Hello Bill, We used SIMBAD, as Kay previously suggested,to figure out the contaminants.
I will keep this in mind. Thanks, Shen On Fri, Nov 15, 2019 at 5:44 AM SHEPARD William < william.shep...@synchrotron-soleil.fr> wrote: > Hi Shen, > > This comment is a too late to help, but out of interest, did you run > SIMBAD, which checks for contamiants, on your data? It's another thing one > do when the MR solutions are very mediocre. > > Cheers, > Bill > > > ------------------------------ > *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Eleanor > Dodson [0000176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk] > *Sent:* 14 November 2019 20:57 > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] molecular > replacement_protein-glycan complex > > 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! >>> > >>> >Thanks, >>> > >>> >Shenyuan Xu >>> > >>> >######################################################################## >>> > >>> >To unsubscribe from the CCP4BB list, click the following link: >>> >https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 >>> <https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1&d=DwMFaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=DivhmpKd5Pwuefz41rsUIvaEj3_QOwMPpIv44-xpi1k&s=tfgHlzCP0-vbR3kmX7qNza4dK5ot2hqFhwDLegNgWq8&e=> >>> > >>> >>> ######################################################################## >>> >>> To unsubscribe from the CCP4BB list, click the following link: >>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 >>> <https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1&d=DwMFaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=DivhmpKd5Pwuefz41rsUIvaEj3_QOwMPpIv44-xpi1k&s=tfgHlzCP0-vbR3kmX7qNza4dK5ot2hqFhwDLegNgWq8&e=> >>> >>> >>> ------------------------------ >>> >>> To unsubscribe from the CCP4BB list, click the following link: >>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 >>> <https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1&d=DwMFaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=DivhmpKd5Pwuefz41rsUIvaEj3_QOwMPpIv44-xpi1k&s=tfgHlzCP0-vbR3kmX7qNza4dK5ot2hqFhwDLegNgWq8&e=> >>> >>> >>> ------------------------------ >>> >>> To unsubscribe from the CCP4BB list, click the following link: >>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 >>> <https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1&d=DwMFaQ&c=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=DivhmpKd5Pwuefz41rsUIvaEj3_QOwMPpIv44-xpi1k&s=tfgHlzCP0-vbR3kmX7qNza4dK5ot2hqFhwDLegNgWq8&e=> >>> >>> ------------------------------ >>> >>> 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 >>> >> >> ------------------------------ >> >> 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 > > ------------------------------ > > 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
