Need help in refining my twinned P6322(apparent) dataset
Hey Phenix community, Here are the details of my current situation - any assistance in the matter would be greatly appreciated as I've been struggling mightily for the past month trying to get this structure refined (to no avail). Details: -3.0 angstrom dataset from a membrane protein crystal -reduced in a P3 Bravis lattice using HKL2000 -Pointless and Xtriage suggest P622 symmetry -Solved structure via MR - ran Phaser in all P622 based space groups, found 1 convincing solution in P6322 (LLG ~110) which makes biological sense (1 monomer in ASU that lies on the screw axis to recapitulate the biological trimer) -31% sequence identical search molecule -no pseudotranslation (via Xtriage) Xtriage suggests that the P6322 dataset is nearly perfectly twinned (twin fraction = 0.49). Mindful of the fact that there are no twin operators for P6322, I'm thinking that the true space group is P321, P312 or P63 and I've tried a number of things listed below: 1. Refined in P6322 (simulated annealing+individual B). Missing side chains and incorrectly modelled loops apparent in maps. Start R=0.49, Rfree=0.5. Refinement stalls at R=0.41, Rfree=0.45 after building. 2. Ran Phaser in P312 (using unrefined model) - no solution. 3. Ran Phaser in P321 (using unrefined model). One nice solution (LLG ~220) - lattice looks very similar to P6322 crystal packing. Twin refinement in phenix using operator suggested by Xtriage (simulated annealing + individual B) results in R=0.38, Rfree=0.41 (no rebuilding performed). 4. Ran Phaser in P63 (using unrefined model). Three solutions (LLGs all ~210) - solution #3 looks very similar to P6322 crystal packing. Twin refinement in phenix using operator suggested by Xtriage of solution #3 gives R=0.37, Rfree=0.40 (no rebuilding performed). Solutions #1 and #2 do not give packing arrangements that look like the P6322 solution. Twin refinement of Sol 1 and 2 give higher R factors. So I'm at a loss as to which space group my crystal truly belongs to, and where I should be focusing my refinement. The maps all look similar before and after DM. Thanks for any assistance or recommendations that you might have! -- John E. Pak, Ph.D. Postdoctoral Associate, Stroud Lab Department of Biochemistry& Biophysics UCSF MC2240 Genentech Hall Room S414 600-16th St. San Francisco, CA 94158-2517 Lab #: 415-476-3937 Fax #: 415-476-1902 Cell #: 415-215-0048
John, Have you tried Zanuda to resolve the space group ambiguity? F On Mar 15, 2011, at 12:55 AM, John Pak wrote:
Hey Phenix community,
Here are the details of my current situation - any assistance in the matter would be greatly appreciated as I've been struggling mightily for the past month trying to get this structure refined (to no avail).
Details:
-3.0 angstrom dataset from a membrane protein crystal -reduced in a P3 Bravis lattice using HKL2000 -Pointless and Xtriage suggest P622 symmetry -Solved structure via MR - ran Phaser in all P622 based space groups, found 1 convincing solution in P6322 (LLG ~110) which makes biological sense (1 monomer in ASU that lies on the screw axis to recapitulate the biological trimer) -31% sequence identical search molecule -no pseudotranslation (via Xtriage)
Xtriage suggests that the P6322 dataset is nearly perfectly twinned (twin fraction = 0.49). Mindful of the fact that there are no twin operators for P6322, I'm thinking that the true space group is P321, P312 or P63 and I've tried a number of things listed below:
1. Refined in P6322 (simulated annealing+individual B). Missing side chains and incorrectly modelled loops apparent in maps. Start R=0.49, Rfree=0.5. Refinement stalls at R=0.41, Rfree=0.45 after building.
2. Ran Phaser in P312 (using unrefined model) - no solution.
3. Ran Phaser in P321 (using unrefined model). One nice solution (LLG ~220) - lattice looks very similar to P6322 crystal packing. Twin refinement in phenix using operator suggested by Xtriage (simulated annealing + individual B) results in R=0.38, Rfree=0.41 (no rebuilding performed).
4. Ran Phaser in P63 (using unrefined model). Three solutions (LLGs all ~210) - solution #3 looks very similar to P6322 crystal packing. Twin refinement in phenix using operator suggested by Xtriage of solution #3 gives R=0.37, Rfree=0.40 (no rebuilding performed). Solutions #1 and #2 do not give packing arrangements that look like the P6322 solution. Twin refinement of Sol 1 and 2 give higher R factors.
So I'm at a loss as to which space group my crystal truly belongs to, and where I should be focusing my refinement. The maps all look similar before and after DM.
Thanks for any assistance or recommendations that you might have! --
John E. Pak, Ph.D. Postdoctoral Associate, Stroud Lab Department of Biochemistry& Biophysics
UCSF MC2240 Genentech Hall Room S414 600-16th St. San Francisco, CA 94158-2517
Lab #: 415-476-3937 Fax #: 415-476-1902 Cell #: 415-215-0048
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--------------------------------------------- Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
John, The test here would be "which maps looks better" and if there isn't any obvious interpretable difference density for a missing/modified part of the structure you might generate some by leaving out a well-ordered element of the structure prior to the first pass through refinement in each of your P321 and P63 cases. Personally I would rebuild both P321 and P63 structures at the same time, and see if any meaningful differences manifest. That having been said, I want to raise the unfortunate possibility that you're really in P3 and doubly twinned. As far as I know phenix.refine does not handle that so it's difficult to test for. However I did have one case that appeared to fit within that scheme (also in trigonal). If you didn't generate your Free R flags within Phenix itself (which typically handles this correctly), you need to make sure that they obey point group 622 symmetry even in lower symmetry trials, to minimize R-free biasing due to NCS and twinning. Phil Jeffrey Princeton On 3/15/11 2:55 AM, John Pak wrote:
Hey Phenix community,
Here are the details of my current situation - any assistance in the matter would be greatly appreciated as I've been struggling mightily for the past month trying to get this structure refined (to no avail).
Details:
-3.0 angstrom dataset from a membrane protein crystal -reduced in a P3 Bravis lattice using HKL2000 -Pointless and Xtriage suggest P622 symmetry -Solved structure via MR - ran Phaser in all P622 based space groups, found 1 convincing solution in P6322 (LLG ~110) which makes biological sense (1 monomer in ASU that lies on the screw axis to recapitulate the biological trimer) -31% sequence identical search molecule -no pseudotranslation (via Xtriage)
Xtriage suggests that the P6322 dataset is nearly perfectly twinned (twin fraction = 0.49). Mindful of the fact that there are no twin operators for P6322, I'm thinking that the true space group is P321, P312 or P63 and I've tried a number of things listed below:
1. Refined in P6322 (simulated annealing+individual B). Missing side chains and incorrectly modelled loops apparent in maps. Start R=0.49, Rfree=0.5. Refinement stalls at R=0.41, Rfree=0.45 after building.
2. Ran Phaser in P312 (using unrefined model) - no solution.
3. Ran Phaser in P321 (using unrefined model). One nice solution (LLG ~220) - lattice looks very similar to P6322 crystal packing. Twin refinement in phenix using operator suggested by Xtriage (simulated annealing + individual B) results in R=0.38, Rfree=0.41 (no rebuilding performed).
4. Ran Phaser in P63 (using unrefined model). Three solutions (LLGs all ~210) - solution #3 looks very similar to P6322 crystal packing. Twin refinement in phenix using operator suggested by Xtriage of solution #3 gives R=0.37, Rfree=0.40 (no rebuilding performed). Solutions #1 and #2 do not give packing arrangements that look like the P6322 solution. Twin refinement of Sol 1 and 2 give higher R factors.
So I'm at a loss as to which space group my crystal truly belongs to, and where I should be focusing my refinement. The maps all look similar before and after DM.
Thanks for any assistance or recommendations that you might have!
On Tue, 2011-03-15 at 12:12 -0400, Phil Jeffrey wrote:
That having been said, I want to raise the unfortunate possibility that you're really in P3 and doubly twinned.
As in P3 with dimer sitting on the axis and resulting in apparent 6-fold symmetry (120+180-360=-60)? -- "I'd jump in myself, if I weren't so good at whistling." Julian, King of Lemurs
On 3/15/11 1:18 PM, Ed Pozharski wrote:
On Tue, 2011-03-15 at 12:12 -0400, Phil Jeffrey wrote:
That having been said, I want to raise the unfortunate possibility that you're really in P3 and doubly twinned.
As in P3 with dimer sitting on the axis and resulting in apparent 6-fold symmetry (120+180-360=-60)?
In this particular case I'd envision it as potentially four crystallographic trimers formed by crystallographic 3-folds, giving 4 unique monomers per asymmetric unit, with two two-fold NCS elements, both perpendicular to the 3-fold, and spaced 60 degrees apart (doesn't this beget 622?). Actually you could probably do this with 3 monomers, but then you might get some apparent statistical disorder of domains when treating the data as either P321 or P63 where the asymmetric unit volume is halved. In my case I saw something that was at first blush "P321" and pseudo P622, had one "good" domain and the other half of the asymmetric unit exhibited statistical disorder that was the superposition of two domains overlapping on themselves related by a twin operator. This interpretation was made possible by looking at the SeMet peaks in the MAD map - it's not something that would be easy to come by from looking at a model-phased map. I felt it was better interpreted as P3 -> P321 -> P622 doubly twinned, because attempts at single twin refinement and handling the statistical disorder did not yield a more refinable structure. But I'm not sure how to prove it one way or the other.
participants (4)
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Ed Pozharski -
Francis E Reyes -
John Pak -
Phil Jeffrey