Hello, at that resolution, the refinement of anisotropic atomic B-factors is absolutely required, as is the modelisation of alternate conformations for surface residues. Optimize also the weight of different restraints (for exemple on B-factors) in order to get the lowest Rfree. Best Wim
De: "Robbie Joosten" <[email protected]> À: [email protected] Envoyé: Mardi 9 Octobre 2018 20:02:17 Objet: Re: [ccp4bb] Issue with high Rfree (0.25) for a high-resolution dataset (1.05 Ang) Hi Hugo, Perhaps you should play with your refinement strategy. Use a decent number of cycles and a sensible restraint weight (something that gives you rmsZ < 1.0 and good R-factors). Anisotropic B-factors are probably needed and make your model as complete as your maps allow. You could try pdb-redo to see if this can help you on your way. Cheers, Robbie On 9 Oct 2018 19:12, Guto Rhys <[email protected]> wrote: 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 | 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
