teaching and education
How to assign a (3 + 1)-dimensional
to an incommensurately modulated biological macromolecular crystalaThe Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198, USA, and bDepartment of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 985870 Nebraska Medical Center, Omaha, NE 68198, USA
*Correspondence e-mail: gborgstahl@unmc.edu
Periodic crystal diffraction is described using a three-dimensional (3D) q vectors for processing. Incommensurately modulated biological macromolecular crystals have been frequently observed but so far have not been solved. The authors of this article have been spearheading an initiative to determine this type of The first step toward structure solution is to collect the diffraction data making sure that the satellite reflections are well separated from the main reflections. Once collected they can be integrated and then scaled with appropriate software. Then the assignment of the is needed. The most common form of modulation is in only one extra direction and can be described with a (3 + 1)D The (3 + 1)D groups for chemical crystallographers are fully described in Volume C of International Tables for Crystallography. This text includes all types of elements found in small-molecule crystals and can be difficult for structural biologists to understand and apply to their crystals. This article provides an explanation for structural biologists that includes only the subset of biological symmetry elements and demonstrates the application to a real-life example of an incommensurately modulated protein crystal.
and 3D space-group symmetry. Incommensurately modulated crystals are a subset of aperiodic crystals that need four to six dimensions to describe the observed diffraction pattern, and they have characteristic satellite reflections that are offset from the main reflections. These satellites have a non-integral relationship to the primary lattice and requireKeywords: protein X-ray crystallography; aperiodic crystals; incommensurate modulation; superspace groups.
1. Introduction
Our laboratory is interested in solving the structures of incommensurately modulated protein crystals. These crystals have a fascinating diffraction pattern with satellite reflections surrounding the main reflections. Commensurate and incommensurate macromolecular crystallography, with examples of such effects, as well as ) and are also discussed in Chapter 8 of Rupp's Biomolecular Crystallography text book (Rupp, 2010). This paper concerns our symmetry analysis of the diffraction from (3 + 1)-dimensionally incommensurately modulated crystals of profilin:actin (PA) (Lovelace et al., 2008; Porta et al., 2011). This publication relies heavily on our study of an article by van Smaalen (2005), Chapters 1, 2 and 3 of van Smaalen's textbook on Incommensurate Crystallography (van Smaalen, 2007) and International Tables for Crystallography, Volume C, Chapter 9.8, Incommensurate and Commensurate Modulated Structures, by Janssen et al. (1999). We also studied Schönleber's lectures on Introduction to Superspace Symmetry from the Workshop on Structural Analysis of Aperiodic Crystals held in Bayreuth, Germany, and an article by Wagner & Schönleber (2009). Although these are excellent sources, they were written for small-molecule crystallographers and physicists and use language and examples that are not encountered in macromolecular crystallography. Therefore, we decided to write this paper for the next biological crystallographer who chooses to solve a modulated crystal, so that it will not be so difficult for them to understand and to confidently assign their to the crystal diffraction data.
and multiple crystal cases were reviewed by Helliwell (2008In this article the nomenclature common to periodic three-dimensional (3D) crystals is used with adaptations to a fourth dimension as needed (Janssen et al., 1999). It is noteworthy that in much of the aperiodic literature another formalism is used, where subscripts i = 1, 2, 3 are used to indicate the space directions (van Smaalen, 2007). This makes it easier to add more dimensions as needed. Hence, the symbols (a, b, c), (x, y, z), (hkl) and (α, β, γ) used in this publication correspond to (a1, a2, a3), (x1, x2, x3), (h1h2h3) and (σ1, σ2, σ3), respectively, in aperiodic crystallography. Vectors are in bold and scalar coefficients are in italics. This is pointed out here to help avoid confusion when reading the aperiodic literature.
Crystal periodicities can be categorized into three types (Fig. 1). The first is the most common case, where the crystal is periodic and the unit-cell contents are duplicated closely by the lattice translations (Fig. 1a). The second type is the case of a commensurate modulation. Here, the spacing of the satellite reflections relative to the main reflections is a rational value. The diffraction pattern can be indexed and integrated with any protein crystallography data reduction software with three integer indices as a In Fig. 1(b), the q vector which is used in aperiodic crystallography to index the satellite reflections relative to the main reflection has a rational value of 0.25 (or 1/4) and the modulation of the structure repeats every four unit cells. This means that the lattice parameters for indexing the satellite reflections are integer multiples (1, 2,…, n) and the can be described with a (Wagner & Schönleber, 2009). The third type is the case of an incommensurately modulated crystal. Here, at least one component of the q vector is irrational and cannot be calculated with a simple fraction (Fig. 1c). An accurate description of an incommensurately modulated crystal can only be obtained by describing the diffraction pattern with q vectors.
When an incommensurately modulated diffraction pattern is observed in protein crystallography, the sample is typically discarded in favour of a better behaving sample that can be processed with standard macromolecular crystallography software. As a consequence, incommensurately modulated macromolecular crystals are rarely reported and these types of structural modulations in the context of a macromolecular crystal are poorly understood. PA crystals can be chemically induced to form a peculiar incommensurately modulated diffraction pattern. More than 28 years ago (Schutt et al., 1989) it was found that when PA crystals are driven to a boundary by exposing the crystals to conditions known to promote actin filament formation they transform into an incommensurately modulated state that is thought to contain a of structural intermediates. By varying the solution conditions PA can be crystallized in either an `open' or a `closed/tight' state that corresponds to the nucleotide binding site opening and closing (Chik et al., 1996; Porta, 2011; Schutt et al., 1993). These two states are accompanied by a change in the c unit-cell dimension from 186 to 172 Å, respectively. Incommensurate diffraction was obtained using precession photography at room temperature from either open or closed states by shifting the pH to 6.0, a condition known to cause profilin to diffuse away from actin and actin filaments to form in vitro (Carlsson, 1979; Oda et al., 2001; Chik, 1996). This research provided the foundation for our continued studies.
Incommensurate modulations within crystals are a result of a displacement modulation that forms but does not align with the spacing of the basic ). In the periodic state, all reflections can be indexed by the three integer indices h, k and l such that
In the resulting diffraction pattern satellite reflections appear near the normal main reflections (see Fig. 2where a*, b* and c* are the vectors of the main reflections and basic With satellite reflections, the diffraction pattern becomes (3 + d) dimensional, where d is the number of satellite directions. The most common form of modulation is in only one extra direction (d = 1), and the diffraction patterns for these crystals have satellite reflections on either side of the main reflection (see Figs. 2a and 2b). This is called a (3 + 1)D modulated crystal. The diffraction pattern for this case can be indexed by the introduction of a single q vector such that
The positions of the satellite reflections are given by the q vector
A modulation wave can be parallel to one of the q coefficients in (3) would be zero. In more complicated cases two or three of the q coefficients can be nonzero (Fig. 2b). Also, multiple-order satellites evenly spaced from the main reflections can exist (see Figs. 2b and 2c). This is represented by the integer value m in equation (2). Interestingly satellites and multi-order satellites are predominantly in the high-resolution bins of data (see Fig. 5 of Lovelace et al., 2010). In 2008, the Borgstahl laboratory was able to reproduced the incommensurately modulated PA crystals from the Schutt laboratory and measured a single-rotation-style diffraction image from a room-temperature protein crystal (Lovelace et al., 2008). The data were indexed and the first q vector was measured for a macromolecular crystal. Research progress was hindered by the reversibility of the modulation at room temperature, perhaps due to crystal heating or radiation damage from the SuperBright FRE X-ray generator, which prevented the collection of a full set of diffraction data. We have since learned to cryocool crystals that were first crosslinked with acidic glutaraldehyde at room temperature and then cryopreserved with sodium formate (named xMod1 and xMod2, Table 1) and more recently not crosslinked and cryopreserved with D-glucose (gMod3, Table 1).
vectors, and in this case two of the scalar
‡. |
All of the (3 + d)D groups have been tabulated for d = 1, 2 or 3. For d = 1 there are 775 groups, for d = 2 there are 3338 groups and for d = 3 there are 12 584 groups (Stokes et al., 2011). A web site has been developed for searching all 775 (3 + 1)D groups listed in International Tables for Crystallography (https://it.iucr.org/resources/finder/; Orlov et al., 2008). These numbers are greatly reduced for biological crystals as there are only 65 chiral (or biological) three-dimensional space groups. Then there are only 135 (3 + 1)D, 368 (3 + 2)D and 1019 (3 + 3)D chiral groups (van Smaalen et al., 2013). An excellent primer to the three-dimensional space groups was written by Dauter & Jaskolski (2010) and can be used to review the symmetry elements found in protein crystals (Dauter & Jaskolski, 2010). Modulated PA crystals have a (3 + 1)D-type because they are modulated in only one direction.
The three cryocooled modulated PA data sets (Table 1) all have basic three-dimensional unit cells like that of the PA open-state crystals and have satellite reflections along b* (Fig. 3). The crystals differ in their resolution of diffraction and the extent of modulation, as indicated by their q vector and satellite intensity strength. The q spacing of the satellite from the main reflections varies from 0.2628 to 0.2829. A demonstration of their similarity to and differences from each other and from open-state crystals was made by calculating Rmerge between data sets (Table 2). The easiest Rmerge to calculate employs the main reflections only. This statistic demonstrates that the crystals are not isomorphous with each other. When the satellite intensities are added to the main reflections the Rmerge values improve and fall in a range of 25–37%, still not isomorphous. Clearly the three modulated structures are significantly different from each other and from the periodic crystal. The intensity of the modulation is also indicated by the strength of the satellite intensities (e.g. in Table 1 I/σ for the satellite reflections ranks their strength as follows: gMod3 > xMod2 = xMod1).
We have three cryocooled incommensurately modulated PA structures to solve of varying modulation strength (Fig. 3). When we look at the gMod3 crystal more closely in pseudo-precession photographs, it can be seen how the satellites relate to the main reflections (Fig. 4). Satellites are not always present (green rectangles, Fig. 4a), do not have to be of equal intensity (red rectangles, Fig. 4a) and can extinguish the main reflections (blue rectangles, Fig. 4a). The relative intensities between the satellite and the main reflections are analysed by resolution bin in Table 3 for the gMod3 crystal (see also Fig. 7 and Table 2 of Porta et al., 2011). The ratio of the satellite to main reflection intensity is lower in low-resolution bins and increases in the high-resolution bins. This is a general feature of modulated PA crystals.
‡Cases where Isatellite > Imain were excluded from the calculation. A similar table for the xMod1 crystal was published by Porta et al. (2011). |
2. Assignment of a to a protein crystal
A general procedure for the assignment of a et al. (1999). These steps are analysed here with our PA diffraction data and the description of the process is streamlined to include only the symmetry elements found in chiral molecule crystals. Hopefully this example will make these methods more accessible to protein crystallographers.
is given by Janssen2.1. Determine the and crystallographic point group
The Laue group of the diffraction pattern is the ). For biological crystals there are 11 Laue symmetry classes and 11 chiral crystallographic point groups (32 point groups for small molecular crystals). These are triclinic 1, monoclinic 2, orthorhombic 222, tetragonal 4 or 422, trigonal 3 or 32, hexagonal 6 or 622, and cubic 23 or 432 (Table S1 in the supporting information). Processing of the main diffraction data with D*TREK (Table 4) or with Eval15 (Table 5) shows that the is mmm and the is 222 (Pflugrath, 1999; Schreurs et al., 2010). There are only 23 (3 + 1)D groups with this symmetry.
in three dimensions that transforms every diffraction peak into a peak of the same intensity (except for deviations from Friedel's law caused by dispersion) (Rupp, 2010
|
|
2.2. Find the basic for the main reflections and a modulation wavevector
The main reflections are separated from the satellites, usually by intensity, and indexed. Reflection Fig. S2). Note that only noncubic classes are possible for (3 + 1)D modulations because a one-dimensional incommensurate modulation is incompatible with cubic symmetry. The satellites are usually assigned to the main reflections (can be extinct) that they are closest to. Then the direction and dimensions of the q vector are determined by fitting the satellites. If possible, it is preferable to place the q vector along a vector.
are used to select the for the main reflections (PA crystals are of the primitive orthorhombic , solution 11). Centring-type P orthorhombic has a low least-squares residual almost as low as P triclinic or P monoclinic. C centring is ruled out by the large least-squares residual. Eval15 processing also selects primitive orthorhombic as the (Table 5). This narrows the assignment down to 15 groups. Eval15 was used to define the q vector, which is in the direction of b*, for each crystal (Porta et al., 2011). We note that the magnitude of the q vector for xMod1 is close to 2/7 = 0.2857… and so 2/7 was used as an approximation when the diffraction was reindexed for display as a pseudo-precession in Fig. 4 (see also Fig. S1).
This can be seen in the analysis of just the main reflections (Table 42.3. Determine the 3D of the average structure
The average structure is commonly found by using the main reflections only and corresponds to averaging the contents of several unit cells in three dimensions. The and 5 show that the three-dimensional is P222. The three-dimensional for the average structure is determined from the main reflections. In our case, checks for centring rule out C, F or I and the lattice is primitive. along h, k and l (Fig. 4) indicate the presence of screw axes along all three dimensions. Since the data are of fairly low resolution the assignment of the was checked by performing with just the main reflections using MOLREP (Table 6) (Vagin & Teplyakov, 2000). This settles any uncertainty and the 3D of the average structure is P212121.
of the average structure is determined from the main reflections. This helps make a good choice for the starting structure in Tables 4
|
At this point a REFMAC crystallographic R values of 27–28% (Murshudov et al., 1997). The electron density of the average structure (Fig. 5) reveals that some parts of the structure are modulated more than others. The average electron density for profilin and subdomains 1 and 3 of actin are fairly well ordered. Actin domains 2 and 4 have very weak density, and this indicates that their motions are more dramatic in the modulation wave. The modulation function in actin appears to be more pronounced than that in profilin.
of the average structure can be performed and the resulting electron density observed. The average structures refined withAn illustration of the crystal contacts in PA crystals shows how the modulated regions correspond to the crystal directions (Fig. 6). For the PA case, the indexing showed that the modulation is along b (collinear with y) in the crystal, which corresponds to an `actin ribbon' formed by the (Schutt et al., 1991). It is likely that the protein undergoes a conformational change that affects the neighbouring PA molecules in such a way as to produce the observed modulation in the diffraction pattern (Schutt et al., 1991). The structural basis for the modulated PA diffraction pattern has not yet been determined.
2.4. Identification of the (3 + 1)D type
The (3 + 1)D α, β, γ of q. Next we find the compatible with the previously derived results and with the special observed in the diffraction pattern. In Table S2 there are 15 orthorhombic (3 + 1)D groups (Nos. 16.1–19.1). From the main reflections we know our lattice is primitive and there is no centring. There are three screw axes. Applying the screw axes narrows the selection down to one and the for the incommensurately modulated PA crystals is number 19.1 with notation P212121(00γ). There are actually three related versions of this P212121(α00), P212121(0β0) and P212121(00γ). The direction of the modulation is shown by the position of the coefficients. For PA the orientation is P212121(0β0). The symmetry operators need to be modified to work with modulation along this axis relative to c* as reported in the tables. Details of this transformation were reported by Lovelace et al. (2013).
is determined by the 3D and the components3. Conclusions
After integration of reflections in Eval15 the unit-cell dimensions and q vector length and direction are known. The final test of the assignment comes in the next step when it is applied to the integrated diffraction data via the SADABS software (Sheldrick, 1996). The program workflow is presented in Fig. 3 of Porta et al. (2011). It can be seen in Table 1 that the Rsym values obtained from SADABS look reasonable and are quite good for the well measured data of <4 Å resolution.
During data collection it was noticed that particularly strong satellite reflections were associated with extinguished main reflections (Fig. 4). As it turns out, this is indicative of large movements in the structural modulation (Janssen et al., 1999). It is interesting to note that, when normal periodic actin in PA crystals undergoes a transition from the `open' to `tight' state, the unit-cell dimension c changes by 14 Å, yet the crystals are stable (Chik, 1996). It is therefore possible that the structural transitions needed to bring about such a large modulation might be on a similar scale, especially those involving actin subdomains 1 and 4. of the incommensurate PA structures will inevitably shed light on the nature of these higher-order actin structures and provide insight into the early stages of actin filament formation. This is the next step in our research and involves further software development for crystallographic of a protein in a (3 + 1)D group.
Supporting information
Supporting information file. DOI: https://doi.org/10.1107/S1600576717007294/gj5178sup1.pdf
Acknowledgements
We would like to thank Clarence Schutt at Princeton University and Uno Lindberg at Stockholm University for introducing us to PA crystals and many useful discussions. We thank Dr Sander Van Smaalen at the Universität Bayreuth, and Dr Václav Petříček, Dr Michal Dušek and Dr Lukáš Palatinus at the Academy of Sciences of the Czech Republic, Prague, for being patient teachers. We thank Dr James Holton at Lawrence Berkley National Laboratory for help in making the pseudo-precession figures and Kelly Jordan for help in constructing tables.
Funding information
Funding for this research was provided by: National Science Foundation, Division of Molecular and Cellular Biosciences (award Nos. MCB-0718661, MCB-1518145, CNIC-IIA-1404976); Nebraska Research Initiative.
References
Carlsson, L. (1979). PhD thesis, Uppsala University, Sweden. Google Scholar
Chik, J. K. (1996). PhD thesis, Princeton University, NJ, USA. Google Scholar
Chik, J. K., Lindberg, U. & Schutt, C. E. (1996). J. Mol. Biol. 263, 607–623. CrossRef CAS PubMed Web of Science Google Scholar
Dauter, Z. & Jaskolski, M. (2010). J. Appl. Cryst. 43, 1150–1171. Web of Science CrossRef CAS IUCr Journals Google Scholar
Helliwell, J. R. (2008). Crystallogr. Rev. 14, 189–250. Web of Science CrossRef CAS Google Scholar
Holton, J. M. (2008). Acta Cryst. A64, C77. CrossRef IUCr Journals Google Scholar
Holton, J. M., Nielsen, C. & Frankel, K. A. (2012). J. Synchrotron Rad. 19, 1006–1011. Web of Science CrossRef CAS IUCr Journals Google Scholar
Janssen, T., Janner, A., Looijenga-Vos, A. & Wolff, P. M. D. (1999). International Tables for Crystallography, Vol. C, Mathematical, Physical and Chemical Tables, edited by A. J. C. Wilson & E. Prince, pp. 899–947. Dordrecht: Kluwer Academic Publishers. Google Scholar
Lovelace, J. J., Murphy, C. R., Daniels, L., Narayan, K., Schutt, C. E., Lindberg, U., Svensson, C. & Borgstahl, G. E. O. (2008). J. Appl. Cryst. 41, 600–605. Web of Science CrossRef CAS IUCr Journals Google Scholar
Lovelace, J. J., Simone, P. D., Petrícek, V. & Borgstahl, G. E. O. (2013). Acta Cryst. D69, 1062–1072. Web of Science CrossRef IUCr Journals Google Scholar
Lovelace, J. J., Winn, M. D. & Borgstahl, G. E. O. (2010). J. Appl. Cryst. 43, 285–292. Web of Science CrossRef CAS IUCr Journals Google Scholar
Murshudov, G. N., Vagin, A. A. & Dodson, E. J. (1997). Acta Cryst. D53, 240–255. CrossRef CAS Web of Science IUCr Journals Google Scholar
Oda, T., Makino, K., Yamashita, I., Namba, K. & Maéda, Y. (2001). Biophys. J. 80, 841–851. Web of Science CrossRef PubMed CAS Google Scholar
Orlov, I., Palatinus, L. & Chapuis, G. (2008). J. Appl. Cryst. 41, 1182–1186. Web of Science CrossRef CAS IUCr Journals Google Scholar
Pflugrath, J. W. (1999). Acta Cryst. D55, 1718–1725. Web of Science CrossRef CAS IUCr Journals Google Scholar
Porta, J. (2011). PhD thesis, University of Nebraska Medical Center, Omaha, NE, USA. Google Scholar
Porta, J. C. & Borgstahl, G. E. (2012). J. Mol. Biol. 418, 103–116. Web of Science CrossRef CAS PubMed Google Scholar
Porta, J., Lovelace, J. J., Schreurs, A. M. M., Kroon-Batenburg, L. M. J. & Borgstahl, G. E. O. (2011). Acta Cryst. D67, 628–638. Web of Science CrossRef CAS IUCr Journals Google Scholar
Rupp, B. (2010). Biomolecular Crystallography: Principles, Practice, and Application to Structural Biology. New York: Garland Science/Taylor and Francis Group. Google Scholar
Schreurs, A. M. M., Xian, X. & Kroon-Batenburg, L. M. J. (2010). J. Appl. Cryst. 43, 70–82. Web of Science CrossRef CAS IUCr Journals Google Scholar
Schutt, C., Lindberg, U. & Myslik, J. (1991). Nature, 353, 508. CrossRef PubMed Web of Science Google Scholar
Schutt, C. E., Lindberg, U., Myslik, J. & Strauss, N. (1989). J. Mol. Biol. 209, 735–746. CrossRef CAS PubMed Web of Science Google Scholar
Schutt, C. E., Myslik, J. C., Rozycki, M. D., Goonesekere, N. C. & Lindberg, U. (1993). Nature, 365, 810–816. CrossRef CAS PubMed Web of Science Google Scholar
Sheldrick, G. M. (1996). SADABS. University of Göttingen, Germany. Google Scholar
Smaalen, S. van (2005). Z. Kristallogr. 219, 681–691. Google Scholar
Smaalen, S. van (2007). Incommensurate Crystallography. Oxford University Press. Google Scholar
Smaalen, S. van, Campbell, B. J. & Stokes, H. T. (2013). Acta Cryst. A69, 75–90. Web of Science CrossRef IUCr Journals Google Scholar
Stokes, H. T., Campbell, B. J. & van Smaalen, S. (2011). Acta Cryst. A67, 45–55. Web of Science CrossRef CAS IUCr Journals Google Scholar
Vagin, A. & Teplyakov, A. (2000). Acta Cryst. D56, 1622–1624. Web of Science CrossRef CAS IUCr Journals Google Scholar
Wagner, T. & Schönleber, A. (2009). Acta Cryst. B65, 249–268. Web of Science CSD CrossRef CAS IUCr Journals Google Scholar
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