

research papers

Partial rotational lattice order–disorder in stefin B crystals
aDepartment of Biochemistry and Molecular and Structural Biology, Josef Stefan Institute,
Jamova 39, SI-1000 Ljubljana, Slovenia, and bCentre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins,
Jamova 39, SI-1000 Ljubljana, Slovenia
*Correspondence e-mail: [email protected]
At present, the determination of crystal structures from data that have been acquired from twinned crystals is routine; however, with the increasing number of crystal structures additional I4 and I422, yet one crystal exhibited a notable rejection rate in the higher symmetry An explanation for this behaviour was found once the crystal structures had been solved and refined and the electron-density maps had been inspected. The lattice of stefin B crystals is composed of five tetramer layers: four well ordered layers which are followed by an additional layer of alternatively placed tetramers. The presence of alternative positions was revealed by the inspection of electron-density score maps. The well ordered layers correspond to the crystal symmetry of I422. In addition, the positions of the molecules in the additional layer are related by twofold rotational axes which correspond to I422; however, these molecules lie on the twofold axis and can only be related in a statistical manner. When the occupancies of alternate positions and overlapping are equal, the indeed fulfills the criteria of I422; when these occupancies are not equal, the lattice only fulfills the criteria of I4.
disorders are being discovered. Here, a previously undescribed partial rotational order–disorder that has been observed in crystals of stefin B is described. The diffraction images revealed normal diffraction patterns that result from a regular The data could be processed in space groupsKeywords: crystal disorder; rotational order–disorder; twinning; stefin B.
PDB reference: stefin B, 4n6v
1. Introduction
In the last two decades, there has been an exponential growth in the number of protein
crystal structures that have been deposited in the Protein Data Bank (PDB; Berman
et al., 2000). This increased deposition rate has been facilitated by advances in instrumentation,
software development and novel high-throughput approaches that have been introduced
in structural biology (Stevens et al., 2001
; Terwilliger, 2011
). The increasing number of crystal structures has provided insight into many important
biological processes which could not be provided by other approaches. With the increasing
rate of crystals with lattice disorders are more commonly observed; however, data are also
discarded or lattice disorders remain undetected. The most common type of disorder is (Yeates, 1997
), although other less frequent disorders, as indicated by diffraction disorders, have also been observed (Helliwell,
2008
). can be detected from scaling statistics and should now be routinely recognized and
considered during using software programs such as SHELXL (Sheldrick, 2008
), CNS (Brünger et al., 1998
), PHENIX (Adams et al., 2010
) and REFMAC (Murshudov et al., 2011
). Despite wide awareness of there remain instances in which the was not detected by the depositors. An analysis of the 11 367 structures with structure
factors that were deposited before February 2004 indicated that there were at least
78 instances with a high degree of confidence of (Lebedev et al., 2006
), whereas a recent analysis indicated that the number of twinned structures may be
as high as 3% (Afonine et al., 2010
).
Among other ; Dornberger-Schiff & Grell-Niemann, 1961
). These OD structures can be detected during data collection because of the modulated
intensities and linearly diffuse profiles of several classes of reflections. The OD
phenomenon has also been observed in various protein crystals, and in a few instances
has resulted in successful (Trame & McKay, 2001
; Kamtekar et al., 2004
; Wang, Kamtekar et al., 2005
; Wang, Rho et al., 2005
; Hwang et al., 2006
; Rye et al., 2007
; Tanaka et al., 2008
; Zhu et al., 2008
; Hare et al., 2009
; Tsai et al., 2009
). Recently, Pletnev et al. (2009
) reported the structure of the fluorescent protein FP480, in which the random distribution
of alternatively orientated tetramers created a statistically averaged I422 symmetry. Pletnev and coworkers named this disorder a `rotational order–disorder
structure'. In addition, the structure of human carbonic anhydrase was first identified
as a rotational order–disorder structure (Robbins et al., 2010a
); however, this identification was later corrected and refined as a structure with
an alternative selection of molecules in the (Robbins et al., 2010b
).
During our attempts to crystallize various oligomeric forms of the cysteine protease
inhibitor human stefin B, which is used as a model protein to study amyloid fibrillation
(Zerovnik et al., 2002) and amyloid-membrane perforation (Rabzelj et al., 2008
), several data sets were collected. The diffraction images indicated a nicely ordered
Yet, the data set from crystal 1 merged in I4, whereas the data set from crystal 2 was successfully merged in I422. In both instances, the first molecular-replacement solution resulted in an incomplete
structure. Specifically, four well ordered layers were separated by an empty space
that extended across the entire crystal. Here, we describe the structure solution,
and crystal packing of monomeric molecules of stefin B, which was identified as a
with rotational order–disorder packing.
2. Materials and methods
2.1. Preparation and crystallization
Stefin B was expressed as described previously (Rabzelj et al., 2005). cDNA of stefin B coding for the complete sequence of the protein from Met1 to Phe98
was inserted into pET-11a vector. Stefin B was expressed in Escherichia coli BL21(DE3) cells after induction with IPTG and was purified using papain Sepharose
and The expressed protein differs from the wild-type protein (GenBank accession No. AAH10532.1)
at one residue. Namely, the cysteine at position 3 was replaced by serine to prevent
the formation of disulfide-linked dimers. Stefin B with this exact sequence has not
been crystallized before.
The monomers were isolated using −1 in 10 mM Tris–HCl, 100 mM NaCl pH 7.5.
and concentrated to 19 mg mlThe crystals, which were rectangular cuboids with dimensions of 0.2 × 0.2 × 0.5 mm, were obtained by sitting-drop vapour diffusion using 1 µl protein solution mixed with 1 µl reservoir solution (0.1 M CAPS buffer pH 10.5, 0.2 M Li2SO4, 2 M ammonium sulfate) and equilibrated against 1 ml of the same reservoir solution for a few days at 293 K. All crystals grew in identical conditions. The crystals appeared to be single and well shaped. Crystals were transferred into cryobuffer [reservoir solution supplemented with 20%(v/v) glycerol] for 5 s prior to flash-cooling in liquid nitrogen.
2.2. Data processing
Two sets of diffraction data (data sets 1 and 2) were collected on beamlines BM14
at the ESRF, Grenoble and PX14.1 at BESSY, Berlin (Mueller et al., 2012), respectively, at 100 K. The diffraction images were processed with HKL-2000 (Otwinowski & Minor, 1997
) or XDSApp (Krug et al., 2012
; Kabsch, 2010
).
Diffraction data were collected from several crystals; however, only two data sets
were chosen for this structural analysis. Both crystals 1 and 2 diffracted to 1.8 Å
resolution. 360 images with an oscillation of 1° per image were collected from crystal
1, while only 180 images (with the same oscillation) were collected from crystal 2
to 1.8 Å resolution owing to the diminishing diffraction quality. The observed diffraction
patterns indicated the presence of a single , Supplementary Figs. S1 and S2). The data could be scaled in several space groups
(C2, F222, I4 and I422), which all gave reasonable statistics, with I422 being the highest symmetry The Rmerge and numbers of rejections for both sets in various space groups are shown in Table
1
. To compensate for the differences in the data completeness in higher resolution
shells for the two crystals, only data truncated to 2.5 Å resolution were included
in this comparison; elsewhere all data were used. The comparison shows that the data
from crystal 2 could be scaled with approximately the same rejection rate in space
groups I422 and I4, whereas the data from crystal 1 were merged in I422 with a considerably higher number of rejected reflections and an almost doubled
Rmerge compared with I4. The data-collection and scaling statistics for both sets are summarized in Table
2
. Diffraction images are made available to the community by the TARDIS web server
(https://tardis.edu.au/).
|
|
![]() |
Figure 1 The diffraction pattern of stefin B crystal 1. (a) Complete diffraction image, (b) enlarged section and (c) the same enlarged section after integration with HKL-2000, containing the positions of predicted reflections with h, k and l indices shown. |
Because Table 1 shows intriguing scaling of the data, the data were checked for possible The intensity statistics showed a nearly perfect shape of the N(Z) plot (Fig. 2
), a Wilson ratio 〈I2〉/〈I〉2 of greater than 2 and 〈L2〉 close to 0.5 for both sets (Table 3
), excluding the possibility of twinning.
|
![]() |
Figure 2 Twinning statistics. The experimental N(Z) plots calculated from the measured data for data set 1 (blue crosses) and data set 2 (red dots). The exponential and sigmoidal black lines correspond to nontwinned and perfectly twinned theoretical instances, respectively. |
H-test (Yeates, 1997) and the Britton test (Fisher & Sweet, 1980
) did indicate perfect However, the use of these two tests is not suitable in this particular case. These
two tests are based on the comparison of intensities within pairs of potentially twin-related
reflections. Since the only possible for I4 is k, h, −l, which corresponds to the present in I422, and since our data could also be merged in I422, these intensities were indeed almost equal. Therefore, resolution of the possible
crystal was postponed until analysis of the refined structure.
In addition, the largest off-origin peaks in the
are 5.4 and 5.8% of the origin peak for data sets 1 and 2, respectively, indicating the absence of significant pseudotranslation.2.3. in I422 using data set 2
The structures were solved by Phaser crystallographic software (v.2.5.2; McCoy et al., 2007) from the CCP4 suite (v.6.3.0; Winn et al., 2011
). was performed with REFMAC5 (Murshudov et al., 2011
) and MAIN (Turk, 2013
). Additionally, real-space model corrections and molecular manipulations were performed
with Coot (Emsley et al., 2010
) and MAIN (Turk, 2013
). The data were verified with phenix.xtriage (Adams et al., 2010
).
Systematic absences clearly indicated the absence of a screw axis. Initial 1stf; Stubbs et al., 1990) with data set 2 in I422 found four positions in the Electron density for all four molecules was well defined. These four molecules formed
four continuous layers that were parallel to the ab plane of the which was intersected by one layer of empty space that was located directly on the
twofold axis. Although a Matthews coefficient analysis did not allow us to ambiguously
determine the number of molecules in the (the Matthews coefficients were 3.31, 2.65 and 2.21 Å3 Da−1, respectively, for four, five and six molecules in the asymmetric unit), it was evident
from the crystal packing alone that there should be more than four molecules in the
to build a three-dimensional crystal lattice.
The R factor of 0.30 with B factors of between 20 and 30 Å2 for all four molecules. Visual inspection of electron-density maps of the molecular-replacement solution with four stefin B molecules revealed uninterpreted density within the empty layer. However, the resulting density maps were not clear enough to build the missing molecules. In part, this was a consequence of the potential overlap that resulted from the twofold crystal symmetry axis that passed through this empty layer. Therefore, we decided to solve the structure by in P1.
of this partial model resulted in an2.4. in P1 using data set 1
B factor of between 25 and 30 Å2 for the eight initially placed tetramers, whereas for the ninth and tenth tetramers the B factor was significantly higher (>50 Å2).
with a monomer as a search model resulted in 32 positions, which formed eight tetrameric rings in four continuous layers and an empty layer. Additional molecular-replacement attempts using the positions of the first eight tetramers as a fixed solution and a tetramer as a search model resulted in placement of the ninth and tenth tetramers. of this model resulted in an averagedOwing to the weak density in the region of the ninth and tenth tetramers, we inspected
the position of the additional tetramers using score maps as implemented in MAIN (Turk, 2013). These maps confirmed that the placement of the ninth and tenth tetramers was indeed
correct; however, the score maps, as well as the density around the models (shown
in blue in Fig. 3
a), revealed some additional unexplained density which indicated the presence of additional
helices. Because the stefin B structure contains only one helix, these additional
unassigned helical density regions could only be explained by two additional tetramers
which overlapped with the already positioned ninth and tenth tetramers by twofold
symmetry.
![]() |
Figure 3 Density evidence for structural disorder. The pairs of overlapping molecules 0A, 0B and 5A, 5B are shown in red and blue, respectively. Score OMIT maps around molecules A and B are shown in grey and black, respectively. (a) Score OMIT 2Fo − Fc map around the helices of tetramers 0A and 0B. The score OMIT map was calculated by omitting both overlapping tetramers 0A and 0B. (b) Score OMIT Fo − Fc map for intersecting strands of molecule 0. The OMIT maps were calculated by omitting the 0A and 0B tetramers/molecules separately and were combined into one image. |
Because the coordinate-system origin of P1 was chosen to superimpose with the solutions for the origins of space groups I4 and I422, we could exploit the twofold rotational axis which was present in the empty layer
in the I422 and modelled the alternate conformations of the ninth and tenth monomers. Indeed,
in these alternative positions the newly placed helices of the models that are shown
in red perfectly superimposed on the unassigned helical density (Fig. 3a).
2.5. and model rebuilding
Occupancy REFMAC against data set 1 was performed by screening different occupancies of the alternatively positioned molecules and by comparing their average B factors after until a match was found. The occupancy implemented in MAIN was performed by defining the two overlapping molecules as members of one overlapping group and by restraining the average B factor of these two molecules to the rest of the structure.
withStructure solution in P1 revealed that the entire structure, which included the alternatively placed tetramers,
still corresponds to the symmetry operators of I4. Therefore, to continue, was performed in steps in I4 using NCS restraints between individual stefin B molecules. Firstly, only eight
molecules (1–4 and 6–9) in the four initial layers were refined, and solvent molecules
in their vicinity were added. The stefin B molecules in layer 0 (molecules 0A, 0B, 5A and 5B) were then added in both overlapping positions, with approximate occupancies deduced
from their correlation with the electron-density maps using MAIN. The structures of the alternatively placed molecules were not manually corrected
owing to ambiguous density; however, these structures were updated from the other
molecules using the NCS operators. After and the further addition of solvent molecules, occupancy was performed by two independent approaches as described in §2. Both approaches gave similar results. The corresponding occupancies are summarized
in Table 4
. After a few additional cycles of manual structure improvement and the final Rwork and Rfree factors were 0.19 and 0.23, respectively (Table 5
).
|
|
The electron densities for the alternatively placed molecules 1 and 6 were well defined
in the β-strand and helix regions (as confirmed by the OMIT maps in Fig. 3b), whereas the density in the loop regions was weaker. Additionally, there are some
peaks that overlap with both alternatively placed molecules, which are most likely
to correspond to partially occupied solvent molecules (not modelled).
The coordinates and structure factors for data set 1 were deposited in the Protein Data Bank under accession code 4n6v, whereas a download link for diffraction images and the partially refined structure of data set 2 are included in the Supporting Information.
3. Results
3.1. Structure description
The crystals of human stefin B contained the complete sequence of the protein from
Met1 to Phe98. The P1 contains 40 molecules, whereas the I4 and I422 cells contain 80 molecules, with ten or five molecules in the respectively. The stefin B molecules share the cystatin fold, which is similar to
that determined in the of the complex with papain and which is composed of a five-stranded β-sheet that is packed against an α-helix. Cystatins are wedge molecules that utilize the N-terminus and two binding
loops for binding into the active-site cleft of cysteine (Stubbs et al., 1990).
The non-overlapping stefin B molecules are well defined by the electron-density maps.
The density did not enable us to ambiguously model the first seven residues that were
positioned in the proximity of the fourfold symmetry axis. The Pro36 residue and Pro74–Pro79
loop are well defined in molecules 4 and 6 only, whereas in the other molecules their
positions and conformations are not unambiguous; therefore, these characteristics
were acquired from molecule 6 using the NCS operators. A total of 19 residues were
modelled in alternate conformations. Residues Val48 and Asp77 are Ramachandran plot
outliers. Val48 is unambiguously defined by the electron-density maps, whereas Asp77
lies in an less ordered region between Pro74 and Lys78 which has continuous density
corresponding to the main chain. However, the density maps are rather featureless
and do not enable exact positioning of the Cβ and O atoms. Interestingly, an energetically unfavourable conformation of Val48 lies
in the region where domain swapping occurs (Jenko Kokalj et al., 2007).
All eight non-overlapping molecules are highly similar. The r.m.s.d. of all Cα atoms between any pair of these structures is lower than 0.32 A. When compared with
the structure of stefin B in complex with papain (Stubbs et al., 1990), the r.m.s.d.s are slightly higher and are in the range between 0.44 and 0.52 A.
The structure that was determined from crystal 2 is identical to that of crystal 1; however, the occupancy of the alternatively placed molecules is different (close to 0.5 for all four molecules), which allows scaling in I422 with a low number of rejections. The structure from crystal set 2 was not deposited owing to the lower quality of the diffraction data, which resulted in a less well defined structure. In particular, the areas of the first and second binding loop around residues Pro36 and Pro79 were not visible in the electron-density maps.
3.2. Crystal packing
The
is composed of monomers that are packed in tetrameric rings (as also called tetramers) positioned on top of each other along the fourfold axis. When viewed from the side, the tetramers have a plate-like shape; therefore, we marked these tetramers with the symbol `('.In tetramers, the first binding loop, 46-QVVAG-50, packs against the groove that was
formed at the side of the next molecule in the ring. The disordered N-termini fill
the area in the centre around the fourfold crystal symmetry axis, whereas the residues
of the second binding loop (74-PHENKP-79) are positioned on the et al., 2001) and in the structure of the domain-swapped tetramer of human stefin B (Jenko Kokalj
et al., 2007
.) Additionally, the second binding loop in stefin B was involved in the tetrameric
loop exchange by the handshake mechanism of His75 in the latter structure. Hence,
the packing that is described here does not contain any similarity to these packing
and swapping mechanisms. Additionally, assembly analysis using PISA (Krissinel & Henrick, 2007
) indicates that the tetramers are not a biologically relevant assembly.
Pairs of tetrameric rings are packed together forming three types of octamers, two
of which have 422 a) is formed from two pairs of tetrameric rings of molecules 1–2 and 8–9 [denoted `)('],
whereas the second 422-type octamer (green, Fig. 4
b) is formed from two pairs of tetrameric rings of chains 3–4 and 6–7 [denoted `()'].
Both twofold axes of the `()' and `)(' octamers lie parallel to the ab plane. In the first type of octamer the twofold axis contacts are in the region 11–14
with the sequence PATA, whereas in the second type the twofold axis contacts are in
the region 85–88 with the sequence YQTN. These two interacting regions are positioned
on opposite sides of the stefin B molecules and are marked in red and blue, respectively,
in Fig. 4
. Hence, the stacking of the two tetrameric rings differs in these two types of octamers.
![]() |
Figure 4 Three types of octamers are shown. (a) Octamer `)(', (b) octamer `()' and (c) octamer `(('. Green and black lines represent twofold and fourfold rotational axes, respectively. Molecules from octamers in (a) and (b) are shown in grey and green, respectively. The octamer in (c) is at the interface of the the octamers in (a) and (b). The colours of the molecules correspond to the colours in the octamers in (a) and (b). The fourfold rotational axis is crystallographic, whereas the twofold axes are operations relating to the molecules in the octamers in (a) and (b), but not that in (c). The two regions that are involved in the interactions between the tetramers are shown in blue (11-PATA-14) and red (85-YQTN-88). |
At the interface between the 422-type octamers, layers from two neighbouring tetramers
build the third octamer type, in which the PATA and YQTN regions interact with each
other [denoted `(('; Fig. 4c]. Together, these octamers form stacks, which are termed stacks 1 and 2. The cross
422-type octamer interaction, PATA–YQTN, is characterized by a hydrogen bond between
the NH group of Ala14 and the carbonyl group of Tyr85, whereas no hydrogen-bonding
contacts are present in the PATA–PATA and YQTN–YQTN interfaces. Horizontally, the
two antiparallel stacks of octamers make contacts in the outer tetramer layers 1 and
4, whereas the molecules from the intermediate two tetrameric layers 2 and 3 interact
only vertically with molecules within the stack.
In the crystal (Fig. 5), the 422-type octamers build two octamer layers which correspond to the four vertical
layers of tetramers that were initially found by These two octameric layers are packed together such that two pairs of octamers pack
antiparallel within each layer, with each vertical pair in a different stack. In these
alternative positioning of octamers, the green octamers always neighbour grey octamers
and vice versa. The same octamer type always has five octamer neighbours of the other type, specifically
four around it within the same layer and one above or below within the same stack.
The octamers in the layers are not related by proper NCS symmetry. A view perpendicular
to the ab plane of the crystal into tetramer layers 1 and 2 shows (Figs. 5
a and 5
b) that the tetramers are oriented differently. However, in the crystal, the two octamer
layers are related by the which corresponds to the twofold axis that is present in I422.
![]() |
Figure 5 Crystal lattice organization. (a, b) Tetramer layers 1 and 2 of the crystal expanded into the surroundings by crystal symmetry operators. Molecules 1 and 2 are shown in green and molecules 6 and 7 are shown in grey, which correspond to the colours that are used in Fig. 4 ![]() |
As shown in Fig. 5(c), the average positions of the Cα atoms of two tetramers in the stack are 25.0 ± 0.5 Å apart; however, the centres
of the molecules in the stacks are shifted 3 Å in opposite directions along the c axis, which corresponds to the z coordinate. The centre of molecule 9, which is the last in the second stack, is at
103 Å, which is ½c − 25 Å, whereas the centre of molecule 4, which is the last in the first stack, is
at 100 Å; thus, the centre of molecule 4 is 3 Å lower than ½c − 25 Å. The same difference is found between the z coordinates of the centres of molecules 1 and 6. However, in the ab plane at z = 0 and z = c/2 the two stacks provide different spacing, which is reflected in the z coordinate of the molecules in the overlapping layer. In layer 0, there is less space
between the first stack than between the second stack. As a consequence, the centres
of overlapping molecules of the first stack, 0A and 0B, are at 0.1 and −0.1 Å, respectively, whereas the centres of overlapping molecules
of the second stack, 5A and 5B, are 2.8 and −2.8 Å apart, respectively. At z = c/2, the situation is the reverse.
In layers 0 and 5, a direct vertical connection between two stacks is formed by tetramer
0 (Fig. 6a), which makes a connection between tetramers 9 and 1. Owing to the crystal symmetry
between the connecting molecules 1 and 9, a symmetrical interaction of the PATA regions
from both of the tetramers is available. Therefore, tetramer 0 has two possible energetically
equivalent positions, making a `)(' octamer with tetramer 1 and a `((' octamer with
tetramer 9 (Fig. 6
a, centre) or vice versa (Fig. 6
a, right). In contrast, tetramer 5 does not connect both stacks owing to the 6 Å separation
of the stacks (Fig. 6
b).
![]() |
Figure 6 Observed orientations of the alternately positioned tetramers 0 (a) and 5 (b). The overlaid configuration is shown on the left, whereas the packing of individual molecules is shown in the centre and on the right. The colour code from Fig. 5 ![]() ![]() |
Therefore, tetramer 5 has two possible equivalent positions, assembling a `((' octamer
with tetramer 4 with no contacts with tetramer 6 (Fig. 6b, centre) or vice versa (Fig. 6
b, right).
Although tetramer 0 directly connects both stacks vertically, the increased separation between tetramers 4 and 6 allows tetramer 5 to choose between the matching `()' or cross `((' interfaces. The crystal packing suggests that the hydrogen bond that bridges the `((' PATA–YQTN packing is the favoured attachment; however, the `((' interface cannot be excluded. The higher average B factor of tetramer 5 and the lower quality of the corresponding electron-density map around these molecules suggest that they are the consequence of the lack of the second crystal contact. As the stability does not depend on these molecules, one can not exclude the possibility that site 5 is not fully occupied or that there are four possibilities for the packing of tetramer 5 into this region.
Hence, the analysis suggests that packing alone is not responsible for differentiation
between the two possible space groups. When the occupancy of the overlapping tetramers
0A, 0B and 5A, 5B is equal, then the twofold rotational axis (shown with a green line in Fig. 5c) becomes the crystallographic twofold axis, thereby changing the from I4 to I422, corresponding to crystal 2.
4. Discussion
The determination of macromolecular structures from crystals that exhibit I422 indicated that there is only a single, well ordered In addition, a found no support in the diffraction patterns (Fig. 1, Supplementary Figs. S1 and S2). Initial molecular-replacement attempts in space
groups with higher symmetry (I422, I4, F222 and C2) resulted in unreasonable crystal packing; therefore, the most important parts of
the were performed in P1. This confirmed that the should be at least I4.
Nevertheless, potential I4 which used the twofold symmetry operator (k, h, −l) had to be explored.
inMerohedral and Table 3
. Because the positions of eight of the ten molecules in the of I4 were consistent with the I422 symmetry, the tests may not be sensitive enough to indicate therefore, we decided to analyze the crystal packing to find support for the twining or to reject this phenomenon.
4.1. Perfect or partial rotational order–disorder
Twins are regular aggregates consisting of crystals of the same species joined together in some definite mutual orientation
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Figure 7 Two possible twinning scenarios. The colour of the molecules corresponds to the colour code used in Fig. 5 ![]() |
In the first A–5A, which are shown in blue in Fig. 7, and of molecules 0B–5B, which are shown in red, is identical, whereas in the second scenario the pairs with
the same occupancy should be 0B–5A and 0A–5B. The occupancies of these molecules refined against the data for crystal 2 are 0.53,
0.47, 0.51 and 0.49 for molecules 0A, 0B, 5A and 5B, respectively. One might disregard the small differences, consider the occupancies
to be equal and conclude that is a plausible explanation for the from data set 2 (which could be merged in I422 without considerable rejection of data). However, one cannot disregard the differences
between the occupancies of the same molecules in crystal 1, which were 0.58, 0.42,
0.51 and 0.49 for molecules 0A, 0B, 5A and 5B, respectively. These differences are too large to accept the potential scenario. Hence, we can conclude that the observed crystal disorder is not but rotational order–disorder which was produced by random combinations of unit cells
with different positions of tetramers 0 and 5 (as shown in Fig. 7
) in an otherwise well arranged crystal network.
4.2. How can such disorders be identified?
After a sufficient number of instances have been elaborated, the protocol may then be standardized; however, our report is only the second of such a
disorder. The main question is how soon in the determination can such a crystal disorder be detected and be properly considered: at the analysis of diffraction data, when solving the or during model building and refinement?In the work of Pletnev et al. (2009), the presence of extremely weak and diffuse reflections and an impossibly small
indicated the presence of a type of crystal disorder, whereas in our instance the
diffraction images did not provide any indication of the presence of an additional
nor was crystal disorder evident from the data processing and its analysis. Data
sets from both stefin B crystals could be scaled in several space groups with acceptable
scaling statistics.
Molecular replacement already worked satisfactorily in I422. Only after inspection of the crystal packing did it become apparent that the solution resulted in packing which could not produce a stable three-dimensional connected Returning to the P1 enabled us to build a stable however, the resulted in significantly higher B values and significantly more ambiguous densities of the two last placed tetramers. Only the inspection of score maps, as implemented in MAIN, enabled us to determine the alternative positions of the last two tetramers. Hence, visual map inspection, together with the score maps, which can average local density by the convolution theorem, were crucial steps in identifying the double occupancy within a layer of the crystal lattice.
The final conclusion regarding the correct
and potential crystal was deduced from comparison and analysis of the occupancies of overlapping molecules. This result indicates that the structures had to be refined in alternative space groups and that the structural data had to be analyzed before a final conclusion could be made regarding the correct and twin operators. To conclude, the analysis of each step, and all of them together, was crucial for determining this with order–disorder.5. Conclusions
The I4 demonstrates that protein crystals can contain a partial rotational order–disorder structure. In such instances, only part of the molecules in an is present in several overlapping orientations. Such an ordered disorder cannot be detected in the diffraction pattern or by scaling statistics owing to the regular crystal network of the rest of the structure.
of stefin B inIn macromolecular crystallography, it is a common practice to discard crystals that do not produce satisfactory solutions (such as an empty layer) and to continue with data collection from other crystals or even return to the wet laboratory until a crystal is found that enables the smooth resolution of a
Perhaps this work will encourage crystallographers to realise that different disorders may be more frequent than previously thought and that the strategy to continue with the data collection from multiple crystals with different percentages of disorders until a crystal with a low percentage of irregularities is found is not the only route to structure solution.Supporting information
PDB reference: stefin B, 4n6v
Supporting Information. DOI: https://doi.org/10.1107/S1399004714000091/yt5064sup1.pdf
Acknowledgements
This paper is dedicated to the memory of my (D.T.) friend and collaborator Nobuhiko Katunuma. Andreja Doberšek and Dr Mira Polajnar are gratefully acknowledged for their contribution to sample preparation and crystallization. This work was made possible by financial support from the Structural Biology Program (ARRS P1-0048) and Proteolysis and its Regulation Program (ARRS P1-0140) and the Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins (CIPKEBIP).
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