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Incorporation of copper ions into crystals of T2 copper-depleted laccase from Botrytis aclada

aLaboratory of Enzyme Engineering, A. N. Bach Institute of Biochemistry, Leninsky pr. 33, Moscow 119071, Russian Federation, bEngelhardt Institute of Molecular Biology, Vavilova str. 32, Moscow 119991, Russian Federation, cDepartment of Food Science and Technology, BOKU – University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Wien, Austria, and dRSC `Kurchatov Institute', Acad. Kurchatov sq. 1, Moscow 123182, Russian Federation
*Correspondence e-mail: e.m.osipov@gmail.com

Edited by N. Sträter, University of Leipzig, Germany (Received 9 July 2015; accepted 29 October 2015; online 18 November 2015)

Laccases belong to the class of multicopper oxidases catalyzing the oxidation of phenols accompanied by the reduction of molecular oxygen to water without the formation of hydrogen peroxide. The activity of laccases depends on the number of Cu atoms per enzyme molecule. The structure of type 2 copper-depleted laccase from Botrytis aclada has been solved previously. With the aim of obtaining the structure of the native form of the enzyme, crystals of the depleted laccase were soaked in Cu+- and Cu2+-containing solutions. Copper ions were found to be incorporated into the active site only when Cu+ was used. A comparative analysis of the native and depleted forms of the enzymes was performed.

1. Introduction

Laccases (EC 1.10.3.2; benzenediol:oxygen oxidoreductases) belong to the class of multicopper oxidases catalyzing the oxidation of phenols accompanied by the reduction of molecular oxygen to water. Laccases are composed of two or three structurally similar cupredoxin-like domains. These enzymes have been well studied both by biochemical (Baldrian, 2006[Baldrian, P. (2006). FEMS Microbiol. Rev. 30, 215-242.]) and structural (Hakulinen & Rouvinen, 2015[Hakulinen, N. & Rouvinen, J. (2015). Cell. Mol. Life Sci. 72, 857-868.]) methods. Owing to their broad substrate specificity and the formation of non­toxic products, laccases are of interest in biotechnology (Xu, 2002[Xu, F. (2002). In Encyclopedia of Bioprocess Technology, edited by M. C. Flickinger & S. W. Drew. New York: John Wiley & Sons. doi:10.1002/0471250589.ebt125.]).

The active site of laccases comprises four Cu atoms classified into three types depending on their spectroscopic characteristics (Solomon et al., 1996[Solomon, E. I., Sundaram, U. M. & Machonkin, T. E. (1996). Chem. Rev. 96, 2563-2606.]). The type 1 copper ion can be detected in the ESR spectrum and gives a characteristic absorption at around 600 nm. This ion has a trigonal planar coordination through the N atoms of two histidines and the S atom of one cysteine. Type 2 and type 3 copper ions form a T2/T3 cluster responsible for the reduction of molecular oxygen to water. The type 2 copper ion is detected in the ESR spectrum and does not show absorption in the optical region. Two type 3 copper ions are antiferromagnetically coupled through an oxygen-containing ligand and consequently do not exhibit an ESR signal, but show an absorption maximum at around 330 nm. The type 2 copper ion adopts a square-planar coordination geometry with two histidines and one or two oxygen-containing ligands. Each type 3 copper ion is coordinated in a tetrahedral geometry by three histidines and one oxygen-containing ligand, through which it is linked to another type 3 copper ion.

The activity of laccase preparations depends on the number of Cu atoms per enzyme molecule. Thus, one way of investigating laccases is to study enzyme preparations in which the copper ions have been partially removed. The type 2 copper ion is more easily removed from the laccase molecule compared with the other copper ions (Malkin et al., 1969[Malkin, R., Malmström, B. G. & Vänngård, T. (1969). Eur. J. Biochem. 7, 253-259.]). On the one hand, procedures have been developed for the preparation of inactive type 2 copper-depleted forms of the enzyme based on treatment with copper chelators in the presence of reducing agents (Reinhammar & Oda, 1979[Reinhammar, B. & Oda, Y. (1979). J. Inorg. Biochem. 11, 115-127.]; Koroleva et al., 2001[Koroleva, O. V., Stepanova, E. V., Gavrilova, V. P., Biniukov, V. I. & Pronin, A. M. (2001). Biochemistry, 66, 960-966.]). On the other hand, the partial loss of Cu atoms from the active site can occur spontaneously during the production and storage of laccases.

In some X-ray diffraction structures of laccases which had not been subjected to a copper-chelation procedure, the type 2 copper ion was either absent or had partial occupancy (Glazunova et al., 2015[Glazunova, O. A., Polyakov, K. M., Fedorova, T. V., Dorovatovskii, P. V. & Koroleva, O. V. (2015). Acta Cryst. D71, 854-861.]; Ducros et al., 1998[Ducros, V., Brzozowski, A. M., Wilson, K. S., Brown, S. H., Østergaard, P., Schneider, P., Yaver, D. S., Pedersen, A. H. & Davies, G. J. (1998). Nature Struct. Mol. Biol. 5, 310-316.]; Osipov et al., 2014[Osipov, E., Polyakov, K., Kittl, R., Shleev, S., Dorovatovsky, P., Tikhonova, T., Hann, S., Ludwig, R. & Popov, V. (2014). Acta Cryst. D70, 2913-2923.]). The restoration of depleted forms of laccases with a simultaneous increase in their activity is accomplished by treatment of the enzyme with copper salts. The insertion of a type 2 copper ion into fungal, plant and bacterial laccases using Cu+ ions has been described in the literature (Malkin et al., 1969[Malkin, R., Malmström, B. G. & Vänngård, T. (1969). Eur. J. Biochem. 7, 253-259.]; Reinhammar & Oda, 1979[Reinhammar, B. & Oda, Y. (1979). J. Inorg. Biochem. 11, 115-127.]; Koroleva et al., 2001[Koroleva, O. V., Stepanova, E. V., Gavrilova, V. P., Biniukov, V. I. & Pronin, A. M. (2001). Biochemistry, 66, 960-966.]). The X-ray crystal structure of the fungal type 2 copper-depleted laccase from Coriolopsis caperata showed that a copper ion was inserted into the active site only when Cu+ ions were used, whereas the use of Cu2+ ions did not give the desired result (Glazunova et al., 2015[Glazunova, O. A., Polyakov, K. M., Fedorova, T. V., Dorovatovskii, P. V. & Koroleva, O. V. (2015). Acta Cryst. D71, 854-861.]). However, it was demonstrated that the incorporation of a type 2 copper ion into the bacterial laccase from Bacillus subtilis occurs in the presence of either Cu+ or Cu2+ ions (Durão et al., 2008[Durão, P., Chen, Z., Fernandes, A. T., Hildebrandt, P., Murgida, D. H., Todorovic, S., Pereira, M. M., Melo, E. P. & Martins, L. O. (2008). J. Biol. Inorg. Chem. 13, 183-193.]).

The laccase from the ascomycete Botrytis aclada has previously been isolated and characterized biochemically (Kittl et al., 2012[Kittl, R., Mueangtoom, K., Gonaus, C., Khazaneh, S. T., Sygmund, C., Haltrich, D. & Ludwig, R. (2012). J. Biotechnol. 157, 304-314.]). The structures of this enzyme (hereafter referred to as T2D) and its L499M mutant were determined at 1.7 Å resolution (Osipov et al., 2014[Osipov, E., Polyakov, K., Kittl, R., Shleev, S., Dorovatovsky, P., Tikhonova, T., Hann, S., Ludwig, R. & Popov, V. (2014). Acta Cryst. D70, 2913-2923.]). Although the laccase was not subjected to a depletion procedure, the type 2 copper ion was absent in both structures and the residue His429, i.e. one of the two histidines that are involved in the coordination of the type 2 copper ion, points towards Cu3α.

The aim of the study presented here is to determine the structure of B. aclada laccase containing the complete set of copper ions. For this purpose, crystals of the type 2 copper-depleted form of B. aclada laccase were soaked in solutions containing Cu+ or Cu2+ ions. Restoration of the native form of the enzyme was only observed in the experiment employing Cu+ ions.

2. Materials and methods

2.1. Purification and crystallization of laccase

Laccase from B. aclada (Table 1[link]) was recombinantly expressed in the yeast Pichia pastoris and purified as described by Kittl et al. (2012[Kittl, R., Mueangtoom, K., Gonaus, C., Khazaneh, S. T., Sygmund, C., Haltrich, D. & Ludwig, R. (2012). J. Biotechnol. 157, 304-314.]). Attempts to obtain crystals of the native form of laccase failed. Therefore, the enzyme was deglycosyl­ated (Osipov et al., 2014[Osipov, E., Polyakov, K., Kittl, R., Shleev, S., Dorovatovsky, P., Tikhonova, T., Hann, S., Ludwig, R. & Popov, V. (2014). Acta Cryst. D70, 2913-2923.]). The crystallization conditions have been described in detail in a previous study (Osipov et al., 2014[Osipov, E., Polyakov, K., Kittl, R., Shleev, S., Dorovatovsky, P., Tikhonova, T., Hann, S., Ludwig, R. & Popov, V. (2014). Acta Cryst. D70, 2913-2923.]). Crystals were grown by the vapour-diffusion technique. A 1.8 M solution of ammonium sulfate in water was used as the reservoir solution. The protein solution consisted of 20 mg ml−1 protein in 25 mM sodium acetate buffer pH 5.0. A 2 µl drop composed of equal volumes of the protein and reservoir solutions was used (Table 2[link]). Crystals appeared within 3 d and reached maximum dimensions of 0.2 × 0.1 × 0.05 mm in one month.

Table 1
Macromolecule-production information

Source organism B. aclada
Forward primer 5BAPml1 5′-ATACACGTGCAAGATGAAGTATTTCACAGTCTTTACTGC-3′
Reverse primer 3BAXba1 5′-ATATCTAGACTTAAATTCCAGAATCGTCCTC-3′
Expression vector pPICZB and pPICZαA
Expression host P. pastoris
Complete amino-acid sequence of the construct produced MKYFTVFTALTALFAQASASAIPAVRSTLTPRQNTTASCANSATSRSCWGEYSIDTNWYDVTPTGVTREYWLSVENSTITPDGYTRSAMTFNGTVPGPAIIADWGDNLIIHVTNNLEHNGTSIHWHGIRQLGSLEYDGVPGVTQCPIAPGDTLTYKFQVTQYGTTWYHSHFSLQYGDGLFGPLIINGPATADYDEDVGVIFLQDWAHESVFEIWDTARLGAPPALENTLMNGTNTFDCSASTDPNCVGGGKKFELTFVEGTKYRLRLINVGIDSHFEFAIDNHTLTVIANDLVPIVPYTTDTLLIGIGQRYDVIVEANAAADNYWIRGNWGTTCSTNNEAANATGILRYDSSSIANPTSVGTTPRGTCEDEPVASLVPHLALDVGGYSLVDEQVSSAFTNYFTWTINSSSLLLDWSSPTTLKIFNNETIFPTEYNVVALEQTNANEEWVVYVIEDLTGFGIWHPIHLHGHDFFIVAQETDVFNSDESPAKFNLVNPPRRDVAALPGNGYLAIAFKLDNPGSWLLHCHIAWHASEGLAMQFVESQSSIAVKMTDTAIFEDTCANWNAYTPTQLFAEDDSGI

Table 2
Crystallization

Method Vapour diffusion
Plate type VDX
Temperature (K) 298
Protein concentration (mg ml−1) 20
Buffer composition of protein solution 25 mM sodium acetate pH 5.0
Composition of reservoir solution 1.8 M ammonium sulfate
Volume and ratio of drop 2 µl, 1:1
Volume of reservoir 500 µl

2.2. Preparation of complexes of T2D with Cu+ and Cu2+

The complexes were obtained by soaking crystals of the enzyme in reservoir solution containing 0.6 mM (saturated solution) CuCl (T2D+Cu+ complex) or 10 mM CuSO4 (T2D+Cu2+ complex). The soaking times were 10 min and 1 d, respectively.

2.3. Data collection and processing

X-ray data sets for T2D+Cu+ and T2D+Cu2+ were collected on the K4.4e beamline at the Belok station at the Kurchatov synchrotron-radiation source at 100 K under a nitrogen flow at a wavelength of 0.98 Å using a Rayonix SX165 detector. Before X-ray data collection, the crystals were placed for 5 s in reservoir solution supplemented with 20%(v/v) glycerol. The X-ray data sets were processed using the XDS package (Kabsch, 2010a[Kabsch, W. (2010a). Acta Cryst. D66, 125-132.],b[Kabsch, W. (2010b). Acta Cryst. D66, 133-144.]). The data-collection statistics are summarized in Table 3[link].

Table 3
Data collection and processing

Values in parentheses are for the outer shell.

Data set T2D+Cu+ T2D+Cu2+
Beamline Beamline K4.4e, Kurchatov SNC
Wavelength (Å) 0.98
Temperature (K) 100
Detector Rayonix SX165 CCD
Rotation range per image (°) 1.0
Total rotation range (°) 154.0 119.0
Space group C2
a, b, c (Å) 69.4, 113.1, 79.6 70.1, 113.9, 80.0
α, β, γ (°) 90, 109.0, 90 90, 108.8, 90
Mosaicity (°) 0.37 0.26
Resolution range (Å) 30–2.30 (2.44–2.30) 30–1.83 (1.94–1.83)
Total No. of reflections 81583 (12203) 124585 (16545)
No. of unique reflections 25106 (3913) 50252 (7615)
Completeness (%) 96.7 (94.6) 96.1 (90.5)
Multiplicity 3.3 (3.1) 2.5 (2.2)
I/σ(I)〉 16.3 (2.3) 20.8 (5.1)
Rmeas 0.071 (0.60) 0.039 (0.23)
Overall B factor from Wilson plot (Å2) 37.7 24.4

2.4. Structure solution and refinement

The crystals of the complexes were isomorphous to the crystals of T2D. The structures were refined with REFMAC5 (Murshudov et al., 2011[Murshudov, G. N., Skubák, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F. & Vagin, A. A. (2011). Acta Cryst. D67, 355-367.]). All crystallographic calculations were carried out using the CCP4 suite (Winn et al., 2011[Winn, M. D. et al. (2011). Acta Cryst. D67, 235-242.]). During the refinement of the copper ions with full occupancy, the Fo − Fc difference map contained essential peaks in the region of the copper ions and the B factors of the copper ions exceeded the B factors of the ligands. Therefore, in the final stages of refinement the occupancies of the copper ions were refined manually. Manual correction of the occupancies was followed by B-factor refinement. In the final model, the B factors of the copper ions were approximately equal to the B factors of the ligands, and the FoFc difference map did not contain essential peaks in the region of the copper ions. Visual inspection and manual rebuilding of the models was carried out using the Coot interactive graphics program (Emsley et al., 2010[Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. (2010). Acta Cryst. D66, 486-501.]). Water molecules were manually added to the structures based on analysis of the difference electron-density maps. The quality of the structures was evaluated with SFCHECK (Vaguine et al., 1999[Vaguine, A. A., Richelle, J. & Wodak, S. J. (1999). Acta Cryst. D55, 191-205.]) and PROCHECK (Laskowski et al., 1993[Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. (1993). J. Appl. Cryst. 26, 283-291.]). The figures were drawn with CCP4mg (McNicholas et al., 2011[McNicholas, S., Potterton, E., Wilson, K. S. & Noble, M. E. M. (2011). Acta Cryst. D67, 386-394.]). MolProbity (Chen et al., 2010[Chen, V. B., Arendall, W. B., Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S. & Richardson, D. C. (2010). Acta Cryst. D66, 12-21.]) was used for Ramachandran analysis. Structure-solution and refinement statistics are summarized in Table 4[link].

Table 4
Structure solution and refinement

Values in parentheses are for the outer shell.

Data set T2D+Cu+ T2D+Cu2+
Resolution range (Å) 30–2.30 (2.44–2.30) 30–1.83 (1.94–1.83)
Completeness (%) 97.2 96.3
No. of reflections, working set 23801 (1706) 47673 (3062)
No. of reflections, test set 1292 (111) 2579 (187)
Final Rcryst 0.181 (0.278) 0.162 (0.241)
Final Rfree 0.233 (0.347) 0.204 (0.302)
No. of non-H atoms
 Protein 4087 4208
 Ion 5 3
 Water 155 429
 Other 197 197
 Total 4444 4837
R.m.s. deviations    
 Bonds (Å) 0.011 0.016
 Angles (°) 1.62 1.92
Average B factors (Å2)
 Protein 38.6 23.3
 Ion 38.3 21.2
 Water 35.8 29.8
 Other 58.9 37.7
Ramachandran plot
 Favoured (%) 96.2 96.8
 Allowed (%) 3.6 3.2

3. Results and discussion

The structure of T2D+Cu2+ was solved at 1.8 Å resolution. The crystals of T2D+Cu2+ have the same qualities as the initial crystals of T2D. Since the structure of T2D+Cu2+ is almost identical to that of T2D, it was not deposited in the PDB. The structures of T2D and T2D+Cu2+ superimposed with an r.m.s.d. of 0.09 Å using the coordinates of 539 equivalent Cα atoms. Soaking T2D crystals for 24 h in a solution containing Cu2+ ions at high concentrations did not lead to the insertion of a copper ion into the T2/T3 cluster.

The complexes were obtained using crystals grown in the same drop. During the preparation of the T2D+Cu+ crystals the quality of the crystals visually deteriorated despite the low concentration of Cu+ ions and the short period of soaking. The structure of T2D+Cu+ was solved at 2.3 Å resolution. The reduction in the diffraction limit was accompanied by an increase in the mosaicity (0.38° for the crystals of T2D+Cu+ versus 0.25° for the crystals of T2D). Superimposition of the T2D+Cu+ and T2D structures using the coordinates of 525 equivalent Cα atoms gave an r.m.s.d. of 0.16 Å. Residues 1–14 (the numbering given is according to the T2D structure) and residues 405–408 were not located in the electron-density map for T2D+Cu+. The latter residues are also not observed in the electron-density maps for T2D and T2D+Cu2+.

The main differences in the structures of T2D+Cu+ and T2D are related to the location of a copper ion in the type 2 site and the orientation of the side chain of His429 (Fig. 1[link]). It should be noted that in the structure of T2D Cu2 is completely absent and the sum of occupancies of the copper ions in T2D (2.6) is almost equal to the content of copper ions per molecule for the enzyme in solution as determined by mass spectrometry (Osipov et al., 2014[Osipov, E., Polyakov, K., Kittl, R., Shleev, S., Dorovatovsky, P., Tikhonova, T., Hann, S., Ludwig, R. & Popov, V. (2014). Acta Cryst. D70, 2913-2923.]). In the T2D+Cu+ structure the type 2 copper ion has a square-planar coordination formed by the His87 N and His429 N atoms and the O atom of a water molecule (Table 5[link]). This situation is typical for all laccases containing type 2 copper ions. In the T2D structure His429 is coordinated to Cu3α by the Nδ atom (Osipov et al., 2014[Osipov, E., Polyakov, K., Kittl, R., Shleev, S., Dorovatovsky, P., Tikhonova, T., Hann, S., Ludwig, R. & Popov, V. (2014). Acta Cryst. D70, 2913-2923.]). Thus, the removal of type 2 copper ions could be associated with changes in the orientation of the side chain of His429. It should be noted that the occupancy of the type 2 copper ion is lower than the occupancy of the type 3 copper ions. Inter­atomic distances, B factors and occupancies for copper ions in the T2/T3 cluster and their respective ligands are shown in Table 5[link]. The lower occupancy of the type 2 copper ion is also clearly visible in the electron-density map of T2D+Cu+ (Fig. 1[link]a).

Table 5
Interatomic distances and temperature factors in the T2/T3 clusters of T2D+Cu+ and T2D+Cu2+

Occupancies for copper ions are given in parentheses.

    T2D+Cu+ T2D+Cu2+
      B factor (Å2)   B factor (Å2)
Atom A Atom B AB distance (Å) Atom A Atom B AB distance (Å) Atom A Atom B
Cu3α His133 N 2.06 34.2 (0.8) 31.4 2.12 20.8 (0.8) 19.2
His431 N 1.95 28.3 2.04 15.0
His488 N 1.91 30.4 2.03 20.0
His429 Nδ 2.8 24.3
His429 Cδ 3.35 29.5
W1 2.03 26.7 1.90 24.5
Cu3β His89 Nδ 2.04 30.5 (0.8) 34.6 2.02 22.7 (0.8) 16.0
His131 N 2.01 28.6 2.05 19.6
His490 N 2.10 33.2 2.11 18.8
W1 2.75 26.7 2.79 24.5
Cu2 His87 N 1.96 35.6 (0.7) 32.0
His429 N 1.77 29.9
W1 3.38 26.7
W2 2.54 35.1
[Figure 1]
Figure 1
Structures of the T2/T3 copper sites in (a) T2D+Cu+ and (b) T2D. The amino-acid residues are shown as ball-and-stick models and are coloured according to atom type. The C atoms of the His residues coordinated to the Cu3α, Cu3β and Cu2 ions are shown in dark green, light green and gold, respectively. The oxygen ligands and copper ions are shown as red and orange spheres, respectively. The 2FoFc electron-density maps (at the 6σ level) for copper ions are shown in blue. Coordination bonds are indicated by black dashed lines.

In the structures of T2 copper-depleted laccases from Coprinus cinereus (Ducros et al., 1998[Ducros, V., Brzozowski, A. M., Wilson, K. S., Brown, S. H., Østergaard, P., Schneider, P., Yaver, D. S., Pedersen, A. H. & Davies, G. J. (1998). Nature Struct. Mol. Biol. 5, 310-316.]), Coriolopsis gallica (De la Mora et al., 2012[De la Mora, E., Lovett, J. E., Blanford, C. F., Garman, E. F., Valderrama, B. & Rudino-Pinera, E. (2012). Acta Cryst. D68, 564-577.]) and Trametes hirsuta (Polyakov et al., 2009[Polyakov, K. M., Fedorova, T. V., Stepanova, E. V., Cherkashin, E. A., Kurzeev, S. A., Strokopytov, B. V., Lamzin, V. S. & Koroleva, O. V. (2009). Acta Cryst. D65, 611-617.]) a histidine residue equivalent to His429 also forms a weak coordination bond to Cu3α.

In addition, the electron-density map of the T2D+Cu+ complex has a peak in the vicinity of His81 at a distance of 2.1 Å from the N atom. This peak was interpreted as a copper ion with an occupancy of 0.2.

It should be noted that the incorporation of copper into the T2 site was unambiguously determined by the X-ray diffraction data. In the study of laccase from B. aclada it was shown that the incorporation of a type 2 copper ion only occurs in experiments with Cu+ ions. In the case of laccase from C. caperata, reconstitution was also observed only with the use of Cu+ salts (Glazunova et al., 2015[Glazunova, O. A., Polyakov, K. M., Fedorova, T. V., Dorovatovskii, P. V. & Koroleva, O. V. (2015). Acta Cryst. D71, 854-861.]). Since the active sites of laccases from different organisms have similar structures, it can be suggested that only Cu+ ions can be efficiently incorporated into the type 2 copper-depleted active sites of all laccases. This is in good agreement with the fact that Cu+ ions are inserted into the type 2 sites of depleted laccases (Malkin et al., 1969[Malkin, R., Malmström, B. G. & Vänngård, T. (1969). Eur. J. Biochem. 7, 253-259.]; Reinhammar & Oda, 1979[Reinhammar, B. & Oda, Y. (1979). J. Inorg. Biochem. 11, 115-127.]). It has been shown by ESR spectroscopy and kinetic measurements that both Cu+ and Cu2+ ions are inserted into the bacterial laccase from B. subtilis (Durão et al., 2008[Durão, P., Chen, Z., Fernandes, A. T., Hildebrandt, P., Murgida, D. H., Todorovic, S., Pereira, M. M., Melo, E. P. & Martins, L. O. (2008). J. Biol. Inorg. Chem. 13, 183-193.]). However, spectroscopic evidence for the incorporation of copper ions into the enzyme was only presented for Cu+; the kinetic data show that copper ions are more efficiently inserted as Cu+ ion into the laccase from B. subtilis.

Supporting information


Acknowledgements

The authors thank Dr Tatiana Safonova for critical reading, translation and helpful suggestions. The work has been financially supported by the Russian Scientific Foundation (project No. 14-24-00172, crystallization and structure solution) and the Austrian Science Fund (FWF, project P25148-B20).

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