Online supporting materials for:
On the validation of crystallographic symmetry and the quality of
structures
Jimin Wang*
Contents: 7 sections on specific responses to reviewers, supplementary
analytic procedures, discussion, and notes, 3 supplemental Figures, 9
supplemental Tables, and 46 supplemental references.
Supplemental Text
1. Introduction
The intended readership of this manuscript is for the entire structural biology, not just for
those experts in crystallography. All highly technical non-essential details have been
removed from the main text for clarity, and included in this online supporting materials
section when necessary. This material includes some of technical responses to specific
requests by reviewers of this manuscript as well as supplementary information to the
main text as stated in the first sentence of each section.
2. Direct methods and symmetry downshifting
One reviewer requested more information on direct methods for phasing procedures in
regard to Miao’ algorithm. There are two classes of direct methods, one pure probability
approach in reciprocal space and the other, being a class of dual-space “chargingflipping” algorithms that include classic solvent-flattening procedure used in
macromolecular crystallography. Miao’s procedure belongs to the dual-space “chargingflipping” algorithms.1,2 More specifically, Miao’s procedure is to numerically determine
unknown values of electron density for properly sized pixels inside the protein boundary
using about 2.6 equations on average relating the observed amplitudes to every unknown
density value through Fourier transformation. This procedure can determine solvent
boundary itself, but not very sufficiently. This procedure works better when a prior
solvent boundary is supplied. Different dual-space procedures differ in speed by several
orders of magnitudes, some of which become impractical when dealing with over
millions of independent observations such as in the C2 crystals of E. coli tetrameric
catalase that is currently pursued for phase solutions in the author’s laboratory. Many
other programs such as ACORN do not require any prior information about the structure,
but often require high resolution.3 When a subset of positioned atoms is used for starting
trial phases, these programs require data at much lower resolution. For example, the
crystal of C2 E. coli catalase diffracted to a resolution of 1.4 Å with the <I/I> value of
0.5 in in the highest resolution shell. We processed the data to a resolution of 1.3 Å and
extended to 1.0 Å resolution using Wilson-expected average values for phase solution. In
this case, the direct method using the program ACORN readily resulted in interpretable
electron density maps when starting phases were seeded by as few as 6.7% positioned
atoms (out of about 30,000 atoms in the asymmetric unit of the unit cell). The success of
this example suggests that a large fraction of the structures in the PDB can be phased
using a variety of direct methods, a subject that is beyond the scope of this manuscript.
Another application of direct methods for phase retrieval highly relevant to this
study on the quality of data is that of 4HYO.4 Using the re-processed data of the correct
P4212 space group at a resolution of 1.65 Å and then extended to 1.0 Å using Wilsonexpected average values, the complete phase retrieval was possible using ACORN
starting a properly positioned, single generic poly-alanine helix model. This was not
possible using the original P1 4HYO data. When positioned C atoms for the entire chain
in P4212 and those for 8 chains in P1 were used as starting trial phases, complete phase
retrieval using ACORN was possible for both space groups. However, the maps in P4212
with the better quality of the data were clearly better than those in P1 (Fig. S1). This
difference disappeared when the reprocessed P4212 data were re-expanded to P1 because
of the newly improved quality of 8-fold reciprocal space averaged P1 data relative to the
original P1 data. Details of this study will be described elsewhere.
Symmetry downshifting is often a preferred method for dual-space direction
methods of phasing because crystals of macromolecules have a solvent region where
randomly placed atoms cannot provide good starting trial phases that would eventually
lead to the correct solution.2 Yet, such impossible trial phases lead to a time-consuming,
very slowly converging process. In fact, these impossible trials often consume over 95%
of the total CPU whereas possible trials consume less than 5%. After symmetry
downshifting to P1, any point in the unit ccell now becomes a possible-solution trial,
which greatly speeds up the computation time. Even when using this specific symmetry
downshifting procedure, the data must be first processed in the correct symmetry of the
space group before they are symmetry downshifted. Of course, one solution for phases is
found, the resulting electron density maps will reproduce the correct symmetry, and
structure refinement has to be carried out in the correct space group.
2. Intensity analysis for symmetry and errors in calculated anomalous amplitudes
This section is supplementary to the main text. Self-rotation function (SRF) were
calculated using CCP4 MolRep, and were sorted in the descending order for further
analysis (Table S1).5,6 The top entries in the resulting list were selected for individual
examination and structure re-refinement with properly selected CV sets after the data
were rescaled in the correct higher-symmetry space groups (Table S2). During atomic rerefinement, it was found that the quality of the repositioned original models was rather
poor and refinement often stalled at an early stage. Re-refinement succeeded only after
alternating steps of geometry idealization and atomic refinement were carried out.
Some of these entries retrieved from the PDB database included the observed
anomalous signals using which better experimental maps could be calculated using
symmetry-averaged data in the correct space groups than was possible in the incorrect P1.
However, it is noted that many entries include an extra column of information about
estimated anomalous signals calculated using refined coordinates, which were incorrectly
treated by the cif2mtz script as being observed signals.5,7 Difference Fourier maps using
such estimated anomalous signals and calculated phases from re-refined models in the
correct space groups showed that these estimated signals could not provide the correct
information about the location heavy atom sites most cases test. This is consistent with
the fact that an addition of small anomalous differences to the observed amplitudes to
generate estimated amplitudes for Friedel mates requires accurate phases, which are not
obtainable with symmetry downshifting. Such estimated amplitudes vary from one run of
2
structure refinement to the next, and indirectly contain phase information from models.
When such estimated amplitudes are used for structure refinement, the refinement is
actually against the calculated structure factors even though the mean values of the
Friedel amplitudes are constrained to the observed amplitudes. Such procedure is
completely invalid!
All Rsymm<0.15, calculated using XPREP of the Shelx suite,8 are tabulated for
statistical analysis (Table S3). Of course, entries with Rsymm>0.15 can still be in higher
symmetry space groups. For example, in a recent paper, all weak-intensity highresolution were included for NCS averaging in which Rmeas is as high as 0.24,9 in which
case, Rmeas was carefully monitored as a function of resolution to ensure that the correct
space group was identified on the basis of low Rsymm values at low resolution. On the
other hand, many entries in the database alleged in higher symmetry space groups with
Rmerging higher than 0.20 may result from incorrect symmetry averaging and actually
belong to lower symmetry space groups, which is on the opposite side of the spectrum of
problems discussed in this study and beyond the scope of this study. Such incorrect
symmetry merging would result in unusual twinning statistics in space groups (such as
non-special orthogonal space group), even when the original crystals may not have
twinning problems.
3. The closely related isomorphous structures of 3LDC, 4HYO, and 4HZ3
This section is supplemental to the main text. The re-refined 4HYO structure at 1.65 Å
resolution is nearly identical to the 3LDC structure at 1.45 Å resolution published by
Jiang and colleagues in the same journal two years earlier with an exception of being at
lower resolution.4,10 In fact, an overall amplitude difference R-factor is only 8.2% for all
the data to 1.65-Å resolution between the 4HYO and 3LDC data (Fig. 4). An overall
isomorphous difference between 4HYO and 4HZ3 is 13.0% (Fig. 4).4 With such small
isomorphous differences, the proper way to determine these closely related structures is
to use the isomorphous difference Fourier method, not independent molecular
replacement. An isomorphous difference Fourier map between the observed 4HYO and
4HZ3 data showed that (i) there was no evident for the claimed binding of tetraammonium antimony in 4HZ3,4 and (ii) K+ sites in the central channel of the 4HYO
structure were not fully occupied (Fig. 4). An isomorphous difference Fourier map
between the observed 4HYO and 3LDC data again showed that K+ sites were not fully
occupied in 4HYO (Fig. 4). These results are consistent with earlier observations made
by Jiang and colleagues that occupancies of K+ in binding sites can be reduced by
lowering K+ concentration in crystal stabilization solution, initially at the third K+ site.10
When K+ fully occupied in the third site in the model for 4HYO in this re-refinement, a
hole at this site in residual Fobs-Fcalc maps was over 7 σ where Fobs and Fcalc denote the
observed and calculated amplitudes, respectively. The hole was reduced below noise
level when the occupancy was reduced by 20%.
4HYO is one of many structures in the PDB that simply reproduced the alreadypublished structures using the symmetry downshifting procedure with improved R free
values as new structures. These structures were often determined using independent
molecular replacement method, rather than isomorphous difference Fourier method.
More often, different crystallographic origins were preferentially chosen to make them
look really different from each other for non-specialists even when such pairs of
3
structures are actually nearly identical to one another, some of which entries are truly
different and have been properly categorized as having closely related entries in the
PDB.11-14
4. Experimental errors and selection of reflections for the CV set in P1
This section is a response to a request of one reviewer on the issue why and how the thin
slice method of selecting reflections into the CV set does not work in P1 in addition to
evidence that has already been provided in the main text. An analysis of the 3MUU data
for a viral glycoprotein E2 structure was carried out using the procedures described in the
main text at the time when the 3MUU structure was published because we attempted to
use this E2 model to determine the structure of an E2 protein structure from bovine viral
diarrhea virus.15,16 Symmetry R-factors for 3MUU were analyzed for each of the three
potential dyads and the 3-fold axis, summarized in Table S9. It is clearly that it actually
belongs to C2, not P1.
To further explain why the selection of reflections in the CV set using a thin slice
method of constant resolution shells in P1 for 4HYO did not work (Fig. 4), the 3MUU
entry was chosen for demonstration because 3MUU had larger variations in the
unconstrained unit cell parameters due to reduced resolution than that of 4HYO.4,15 The
crystals used to obtain 3MUU belonged to C2, a subgroup of the closely related
hexagonal crystal form P321, but the structure it yielded was refined in P1.15 This highto-low symmetry downshifting made it appear that some CS or NCS-related reflections
actually differ in resolution because unit cell dimensions were allowed to vary when the
data were reprocessed. The P1 unit cell parameters obtained for 3MUU are: a=67.482 Å,
b=158.430 Å, c=160.676 Å, =60.42°, =89.80°, and =89.65°; but for the closely
related P321 unit cell, which was initially used to generate experimental maps, the
parameters are a=b=159.8 Å, c=68.0 Å, ==90°, and =120°. In 3MUU, an appropriate
selection of reflections for inclusion in the CV set should be done in the highest pseudoP321 symmetry, not in P1.
If a selection of the CV set were done in the P1 3MUU data, one ought to take
into account experimental errors in cell parameters. Because of 1% differences in
b=158.430 Å ≠ c=160.676 Å in P1, the resolution of all 3-fold NCS-related mates of
reflections that ought to have the same resolution now also differs by as much as 1% of
their resolution values, for example between pairs of (0k0) and (00l) of the same index
numbers (i.e., k=l). This resolution difference is amplified rapidly in high resolution. For
example, the resolution of the reflection (0,10,0) is 15.8 Å and its 3-fold NCS-related
mate (0,0,10) is 16.1 Å. The resolution of the reflection (0,50,0) is 3.16 Å and its 3-fold
NCS-related mate (0,0,50) is 3.35 Å. If one does not use NCS geometric relationship of
indexes for selection of reflections in the CV set (i.e., not used pseudo-P321 symmetry),
instead using a thin slice method of a constant resolution shell (i.e., in P1 symmetry), the
thickness of the resolution shell must cover all reflections within the resolution range
between both (0,50,0) and (0,0,50), i.e., between 3.16 and 3.35 Å. With such large unit
cell where the Bragg reflections severely overlap in reciprocal space in resolution, the
reflections between 3.35 and 3.16 Å resolution shell are far greater than 5% of the data.
At the same time, all other reflections within this resolution shell will have their own 3fold NCS-related mates located outside this shell, and they demand a further expansion of
new boundaries of the resolution shell to include all of their 3-fold NCS-related mates,
4
and so on. This is why the thin slice method of selecting reflections for inclusion of the
CV set in P1 will never work as long as experimental errors on cell parameters exist. Of
course, it might work if experimental errors do not exist or the unit cell parameters are
properly constrained to be identical. Thus, in the presence of experimental errors for cell
parameters as explained in the above example, the probability of each pair of 2-fold
NCS-related mates at high resolution shells simultaneously being selected into the CV set
is (5%)2 and the probability of each triplet of 3-fold NCS-related mates in the same CV
set is (5%)3 and so on when an ultra thin slice method is used. Thus, the probability of
any reflections in the CV set being related one in the working set is 1-(5%)2=97.5% or
higher. This violates the requirement that the two sets of reflections be independent.17
Experimental errors of cell parameters in macromolecular crystallography are
often ignored in structure refinement in many cases. For example, an overall amplitude
difference between 4HYO and 3LDC data is only 8.2% for all the data to a resolution of
1.65Å. Yet, their unit cell parameters differ by as much as 0.6%. This cell-parameter
difference was due to numerical instability in data processing, not actual differences. This
numerical error can cause the entire molecule to expand or contract, including bond
distances that would now be incorrectly used as restraints in structure refinement. Many
investigators over-trusted unit cell parameters produced by the data processing programs,
even with diffraction data at a resolution of about 5.0 Å.12
We recently analyzed the unit cell parameters/detector distance during the data
processing of 1.3-Å resolution of C2 crystal of E. coli catalase. The data were collected
using ultra-thin oscillation range of 0.2° per image and processed in 25 images per sector,
which corresponds 5° of reciprocal space. We found that the detector distance changed as
much as 0.5% from the first zone to the last zone with corresponding expansion and
contraction of unit-cell parameters. The numerical instability in data processing is
intrinsic to currently implemented data collection/processing methods: one could not get
accurate three-dimensional cell parameters from two-dimensional data covering only 5°
of reciprocal space even at a resolution of 1.3 Å. These procedures need to improve.
5. Asymptotic R-factor values
This section is supplementary to the main text. Evans and Murshudov have shown in an
elegant theoretical studies that when a model is refined against the Wilson-expected
average values of the data in the highest resolution shells, the asymptotic value of Rfactor is about 42.3%,18 This implies when a model has successfully explained the
Wilson-expected values of the data, the asymptotic value of R-factor is about 42.3%. This
value is reduced to 29.1% in the presence of perfect merohedry twinning, and is typically
between these two values in the presence of any other special or non-special NCS. Thus,
the original TTHRNP 3DXJ structure discussed in the text had an Rfree value of 28.9% in
the presence of incorrectly proposed perfect merohedral twinning,19 and this value is only
0.2% better than its asymptotic value using the Wilson-expected amplitudes for structure
refinement instead of using real data. Thus, it is almost certain that in the presence of
such special NCS, a structure with R-factor of about 30% can be completely wrong.
6. NCS restraints versus CS constraints in structure refinement, and multi-start and
multi-copy structure refinement
5
This section is mainly a response to a request of one reviewer on how special NCS and
non-special NCS behaved differently in structure refinement such as composite-omit
maps. There are several important distinctions between non-special and special NCS.
Downshifted symmetry cannot distinguish the correct and an incorrect model, always
results in model bias even when the model is incorrect. For example, a special
translational NCS is related by a vector of approximately (0,0,1/2) and will cause the
intensities of reflections in l=odd layers to be near zero and in l=even layers to have the
same distribution as a structure without any NCS. A placement of any unrelated molecule
in asymmetric unit (for example, RB69 DNA polymerase into a hypothetic TTHRNP unit
cell) will result in completely model-biased densities for this molecule in a new location
that differs by this vector, even though this molecule is not physically present (or is
omitted) in the new location. Non-special NCS such as 222-NCS in asymmetric unit does
not have such model bias in omitted-maps. In fact, with sufficiently high solvent content,
averaging using non-special 222-NCS can result in correct phases even though starting
models were completely wrong. Thus, there is no model bias in composite-omit maps in
the presence non-special 222 NCS.
NCS restraints after symmetry downshifting differ from strict CS or true NCS
constraints in two important respects. First, molecules that are related by true NCS
restraints have conformations that are similar but not necessarily identical. Second, true
NCS restraints do not impose any geometric constraints on where the NCS axis is located
or how it is oriented. However, the symmetry in data sets that is caused by CS of data
will not be faithfully reproduced in the amplitudes computed for the structures obtained
after CS has been reassigned as NCS unless strict NCS constraints are imposed, which
will require that NCS-related molecule be all but perfectly identical, and that NCS axes
are very closely aligned with unit cell axes. To resolve this somewhat paradoxical
problem, some authors introduced twinning to account for the CS that is lost when the
expand data into lower symmetry space groups.19-29 Another attempted the restoration of
the lost CS by introducing multiple conformations of the entire asymmetric unit.30
Symmetry downshifting is another type of multi-copy refinement without
involving rotational symmetry, which method is known to reduce both working and free
R-factors even when the model being refined is completely wrong.31 In the case of the
first symmetry downshifting issue I addressed,17 the original authors quadrupled the
number of atoms in the model: 2-fold from symmetry downshifting and 2-fold from
multiple conformations of the entire asymmetric unit (or the unit cell), resulting in 4-copy
of the model.30 There is an important distinction between multi-start refinement32 and
multi-copy refinement33 in that the former generates better calculated phases that do not
bias towards individual models so that a better model can be rebuilt. The latter assumes
that an ensemble of models is a better representation of what is in crystal, including an
averaged structure. However, an averaged structure has no physical meaning at all.
During the last steps of re-refinement of the 1EGW and 1JXO structures (Table
S5-8),34,35 Rfree values varied about 0.2%. An attempt was made in the last steps of the
1JXO re-refinement to automatically refine 5 residues at a time throughout the entire
structure in real-space refinement using graphics programs. This resulted in a decrease of
Rfree values by more than 2% but without substantial decrease of R working so that the gap
between them reduced. This decrease of Rfree values does not suggest an improved
quality, but rather results from an invalid approach for using reflections the CV set when
6
reflections in the CV set were included in map calculations for real-space refinement, a
serious problem arises which will be addressed elsewhere. These calculations suggest that
the surfaces of multi-parameter minimization functions for reflections in both working
and CV sets have many local minima, and reductions in both working and free R-factors
may result from being trapped inside these local minima, not necessarily from improved
quality.
In 2005, I first addressed problems caused by symmetry downshifting in a brief
communication arising from an original publication.17,30 It is unfortunate that the original
authors and many other structural biologists mistook the journal policy that the original
authors should have the last word in reply in a brief communication arising format as a
vindication of symmetry downshifting based on improved Rfree values and improved
composite-omit maps. 17,30 These authors continued to carry out symmetry-downshifting
and quadrupling atoms in asymmetric unit to publish additional structures from the same
crystal form afterwards,36 followed shortly by an explosion of symmetry shifting entries
in the PDB (Table S1). Now, there are more than over 200 entries in the PDB whose
symmetry has been downshifted to P1, over 560 to P21, over 180 to C2, over 240 to
P212121 and so on (Table 1, S3).
7
Supplemental Figures
Figure S1. Electron density maps generated using ACORN using normalized extended
structure factors. (a) Using merged P4212 data for 4HYO starting with C atoms only.
(b) Using the original P1 4HYO data starting with C atoms for all 8 chains. Maps were
contoured at 3.0 , and arrows indicate noticeable differences between the two maps.
Figure S2. Comparison of observed (blue) and calculated (red) axial reflections along the
(00l) direction. The calculated amplitudes from the incorrectly assigned P32 space group
failed to reproduce the observed P65 systematic absences in the 1SMY structure.
8
Figure S3. Distribution of intensities (A) and intensity/sigma ratios (B) of (0k0) axial
reflections in the re-merged 1EGW data. Re-refinement has confirmed the correct space
group is P21, as supported by the intensity distribution (A), but not by the I/I
distribution (B). Violations of systematic absences in (B) are likely due to errors in the
original data processing.
Figure S4. The re-refined 1EGW structure in P21 with one complex per asymmetric unit
in two orthogonal views. Three additional residues were built in the protein. When the
DNA duplex is rotated around its pseudo-dyad, resulting in two different orientations,
only three bases per strand have different identities.
9
Supplemental Tables
Table S1. P1 unit cell parameters, and location and heights of self-rotation function peaksa
PDB(year)
1Z9O(2005)
4HZ3(2013)
1WW5(2005)
4NHZ(2013)
4HYO(2013)
3EBN(2009)
1WW4(2005)
4EY0(2012)
3TQS(2011)
3RL0(2011)
3OVJ(2011)
3MU6(2012)
3I3U(2009)
3H0R(2009)
3H0M(2009)
2CJF(2007)
1ZBL(2005)
1JXO(2001)
1EGW(2000)
4LOH(2013)
4IT7(2013)
3VXO(2012)
3RFU(2011)
3HTM(2009)
3CS2(2008)
2O4Q(2008)
1ZLJ(2005)
1WW6(2005)
1R52(2003)
4INH(2013)
4GM3(2013)
4F21(2013)
4DX9(2013)
3NY2(2010)
3MGO(2010)
3MD2(2010)
3FGQ(2009)
4LL7(2014)
3HZ2(2009)
3EVX(2009)
2PIH(2007)
2DES(1993)
4K4X(2013)
4J1R(2013)
4H82(2012)
3S2G(2011)
3RK2(2011)
3R05(2011)
3O37(2010)
3MUU(2010)
3K9M(2010)
3FIX(2011)
3FD2(2009)
3F7F(2009)
2PMC(2008)
Res(Å)
1.9
1.7
2.2
1.9
1.65
2.4
2.3
2.8
1.98
3.8
1.8
2.43
2.8
3.0
2.8
1.95
2.2
2.3
1.5
2.25
2.10
2.61
3.2
2.5
1.95
1.95
2.00
2.2
2.89
1.70
3.39
2.5
2.99
2.61
2.30
2.20
2.09
2.31
1.86
2.54
2.10
1.50
2.37
2.70
1.90
2.30
2.20
2.95
2.00
3.29
2.61
2.30
2.69
2.60
2.69
Footnotes:
a(Å)
50.048
43.965
47.623
96.998
44.035
51.395
47.264
54.900
38.007
53.745
9.536
41.567
43.706
127.377
128.248
195.755
36.897
45.009
41.371
36.500
43.510
51.120
44.150
36.800
55.295
55.295
33.069
47.506
57.816
56.335
48.943
50.997
75.622
29.390
63.211
29.246
46.017
28.069
29.300
41.368
29.322
18.010
61.001
67.516
39.900
68.080
27.627
61.030
35.667
67.482
62.122
42.229
41.932
52.680
34.760
b(Å)
50.033
63.396
57.648
99.359
63.452
51.350
57.078
59.200
57.895
127.357
26.008
61.622
60.287
130.411
129.856
195.730
44.983
53.410
60.696
59.209
44.499
66.382
72.980
88.700
68.299
68.299
60.488
57.344
75.599
66.917
105.985
64.416
122.207
61.456
79.322
37.748
51.953
49.576
54.202
64.537
52.201
18.830
61.013
67.441
98.860
92.472
39.769
114.574
63.848
158.430
31.078
60.915
41.933
117.478
53.672
c(Å)
90.287
63.311
64.643
108.734
63.477
51.390
64.306
79.200
112.938
142.725
25.803
61.478
60.319
153.973
155.069
239.680
62.280
63.262
63.987
59.205
44.582
87.316
329.950
88.700
90.030
90.030
74.226
64.471
91.327
100.459
120.907
139.039
135.269
72.806
87.146
122.221
80.316
149.950
54.179
66.422
55.448
26.750
195.137
116.422
47.130
116.855
102.275
160.129
79.299
160.676
70.937
72.116
70.960
146.287
65.641
(°)
(°)
(°)
(°)
(°)
(°)
90.01
89.99
89.96
89.98
90.03
112.22
89.98
90.00
90.00
107.49
62.28
114.12
120.04
89.89
90.01
65.84
83.63
89.96
115.18
83.98
90.00
112.30
89.96
90.80
90.05
90.05
89.90
90.16
114.44
89.99
90.00
94.89
89.97
65.05
90.00
89.98
90.09
89.96
85.81
90.06
61.85
92.60
90.02
90.13
90.03
106.28
83.38
89.60
89.92
60.42
89.95
101.19
107.21
89.94
90.22
90.00
89.95
111.65
89.97
89.99
112.00
111.73
90.00
90.01
90.01
88.59
89.99
89.96
90.21
89.96
65.89
89.90
90.00
89.99
85.83
89.99
89.88
90.04
89.30
100.42
100.42
89.91
111.62
108.43
90.09
89.76
90.12
89.99
89.98
90.00
90.00
90.05
84.67
74.31
89.95
90.64
100.50
89.98
90.08
111.95
90.00
89.94
89.98
89.99
89.80
104.45
90.10
95.43
89.77
102.92
60.03
90.00
90.14
89.99
89.99
104.36
90.22
90.00
104.34
90.05
88.45
89.95
90.03
89.95
90.11
89.97
65.69
91.78
90.00
85.87
90.00
90.01
90.22
89.90
89.96
89.96
90.99
89.88
89.98
75.93
90.03
89.96
108.11
90.01
90.02
112.53
97.12
89.98
74.32
105.18
90.01
94.90
78.36
81.11
89.98
89.97
89.87
87.96
89.80
89.65
89.90
89.97
109.44
89.89
90.18
90.0
90.0
90.0
90.0
90.0
90.0
90.0
90.0
180.0
90.0
148.9
90.0
90.0
90.0
90.0
180.0
90.0
180.0
90.0
137.9
180.0
90.0
90.0
90.0
90.0
90.0
180.0
180.0
180.0
180.0
90.0
90.0
90.0
90.0
180.0
180.0
180.0
180.0
90.0
180.0
90.0
88.9
180.0
180.0
90.0
90.0
90.0
90.0
90.0
90.0
90.0
90.0
90.0
90.0
90.0
-180.0
180.0
-89.9
180.0
0.0
-121.5
-90.0
-0.0
0.0
-180.0
89.9
180.0
0.0
89.9
-89.9
0.0
179.8
0.0
-180.0
90.1
0.0
90.0
-180.0
180.0
-90.0
-90.0
0.0
0.0
0.0
0.0
-89.8
-0.0
90.1
-90.0
0.0
0.0
0.0
0.0
0.3
0.0
89.8
-89.4
0.0
0.0
90.0
-0.0
-0.0
-180.0
179.8
-90.0
-90.1
180.0
56.5
-0.0
-90.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
60.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
90.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
180.0
SR-Fun
1.00
0.99
0.99
0.98
0.98
0.98
0.98
0.97
0.97
0.97
0.97
0.97
0.97
0.97
0.97
0.97
0.97
0.97
0.97
0.96
0.96
0.96
0.96
0.96
0.96
0.96
0.96
0.96
0.96
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.94
0.94
0.94
0.94
0.94
0.93
0.93
0.93
0.93
0.93
0.93
0.93
0.93
0.93
0.93
0.93
0.93
0.93
a
The date of structure entries corresponds either the publication date or the deposition date when there is no publication
associated with them. The self-rotation peak heights (SF-Fun) are relative to the crystallographic origin peak. X-ray fiber
diffraction structures and the inverted Fourier transformed structure factors from electron microscopic structures were
excluded for analysis. Errors in some deposited structure factor/intensity data existed but should not affect the overall
conclusions of systematical analysis. For example, the first entry 1Z9O deposition in the table has two sets of structure factors,
the first including the cross-validation set and intensity data. However, the cross-validation set had a uniform value and
intensity data fields were blank. Moreover, the second set was a duplicate of the first set without intensity fields; and the
deposited structure factors were actually only at 2.78 Å resolution, and did not corresponding to the resolution of the reported
structure.
11
Table S2. Identification of correct space groups from incorrect P1 space groups
Accession Code
Reference citation number
3O37
37
4HYO
4
4HZ3
4
1Z9Ob
38
1WW5
39
3EBN
40
4EY0
41
3RL0
42
4LL7
43
3MUU
15
3OVJ
44
1ZBL
45
1JXO
34
1EGW
35
4LOH
46
3RFU
47
3HTM
48
4DX9
49
3NY2
50
3RK2
42
3F7F
43
Original Incorrect P1
a,b,c (Å)
,, (°)
Resolution (Å)/Rwork (%)
35.667; 63.848; 79.299
89.92; 89.99; 89.80
2.0 Å/22.7%
44.035; 63.452; 63.477
90.03; 89.99; 89.99
1.65Å/16.5%
43.965; 63.396; 63.311
89.99; 89.95; 90.00
1.70Å/17.4%
50.048; 50.033; 90.287
90.01; 90.00; 60.03
1.90Å/21.9%
47.623; 57.648; 64.643
89.96; 111.65; 90.14
2.20Å/19.8%
51.395; 51.350; 51.390
112.22, 112.00, 104.46
2.4Å/20.8%
54.900; 59.200; 79.200
90; 90; 90
2.8Å/19.9%
53.745; 127.357; 142.725
107.49; 90.01; 90.05
3.80Å/30.8%
28.069; 49.576; 149.950
89.96; 84.67; 89.98
2.31Å/23.1%
67.482; 158.430; 160.676
60.42; 89.80; 89.65
3.29Å/24.0%
9.536; 26.008; 25.803
62.28; 88.59; 88.45
1.80Å/20.7%
36.897; 44.983; 62.280
83.63; 89.90; 65.69
2.20Å/21.7%
45.009; 53.410; 63.262
89.96; 90.00; 91.78
2.30Å/22.0%
41.371; 60.696; 63.987
115.18; 89.99; 90.00
1.50Å/20.6%
36.500; 59.209; 59.205
83.98; 85.83; 85.87
2.25Å/18.8%
44.150; 72.980; 329.950
89.96; 90.04; 90.22
3.20Å/23.6%
36.800; 88.70; 88.700
90.80; 89.30; 89.90
2.50Å/21.7%
75.622; 122.207; 135.269
89.97; 89.99; 108.11
2.99Å/25.5%
29.390; 61.456; 72.806
65.05; 89.98; 90.01
2.61Å/23.3%
27.627; 39.769; 102.275
83.38; 89.94; 89.87
2.20Å/23.0%
52.680; 117.478; 146.287
89.94; 89.77; 89.89
2.60Å/23.2%
Transformation
Matrix
[1 0 0]
[0 1 0]
[0 0 1]
[0 1 0]
[0 0 1]
[1 0 0]
[0 1 0]
[0 0 1]
[1 0 0]
[0 1 0]
[-1 0 0]
[0 0 1]
[0 1 0]
[-1 0 -2]
[0 1 0]
[0 -1 -1]
[1 0 1]
[-1 -1 0]
[1 0 0]
[0 1 0]
[0 0 1]
[0 1 0]
[-1 0 0 ]
[0 0 1]
[1 0 0]
[1 0 -2]
[0 1 0]
[0 -1 2]
[0 1 0]
[-1 0 0]
[0 1 1]
[0 1 -1]
[-1 0 0]
[1 -2 0]
[1 0 0]
[0 0 1]
[-1 0 0 ]
[0 0 -1]
[0 -1 0]
[0 -1 0]
[-1 0 0 ]
[0 0 -1]
[0 1 1]
[0 1 -1]
[-1 0 0]
[1 0 0]
[0 1 0]
[0 0 1]
[0 1 1]
[0 1 -1]
[-1 0 0]
[-1 0 0]
[-1 -2 0]
[0 0 1]
[0 -1 0]
[0 -1 2]
[-1 0 0]
[0 1 0]
[1 0 0]
[0 0 -1]
[1 0 0]
[0 1 0]
[0 0 1]
New Cell Parameters
a,b,c (Å)
,, (°)
Rwork/Rfree (%)
35.667; 63.848; 79.299
90; 90; 90
14.2%/22.7%
63.452; 63.477; 44.035
90; 90; 90
12.6%/17.5%
63.452; 63.477; 44.035
90; 90; 90
14.7%/19.1%
50.040; 50.040; 90.287
90; 90; 120
19.7%/29.0% (2.7Å)b
47.623; 120.166; 57.648
90; 90; 90
19.0/24.5%
57.288; 57.477; 63.001
90; 90; 90
18.1%/25.7%
54.900; 59.200; 79.200
90; 90; 90
19.9%/29.2%
142.725; 53.745; 127.357
90; 107.493; 90.
Correct Space Groupa
RPIM(%)
RSymm(%)
279.478; 158.430; 67.482
90; 90.32; 90
19.8%/25.9%
44.345; 26.794; 9.536
90; 91.73; 90
P212121
5.8%
9.3%
P4212
1.0%
2.5%
P4212
1.5%
3.7%
P3121
2.2%
7.1%
C2221
3.1%
5.1%
I222
3.0%
4.8%
P21212
5.0%
8.0%
P21
4.5%
7.4%
C2221
6.0%
9.4%
C2
7.1%
12.1%
C2
10.2%
81.989; 36.897; 62.280
90; 96.95; 90
C2
4.6%
45.009; 63.262; 53.410
90; 91.78; 90
17.6%/23.5%
60.696; 41.371; 63.987
90; 115.18; 90
14.2%/18.3%
88.011; 79.221; 36.500
90; 95.59; 90
P21
1.8%
44.150; 72.980; 329.950
90; 90; 90
P212121
5.8%
124.562; 126.313; 36.80
90; 90; 90
C2221
5.2%
75.622; 232.306; 135.269
90; 90; 90
C2221
7.0%
61.456; 132.023; 29.30
90; 90; 90
C222
8.7%
39.769; 27.627; 102.275
90; 96.62; 90
P2
4.3%
52.680; 117.478; 146.287
90; 90; 90
P22121
6.7%
28.069; 298.603; 49.576
90; 90; 90
P2
5.3%
C2
2.5%
Footnotes:
a
The top seven entries and three other entries have been re-refined, additional five
are still being partially re-refined, and the last ten entries included only Rsymm values.
b.
See footnote a of Table S1.
Table S3. R-symmetry of the experimental data of crystals assigned to P1 that have special NCSa
PDB
1W9S
3VQR
1TQE
2DDS
1TKU
4LOH
3UXJ
1P7H
2YWV
1W9T
1JXO
2D3E
3SJO
2PMC
3U5P
3UXV
3TQS
1U8T
2O4Q
3OJV
1TKS
1YUP
2DDR
1V40
1VG8
3KDM
4GUN
4NMU
3BX8
3I4W
2AEN
4J4Y
1WW6
3N9R
1ZLJ
2R8E
3D4G
3KSE
3U5O
3BP9
3FD2
3IA0
1ZBL
3MFB
4BBF
4BBE
1R52
3K2C
4NHZ
3IHF
2A8Y
3EBN
<I/I>
23.97
35.16
12.96
16.82
11.25
29.33
18.65
14.92
15.65
14.38
15.59
29.80
13.31
14.72
17.30
14.45
12.97
21.39
11.60
22.73
13.66
9.00
9.38
14.43
9.91
14.11
10.15
15.89
10.64
8.31
11.19
15.84
37.87
7.31
10.70
19.65
11.02
14.29
25.64
10.51
9.71
4.88
12.73
11.48
10.51
7.51
16.12
11.95
7.52
23.39
8.67
11.41
Lattice
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
P2
C2
P2
P2
P422
C2
P2
C2
P2
C2
C2
P2
C2
P2
C2
C2
P2
P2
C2
P2
P2
C2
C222
P2
P2
I422
P422
R3
P422
C2
C2
P222
C2
P2
C2
C2
I222
P2
P2
P222
C2
I222
Rsymm
0.018
0.019
0.022
0.023
0.024
0.024
0.025
0.026
0.026
0.027
0.028
0.029
0.029
0.030
0.030
0.030
0.031
0.032
0.033
0.033
0.034
0.034
0.034
0.035
0.035
0.035
0.035
0.036
0.037
0.037
0.038
0.038
0.039
0.039
0.040
0.040
0.040
0.040
0.040
0.041
0.041
0.041
0.042
0.042
0.042
0.043
0.044
0.044
0.044
0.045
0.046
0.046
PDB
3MER
1T91
3S19
4IT7
4LGG
1AAR
1U0D
3D6E
2OQY
2DYK
3BS0
1WW5
3BFV
3GUW
3SQP
4OIJ
1SQL
2H6L
3HTM
3PQJ
1EGW
2DDT
3GND
3H7N
3O33
4IFC
2AG5
2C7N
2QGQ
2YZJ
2Z66
1C03
2Q5C
3HQM
3RFU
4KMP
4N0O
2W89
3V1D
4DRW
4DX9
1UKP
3LK4
2WVA
3VEK
4MJM
3EXA
4N21
1R19
3F7F
3MGO
3VXO
<I/I>
15.72
11.29
21.97
8.90
12.59
0.52
18.73
15.70
13.68
12.64
12.63
32.88
9.28
8.16
29.02
9.03
9.70
11.81
23.16
13.35
9.95
15.40
6.85
25.87
18.35
9.08
11.98
13.33
10.70
6.32
8.70
11.68
15.16
20.68
11.59
10.57
18.93
13.00
11.11
7.03
10.66
5.71
13.95
11.42
9.17
10.49
11.70
8.06
11.79
11.34
19.88
14.87
Lattice
C2
C2
C2
P222
P2
C2
P2
C2
I422
C2
P2
C222
C2
P2
C2
C2
P2
R3
C222
P2
P2
C2
C222
P222
C2
C2
C2
C2
C222
R3
P222
I222
C2
C2
P222
C2
C2
C2
P222
C2
C222
P2
P222
P2
C2
I422
P2
R3
C2
P222
P222
C2
Rsymm
0.046
0.047
0.047
0.047
0.047
0.048
0.048
0.048
0.049
0.050
0.050
0.051
0.051
0.051
0.051
0.051
0.052
0.052
0.052
0.052
0.053
0.053
0.053
0.053
0.053
0.053
0.054
0.055
0.055
0.056
0.056
0.057
0.057
0.058
0.058
0.058
0.058
0.059
0.059
0.059
0.059
0.060
0.061
0.062
0.062
0.062
0.063
0.063
0.066
0.066
0.066
0.066
PDB
4K4X
1SED
3F9X
3WHK
4K4U
4K4V
3R6V
1YNF
2XH0
4J81
2C91
3L89
4INH
3WGJ
4EFR
2X1G
4JYM
2DRM
4E4M
1HV4
4PW5
2ZDC
3CFK
3R0R
3GK0
4EY0
2YHP
2YHQ
3WGN
1WW4
2PIH
3EVX
2IVK
4H82
4OIK
2A1F
2BS1
3R6Q
4K4T
4K4Z
2XLL
3K9M
3RZP
2CJF
3ES8
3S2F
3UDU
3H2V
2V8V
3NMW
3O37
3U58
<I/I>
7.16
10.42
20.32
11.94
7.89
8.95
8.08
8.56
9.61
15.46
8.24
9.01
10.47
18.46
13.93
5.07
4.61
14.01
5.72
20.75
6.35
10.12
11.29
8.76
10.18
6.36
4.51
4.51
11.91
26.88
10.47
10.42
13.70
6.65
4.19
6.96
12.28
10.21
7.10
6.62
12.21
8.69
8.09
6.08
9.01
8.22
11.94
12.30
6.67
5.41
14.94
16.29
Lattice
C222
R3
P2
P2
C2
C2
P2
C2
P2
C2
C2
P2
P2
C2
C2
P2
P2
P222
C2
C2
P2
R3
P222
P2
C2
P222
P2
P2
C2
C222
C2
P2
C2
P2
C2
C2
C2
P2
C2
C222
P222
P2
C2
C2
I422
P2
P2
P2
C2
P2
P222
I422
Rsymm
0.066
0.067
0.067
0.067
0.067
0.067
0.068
0.069
0.069
0.069
0.070
0.071
0.072
0.073
0.073
0.074
0.074
0.075
0.075
0.076
0.076
0.077
0.077
0.077
0.078
0.079
0.081
0.081
0.081
0.082
0.082
0.083
0.084
0.085
0.085
0.086
0.086
0.087
0.087
0.087
0.088
0.088
0.088
0.089
0.089
0.089
0.089
0.090
0.091
0.092
0.092
0.092
PDB
3B4V
4F21
4GM3
1FFP
4EIL
3CJH
4J1R
4JYP
4LL7
4BI5
2RH0
2XI9
4K50
3OVJ
3S2G
4N9G
1FFO
4K4S
2XTQ
3UKF
3VO8
4HLY
4K4W
1FFN
1UKO
3OA8
4PW7
4J82
1YAJ
4LY6
2WH8
4LZZ
3MGT
1M9X
3V4E
2VCV
3DA7
2X7L
2IJZ
4FMY
3OA6
2POS
2VI5
4GEZ
4N9F
3HZ2
4M6D
3RCD
2GLJ
3EHR
<I/I>
9.97
5.68
6.80
7.93
8.11
9.72
7.18
6.25
13.36
5.63
4.88
15.18
5.17
3.55
6.53
10.75
5.36
6.41
6.04
5.84
16.98
12.15
8.84
4.07
27.43
7.01
6.83
11.88
6.34
5.34
11.71
6.56
25.91
9.94
22.88
7.53
22.49
7.44
4.01
9.87
6.67
17.96
9.79
5.92
10.07
21.24
6.48
7.65
2.94
14.39
Lattice
P2
P2
P222
C2
P222
C2
C222
P2
C222
P2
C2
C2
P2
C2
P2
P2
C2
C2
C2
P2
C2
C2
C2
C2
P2
C2
P422
C2
P2
P2
C2
P2
P222
P2
R3
P2
C2
C2
R3
C2
P2
C2
C2
C2
C2
F222
R3
P2
P2
I222
Rsymm
0.094
0.094
0.094
0.095
0.095
0.096
0.096
0.096
0.097
0.100
0.101
0.101
0.101
0.102
0.103
0.107
0.108
0.108
0.109
0.109
0.109
0.109
0.110
0.111
0.113
0.113
0.113
0.115
0.117
0.117
0.118
0.119
0.120
0.122
0.124
0.126
0.127
0.129
0.130
0.130
0.131
0.133
0.134
0.134
0.141
0.142
0.142
0.144
0.145
0.149
Footnotes:
a
Entries with Rsymm of zero using symmetry-expanded data for atomic refinement were omitted in this table.
Table S4. Refinement statistics of the revised TthRNAP holoenzyme structures
Myxopyronin complex
[3DXJ]a
P65 [P32]
3.0 (3.00-3.07)b
149,560
5.33 [3.33]
20.3(41.6) [23.5]
23.4(39.7) [28.9]
28,078 [56,018]
1 [2]
1 [2]
0
27,948 [55,882]
62 [68]
0.009 [0.008]
1.31 [1.57]
(-h-k,k,-l) [(-h,-k,l)]
(4.4) [50]
45.6
PDB ID
Space Group
Resolution (Å)
# Reflections
RO2Ac
R-factor (%)
Free R-factor (%)
Total # Atoms
# Complex
# Myx
# ppGpp
# Protein Atoms
# H2O/Ions
rmsd Bond (Å)e
rmsd Angle (°)e
Twin Operation
Twin Fractions (%)f
Rmerging for twinning (%)g
Footnotes:
a.
b.
c.
d.
e.
f.
g.
Alarmone ppGpp complex
[1SMY]
P65 [P32]
2.71 (2.71-2.78)
198,576
6.99 [4.67]
26.6(38.5) [18.6]
28.6(38.6) [26.6]
28,419 [53,552]
1 [2]
0
1 [2]
28,098 [42,518]
285 [10,962d]
0.009 [0.013]
1.28 [2.08]
(-h-k,k,-l) [(-h,-k,l)]
(12.8) [50]
41.0
Statistics for the original structures are shown in square brackets.
Statistics for the highest resolution shell are shown in parenthesis.
RO2A is the observation-to-atom ratio.
1SMY entry contains 1,897 Mg2+ and 34 Zn2+ in addition to 9,031 water molecules, which is 20%
of all atoms, but is missing 16% protein residues.
Root mean squares deviations (rmsd) to ideal bond length or bond angle values.
Only fractions of the minor twinning domain are shown. For fair comparison, we used the original
selection of cross-validation set in P65. Because correlation between the working and crossvalidation sets introduced by the twinning operation relating the pairs of reflections between (h, k,
l) and (-h-k, k, -l), which twinning results in the higher-symmetry pseudo P6522, free R-factors are
generally underestimated. An error of underestimation increases proportionally with the fractions
of the minor twinning domain or inverse proportionally with merging R-factors between paired
reflections related by the twin operations (h, k, l) and (-h-k, k, -l).
The merging R-factors of twinning operation are defined between (h, k, l) and (-h-k, k, -l)
domains.
Table S5. P21 Symmetry R-factors of the 1JXO data
Resolution Range (Å)
99.0-4.95
4.95-3.93
3.93-3.43
3.43-3.12
3.12-2.90
2.90-2.73
2.73-2.59
2.59-2.48
2.48-2.38
2.38-2.30
All
I/I
32.58
36.41
30.65
26.19
22.54
20.07
17.91
15.60
15.57
12.86
27.93
Rsymm
0.029
0.023
0.026
0.025
0.032
0.034
0.041
0.045
0.049
0.54
0.028
Rmeas
0.041
0.032
0.036
0.035
0.046
0.048
0.057
0.064
0.070
0.076
0.040
CC1/2
0.987
0.995
0.992
0.995
0.989
0.993
0.991
0.988
0.986
0.988
Table S6. P21 1JXO re-refinement statistics
Space group
P21
Unit Cell parameters
a=45.009 Å, b=63.262 Å, c=53.410 Å, =91.78°
Resolution
54-2.30 Å (2.359-2.300 Å)
Number of reflections
12,667 (925)
Rworking
17.3%(17.3%)
Rfree
23.6%(22.7%)
Number of atomsa
2,656
Observation/Atom Ratio, RO2A 4.8
<B>
29.8 Å2
Bond length deviations
0.008 Å
Bond angle deviations
1.18°
Ramachandran Plots
Preferred regions
97.6%
Allowed regions
2.1%
Disallowed regions
0.4% (1 residue)
Footnotes:
a
For comparison, the original P1 structure had 4,207 atoms.
Table S7. P21 Symmetry R-factors of the 1EGW data
Resolution Range (Å)
99.0-3.23
3.23-2.56
2.56-2.24
2.24-2.04
2.04-1.89
1.89-1.78
1.78-1.69
1.69-1.62
1.62-1.55
1.55-1.50
All
I/I
16.99
19.58
18.07
14.54
13.88
13.52
12.33
11.89
11.14
9.31
16.93
Rsymm
0.045
0.053
0.069
0.077
0.082
0.091
0.089
0.089
0.095
0.114
0.053
Rmeas
0.050
0.075
0.097
0.109
0.116
0.129
0.126
0.126
0.135
0.161
0.075
CC1/2
0.993
0.995
0.979
0.974
0.979
0.970
0.970
0.985
0.981
0.963
Table S8. P21 1EGW re-refinement statistics
Space group
P21
Unit Cell parameters
a=60.696 Å, b=41.371 Å, c=63.987 Å, =115.18°
Resolution
58-1.5 Å (1.539-1.500 Å)
Number of reflections
43,991 (3,224)
Rworking
14.2% (14.9%)
Rfree
18.2% (18.4%)
Number of atomsa
3,165
Observation/Atom Ratio, RO2A 13.9
<B>
23.5 Å2
Bond length deviations
0.004 Å
Bond angle deviations
1.10°
Ramachandran Plots
Preferred regions
99.2%
Allowed regions
0.8%
Disallowed regions
0%
Footnotes:
a
For comparison, the original P1 structure had 5,996 atoms, which would not allow individual
anisotropic B-factors to be refined. With the correct symmetry, one can now refine individual anisotropic
15
B-factors. Since two orientations of the DNA duplex resulted in only three nucleotides per strand having
two identities for the given sequence, the number of total atoms can be reduced to 2,591 when all
alternative conformers having the same identity are removed for re-refinement. This resulted in slightly
increased Rworking and Rfree of 15.3% and 19.2%, respectively. In this case, the observation/parameter ratio
increased to 1.88 from 1.54. If the observation/parameter ratio is included in validation, it is most likely
that the structure being refined with the least number of atomic parameters has the highest quality.
Unfortunately, the current validation does not the observation/parameter ratio into account such as in Rfree
calculations.
Table S9. Symmetry R-factors of the 3MUU dataa
Resolution Range(Å)
C2
Rsymm
0.063
0.087
0.103
0.119
0.146
0.213
0.279
0.383
0.447
0.510
0.121
P3
Rsymm
0.133
0.191
0.230
0.252
0.288
0.364
0.436
0.523
0.605
0.615
0.224
P321
Rsymm
0.134
0.192
0.232
0.255
0.292
0.371
0.446
0.539
0.624
0.649
0.227
I/I
I/I
I/I
99.0-7.10
54.7
70.0
88.4
7.10-5.63
28.6
34.6
46.4
5.63-4.92
21.5
25.9
35.2
4.92-4.47
17.6
21.1
28.6
4.47-4.15
13.5
16.2
22.1
4.15-3.91
9.4
11.4
15.5
3.91-3.71
6.6
8.2
11.4
3.71-3.55
4.4
5.8
8.0
355-3.41
3.3
3.9
5.3
3.41-3.29
2.1
2.7
3.2
All reflections
17.0
20.3
27.4
Footnotes:
a
Reported Rmerging in P1 was 12.8%. Even without taking multiplicity into account, one of the three
2-fold axes is clearly retained as the crystallographic axis.
16
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