mischa_mach...@med.unc.edu wrote: Not much info to go by... Anyway, if the program 'goes crazy' you either have very different exposure levels, radiation damage leading to non-isomorphism, or you have a trigonal space group (P3xxx or P6xxx) and forgot to make sure all batches are indexed the same way. If you have translated your crystal between batches, HKL2000 won't of course be able to process all batches in one go. If you haven't touched the crystals at all, and alln-in-one processing doesn't work, the parameters at the end of one batch may not be accurate to start off a new batch, which is mostly due to inaccurate goniostats. In that case, you will need to process the batches individually and them combine them during scaling. Hope that helps. MM On Aug 1, 2012, at 8:50 AM, Uma Ratu wrote: Dear All: I collected 5 data sets from one crystal and would like to process them together. Here is how I did: In HKL2000, load the all data sets. Index each set. When I try Intergrate, the program automatically go through the whole data sets there, and do not go through. I then process data sets by loading one at each time. Index, intergrate and scale all go through very smoothly. But when I put them together, the program just goes crazy. Thank you for advice Uma
Re: [ccp4bb] Iron induced reduction of crystal
Take a look at http://en.wikipedia.org/wiki/Great_Oxygenation_Event This might suggest you may have used up the available oxygen. If you would like to try growing your crystals in an oxygen-free environment, we (in Leicester) have a glove box with a Douglas Instruments Oryx 4 robot. I know its not exactly handy for Glasgow, but .. On 1 August 2012 13:53, RHYS GRINTER r.grinte...@research.gla.ac.uk wrote: Hi All, I'm currently working of a protein with a ferredoxin protein with anIron-Sulphur cluster. I was harvesting some crystals the other day and a piece of my scalpel blade broke off and ended up in the well solution. I Sealed the well without noticing, the shard of iron oxidised and the crystals lost most of their red colour: Ordinary crystals: http://s1058.photobucket.com/albums/t401/__Rhys__/?action=viewcurrent=MBPR_Rodcluster2edit.png Crystals from Blade containing well: http://s1058.photobucket.com/albums/t401/__Rhys__/?action=viewcurrent=MBPR_Bleached.png My explanation for this (If someone has a different one that'd be great too) Is that the oxidation of the metallic iron the well, created reducing conditions in the chamber and reduced the iron-sulphur cluster (reduced ferredoxin is much less strongly coloured). Which got me to thinking...Could this be applied as a technique to create reducing conditions in protein crystallography, as the use of reducing agents isn't always practical. Cheers, Rhys Grinter PhD Candidate University of Glasgow
Re: [ccp4bb] Process multiple data sets
Hi I'd process (i.e. index, refine, integrate) each data set individually, check that (at least) they all have the same crystal system, then combine the datasets using Pointless. Then scale with Aimless. Of course, I'd use Mosflm/Pointless/Aimless rather than HKL, but that's another question (pace, ZO, WM, MM!) On 1 Aug 2012, at 14:08, Uma Ratu wrote: The data sets were collected from the same crystal by scan collecting 40 frames from each section. The space group of this crystal is P2. My guess that I may have to index and integrate each set indivadually, and then scale them together. Thanks Uma On Wed, Aug 1, 2012 at 9:01 AM, Machius, Mischa Christian mischa_mach...@med.unc.edu wrote: Not much info to go by... Anyway, if the program 'goes crazy' you either have very different exposure levels, radiation damage leading to non-isomorphism, or you have a trigonal space group (P3xxx or P6xxx) and forgot to make sure all batches are indexed the same way. If you have translated your crystal between batches, HKL2000 won't of course be able to process all batches in one go. If you haven't touched the crystals at all, and alln-in-one processing doesn't work, the parameters at the end of one batch may not be accurate to start off a new batch, which is mostly due to inaccurate goniostats. In that case, you will need to process the batches individually and them combine them during scaling. Hope that helps. MM On Aug 1, 2012, at 8:50 AM, Uma Ratu wrote: Dear All: I collected 5 data sets from one crystal and would like to process them together. Here is how I did: In HKL2000, load the all data sets. Index each set. When I try Intergrate, the program automatically go through the whole data sets there, and do not go through. I then process data sets by loading one at each time. Index, intergrate and scale all go through very smoothly. But when I put them together, the program just goes crazy. Thank you for advice Uma Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH
Re: [ccp4bb] Iron induced reduction of crystal
Hi, To me this looks like a simple redox displacement reaction which has nothing to do with oxygen. E.g. if one puts a piece of iron in a coppersulfate solution, the more noble copper ions take electrons from the less noble iron and will come out of solution as a metal, while the less noble iron will disolve as ions. The same principle is used by plating zinc on iron to prevent rust in e.g. cars. The less noble zinc will provide electrons to protect the more noble iron. By choosing the right metal, one might indeed be able to very precisely reduce redox active groups in proteins. My 2 cents, Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Peter Moody Sent: Wednesday, August 01, 2012 3:30 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Iron induced reduction of crystal Take a look at http://en.wikipedia.org/wiki/Great_Oxygenation_Event This might suggest you may have used up the available oxygen. If you would like to try growing your crystals in an oxygen-free environment, we (in Leicester) have a glove box with a Douglas Instruments Oryx 4 robot. I know its not exactly handy for Glasgow, but .. On 1 August 2012 13:53, RHYS GRINTER r.grinte...@research.gla.ac.uk wrote: Hi All, I'm currently working of a protein with a ferredoxin protein with anIron-Sulphur cluster. I was harvesting some crystals the other day and a piece of my scalpel blade broke off and ended up in the well solution. I Sealed the well without noticing, the shard of iron oxidised and the crystals lost most of their red colour: Ordinary crystals: http://s1058.photobucket.com/albums/t401/__Rhys__/?action=viewcurrent=M BPR_Rodcluster2edit.png Crystals from Blade containing well: http://s1058.photobucket.com/albums/t401/__Rhys__/?action=viewcurrent=M BPR_Bleached.png My explanation for this (If someone has a different one that'd be great too) Is that the oxidation of the metallic iron the well, created reducing conditions in the chamber and reduced the iron-sulphur cluster (reduced ferredoxin is much less strongly coloured). Which got me to thinking...Could this be applied as a technique to create reducing conditions in protein crystallography, as the use of reducing agents isn't always practical. Cheers, Rhys Grinter PhD Candidate University of Glasgow
Re: [ccp4bb] Process multiple data sets
Note that neither Aimless nor Scala will do a particularly good job at scaling data from Denzo or Scalepack, since the output files from Scalepack are missing essential geometrical information. They work well with data from Mosflm or XDS (or Saint) (although AFAIK the XDS Saint scaling programs work perfectly well) However, Pointless may still be useful to check that you have indexed them consistently Phil On 1 Aug 2012, at 14:36, Harry Powell wrote: Hi I'd process (i.e. index, refine, integrate) each data set individually, check that (at least) they all have the same crystal system, then combine the datasets using Pointless. Then scale with Aimless. Of course, I'd use Mosflm/Pointless/Aimless rather than HKL, but that's another question (pace, ZO, WM, MM!) On 1 Aug 2012, at 14:08, Uma Ratu wrote: The data sets were collected from the same crystal by scan collecting 40 frames from each section. The space group of this crystal is P2. My guess that I may have to index and integrate each set indivadually, and then scale them together. Thanks Uma On Wed, Aug 1, 2012 at 9:01 AM, Machius, Mischa Christian mischa_mach...@med.unc.edu wrote: Not much info to go by... Anyway, if the program 'goes crazy' you either have very different exposure levels, radiation damage leading to non-isomorphism, or you have a trigonal space group (P3xxx or P6xxx) and forgot to make sure all batches are indexed the same way. If you have translated your crystal between batches, HKL2000 won't of course be able to process all batches in one go. If you haven't touched the crystals at all, and alln-in-one processing doesn't work, the parameters at the end of one batch may not be accurate to start off a new batch, which is mostly due to inaccurate goniostats. In that case, you will need to process the batches individually and them combine them during scaling. Hope that helps. MM On Aug 1, 2012, at 8:50 AM, Uma Ratu wrote: Dear All: I collected 5 data sets from one crystal and would like to process them together. Here is how I did: In HKL2000, load the all data sets. Index each set. When I try Intergrate, the program automatically go through the whole data sets there, and do not go through. I then process data sets by loading one at each time. Index, intergrate and scale all go through very smoothly. But when I put them together, the program just goes crazy. Thank you for advice Uma Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH
Re: [ccp4bb] Process multiple data sets
Please correct me if I am wrong: The HKL is not good to combine multiple data sets, even they are come from the same crystal? With HKL, I also tried this way: Index, integrate each data set individually, they all have the same space group. Then scale them together. Still, the graph from scale of the whole set look very wired compared with those of individuals. Uma On Wed, Aug 1, 2012 at 9:55 AM, Phil Evans p...@mrc-lmb.cam.ac.uk wrote: Note that neither Aimless nor Scala will do a particularly good job at scaling data from Denzo or Scalepack, since the output files from Scalepack are missing essential geometrical information. They work well with data from Mosflm or XDS (or Saint) (although AFAIK the XDS Saint scaling programs work perfectly well) However, Pointless may still be useful to check that you have indexed them consistently Phil On 1 Aug 2012, at 14:36, Harry Powell wrote: Hi I'd process (i.e. index, refine, integrate) each data set individually, check that (at least) they all have the same crystal system, then combine the datasets using Pointless. Then scale with Aimless. Of course, I'd use Mosflm/Pointless/Aimless rather than HKL, but that's another question (pace, ZO, WM, MM!) On 1 Aug 2012, at 14:08, Uma Ratu wrote: The data sets were collected from the same crystal by scan collecting 40 frames from each section. The space group of this crystal is P2. My guess that I may have to index and integrate each set indivadually, and then scale them together. Thanks Uma On Wed, Aug 1, 2012 at 9:01 AM, Machius, Mischa Christian mischa_mach...@med.unc.edu wrote: Not much info to go by... Anyway, if the program 'goes crazy' you either have very different exposure levels, radiation damage leading to non-isomorphism, or you have a trigonal space group (P3xxx or P6xxx) and forgot to make sure all batches are indexed the same way. If you have translated your crystal between batches, HKL2000 won't of course be able to process all batches in one go. If you haven't touched the crystals at all, and alln-in-one processing doesn't work, the parameters at the end of one batch may not be accurate to start off a new batch, which is mostly due to inaccurate goniostats. In that case, you will need to process the batches individually and them combine them during scaling. Hope that helps. MM On Aug 1, 2012, at 8:50 AM, Uma Ratu wrote: Dear All: I collected 5 data sets from one crystal and would like to process them together. Here is how I did: In HKL2000, load the all data sets. Index each set. When I try Intergrate, the program automatically go through the whole data sets there, and do not go through. I then process data sets by loading one at each time. Index, intergrate and scale all go through very smoothly. But when I put them together, the program just goes crazy. Thank you for advice Uma Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH
Re: [ccp4bb] Process multiple data sets
Hi I don't think Phil or I are saying that HKL is not a good tool to use in this case - but (*) I develop Mosflm, so I have a certain bias. (*) As far as Pointless and Aimless are concerned, to get the best out of them you need to have some geometrical information that the authors of HKL have not included in the reflection output files. Mosflm (and XDS Saint...) do give this information. So if you want to use Pointless and Aimless at any point, it may be better to use a different integration package than HKL (bearing in mind Phil's point about checking your symmetry with Pointless)... On 1 Aug 2012, at 16:16, Uma Ratu wrote: Please correct me if I am wrong: The HKL is not good to combine multiple data sets, even they are come from the same crystal? With HKL, I also tried this way: Index, integrate each data set individually, they all have the same space group. Then scale them together. Still, the graph from scale of the whole set look very wired compared with those of individuals. Uma On Wed, Aug 1, 2012 at 9:55 AM, Phil Evans p...@mrc-lmb.cam.ac.uk wrote: Note that neither Aimless nor Scala will do a particularly good job at scaling data from Denzo or Scalepack, since the output files from Scalepack are missing essential geometrical information. They work well with data from Mosflm or XDS (or Saint) (although AFAIK the XDS Saint scaling programs work perfectly well) However, Pointless may still be useful to check that you have indexed them consistently Phil On 1 Aug 2012, at 14:36, Harry Powell wrote: Hi I'd process (i.e. index, refine, integrate) each data set individually, check that (at least) they all have the same crystal system, then combine the datasets using Pointless. Then scale with Aimless. Of course, I'd use Mosflm/Pointless/Aimless rather than HKL, but that's another question (pace, ZO, WM, MM!) On 1 Aug 2012, at 14:08, Uma Ratu wrote: The data sets were collected from the same crystal by scan collecting 40 frames from each section. The space group of this crystal is P2. My guess that I may have to index and integrate each set indivadually, and then scale them together. Thanks Uma On Wed, Aug 1, 2012 at 9:01 AM, Machius, Mischa Christian mischa_mach...@med.unc.edu wrote: Not much info to go by... Anyway, if the program 'goes crazy' you either have very different exposure levels, radiation damage leading to non- isomorphism, or you have a trigonal space group (P3xxx or P6xxx) and forgot to make sure all batches are indexed the same way. If you have translated your crystal between batches, HKL2000 won't of course be able to process all batches in one go. If you haven't touched the crystals at all, and alln-in-one processing doesn't work, the parameters at the end of one batch may not be accurate to start off a new batch, which is mostly due to inaccurate goniostats. In that case, you will need to process the batches individually and them combine them during scaling. Hope that helps. MM On Aug 1, 2012, at 8:50 AM, Uma Ratu wrote: Dear All: I collected 5 data sets from one crystal and would like to process them together. Here is how I did: In HKL2000, load the all data sets. Index each set. When I try Intergrate, the program automatically go through the whole data sets there, and do not go through. I then process data sets by loading one at each time. Index, intergrate and scale all go through very smoothly. But when I put them together, the program just goes crazy. Thank you for advice Uma Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH
[ccp4bb] MR with Phaser
Dear All: I try to use Phaser to solve the structure by Molecular Replacement. The data set is collected @180 degree. I process the data using HKL, and have resonable good score: rejection (0.05), Linear R-factor (0.038), completeness (98.3), resolution (50-1.5). I then use Phaser to do MR. The parameter setting are: automated search components in asymmetric unit;number of residue 1332; number in asymmetric unit 1 perform search search using ensemble1 number of copies to search for 4 The protein is in tetramer form. I define this by using the residue number (1332) which is 4 x monomer. After run, Phaser only gave 9 partial solutions, and no solution with all components. The resulted PDB contains only dimer form of the protein, not the tetramer. And the first TFZ score is around 2.5, which is too low for MR. I have the report file of data processing and the summary of Phaser attached. Could you please advice which part is wrong, why can I get the tetramer form of the protein? Thank you Uma Crystal - Global Refinement *Space Group* P2 *Resolution Processing* 50.00 - 1.52 *Resolution Scaling*50.00 - 1.52 *a* 79.70 ± 0.01 *b* 125.27 ± 0.01 *c* 83.60 ± 0.01 *alpha* 90.000 ± *beta* 118.02 ± 0.05 *gamma* 90.000 ± *Volume*736873.6 *Mosaicity Range* 0.20 - 0.34 Global Statistics *Crystal* crystal1 *Date* Aug 01, 2012 *Experimenter* KP *Rawdata directory* /home/KP/Desktop/BY/0712/process-0712/data/CD6_7 *Processing directory* /home/KP *Data files*CD6_7_1_0001.cbf[1-180] *Wavelength*0.97920 *Total Number of Reflections* 728105 *Number of Unique Reflections* 220394 *Number of Reflections Marked for Rejection*346 *Percentage of Reflections Marked for Rejection*0.05 *Percentage of Reflections Rejected*0.00 *Linear R-factor in shell 50.00 - 4.13* 0.038 *Change of B-factor*-0.40 - 8.33 *Completeness* 98.3 *Completeness in shell 1.55 - 1.52* 91.9 *Mean I/sigma* 10.5 *Mean I/sigma in shell 1.55 - 1.52* 0.7 *Total Linear R-Merge* 0.094 Site Information *Detector* CCD pilatusCBF *Polarization* 0.99 *X Beam Position* 217.5 *Y Beam Position* 219.5 *Cassette Rotation X* 0.00 *Cassette Rotation Y* 0.00 *Cassette Rotation Z* 0.00 *Yscale*1.0 *Skew* 0.0 *Crossfire X* 0.00 *Crossfire Y* 0.00 *Crossfire XY* 0.00 Scale and B vs. Frame Chi^2 and R-Factor vs. Frame *Frame Number* *Scale* *B* *Chi^2* *R-factor* 1 10.91589-0.40 1.185 0.082 2 10.77627-0.38 1.234 0.082 3 10.53368-0.33 1.162 0.083 4 10.36566-0.27 1.162 0.079 5 10.32651-0.19 1.064 0.077 6 10.18485-0.10 1.030 0.075 7 10.00.001.076 0.076 8 10.051110.101.013 0.078 9 9.92706 0.210.995 0.076 10 9.83898 0.310.952 0.075 11 9.64515 0.420.907 0.077 12 9.61982 0.530.880 0.076 13 9.61376 0.630.885 0.069 14 9.44380 0.740.834 0.073 15 9.44114 0.850.765 0.068 16 9.29782 0.960.834 0.069 17 9.21481 1.080.750 0.067 18 9.12255 1.180.737 0.071 19 9.15504 1.280.798 0.073 20 9.06806 1.380.795 0.076 21 9.03864 1.470.740 0.074 22 8.96390 1.570.793 0.070 23 8.87753 1.660.759 0.073 24 8.81280 1.760.774 0.076 25 8.80572 1.840.777 0.073 26 8.64398 1.930.825 0.079 27 8.56313 2.020.796 0.074 28 8.58220 2.110.720 0.073 29 8.45429 2.190.763 0.074 30 8.43629 2.270.746 0.072 31 8.37449 2.340.821 0.081 32 8.32575 2.410.767 0.069 33 8.23116 2.470.848 0.080 34 8.25275 2.530.717 0.080 35 8.20440 2.570.734 0.073 36 8.12370 2.620.788 0.079 37 8.10550 2.660.777 0.077 38 8.04424 2.700.726 0.077 39 7.98665 2.740.760 0.077 40 7.92014 2.770.692 0.079 41 7.94125 2.810.711 0.079 42 7.89751 2.850.760 0.078 43 7.91178 2.900.650 0.079 44 7.84116 2.940.692 0.078 45 7.80008 2.990.737 0.077 46 7.78514 3.030.658 0.076 47 7.79019 3.070.656 0.077 48 7.77994 3.100.651 0.081 49 7.76673 3.140.567 0.077 50 7.71487 3.180.630 0.079 51 7.64531 3.220.631 0.082 52 7.65363 3.260.586 0.082 53 7.69821 3.310.604 0.080 54 7.67023 3.360.589 0.083 55 7.66701 3.420.550 0.082 56 7.67157 3.470.544 0.080 57 7.66821 3.520.609 0.087 58 7.67582 3.570.550 0.084 59 7.70985 3.630.572 0.086 60 7.69346 3.680.553 0.084 61 7.75456 3.730.550 0.083 62 7.73917 3.790.546 0.082 63 7.76342 3.840.553 0.081 64
Re: [ccp4bb] MR with Phaser
On Wed, Aug 1, 2012 at 11:27 AM, Uma Ratu rosiso2...@gmail.com wrote: The protein is in tetramer form. I define this by using the residue number (1332) which is 4 x monomer. After run, Phaser only gave 9 partial solutions, and no solution with all components. The resulted PDB contains only dimer form of the protein, not the tetramer. And the first TFZ score is around 2.5, which is too low for MR. I have the report file of data processing and the summary of Phaser attached. Could you please advice which part is wrong, why can I get the tetramer form of the protein? Your data are processed as P2, which is much less common (for proteins) than P21, and it looks like you haven't told Phaser to try P21 too. There are many other reasons why MR might not work, but I think it's very likely that the space group is wrong. -Nat
[ccp4bb] Enhancing Crystal Quality
Hi CCP4BBers, I have a protein crystallized with 0.2mM Mg acetate and PEG 8000 or 0.1mM cacodylate, pH 6.5, 0.2 Mg acetate, PEG 3350. Both of the conditions gave triangle pyramid like crystals. I brought the crystals to synchrotron using 30% PEG 400 as cryoprotactant, the resolution was only be able to reach 4A or worse. I have tried changing pH and concentrations of PEG, PEG types. I found out this crystal only grew between pH 6.5~7.5 and PEG types did not change the result of diffraction dramatically. I have also tried the seeding (break it down and reseed in the same condition. Maybe I did it wrong?). It gave me the similar results, not improving. Is there any simple way of improving it before jumping into reengineering the protein. Thanks, Lucas
Re: [ccp4bb] Enhancing Crystal Quality
Have you tried different cryoprotectants? Can make a huge difference. Also, have you shot an xtal at room temp - to see what the intrinsic diffraction limit is? Additive screens? If all else fails you may well need to explore a different expression construct. Tony. Sent from my iPhone On 1 Aug 2012, at 19:52, Yi-Liang Liu yiliang...@gmail.com wrote: Hi CCP4BBers, I have a protein crystallized with 0.2mM Mg acetate and PEG 8000 or 0.1mM cacodylate, pH 6.5, 0.2 Mg acetate, PEG 3350. Both of the conditions gave triangle pyramid like crystals. I brought the crystals to synchrotron using 30% PEG 400 as cryoprotactant, the resolution was only be able to reach 4A or worse. I have tried changing pH and concentrations of PEG, PEG types. I found out this crystal only grew between pH 6.5~7.5 and PEG types did not change the result of diffraction dramatically. I have also tried the seeding (break it down and reseed in the same condition. Maybe I did it wrong?). It gave me the similar results, not improving. Is there any simple way of improving it before jumping into reengineering the protein. Thanks, Lucas
Re: [ccp4bb] Process multiple data sets
I notice one thing with my data sets. The unit cells is slightly different from each other. For example, one has a/b/c @ 79/126/83. The other has a/b/c @ 84/127/90. Although they are collected from the same crystal. Is this the reason that I can't index both with same parameter in HKL? And subsequently, can't integrate and scala together. If so, is there a way that I can fix it? Thank you for your advice Uma On Wed, Aug 1, 2012 at 8:50 AM, Uma Ratu rosiso2...@gmail.com wrote: Dear All: I collected 5 data sets from one crystal and would like to process them together. Here is how I did: In HKL2000, load the all data sets. Index each set. When I try Intergrate, the program automatically go through the whole data sets there, and do not go through. I then process data sets by loading one at each time. Index, intergrate and scale all go through very smoothly. But when I put them together, the program just goes crazy. Thank you for advice Uma
Re: [ccp4bb] Process multiple data sets
Hi Uma, I've used HKL2000 to combine datasets from different crystals, so it's definitely possible to do so (although depending on the data volume it may be better to deal with scalepack directly). There are two things that you don't mention about your data - the (approximate) resolution, and the relationship of your datasets (different angular ranges, different exposure times, or something else?). Unit cell parameters are not always well determined for low resolution datasets, so this could be one cause of your cell variability. If not, check that the usual suspects (wavelength and detector to crystal distance) are correct, especially if they may have changed during data collection. This can be exacerbated if the datasets cover different angular ranges. From your initial description: In HKL2000, load the all data sets. Index each set. When I try Intergrate, the program automatically go through the whole data sets there, and do not go through. It's possible that there's an issue with discontinuities in the orientation matrices for the different datasets. In my hands, mosflm is easier for dealing with integration when you have to explicitly specify the orientation matrix for a dataset/wedge/batch (for example: integrate 20 images, switch the orientation matrix back to the beginning, and integrate another 20 images). Good luck, Pete Uma Ratu wrote: I notice one thing with my data sets. The unit cells is slightly different from each other. For example, one has a/b/c @ 79/126/83. The other has a/b/c @ 84/127/90. Although they are collected from the same crystal. Is this the reason that I can't index both with same parameter in HKL? And subsequently, can't integrate and scala together. If so, is there a way that I can fix it? Thank you for your advice Uma On Wed, Aug 1, 2012 at 8:50 AM, Uma Ratu rosiso2...@gmail.com wrote: Dear All: I collected 5 data sets from one crystal and would like to process them together. Here is how I did: I then process data sets by loading one at each time. Index, intergrate and scale all go through very smoothly. But when I put them together, the program just goes crazy. Thank you for advice Uma
Re: [ccp4bb] Enhancing Crystal Quality
Hi, Thanks for the kindly answers from everyone. I actually haven't tried different cryoprotectants. I might will give a try next time. I usually only use mother liquor+30% PEG400. It is noticeable that it has some patterns (cracks (?)) on the crystal. However, it didn't form icy rings or etc. The diffraction pattern looks funky too. It looks like it is twin and the diffraction spot has tails. Does this indicate the cryoprotectant problem? Lucas On Aug 1, 2012, at 2:11 PM, Antony Oliver wrote: Have you tried different cryoprotectants? Can make a huge difference. Also, have you shot an xtal at room temp - to see what the intrinsic diffraction limit is? Additive screens? If all else fails you may well need to explore a different expression construct. Tony. Sent from my iPhone On 1 Aug 2012, at 19:52, Yi-Liang Liu yiliang...@gmail.com wrote: Hi CCP4BBers, I have a protein crystallized with 0.2mM Mg acetate and PEG 8000 or 0.1mM cacodylate, pH 6.5, 0.2 Mg acetate, PEG 3350. Both of the conditions gave triangle pyramid like crystals. I brought the crystals to synchrotron using 30% PEG 400 as cryoprotactant, the resolution was only be able to reach 4A or worse. I have tried changing pH and concentrations of PEG, PEG types. I found out this crystal only grew between pH 6.5~7.5 and PEG types did not change the result of diffraction dramatically. I have also tried the seeding (break it down and reseed in the same condition. Maybe I did it wrong?). It gave me the similar results, not improving. Is there any simple way of improving it before jumping into reengineering the protein. Thanks, Lucas
