Hi,
If you search with a dimer, then the version of Phaser in CCP4 doesn't know
that there is internal symmetry in your model, and it will find two
solutions that are equivalent by the internal symmetry, but would not be
equivalent if the model were not symmetric. The latest version of Phaser
(which is available in Phenix, and should be in the next CCP4 release)
identifies internal symmetry and would recognise the two solutions as being
equivalent.
You can check this by reading the two solutions into coot, using ssm
superpose to superimpose the first monomer of the first solution on the
second monomer of the second solution, and seeing whether the
transformation is some combination of crystallographic symmetry and an
allowed origin shift.
Best wishes,
Randy Read
On Oct 21 2009, X Xiong, Cellular & Molecular Medicine wrote:
Hi All,
Thanks for all the replies, I would like to add more information, after
reindex it to P21212, the cell parameter is a=88.71, b=116.26, c=55.12,
the molecule is a long rod like head to head dimer with a length of 110Å
(55Å long for each monomer) and we used the dimer to search the
orthorhombic data, two solutions were found as shown in the original
thread which can be both refined to almost the same very good R/Rfree,
coot was used to generated the symmetry related molecules from both
solutions and the two solutions had the same packing but translated along
the b axis by 31.9Å, thus clashed into each other and made a mess in the
overlap region, so I think they are not crystallographically the same
solutions, unless the origin of the cell in P21212 can be placed on
b-axis arbitrarily, and I think Phaser will also prune the
crystallographically same solutions.
Interestingly, the most prominent pseudo-translational peak (1/3 of the
origin peak), has a fractional vector 0.5000 0.2741 0.5000, and the
fraction on b axis = 0.2741*116.26 = 31.8755Å, and that is exactly how
long the two solutions translated along the b-axis, I don't know what
that means, does these information verify this is the second case Eleanor
suggested? if so should I keep the messy overlapped region and set the
rest 0 occupancy to check the density?
Thanks for all the suggestions so far!
Xiaoli
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
