Hi Guto,
I always feel some people are too "greedy" with the resolution they want to achieve. I mostly find that extremely high density is a pain to work with as it's usually accompanied by many dual, triple conformers, a lot of noise in the solvent phase that is often difficult to interpret, etc... Often you spend more time on a high resolution structure that clearly shows your protein, as opposed to a low resolution structure where it's difficult to interpret parts of the map. Also, in most cases you REALLY don't need a 1 Ang map to clearly show the overall structure of your protein, ligands, first shell of solvent molecules on the surface of your protein, etc... Your completeness is 90% in your high resolution shell, which is fine, but have you checked you can clearly see most reflections for h, k and l? Maybe you're missing many reflections for one of them. I would at least try cutting your data back to 1.1 or 1.2 Ang, as it might dramatically improve your R factors and still show everything you want to show. Also, did you try TLS refinement? Best regards, Tony ------------------------------------------------------ Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE e-mail: antonio.ar...@path.ox.ac.uk<mailto:antonio.ar...@path.ox.ac.uk> Tel: 00 +44 1865 285655 Links to my public profiles: ResearchGate<https://www.researchgate.net/profile/Antonio_Ariza> LinkedIn<https://www.linkedin.com/in/antonioariza1> GoogleScholar<https://scholar.google.co.uk/citations?user=9pAIKV0AAAAJ&hl=en> Twitter<https://twitter.com/DrAntonioAriza?lang=en> Check out my latest paper!!! Structural insights into the function of ZRANB3 in replication stress response<http://www.nature.com/articles/ncomms15847> ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Guto Rhys <guto.r...@bristol.ac.uk> Sent: 09 October 2018 18:12 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Issue with high Rfree (0.25) for a high-resolution dataset (1.05 Ang) Hi all, I have a 1.05 Angstrom dataset that I was able to phase but the refined model only has an Rfree of approximately 0.25. The dataset includes 1800 images and, as the crystal did not suffer significantly from radiation damage, comprises all 360 deg. Auto-processing pipelines at diamond light source all suggest I222. I have also indexed the data in iMOSFLM, which has the highest-symmetry Laue group that is least penalised of I422. Subsequent scaling and merging in AIMLESS strongly indicates that I222 is the likely space group (see below). I have ran the refined model through ZANUDA, which has similar R values to lower symmetry space groups (see below). The output from Phenix Xtriage does not find any specific crystal pathologies and if twinning is present it is very low (2 to 4%, see below). The difference map suggests that the model accounts for nearly all the density. Any ideas or direction would be greatly appreciated. Best, Guto AIMLESS Summary Overall InnerShell OuterShell Low resolution limit 27.75 27.75 1.07 High resolution limit 1.05 5.75 1.05 Rmerge (within I+/I-) 0.050 0.078 0.466 Rmerge (all I+ and I-) 0.051 0.080 0.536 Rmeas (within I+/I-) 0.055 0.086 0.591 Rmeas (all I+ & I-) 0.054 0.085 0.613 Rpim (within I+/I-) 0.023 0.034 0.359 Rpim (all I+ & I-) 0.017 0.028 0.288 Rmerge in top intensity bin 0.049 - - Total number of observations 107950 779 1972 Total number unique 11315 88 486 Mean((I)/sd(I)) 19.7 46.2 1.8 Mn(I) half-set correlation CC(1/2) 0.998 0.994 0.796 Completeness 99.1 99.2 90.4 Multiplicity 9.5 8.9 4.1 Anomalous completeness 98.1 100.0 79.1 Anomalous multiplicity 5.0 6.4 2.2 DelAnom correlation between half-sets -0.067 0.286 0.097 Mid-Slope of Anom Normal Probability 0.789 - - Estimate of maximum resolution for significant anomalous signal = 1.14A, from CCanom > 0.15 Estimates of resolution limits: overall from half-dataset correlation CC(1/2) > 0.30: limit = 1.05A == maximum resolution from Mn(I/sd) > 1.50: limit = 1.05A == maximum resolution from Mn(I/sd) > 2.00: limit = 1.07A Estimates of resolution limits in reciprocal lattice directions: Along h axis from half-dataset correlation CC(1/2) > 0.30: limit = 1.06A from Mn(I/sd) > 1.50: limit = 1.09A Along k axis from half-dataset correlation CC(1/2) > 0.30: limit = 1.11A from Mn(I/sd) > 1.50: limit = 1.13A Along l axis from half-dataset correlation CC(1/2) > 0.30: limit = 1.05A == maximum resolution from Mn(I/sd) > 1.50: limit = 1.05A Anisotropic deltaB (i.e. range of principal components), A^2: 6.40 Average unit cell: 29.12 29.26 55.50 90.00 90.00 90.00 Space group: I 2 2 2 Average mosaicity: 0.36 AIMLESS Laue Group prediction Laue Group Lklhd NetZc Zc+ Zc- CC CC- Rmeas R- Delta ReindexOperator = 1 I m m m *** 0.987 6.25 9.19 2.94 0.92 0.29 0.07 0.49 0.0 [h,k,l] 2 I 1 2/m 1 0.004 4.36 9.32 4.96 0.93 0.50 0.07 0.30 0.0 [-h,-k,l] 3 I 1 2/m 1 0.004 4.14 9.24 5.10 0.92 0.51 0.07 0.30 0.0 [k,-h,l] 4 I 1 2/m 1 0.004 4.05 9.23 5.18 0.92 0.52 0.06 0.31 0.0 [h,-l,k] 5 I 4/m m m 0.000 6.35 6.35 0.00 0.64 0.00 0.22 0.00 0.3 [h,k,l] 6 I 4/m 0.000 0.90 6.83 5.93 0.68 0.59 0.17 0.26 0.3 [h,k,l] 7 P -1 0.000 3.69 9.36 5.67 0.94 0.57 0.06 0.26 0.0 [-h,k,1/2h-1/2k-1/2l] 8 I 1 2/m 1 0.000 0.08 6.40 6.33 0.64 0.63 0.23 0.22 0.3 [-1/2h+1/2k-1/2l,-h-k,-1/2h+1/2k+1/2l] 9 F m m m 0.000 -0.43 6.17 6.60 0.62 0.66 0.24 0.20 0.3 [h+k,-h+k,l] 10 I 1 2/m 1 0.000 0.75 6.89 6.14 0.69 0.61 0.22 0.22 0.3 [-1/2h-1/2k-1/2l,h-k,-1/2h-1/2k+1/2l] Xtriage Summary Twinning and intensity statistics summary (acentric data): Statistics independent of twin laws <I^2>/<I>^2 : 2.126 (untwinned: 2.0, perfect twin: 1.5) <F>^2/<F^2> : 0.774 (untwinned: 0.785, perfect twin: 0.885) <|E^2-1|> : 0.761 (untwinned: 0.736, perfect twin: 0.541) <|L|>, <L^2>: 0.490, 0.323 Multivariate Z score L-test: 1.303 The multivariate Z score is a quality measure of the given spread in intensities. Good to reasonable data are expected to have a Z score lower than 3.5. Large values can indicate twinning, but small values do not necessarily exclude it. Statistics depending on twin laws ----------------------------------------------------------------------------------- | Operator | type | R_abs obs. | R_abs calc. | Britton alpha | H alpha | ML alpha | ----------------------------------------------------------------------------------- | k,h,-l | PM | 0.469 | 0.478 | 0.016 | 0.041 | 0.022 | ----------------------------------------------------------------------------------- Patterson analyses - Largest peak height : 13.667 (corresponding p value : 0.04780) The largest off-origin peak in the Patterson function is 13.67% of the height of the origin peak. No significant pseudotranslation is detected. The results of the L-test indicate that the intensity statistics behave as expected. No twinning is suspected. Zanuda output Step 2. Refinements in subgroups. There are 5 subgroups to test. current time: Jan 08 16:20 GMT expected end of job: Jan 08 16:28 GMT ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ | >> 5 | I 2 2 2 | 0.0005 | -- | -- | -- | --------------------------------------------------------------------- | 1 | P 1 | 0.0731 | 0.3242 | 0.2842 | 0.2768 | | 2 | C 1 2 1 | 0.0755 | 0.3796 | 0.2899 | 0.2799 | | 3 | C 1 2 1 | 0.0727 | 0.3385 | 0.2909 | 0.2806 | | 4 | C 1 2 1 | 0.0729 | 0.3398 | 0.2900 | 0.2813 | | 5 | I 2 2 2 | 0.0707 | 0.3582 | 0.2976 | 0.2802 | --------------------------------------------------------------------- | << 1 | P 1 | 0.0731 | 0.3242 | 0.2842 | 0.2768 | ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ Step 3. Refinement of the best model. Candidate symmetry elements are added one by one. current time: Jan 08 16:25 GMT expected end of job: Jan 08 16:28 GMT ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ | >> 1 | P 1 | 0.0731 | 0.3242 | 0.2842 | 0.2768 | --------------------------------------------------------------------- | 1 | P 1 | 0.0730 | 0.3243 | 0.2842 | 0.2768 | | 4 | C 1 2 1 | 0.0751 | -- | 0.2896 | 0.2826 | | 5 | I 2 2 2 | 0.0737 | -- | 0.2970 | 0.2801 | --------------------------------------------------------------------- | << 5 | I 2 2 2 | 0.0737 | -- | 0.2970 | 0.2801 | --------------------------------------------------------------------- R-factor in the original subgroup is (almost) the best. The original spacegroup assignment seems to be correct. ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
