Dear Phenix Users, I'm currently using version: Version: 1.4 Release tag: 3 cctbx tag: 2008_12_07_1353 Platform: mac-intel-osx osx-10.5.6 I am working on characterising substrate/inhibitor interactions of a protein, the structure has been solved many times so its unit cell dimensions, contents (1 molecule per ASU) and space group (P3121) are well characterized. However, I have recently encountered several crystals that appear exhibit merohedral twinning, and am having trouble converging the R-factors to reasonable levels. The details of one such data set is described below: Diffraction to ~1.9A was recorded using a high and low resolution pass of 180º each. When processed with HKL2000 separately or together, Scalepack wanted to reject huge amounts of my data (~20%). I was forced to increase the error scale factor to 2 and adjust my error model to the very high R-factors of the low-resolution shells. The summary table from my final round of scalepack is: Shell Lower Upper Average Average Norm. Linear Square limit Angstrom I error stat. Chi**2 R-fac R-fac 50.00 4.09 7648.3 1498.7 182.7 1.030 0.376 0.424 4.09 3.25 3885.1 568.9 64.1 1.410 0.416 0.480 3.25 2.84 1609.7 225.0 26.1 1.227 0.451 0.517 2.84 2.58 807.9 97.1 25.3 1.958 0.439 0.561 2.58 2.39 496.6 59.7 22.0 1.060 0.257 0.268 2.39 2.25 335.5 53.0 18.1 0.717 0.275 0.238 2.25 2.14 221.7 19.0 17.4 2.389 0.354 0.298 2.14 2.05 143.1 19.5 17.5 1.537 0.459 0.351 2.05 1.97 87.0 20.2 17.1 1.079 0.663 0.480 1.97 1.90 54.6 20.3 16.7 1.073 0.990 0.696 All reflections 1588.3 269.2 41.9 1.350 0.400 0.444 In addition, many systematic absences appear to be present: Intensities of systematic absences h k l Intensity Sigma I/Sigma 0 0 5 177.7 56.7 3.1 0 0 7 255.5 70.6 3.6 0 0 8 264.8 73.2 3.6 0 0 10 595.0 164.2 3.6 0 0 11 2343.5 1289.9 1.8 0 0 13 321.3 125.4 2.6 0 0 14 66.5 21.8 3.1 0 0 16 238.0 76.3 3.1 0 0 17 394.3 109.1 3.6 0 0 19 13.0 7.1 1.8 0 0 20 650.1 179.5 3.6 0 0 22 199.6 55.6 3.6 0 0 23 953.9 372.0 2.6 0 0 25 192.2 62.2 3.1 0 0 26 1038.7 405.0 2.6 0 0 28 96.3 28.9 3.3 0 0 29 2303.0 897.0 2.6 0 0 31 12.0 11.5 1.0 0 0 32 1622.4 516.5 3.1 0 0 34 11.5 13.8 0.8 0 0 35 22.8 14.8 1.5 0 0 37 27.9 18.0 1.5 0 0 38 154.3 45.6 3.4 0 0 40 55.8 27.8 2.0 0 0 41 35.5 23.7 1.5 0 0 43 -25.3 34.4 -0.7 0 0 44 67.7 32.1 2.1 The data has an overall completeness of 99.7%. When I run the data trough xtriage, it tells me: Statistics depending on twin laws ----------------------------------------------------------------- | Operator | type | R obs. | Britton alpha | H alpha | ML alpha | ----------------------------------------------------------------- | -h,-k,l | M | 0.307 | 0.301 | 0.256 | 0.022 | ----------------------------------------------------------------- Patterson analyses - Largest peak height : 5.288 (corresponding p value : 0.86552) The largest off-origin peak in the Patterson function is 5.29% of the height of the origin peak. No significant pseudotranslation is detected. The results of the L-test indicate that the intensity statistics are significantly different than is expected from good to reasonable, untwinned data. As there are twin laws possible given the crystal symmetry, twinning could be the reason for the departure of the intensity statistics from normality. It might be worthwhile carrying out refinement with a twin specific target function. I also estimated the twin fraction with the following server: http://nihserver.mbi.ucla.edu/Twinning/ And found it to about 20% (using data from 6-3A). After phasing the data with rigid body refinement in Refmac with intensity based twin refinement (brings both R-factors to ~36%), I have tried doing a twin refinement with Phenix: %phenix.refine model.pdb data.mtz twin_law="-h,-k,l" strategy=rigid_body+individual_sites+group_adp The R_factors consistently diverge to the following values: Final R-work = 0.3392, R-free = 0.4360 Simulated annealing/TLS/ordered solvent/messing with wxc/wxu weights does not seem to improve the situation. I have also tried this with and without prior detwinning with the CCP4 program "Detwin", with similar results. Reindexing to -h,-k,l gives similar R-factors after rigid body refinement but after refinement with Phenix, things get even worse: Final R-work = 0.3919, R-free = 0.5059 I was surprised to see that the fit to the map using both indexing conventions are comparable even though this should effectively reverse the hand of the structure. I assume this is because twin refinement is basically doing the job of "partial reindexing" on those parts of the lattice which are twinned? I have been working on this for about a week now and can't for the life of me find a way forward. If anyone can offer any suggestions It would greatly be appreciated. Im slowly beginning to lose my sanity.... BTW I don't think my problems stem from misindexing, although the beam was wondering all over the place during this block of data collection, I have previously solved another structure at the same d and xbeam ybeam values without any trouble. Sorry about the v.long post. Thought it would be better to give too much information rather than not enough =) Many thanks for all your suggestions in advance and for everything I have learned from this community in the past. Cheers, Joe PhD student. _________________________________________________________________ Get personal with Windows. Download a free gift for your PC. http://experience.windows.com
Hi Joe,
Some suggestions
1. Update your phenix version please. This is rather outdated.
2.0. If it doesn't refine in the normal cell / sg, toss this
preconceived notion away and:
2.1. Get your data in P1 (phenix.reflection_file_converter
data.sca--sca=p1.sca --expand_to_p1)
2.2. Do MR in P1
2.3 Assign free flags with phenix.refine (*IMPORTANT*)
2.4 Refine (a bit)
2.5 Run xtriage on the P1 data and provide the model as well (check
the phantastic GUI)
2.6 Check your RvsR stats to get an idea about the 'true' symmetry of the system
2.7 Read the RvsR paper quoted in the log file
2.8 Find out what this all means.
If confused about results, let me know.
HTH
Peter
2010/3/25 Joseph Brock
Dear Phenix Users,
I'm currently using version: Version: 1.4 Release tag: 3 cctbx tag: 2008_12_07_1353 Platform: mac-intel-osx osx-10.5.6
I am working on characterising substrate/inhibitor interactions of a protein, the structure has been solved many times so its unit cell dimensions, contents (1 molecule per ASU) and space group (P3121) are well characterized.
However, I have recently encountered several crystals that appear exhibit merohedral twinning, and am having trouble converging the R-factors to reasonable levels. The details of one such data set is described below:
Diffraction to ~1.9A was recorded using a high and low resolution pass of 180º each. When processed with HKL2000 separately or together, Scalepack wanted to reject huge amounts of my data (~20%). I was forced to increase the error scale factor to 2 and adjust my error model to the very high R-factors of the low-resolution shells. The summary table from my final round of scalepack is: Shell Lower Upper Average Average Norm. Linear Square limit Angstrom I error stat. Chi**2 R-fac R-fac 50.00 4.09 7648.3 1498.7 182.7 1.030 0.376 0.424 4.09 3.25 3885.1 568.9 64.1 1.410 0.416 0.480 3.25 2.84 1609.7 225.0 26.1 1.227 0.451 0.517 2.84 2.58 807.9 97.1 25.3 1.958 0.439 0.561 2.58 2.39 496.6 59.7 22.0 1.060 0.257 0.268 2.39 2.25 335.5 53.0 18.1 0.717 0.275 0.238 2.25 2.14 221.7 19.0 17.4 2.389 0.354 0.298 2.14 2.05 143.1 19.5 17.5 1.537 0.459 0.351 2.05 1.97 87.0 20.2 17.1 1.079 0.663 0.480 1.97 1.90 54.6 20.3 16.7 1.073 0.990 0.696 All reflections 1588.3 269.2 41.9 1.350 0.400 0.444
In addition, many systematic absences appear to be present:
Intensities of systematic absences h k l Intensity Sigma I/Sigma
0 0 5 177.7 56.7 3.1 0 0 7 255.5 70.6 3.6 0 0 8 264.8 73.2 3.6 0 0 10 595.0 164.2 3.6 0 0 11 2343.5 1289.9 1.8 0 0 13 321.3 125.4 2.6 0 0 14 66.5 21.8 3.1 0 0 16 238.0 76.3 3.1 0 0 17 394.3 109.1 3.6 0 0 19 13.0 7.1 1.8 0 0 20 650.1 179.5 3.6 0 0 22 199.6 55.6 3.6 0 0 23 953.9 372.0 2.6 0 0 25 192.2 62.2 3.1 0 0 26 1038.7 405.0 2.6 0 0 28 96.3 28.9 3.3 0 0 29 2303.0 897.0 2.6 0 0 31 12.0 11.5 1.0 0 0 32 1622.4 516.5 3.1 0 0 34 11.5 13.8 0.8 0 0 35 22.8 14.8 1.5 0 0 37 27.9 18.0 1.5 0 0 38 154.3 45.6 3.4 0 0 40 55.8 27.8 2.0 0 0 41 35.5 23.7 1.5 0 0 43 -25.3 34.4 -0.7 0 0 44 67.7 32.1 2.1
The data has an overall completeness of 99.7%.
When I run the data trough xtriage, it tells me:
Statistics depending on twin laws ----------------------------------------------------------------- | Operator | type | R obs. | Britton alpha | H alpha | ML alpha | ----------------------------------------------------------------- | -h,-k,l | M | 0.307 | 0.301 | 0.256 | 0.022 | -----------------------------------------------------------------
Patterson analyses - Largest peak height : 5.288 (corresponding p value : 0.86552)
The largest off-origin peak in the Patterson function is 5.29% of the height of the origin peak. No significant pseudotranslation is detected.
The results of the L-test indicate that the intensity statistics are significantly different than is expected from good to reasonable, untwinned data. As there are twin laws possible given the crystal symmetry, twinning could be the reason for the departure of the intensity statistics from normality. It might be worthwhile carrying out refinement with a twin specific target function.
I also estimated the twin fraction with the following server:
http://nihserver.mbi.ucla.edu/Twinning/
And found it to about 20% (using data from 6-3A).
After phasing the data with rigid body refinement in Refmac with intensity based twin refinement (brings both R-factors to ~36%), I have tried doing a twin refinement with Phenix:
%phenix.refine model.pdb data.mtz twin_law="-h,-k,l" strategy=rigid_body+individual_sites+group_adp
The R_factors consistently diverge to the following values:
Final R-work = 0.3392, R-free = 0.4360
Simulated annealing/TLS/ordered solvent/messing with wxc/wxu weights does not seem to improve the situation. I have also tried this with and without prior detwinning with the CCP4 program "Detwin", with similar results.
Reindexing to -h,-k,l gives similar R-factors after rigid body refinement but after refinement with Phenix, things get even worse: Final R-work = 0.3919, R-free = 0.5059
I was surprised to see that the fit to the map using both indexing conventions are comparable even though this should effectively reverse the hand of the structure. I assume this is because twin refinement is basically doing the job of "partial reindexing" on those parts of the lattice which are twinned?
I have been working on this for about a week now and can't for the life of me find a way forward. If anyone can offer any suggestions It would greatly be appreciated. Im slowly beginning to lose my sanity....
BTW I don't think my problems stem from misindexing, although the beam was wondering all over the place during this block of data collection, I have previously solved another structure at the same d and xbeam ybeam values without any trouble.
Sorry about the v.long post. Thought it would be better to give too much information rather than not enough =)
Many thanks for all your suggestions in advance and for everything I have learned from this community in the past. Cheers,
Joe PhD student.
________________________________ Download a free gift for your PC. Get personal with Windows. _______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb
-- ----------------------------------------------------------------- P.H. Zwart Beamline Scientist Berkeley Center for Structural Biology Lawrence Berkeley National Laboratories 1 Cyclotron Road, Berkeley, CA-94703, USA Cell: 510 289 9246 BCSB: http://bcsb.als.lbl.gov PHENIX: http://www.phenix-online.org CCTBX: http://cctbx.sf.net -----------------------------------------------------------------
On Mar 25, 2010, at 10:13 PM, Joseph Brock wrote:
Shell Lower Upper Average Average Norm. Linear Square limit Angstrom I error stat. Chi**2 R-fac R-fac 50.00 4.09 7648.3 1498.7 182.7 1.030 0.376 0.424 All reflections 1588.3 269.2 41.9 1.350 0.400 0.444
Aside from what Peter said: aren't these R-sym values are far too high for the symmetry (or indexing) to be correct? Or is this allowed when twinning is present? If Scalepack is throwing away that much data, this is also telling you that something else is wrong. I'd start back at the indexing step and re-process in P1, then follow the rest of Peter's suggestions starting from step 2.2 (MR). Also, I'd recomment trying labelit.index (included in any Phenix distribution after 1.5) on the raw images, because it may notice something that the conventional indexing programs miss. As a general rule, you can't always assume that the symmetry will always be the same from crystal to crystal - identical proteins may form completely different crystal lattices depending on crystallization condition or ligand binding or freak chance. -Nat -------------------- Nathaniel Echols Lawrence Berkeley Lab 510-486-5136 [email protected]
didn't look properly.
Nat is correct: the data processing is seriously flawed.
Reduce in P1 and see how that look.s
P
2010/3/25 Nathaniel Echols
On Mar 25, 2010, at 10:13 PM, Joseph Brock wrote:
Shell Lower Upper Average Average Norm. Linear Square limit Angstrom I error stat. Chi**2 R-fac R-fac 50.00 4.09 7648.3 1498.7 182.7 1.030 0.376 0.424 All reflections 1588.3 269.2 41.9 1.350 0.400 0.444
Aside from what Peter said: aren't these R-sym values are far too high for the symmetry (or indexing) to be correct? Or is this allowed when twinning is present? If Scalepack is throwing away that much data, this is also telling you that something else is wrong. I'd start back at the indexing step and re-process in P1, then follow the rest of Peter's suggestions starting from step 2.2 (MR). Also, I'd recomment trying labelit.index (included in any Phenix distribution after 1.5) on the raw images, because it may notice something that the conventional indexing programs miss.
As a general rule, you can't always assume that the symmetry will always be the same from crystal to crystal - identical proteins may form completely different crystal lattices depending on crystallization condition or ligand binding or freak chance.
-Nat
-------------------- Nathaniel Echols Lawrence Berkeley Lab 510-486-5136 [email protected]
_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb
-- ----------------------------------------------------------------- P.H. Zwart Beamline Scientist Berkeley Center for Structural Biology Lawrence Berkeley National Laboratories 1 Cyclotron Road, Berkeley, CA-94703, USA Cell: 510 289 9246 BCSB: http://bcsb.als.lbl.gov PHENIX: http://www.phenix-online.org CCTBX: http://cctbx.sf.net -----------------------------------------------------------------
Here's another possibility. P31 2 1 is a polar space group, which means that flipping the crystal end-for-end will result in the same lattice but different intensities. It's possible that your first sweep was indexed one way and the second sweep the other. This would result in good merging stats for each sweep but the whole thing falling apart when you merge the two together. This seems more likely to me since a twinned crystal should show good merging stats but lousy refinement stats (if the twinning is ignored). Dale Tronrud Joseph Brock wrote:
Dear Phenix Users,
I'm currently using version: Version: 1.4 Release tag: 3 cctbx tag: 2008_12_07_1353 Platform: mac-intel-osx osx-10.5.6
I am working on characterising substrate/inhibitor interactions of a protein, the structure has been solved many times so its unit cell dimensions, contents (1 molecule per ASU) and space group (P3121) are well characterized.
However, I have recently encountered several crystals that appear exhibit merohedral twinning, and am having trouble converging the R-factors to reasonable levels. The details of one such data set is described below:
Diffraction to ~1.9A was recorded using a high and low resolution pass of 180º each. When processed with HKL2000 separately or together, Scalepack wanted to reject huge amounts of my data (~20%). I was forced to increase the error scale factor to 2 and adjust my error model to the very high R-factors of the low-resolution shells. The summary table from my final round of scalepack is: Shell Lower Upper Average Average Norm. Linear Square limit Angstrom I error stat. Chi**2 R-fac R-fac 50.00 4.09 7648.3 1498.7 182.7 1.030 0.376 0.424 4.09 3.25 3885.1 568.9 64.1 1.410 0.416 0.480 3.25 2.84 1609.7 225.0 26.1 1.227 0.451 0.517 2.84 2.58 807.9 97.1 25.3 1.958 0.439 0.561 2.58 2.39 496.6 59.7 22.0 1.060 0.257 0.268 2.39 2.25 335.5 53.0 18.1 0.717 0.275 0.238 2.25 2.14 221.7 19.0 17.4 2.389 0.354 0.298 2.14 2.05 143.1 19.5 17.5 1.537 0.459 0.351 2.05 1.97 87.0 20.2 17.1 1.079 0.663 0.480 1.97 1.90 54.6 20.3 16.7 1.073 0.990 0.696 All reflections 1588.3 269.2 41.9 1.350 0.400 0.444
In addition, many systematic absences appear to be present:
Intensities of systematic absences h k l Intensity Sigma I/Sigma
0 0 5 177.7 56.7 3.1 0 0 7 255.5 70.6 3.6 0 0 8 264.8 73.2 3.6 0 0 10 595.0 164.2 3.6 0 0 11 2343.5 1289.9 1.8 0 0 13 321.3 125.4 2.6 0 0 14 66.5 21.8 3.1 0 0 16 238.0 76.3 3.1 0 0 17 394.3 109.1 3.6 0 0 19 13.0 7.1 1.8 0 0 20 650.1 179.5 3.6 0 0 22 199.6 55.6 3.6 0 0 23 953.9 372.0 2.6 0 0 25 192.2 62.2 3.1 0 0 26 1038.7 405.0 2.6 0 0 28 96.3 28.9 3.3 0 0 29 2303.0 897.0 2.6 0 0 31 12.0 11.5 1.0 0 0 32 1622.4 516.5 3.1 0 0 34 11.5 13.8 0.8 0 0 35 22.8 14.8 1.5 0 0 37 27.9 18.0 1.5 0 0 38 154.3 45.6 3.4 0 0 40 55.8 27.8 2.0 0 0 41 35.5 23.7 1.5 0 0 43 -25.3 34.4 -0.7 0 0 44 67.7 32.1 2.1
The data has an overall completeness of 99.7%.
When I run the data trough xtriage, it tells me:
Statistics depending on twin laws ----------------------------------------------------------------- | Operator | type | R obs. | Britton alpha | H alpha | ML alpha | ----------------------------------------------------------------- | -h,-k,l | M | 0.307 | 0.301 | 0.256 | 0.022 | -----------------------------------------------------------------
Patterson analyses - Largest peak height : 5.288 (corresponding p value : 0.86552)
The largest off-origin peak in the Patterson function is 5.29% of the height of the origin peak. No significant pseudotranslation is detected.
The results of the L-test indicate that the intensity statistics are significantly different than is expected from good to reasonable, untwinned data. As there are twin laws possible given the crystal symmetry, twinning could be the reason for the departure of the intensity statistics from normality. It might be worthwhile carrying out refinement with a twin specific target function.
I also estimated the twin fraction with the following server:
http://nihserver.mbi.ucla.edu/Twinning/
And found it to about 20% (using data from 6-3A).
After phasing the data with rigid body refinement in Refmac with intensity based twin refinement (brings both R-factors to ~36%), I have tried doing a twin refinement with Phenix:
%phenix.refine model.pdb data.mtz twin_law="-h,-k,l" strategy=rigid_body+individual_sites+group_adp
The R_factors consistently diverge to the following values:
Final R-work = 0.3392, R-free = 0.4360
Simulated annealing/TLS/ordered solvent/messing with wxc/wxu weights does not seem to improve the situation. I have also tried this with and without prior detwinning with the CCP4 program "Detwin", with similar results.
Reindexing to -h,-k,l gives similar R-factors after rigid body refinement but after refinement with Phenix, things get even worse: Final R-work = 0.3919, R-free = 0.5059
I was surprised to see that the fit to the map using both indexing conventions are comparable even though this should effectively reverse the hand of the structure. I assume this is because twin refinement is basically doing the job of "partial reindexing" on those parts of the lattice which are twinned?
I have been working on this for about a week now and can't for the life of me find a way forward. If anyone can offer any suggestions It would greatly be appreciated. Im slowly beginning to lose my sanity....
BTW I don't think my problems stem from misindexing, although the beam was wondering all over the place during this block of data collection, I have previously solved another structure at the same d and xbeam ybeam values without any trouble.
Sorry about the v.long post. Thought it would be better to give too much information rather than not enough =)
Many thanks for all your suggestions in advance and for everything I have learned from this community in the past. Cheers,
Joe PhD student.
------------------------------------------------------------------------ Download a free gift for your PC. Get personal with Windows. http://experience.windows.com
------------------------------------------------------------------------
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participants (4)
-
Dale Tronrud -
Joseph Brock -
Nathaniel Echols -
Peter Zwart