Oh dear! On Thu, 14 Nov 2019 at 19:47, Xu, Shenyuan <x...@miamioh.edu> wrote:
> Dear everyone, > > Thanks for all of your suggestions, and especially Kay. > > It turns out I successfully crystallized a contaminant, glutathione > S-transferase, which was used as a tag > to purify my protein. > > Thanks, > > Shen > > > > > On Tue, Nov 12, 2019 at 4:16 AM Harry Powell < > 0000193323b1e616-dmarc-requ...@jiscmail.ac.uk> wrote: > >> 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> *Im Auftrag von *Xu, >> Shenyuan >> *Gesendet:* Montag, 11. November 2019 16:51 >> *An:* CCP4BB@JISCMAIL.AC.UK >> *Betreff:* [EXTERNAL] Re: [ccp4bb] molecular replacement_protein-glycan >> complex >> >> >> *EXTERNAL : *Real sender is 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> 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> 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> 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> 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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