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 [[email protected]] on behalf of Eleanor Dodson [[email protected]] Sent: 14 November 2019 20:57 To: [email protected] Subject: Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] molecular replacement_protein-glycan complex Oh dear! On Thu, 14 Nov 2019 at 19:47, Xu, Shenyuan <[email protected]<mailto:[email protected]>> 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 <[email protected]<mailto:[email protected]>> 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, [email protected]<mailto:[email protected]> 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 <[email protected]<mailto:[email protected]>> Im Auftrag von Xu, Shenyuan Gesendet: Montag, 11. November 2019 16:51 An: [email protected]<mailto:[email protected]> Betreff: [EXTERNAL] Re: [ccp4bb] molecular replacement_protein-glycan complex EXTERNAL : Real sender is [email protected]<mailto:[email protected]> 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 <[email protected]<mailto:[email protected]>> 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 <[email protected]<mailto:[email protected]>> 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 <[email protected]<mailto:[email protected]>> 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 <[email protected]<mailto:[email protected]>> 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
