[phenixbb] Need help in refining my twinned P6322(apparent) dataset

John Pak john.pak at msg.ucsf.edu
Mon Mar 14 23:55:05 PDT 2011


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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