Hi Just my two ha’porth.
Having split spots at high resolution like that is a bit reminiscent of pseudo merohederal twinning, where the actual crystal system is metrically almost tetragonal but really lower symmetry (I’ve seen it a few times with orthorhombic where a~b) - so to begin with, I’d be interested in seeing the whole Pointless log, not just the précis. DISCLAIMER - I haven’t read this thread very closely so I might have missed something that someone else has brought up. Harry -- Dr Harry Powell > On 12 Nov 2019, at 07:56, herman.schreu...@sanofi.com wrote: > > Hi Shen, > > I agree with Eleanor that the split spots will cause worse statistics, but > should not be a reason for molrep to fail. > What I would do: > Molecular replacement with a resolution cut of 3 or 3.5 Å. > Process and run molecular replacement in P1. You may have some tricky > pseudo-symmetry. With 8 molecules, molrep may take somewhat longer, but I > think it is worth a try. > > Best, > Herman > > Von: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK > <mailto:CCP4BB@JISCMAIL.AC.UK>> Im Auftrag von Xu, Shenyuan > Gesendet: Montag, 11. November 2019 16:51 > An: CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK> > Betreff: [EXTERNAL] Re: [ccp4bb] molecular replacement_protein-glycan complex > > EXTERNAL : Real sender is owner-ccp...@jiscmail.ac.uk > <mailto:owner-ccp...@jiscmail.ac.uk> > > > Hello Eleanor, > > I used the solved one as the input model. The space group of the solved > crystals are: P1 21 1, and P 21. > > I will turn the anomalous processing off. > > Thanks, > > Shen > > On Mon, Nov 11, 2019 at 10:40 AM Eleanor Dodson <eleanor.dod...@york.ac.uk > <mailto:eleanor.dod...@york.ac.uk>> wrote: > Why dont you use one of your solved structures as a model? > What space group are the solved ones in? > The split spots are not good, but they are well separated and integration > programs are not bad at such spacings? And your R merges etc look OK. > Just a Q - why turn off the anomalous processing. You dont need to use it, > but at some point it may help you find Ss or Ca or whatever.. > Eleanor > > > On Mon, 11 Nov 2019 at 15:24, Xu, Shenyuan <x...@miamioh.edu > <mailto:x...@miamioh.edu>> wrote: > Dear All, > > Thanks for all of your nice suggestions! (Sorry I did not respond to > individual email to say Thanks.) I have used phaser to go through all > possible space groups in the same point group(choose "all possible in same > pointgroup" in the space group menu). I also try P43212 alone. The best > result I get from phaser is (Top LLG: 26.490, Top TFZ: 6.7, Spacegroup: P41 2 > 2), and refinement ends in the R-work: ~0.54, R-free:~0.57. > > I go back to see the diffraction data, and it seems that spots in the high > resolution bin are severely split (pictures are attached). I feel like the > fail of replacement is caused by this. Is this caused by twinning? > > I am looking forward suggestions! > > Thanks, > > Shenyuan Xu > > On Wed, Nov 6, 2019 at 2:58 PM Kay Diederichs <kay.diederi...@uni-konstanz.de > <mailto:kay.diederi...@uni-konstanz.de>> wrote: > Dear Shenyuan, > > it seems you assumed P41 21 2 is correct but it isn't - did you try P43 21 2 > in molecular replacement? > > The pointless log file mentions this alternative a few lines above the ones > you've quoted. > > If that does not work, try the other P4x 2y 2 space groups. All of this can > be done in a single phaser run (choose "all possible in same pointgroup" in > the space group menu). > > Why did you cut the data at 2.29A? They are very strong in the high > resolution shell! > > HTH, > Kay > > On Wed, 6 Nov 2019 14:29:13 -0500, Xu, Shenyuan <x...@miamioh.edu > <mailto:x...@miamioh.edu>> wrote: > > >Dear all, > > > >I am working on a protein-glycan complex trying to use molecular > >replacement.This protein is a VP8* domain from rotavirus, adopting a > >classical galectin-like fold. I already used MR to solve some other strains > >(apo-form and complex form). However, When I want to use the same model to > >solve this particular strain (sequence identity > 95%), MR seems to be > >failed with R=0.54, Free R = 0.57, and the model does not agree with the > >electron density map once viewed on coot. One difference of this > >protein-glycan complex between others is that the glycan is longer, which > >has six carbohydrate unit. > > > >I used imosflm trying to reindex and use pointless to check the space > >group, it usually ends up with the current choice: > >25_pointless.log > > > >WARNING! one or more zones have data systematically missing from the input > >file > >thus we cannot determine if reflections are truly systematically absent > > > >Best Solution: space group P 41 21 2 > >Reindex operator: [h,k,l] > >Laue group probability: 1.000 > >Systematic absence probability: 0.995 > >Total probability: 0.995 > >Space group confidence: 0.993 > >Laue group confidence 1.000 > > > >WARNING: You will have to resolve the enantiomorphic ambiguity later > > > >Unit cell: 106.78 106.78 138.59 90.00 90.00 90.00 > > > >84.58 to 2.43 - Resolution range used for Laue group search > > > >84.58 to 2.29 - Resolution range in file, used for systematic absence check > > > >Number of batches in file: 1 > > > >The data do not appear to be twinned, from the L-test > >I also use Zanuda to validate the space group, it ends with the same choice: > > > >Step 3. > > Refinement of the best model. > > Candidate symmetry elements are added one by one. > > > > current time: Nov 06 16:17 GMT > > expected end of job: Nov 06 17:06 GMT > > > > ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ > > | >> 3 | C 1 2 1 | 0.3736 | 0.5334 | 0.5044 | 0.5586 | > > --------------------------------------------------------------------- > > | 1 | P 1 | 0.4058 | 0.5318 | 0.5020 | 0.5624 | > > | 5 | P 1 21 1 | 0.4928 | -- | 0.4973 | 0.5701 | > > | 9 | P 21 21 21 | 0.5470 | -- | 0.5011 | 0.5736 | > > | 10 | P 41 21 2 | 0.5749 | -- | 0.5018 | 0.5778 | > > --------------------------------------------------------------------- > > | << 10 | P 41 21 2 | 0.5749 | -- | 0.5018 | 0.5778 | > > --------------------------------------------------------------------- > > > > R-factor in the original subgroup is (almost) the best. > > The original spacegroup assignment seems to be correct. > > > > The statistics of the dataset seems good: > >Summary data for Project: Test Crystal: A Dataset: 1 > > > > Overall InnerShell OuterShell > >Low resolution limit 84.58 84.58 2.41 > >High resolution limit 2.29 7.23 2.29 > > > >Rmerge (within I+/I-) 0.086 0.036 0.564 > >Rmerge (all I+ and I-) 0.088 0.036 0.581 > >Rmeas (within I+/I-) 0.093 0.039 0.610 > >Rmeas (all I+ & I-) 0.092 0.038 0.604 > >Rpim (within I+/I-) 0.036 0.015 0.230 > >Rpim (all I+ & I-) 0.026 0.011 0.165 > >Rmerge in top intensity bin 0.045 - - > >Total number of observations 466972 14511 70743 > >Total number unique 37007 1338 5297 > >Mean((I)/sd(I)) 20.4 44.5 5.0 > >Mn(I) half-set correlation CC(1/2) 0.999 0.999 0.947 > >Completeness 100.0 100.0 100.0 > >Multiplicity 12.6 10.8 13.4 > >Mean(Chi^2) 0.97 0.85 0.86 > > > >Anomalous completeness 100.0 100.0 100.0 > >Anomalous multiplicity 6.5 6.6 6.7 > >DelAnom correlation between half-sets -0.172 -0.180 -0.083 > >Mid-Slope of Anom Normal Probability 0.857 - - > > > >Anomalous flag switched OFF in input, anomalous signal is weak > > > >Estimates of resolution limits: overall > > from half-dataset correlation CC(1/2) > 0.30: limit = 2.29A == > >maximum resolution > > from Mn(I/sd) > 2.00: limit = 2.29A == > >maximum resolution > > from Mn(I/sd) > 2.00: limit = 2.29A == > >maximum resolution > > > >Estimates of resolution limits in reciprocal lattice directions: > > Along h k plane > > from half-dataset correlation CC(1/2) > 0.30: limit = 2.29A == > >maximum resolution > > from Mn(I/sd) > 2.00: limit = 2.29A == > >maximum resolution > > Along l axis > > from half-dataset correlation CC(1/2) > 0.30: limit = 2.29A == > >maximum resolution > > from Mn(I/sd) > 2.00: limit = 2.29A == > >maximum resolution > > > >Anisotropic deltaB (i.e. range of principal components), A^2: 6.03 > > > >Average unit cell: 106.78 106.78 138.59 90.00 90.00 90.00 > >Space group: P 41 21 2 > >Average mosaicity: 0.27 > > > >Minimum and maximum SD correction factors: Fulls 1.00 1.35 Partials > >0.00 0.00 > > > >Any suggestions are welcomed! 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