From the Pattersn peak it seems very likely that you have two
molecules in the asymmetric unit seperated by the very vector that
seperates your two MR solutions, and both MR solutions are correct?
Or is that not possible? Is there no room for 2 molecules in the
asymmetric unit, and the Patterson peak isa function of the "highly
repetitive dimer"
Eleanor
If that is so you need to set the occupancy of any differences in the
solution to 0.00 and check from the maps after refinement if you can see
which copy of the molecule fits the difference density best - it would
nice if you had a TRP/ALA pair of residues or something very distinctive..
Eleanor
X Xiong, Cellular & Molecular Medicine wrote:
Dear Crystallographers,
We got a highly repetitive dimeric protein solved by SeMet-SAD in P21
crystal form, and I am now trying to solve a dataset collected from a
non-reproducible orthorhombic crystal of the same protein using the
structure refined from P21 data.
From the Scala statistics, the orthorhombic crystal diffracted to
2.2Å with
an I/sigma of 3.1 at outmost shell; 98% complete overall, 89% complete
43.4-7.0Å, 99% complete 2.32-2.2Å, no twinning was detected. Due to
the incompleteness at low resolution, it was hard to determine which
orthorhombic space group it is in so data was scaled in P222. Very
strong pseudo-translational symmetry has been detected by
self-Patterson, as shown for reindexed data P21212 (space group later
found by Phaser):
Order No. Site Height/Rms Grid Fractional coordinates
Orthogonal coordinates
1 1 1 128.24 0 0 0 0.0000 0.0000 0.0000
0.00 0.00 0.00
2 13 13 57.51 60 44 38 0.5000 0.2741 0.5000
44.37 31.88 27.55
3 2 2 33.75 0 7 0 0.0000 0.0414 0.0000
0.00 4.82 0.00
4 14 14 16.09 60 50 38 0.5000 0.3150 0.5000
44.37 36.63 27.55
5 12 12 15.75 60 37 38 0.5000 0.2324 0.5000
44.37 27.03 27.55
6 3 3 12.28 0 13 0 0.0000 0.0836 0.0000
0.00 9.72 0.00
7 15 0 7.06 60 57 38 0.5000 0.3574 0.5000
44.37 41.56 27.55
8 4 4 6.18 0 72 0 0.0000 0.4503 0.0000
0.00 52.36 0.00
9 9 9 5.68 5 0 5 0.0410 0.0000 0.0683
3.64 0.00 3.76
10 5 5 5.36 2 20 2 0.0142 0.1254 0.0206
1.26 14.59 1.14
11 11 11 5.33 58 31 38 0.4852 0.1909 0.5000
43.06 22.20 27.55
12 6 6 3.98 5 0 2 0.0435 0.0000 0.0286
3.86 0.00 1.58
13 7 7 3.82 2 27 3 0.0168 0.1659 0.0334
1.49 19.30 1.84
14 8 8 3.68 0 0 5 0.0000 0.0000 0.0722
0.00 0.00 3.98
15 10 10 3.41 60 64 37 0.5000 0.4007 0.4872
44.37 46.59 26.84
Phaser was used to test all possible alternative space groups to find
MR solution using the structure from P21 data:
# Phaser_P222_MosFLM_all_spacegroup
SPACegroup HALL P 2bc 2 #P 2 21 21
SOLU SET RFZ=9.1 TFZ=24.3 PAK=0 LLG=2545 LLG=3718
SOLU 6DIM ENSE ensemble1 EULER 273.097 1.162 88.144 FRAC
-0.03394 0.50659 -0.22125
SOLU SET RFZ=9.1 TFZ=25.0 PAK=0 LLG=2496 LLG=3622
SOLU 6DIM ENSE ensemble1 EULER 91.491 0.850 89.812 FRAC
0.03435 -0.00618 0.00352
and it found 2 solutions with very similar Z-scores and LLG gains, If
I am right they are not crystallographic equivalent, and Phaser checks
that as well.
I reindexed the data to P21212 and Phaser found the same solutions:
# Phaser_Reindexed_P21212_2_solutions
SPACegroup HALL P 2 2ab #P 21 21 2
SOLU SET RFZ=10.0 TFZ=23.0 PAK=0 LLG=3266 LLG=3718
SOLU 6DIM ENSE ensemble1 EULER 88.843 90.063 1.249 FRAC
-0.00661 -0.22126 -0.46598
SOLU SET RFZ=9.9 TFZ=23.2 PAK=0 LLG=3178 LLG=3624
SOLU 6DIM ENSE ensemble1 EULER 270.841 89.977 181.339 FRAC
-0.50634 -0.00350 0.46533
The difference between the two solutions seems to be that the second
solution translated along the longer 21 axis by about ~32Å, I chose
the first solution to re-build and refine, and final R/Rfree I got was
21.6%/26.5%. After that, I hope to solve the ambiguity of which MR
solution is right by running Phaser again with the complete model
(including H2O):
# Phaser_Reindexed_P21212_2_solutions
SPACegroup HALL P 2 2ab #P 21 21 2
SOLU SET RFZ=12.8 TFZ=28.4 PAK=0 LLG=6542 LLG=7649
SOLU 6DIM ENSE ensemble1 EULER 180.156 0.000 0.000 FRAC
-0.50060 -0.00065 0.49998
SOLU SET RFZ=12.8 TFZ=32.3 PAK=0 LLG=6036 LLG=7059
SOLU 6DIM ENSE ensemble1 EULER 179.201 180.000 0.000 FRAC
0.00227 0.22262 -0.50054
It seems that the previous first solution has become the second
solution, while the previous second solution became the first. Refmac
refinement was performed on both solutions (H2O removed) came out from
Phaser,
solution 1 R/Rfree = 24.1/28.9
solution 2 R/Rfree = 24.8/28.7
the previous first solution got slightly worse scores, however, the
R-factors for both solutions are so similar and both of them gave very
similar electron density that I can not figure out which one is the
right solution.
I would very grateful for any advices.
Thanks in advance,
----------------------
Xiaoli Xiong
PhD Candidate
Department of Cellular and Molecular Medicine
School of Medical Sciences
University of Bristol
University Walk
Bristol BS8 1TD, UK
x.l.xi...@bristol.ac.uk
