Dear all,
Thank you for your kind expert suggestions, and sorry for the late
reply, I'm still trying all the suggested approaches.
Some comments:
- I can't use buccaneer yet, since I'm still working to resolve the
structure.
- Robyn Stanfield's suggestion of using Shelxe to try to verify the
validity of the MR solutions was very helpful. I'm trying it a lot,
however without success. Thank you Robyn and all Shelx developers!
- The maps phased with partial molecular replacement solutions look
reasonable, but do not show any sign of the rest of the molecules or of
the missing side chains.
- ContaMiner gave me green lights, so it does not appear to be a
contaminant (thank you Ivan Shabalin).
- Similarly, SAUC indicates there is no other PDB with cell parameters
similar to this data set (thank you Jonathan Cooper).
- I am currently using EPMR to try to resolve the structure by MR (thank
you Roger Rowlett).
- I am not sure if there is translational NCS, as Phil Jeffrey suggested
despite phenix.xtriage's green lights. I'm not experienced with this
issue, so I'm sending the log file to Randy Read as he kindly offered to
help.
I think one of my problems is that the AU is big (at least to me),
apparently containing from 9 to 12 copies of my protein, so the MR
solutions contain less than 10% of the scatters in the AU.
Maps look good when I refine one chain of the MR solutions in Phenix
(blue maps over most of the protein, with reasonable main chain
continuity and very few green or red blobs), but the R values are 0.50
and I can't see any feature suggesting the solution is good (like a
well-defined new side chain).
I will keep trying.
Thank you all again! And please let me know if any ideas cross your wise
minds.
Regards,
Napo
Em 2019-08-30 06:29, Randy Read escreveu:
> Dear Napoleão,
> Yes, I agree with Phil that it looks like a case where there *is*
> translational NCS but it's not being used by Phaser, either because it wasn't
> automatically detected (native Patterson peak just under the default limit?
> more than one off-origin Patterson peak?) or because Phaser was told to
> ignore tNCS. You should look in detail at the relevant section of the
> logfile, and manually override Phaser's automated decision if you think
> there's good evidence for tNCS. I would say that, as Phil noted, the fact
> that two molecules are in basically the same orientation separated by a
> translation is pretty strong evidence. In particular, the fact that placing
> the second molecule in the same orientation gave such a large increase in
> both LLG and TFZ is strong evidence: this tells us that having two molecules
> in the same orientation (even if they're the wrong molecule or in the wrong
> orientation) explains some feature of the data, i.e. the modulation caused by
> tNCS.
>
> I'd be happy to look at the log file for you if you find it hard to
> interpret.
>
> Best wishes,
>
> Randy Read
>
> On 29 Aug 2019, at 17:24, Phil Jeffrey <pjeff...@princeton.edu> wrote:
>
> Are you *sure* there's no translational NCS ?
>
> For example your first molecular replacement solution out of Phenix shows
>
> EULER 293.6 27.7 288.7
> FRAC -0.02 0.02 0.02
> (that's "first molecule at origin in P1")
>
> and
>
> EULER 294.0 27.9 288.8
> FRAC -0.37 0.02 0.02
>
> which is essentially the same orientation, and a translation down one
> crystallographic axis (a*)
>
> And this suggests to me that either Xtriage or Phaser is missing something
> here. Does Phaser find translational NCS in its initial data analysis ?
> Unmodeled translational NCS could cause significant problems with the
> molecular replacement search.
>
> Phil Jeffrey
> Princeton
>
> On 8/29/19 11:28 AM, Napoleão wrote:
> Deal all,
> Sorry for the long post.
> I have a data set obtained from a crystal produced after incubating a
> protease with a protein which is mostly composed by an antiparallel beta
> sheet. I have tried numerous approaches to solve it, and failed. Molecular
> replacement using Phaser, and the protease or the protein as a template
> yields no solution. However, molecular replacement using only part of the
> beta sheet yields LLG=320 TFZ==28.0 (see below).
> The apparently good data extends to 1.9 A, as processed by XDS, and the space
> group is P1 (pointless agree). XDS info below:
> SPACE_GROUP_NUMBER= 1
> UNIT_CELL_CONSTANTS= 44.43 72.29 77.30 97.802 89.939 101.576
> a b ISa
> 9.647E-01 3.176E-03 18.07
> RESOLUTION NUMBER OF REFLECTIONS COMPLETENESS R-FACTOR R-FACTOR
> COMPARED I/SIGMA R-meas CC(1/2) Anomal SigAno Nano
> LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed expected
> Corr
> 1.90 24890 19149 23814 80.4% 58.1% 63.7% 11482
> 0.77 82.2% 63.8* 3 0.694 492
> total 163756 125884 146938 85.7% 10.6% 10.8% 75744
> 3.78 15.0% 99.0* -3 0.761 5834
> Xtriage in Phenix 1.16-3549 gives me all green lights (print below),
> suggesting the data presents no twinning, no translational NCS, no ice rings
> and is not anisotropic.
> http://fullonline.org/science/phenix_xtriage_green.png
> Molecular replacement in Phaser yields single solutions like:
> Solution annotation (history):
> SOLU SET RFZ=3.0 TFZ=* PAK=0 LLG=29 RFZ=2.8 TFZ=8.8 PAK=1 LLG=310 TFZ==27.6
> LLG=320 TFZ==28.0
> SOLU SPAC P 1
> SOLU 6DIM ENSE ensemble1 EULER 293.6 27.7 288.7 FRAC -0.02 0.02 0.02
> BFAC
> -6.03
> SOLU 6DIM ENSE ensemble1 EULER 294.0 27.9 288.8 FRAC -0.37 0.02 0.02
> BFAC
> -6.52
> SOLU ENSEMBLE ensemble1 VRMS DELTA -0.1983 RMSD 0.49 #VRMS 0.21
> or partial solutions like:
> Partial Solution #1 annotation (history):
> SOLU SET RFZ=3.7 TFZ=* PAK=0 LLG=32 RFZ=2.8 TFZ=13.0 PAK=0 LLG=317 TFZ==30.2
> LLG=331 TFZ==30.5 RFZ=2.4 TFZ=7.2 PAK=0 LLG=464 TFZ==18.5 RFZ=2.7 TFZ=5.7
> PAK=1
> LLG=501 TFZ==6.8 LLG=509 TFZ==6.6
> SOLU SPAC P 1
> SOLU 6DIM ENSE ensemble1 EULER 85.4 153.0 138.5 FRAC -0.01 -0.00 -0.00
> BFAC
> -12.30
> SOLU 6DIM ENSE ensemble1 EULER 86.2 153.2 139.5 FRAC -0.36 -0.01 -0.01
> BFAC
> -9.16
> SOLU 6DIM ENSE ensemble1 EULER 83.8 152.3 135.9 FRAC -0.00 0.00 -0.25
> BFAC
> 1.52
> SOLU 6DIM ENSE ensemble1 EULER 191.2 109.1 39.3 FRAC -0.27 -0.01 0.22
> BFAC
> 10.18
> SOLU ENSEMBLE ensemble1 VRMS DELTA -0.0447 RMSD 0.49 #VRMS 0.44
> However, after 1 refinement round in Phenix_Refine (Final: r_work = 0.4881
> r_free = 0.5009) I got densities that are part good and part bad, and if I
> delete the bad parts and refine again, the good parts become bad. Please
> check the prints:
> http://fullonline.org/science/good_part_of_density.png
> http://fullonline.org/science/bad_part_of_density.png
> What is the explanation for these molecular replacement results?
> What else should I try? Arcimboldo takes 2 days+ to run and yields no good
> solution.
> Thank you!
> Regards,
> Napo
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------
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research Tel: + 44 1223 336500
The Keith Peters Building Fax: + 44 1223
336827
Hills Road E-mail:
rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.
www-structmed.cimr.cam.ac.uk [1]
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