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Trigonal crystals of MJ0729 showing different degrees of merohedral twinning that may vary from perfect hemihedral twinning to perfect tetartohedral twinning were obtained upon slight variation of the pH.

CBS domains are small protein motifs, usually associated in tandem, that are implicated in binding to adenosyl groups. Several genetic diseases in humans have been associated with mutations in CBS sequences, which has made them very promising targets for rational drug design. Trigonal crystals of the CBS-domain protein MJ0729 from Methanococcus jannaschii were grown by the vapour-diffusion method at acidic pH. Preliminary analysis of nine X-ray diffraction data sets using Yeates statistics and Britton plots showed that slight variation in the pH as well as in the buffer used in the crystallization experiments led to crystals with different degrees of merohedral twinning that may vary from perfect hemihedral twinning to perfect tetartohedral twinning.

The presence of pseudosymmetry can cause problems in structure determination and refinement. The relevant background and representative examples are presented.

It is not uncommon for protein crystals to crystallize with more than a single molecule per asymmetric unit. When more than a single molecule is present in the asymmetric unit, various pathological situations such as twinning, modulated crystals and pseudo translational or rotational symmetry can arise. The presence of pseudosymmetry can lead to uncertainties about the correct space group, especially in the presence of twinning. The background to certain common pathologies is presented and a new notation for space groups in unusual settings is introduced. The main concepts are illustrated with several examples from the literature and the Protein Data Bank.

A truncated soluble human semicarbazide-sensitive amine oxidase has been crystallized. Data were collected to 2.5 Å from a crystal suffering from twinning, pseudo-symmetry and anisotropy. The structure was solved in space group P43.

Human semicarbazide-sensitive amine oxidase (SSAO) is a homodimeric copper-containing monoamine oxidase that occurs in both a membrane-bound and a soluble form. SSAO is also known as vascular adhesion protein-1 (VAP-1). A truncated soluble form of human SSAO (comprising residues 29–763) was expressed in human embryonic kidney 293 cells and purified to homogeneity. Tetragonal crystals were obtained and a data set extending to 2.5 Å was collected. The crystals are merohedrally twinned and the estimation of the twinning fraction was complicated by pseudo-symmetry and the anisotropic character of the crystals. Using a recently developed method for twinning detection that is insensitive to phenomena such as anisotropy or pseudo-symmetry [Padilla & Yeates (2003 ▶), Acta Cryst. D59, 1124–1130], the twinning fraction was estimated to be 0.3. The structure was eventually solved by molecular replacement in space group P43.

A case of imperfect pseudo-merohedral twinning in monoclinic crystals of fungal fatty acid synthase is discussed. A space-group transition during crystal dehydration resulted in a Moiré pattern-like interference of the twinned diffraction patterns.

The recent high-resolution structures of fungal fatty acid synthase (FAS) have provided new insights into the principles of fatty acid biosynthesis by large multifunctional enzymes. The crystallographic phase problem for the 2.6 MDa fungal FAS was initially solved to 5 Å resolution using two crystal forms from Thermomyces lanuginosus. Monoclinic crystals in space group P21 were obtained from orthorhombic crystals in space group P212121 by dehydration. Here, it is shown how this space-group transition induced imperfect pseudo-merohedral twinning in the monoclinic crystal, giving rise to a Moiré pattern-like interference of the two twin-related reciprocal lattices. The strategy for processing the twinned diffraction images and obtaining a quantitative analysis is presented. The twinning is also related to the packing of the molecules in the two crystal forms, which was derived from self-rotation function analysis and molecular-replacement solutions using a low-resolution electron microscopy map as a search model.

The crystal structure of the dimethyllysine derivative of the E. coli RNA polymerase α subunit C-terminal domain is reported at 2.0 Å resolution.

The α subunit C-terminal domain (αCTD) of RNA polymerase (RNAP) is a key element in transcription activation in Escherichia coli, possessing determinants responsible for the interaction of RNAP with DNA and with transcription factors. Here, the crystal structure of E. coli αCTD (α subunit residues 245–329) determined to 2.0 Å resolution is reported. Crystals were obtained after reductive methylation of the recombinantly expressed domain. The crystals belonged to space group P21 and possessed both pseudo-translational symmetry and pseudo-merohedral twinning. The refined coordinate model (R factor = 0.193, R free = 0.236) has improved geometry compared with prior lower resolution determinations of the αCTD structure [Jeon et al. (1995 ▶), Science, 270, 1495–1497; Benoff et al. (2002 ▶), Science, 297, 1562–1566]. An extensive dimerization interface formed primarily by N- and C-terminal residues is also observed. The new coordinates will facilitate the improved modeling of αCTD-containing multi-component complexes visualized at lower resolution using X-ray crystallo­graphy and electron-microscopy reconstruction.

The title compound, C40H64O12, crystallizes in a pseudo­merohedrally twinned primitive monoclinic cell with similar contributions of the two twin components. There are two symmetry-independent half-mol­ecules of nona­ctin in the asymmetric unit. Each mol­ecule has a pseudo-S 4 symmetry and resides on a crystallographic twofold axis; the axes pass through the mol­ecular center of mass and are perpendicular to the plane of the macrocycle. The literature description of the room-temperature structure of nona­ctin as an order–disorder structure in an ortho­rhom­bic unit cell is corrected. We report a low-temperature high-precision ordered structure of ‘free’ nona­ctin that allowed for the first time precise determination of its bond distances and angles. It possesses an unfolded and more planar geometry than its complexes with encapsulated Na+, K+, Cs+, Ca2+ or NH4 + cations that exhibit more isometric overall conformations.

P212121 crystals of SIV Nef core domain bound to a peptide fragment of the T-cell receptor ζ subunit exhibited noncrystallographic symmetry and nearly perfect pseudo-merohedral twinning simulating tetragonal symmetry. For a different peptide fragment, nontwinned tetragonal crystals were observed but diffracted to lower resolution. The structure was determined after assignment of the top molecular-replacement solutions to various twin or NCS domains followed by refinement under the appropriate twin law.

HIV/SIV Nef mediates many cellular processes through interactions with various cytoplasmic and membrane-associated host proteins, including the signalling ζ subunit of the T-­cell receptor (TCRζ). Here, the crystallization strategy, methods and refinement procedures used to solve the structures of the core domain of the SIVmac239 isolate of Nef (Nefcore) in complex with two different TCRζ fragments are described. The structure of SIVmac239 Nefcore bound to the longer TCRζ polypeptide (Leu51–Asp93) was determined to 3.7 Å resolution (R work = 28.7%) in the tetragonal space group P43212. The structure of SIVmac239 Nefcore in complex with the shorter TCRζ polypeptide (Ala63–Arg80) was determined to 2.05 Å resolution (R work = 17.0%), but only after the detection of nearly perfect pseudo-merohedral crystal twinning and proper assignment of the orthorhombic space group P212121. The reduction in crystal space-group symmetry induced by the truncated TCRζ polypeptide appears to be caused by the rearrangement of crystal-contact hydrogen-bonding networks and the substitution of crystallographic symmetry operations by similar noncrystallographic symmetry (NCS) operations. The combination of NCS rotations that were nearly parallel to the twin operation (k, h, −l) and a and b unit-cell parameters that were nearly identical predisposed the P212121 crystal form to pseudo-merohedral twinning.

Complications to molecular replacement resulting from a poor starting search model, pseudosymmetry, twinning and a high copy number in the asymmetric unit made the determination of the structure of D. desulfuricans (ATCC 29577) flavodoxin in two crystal forms challenging.

The crystal structure of oxidized flavodoxin from Desulfo­vibrio desulfuricans (ATCC 29577) was determined by molecular replacement in two crystal forms, P3121 and P43, at 2.5 and 2.0 Å resolution, respectively. Structure determination in space group P3121 was challenging owing to the presence of pseudo-translational symmetry and a high copy number in the asymmetric unit (8). Initial phasing attempts in space group P3121 by molecular replacement using a poor search model (46% identity) and multi-wavelength anomalous dispersion were unsuccessful. It was necessary to solve the structure in a second crystal form, space group P43, which was characterized by almost perfect twinning, in order to obtain a suitable search model for molecular replacement. This search model with complementary approaches to molecular replacement utilizing the pseudo-translational symmetry operators determined by analysis of the native Patterson map facilitated the selection and manual placement of molecules to generate an initial solution in the P3121 crystal form. During the early stages of refinement, application of the appropriate twin law, (−h, −k, l), was required to converge to reasonable R-factor values despite the fact that in the final analysis the data were untwinned and the twin law could subsequently be removed. The approaches used in structure determination and refinement may be applicable to other crystal structures characterized by these complicating factors. The refined model shows flexibility of the flavin mononucleotide coordinating loops indicated by the isolation of two loop conformations and provides a starting point for the elucidation of the mechanism used for protein-partner recognition.

Based on the minimum shear criterion, a direct and simple method is proposed to calculate twinning elements from the experimentally determined twinning plane for Type I twins or the twinning direction for Type II twins. It is generic and applicable to any crystal structure.

The fundamental theory of crystal twinning has been long established, leading to a significant advance in understanding the nature of this physical phenomenon. However, there remains a substantial gap between the elaborate theory and the practical determination of twinning elements. This paper proposes a direct and simple method – valid for any crystal structure and based on the minimum shear criterion – to calculate various twinning elements from the experimentally determined twinning plane for Type I twins or the twinning direction for Type II twins. Without additional efforts, it is generally applicable to identify and predict possible twinning modes occurring in a variety of crystalline solids. Therefore, the present method is a promising tool to characterize twinning elements, especially for those materials with complex crystal structure.

Twinned crystals of humaan MD-2 are transformed into single crystals with cryoprotectant optimization.

Twinning of crystals causes overlapping of two or more reciprocal lattice points, and hence structure amplitudes for a single crystalline domain are hardly obtained from X-ray diffraction intensities. MD-2 protein forms a stable complex with Toll-like receptor 4 and recognizes bacterial lipopolysaccharide (LPS). Excessive immune responses activated by LPS cause septic shocks. Saccharide-trimmed human MD-2 crystallizes in the tetragonal form with apparent Laue symmetry of 4/mmm, and diffraction intensities from these crystals indicate crystal twinning. The crystal consists of two different domains, A and B. The c A axis of domain A coincides with the c B axis of domain B with a smaller lattice, and the a A axis corresponds to the (a B + b B) axis. This twinning severely imposes difficulty in structure determination. Through optimization of cryoprotectant, domain A was thoroughly transformed into domain B. The crystal containing only domain B is in space group P41212 with one MD-2 molecule in the asymmetric unit. The structure of this form of MD-2 as well as its complex with antiendotoxic lipid IVa was successfully determined using the multiple isomorphous replacement method.

The title compound, [AuCl(C18H21P)], a monomeric two-coordinate gold(I) complex, has been characterized at 100 K as two distinct monoclinic polymorphs, one from a single crystal, (Is), and one from a pseudo-merohedrally twinned crystal, (It). The mol­ecular structures in the two monoclinic [P21/n for (Is) and P21/c for (It)] polymorphs are similar; however, the packing arrangements in the two lattices differ considerably. The structure of (It) is pseudo-merohedrally twinned by a twofold rotation about the a* axis.

In the title compound, C15H10BrClO, the mol­ecule adopts an E configuration with respect to the C=C double bond and the dihedral angle between the aromatic ring planes is 3.98 (16)°. In the crystal, inversion dimers linked by pairs of C—H⋯O bonds are seen and weak π–π stacking [centroid–centroid separation = 3.8776 (19) Å] may further consolidate the structure. The crystal studied was a non-merohedral twin with a ratio of the twin components of 0.9093 (13):0.0907 (13). The twin operation is a twofold rotation around c*.

The title compound, C12H12BrNO2S, was isolated after direct irradiation (hν 350 nm, hexa­ne) of a mixture of stereoisomeric sulfonamides containing a vicinal dibromide and a conjugated diene. This product is one of a group of substrates that has contributed to our understanding of the photoreactivity patterns of non-bridged sulfonamides. The crystal structure was determined from a non-merohedrally twinned data set, where the twin law corresponded to a 180° rotation about the a* axis. The minor twin component refined to a value of 0.176 (3). The conformation of the mol­ecule is planar at one end, as the benzene ring and the adjacent fused five-membered ring are coplanar, and U-shaped at the other end, where the five-membered ring is fused to the heterocyclic six-membered ring containing an allyl bromide group.

The pitfalls of experimental phasing are described.

Developments in protein crystal structure determination by experimental phasing are reviewed, emphasizing the theoretical continuum between experimental phasing, density modification, model building and refinement. Traditional notions of the composition of the substructure and the best coefficients for map generation are discussed. Pitfalls such as determining the enantiomorph, identifying centrosymmetry (or pseudo-symmetry) in the substructure and crystal twinning are discussed in detail. An appendix introduces com­bined real–imaginary log-likelihood gradient map coefficients for SAD phasing and their use for substructure completion as implemented in the software Phaser. Supplementary material includes animated probabilistic Harker diagrams showing how maximum-likelihood-based phasing methods can be used to refine parameters in the case of SIR and MIR; it is hoped that these will be useful for those teaching best practice in experimental phasing methods.

X-ray crystallographic analysis of human inosine triphosphate pyrophosphohydrolase provided the secondary structure and active-site structure at 1.6 Å resolution in an orthorhombic crystal form. The structure gives a framework for future structure–function studies employing site-directed mutagenesis and for the identification of substrate/product-binding sites.

The structure of human inosine triphosphate pyrophosphohydrolase (ITPA) has been determined using diffraction data to 1.6 Å resolution. ITPA contributes to the accurate replication of DNA by cleansing cellular dNTP pools of mutagenic nucleotide purine analogs such as dITP or dXTP. A similar high-resolution unpublished structure has been deposited in the Protein Data Bank from a monoclinic and pseudo-merohedrally twinned crystal. Here, cocrystallization of ITPA with a molar ratio of XTP appears to have improved the crystals by eliminating twinning and resulted in an orthorhombic space group. However, there was no evidence for bound XTP in the structure. Comparison with substrate-bound NTPase from a thermophilic organism predicts the movement of residues within helix α1, the loop before α6 and helix α7 to cap off the active site when substrate is bound.

Corrigendum to Acta Cryst. (2009), E65, o301.

Consideration of a previous unrecognized twinning of the original investigated crystal of the title compound [Kia et al. (2009 ▶). Acta Cryst. E65, o301] led to improved reliability factors and to a slightly higher precision for all geometric parameters. The crystal under investigation was twinned by pseudo-merohedry with [100, 00, 00] as the twin matrix and a refined twin domain fraction of 0.9610 (5):0.0390 (5). The results of the new crystal structure refinement are given here.

The asymmetric unit of the title compound, [H3N(CH2)5NH3]2I[I3]3 or 2C5H16N2 2+·3I3 −·I−, consists of two crystallographically independent pentane-1,5-diaminium dications and two triiodide anions in general positions besides two additional triiodide and two iodide anions located on twofold axes. The compound crystallizes in the centrosymmetric monoclinic space group P2/n. The structure refinement was handicapped by the pseudosymmetry (pseudo-centering) of the structure and by twinning. The crystal structure is composed of two alternate layers, which differ in their arrangement of the pentane-1,5-diaminium dications and the iodide/triiodide anions and which are connected via weak to medium–strong N—H⋯I hydrogen bonds, constructing a complex hydrogen-bonded network.

The structure of the 1918 H1N1 neuraminidase was determined to 1.65 Å from crystals with a lattice-translocation defect using uncorrected, as well as corrected, diffraction data.

Few examples of macromolecular crystals containing lattice-translocation defects have been published in the literature. Lattice translocation and twinning are believed to be two common but different crystal-growth anomalies. While the successful use of twinned data for structure determination has become relatively routine in recent years, structure determination of crystals with lattice-translocation defects has not often been reported. To date, only four protein crystal structures containing such a crystal defect have been determined, using corrected, but not uncorrected, intensity data. In this report, the crystallization, structure determination and refinement of N1 neuraminidase derived from the 1918 H1N1 influenza virus (18NA) at 1.65 Å resolution are described. The crystal was indexed in space group C2221, with unit-cell parameters a = 117.7, b = 138.5, c = 117.9 Å, and the structure was solved by molecular replacement. The lattice-translocation vector in the 18NA crystal was (0, 1/2, 1/2) or its equivalent vector (1/2, 0, 1/2) owing to the C lattice symmetry. Owing to this special lattice-translocation vector in space group C2221, structure refinement could be achieved in two different ways: using corrected or uncorrected diffraction data. In the refinement with uncorrected data, a composite model was built to represent the molecules in the translated and untranslated layers, respectively. This composite structure model provided a unique example to examine how the molecules were arranged in the two lattice domains resulting from lattice-translocation defects.

Crystals of adeno-associated virus serotype 3b, a human DNA virus with promise as a vector for gene therapy, have been grown, diffract X-rays to ∼2.6 Å resolution and are suitable for structure determination in spite of twinning.

Adeno-associated viruses (AAVs) are leading candidate vectors for gene-therapy applications. The AAV-3b capsid is closely related to the well characterized AAV-2 capsid (87% identity), but sequence and presumably structural differences lead to distinct cell-entry and immune-recognition properties. In an effort to understand these differences and to perhaps harness them, diffraction-quality crystals of purified infectious AAV-3b particles have been grown and several partial diffraction data sets have been recorded. The crystals displayed varying levels of merohedral twinning that in earlier times would have rendered them unsuitable for structure determination, but here is shown to be a tractable complication.

In the title compound, [Cu(SO4)(C12H8N2)2]·C3H8O2, the CuII ion is bonded to two chelating 1,10-phenanthroline (phen) ligands and one O atom from a monodentate sulfate ligand in a distorted square-based pyramidal arrangement, with the O atom in a basal site. The two chelating N2C2 groups subtend a dihedral angle of 71.10 (15)°. In the crystal, the solvent mol­ecule forms two O—H⋯O hydrogen bonds to its adjacent complex mol­ecule. The chosen crystal was found to be a racemic twin; the presence of pseudosymmetry in the structure suggests the higher symmetry space group C2/c, but attempts to refine the structure in this space group resulted in an unsatisfactory model and high R and wR values.

Genetic-epidemiological studies on monozygotic (MZ) twins have been used for decades to tease out the relative contributions of genes and the environment to a trait. Phenotypic discordance in MZ twins has traditionally been ascribed to non-shared environmental factors acting after birth, however recent data indicate that this explanation is far too simple. In this paper, we review other reasons for discordance, including differences in the in utero environment, genetic mosaicism, and stochastic factors, focusing particularly on epigenetic discordance. Epigenetic differences are gaining increasing recognition. Although it is clear that in specific cases epigenetic alterations provide a causal factor in disease etiology, the overall significance of epigenetics in twin discordance remains unclear. It is also challenging to determine the causality and relative contributions of environmental, genetic, and stochastic factors to epigenetic variability. Epigenomic profiling studies have recently shed more light on the dynamics of temporal methylation change and methylome heritability, yet have not given a definite answer regarding their relevance to disease, because of limitations in establishing causality. Here, we explore the subject of epigenetics as another component in human phenotypic variability and its links to disease focusing particularly on evidence from MZ twin studies.

Beta toxin is a neutral sphingomyelinase secreted by certain strains of Staphylococcus aureus. This virulence factor lyses erythrocytes in order to evade the host immune system as well as scavenge nutrients. The structure of beta toxin was determined at 2.4-Å resolution using crystals that were merohedrally twinned. This structure is similar to that of the sphingomyelinases of Listeria ivanovii and Bacillus cereus. Beta toxin belongs to the DNase I folding superfamily; in addition to sphingomyelinases, the proteins most structurally related to beta toxin include human endonuclease HAP1, Escherichia coli endonuclease III, bovine pancreatic DNase I, and the endonuclease domain of TRAS1 from Bombyx mori. Our biological assays demonstrated for the first time that beta toxin kills proliferating human lymphocytes. Structure-directed active site mutations show that biological activities, including hemolysis and lymphotoxicity, are due to the sphingomyelinase activity of the enzyme.

In the title compound, C26H19N3O, the dimethyl­ene hydrazine (—C=N—N=C—) unit is approximately planar, the torsion angle around the N—N bond being 162.2 (6)°. The phenyl and benzoyl­phenyl rings at one end of the hydrazine unit are aligned at angles of 9.5 (5) and 88.5 (4)°, respectively, with respect to the hydrazine unit, whereas the benzene ring at the other end is twisted by an angle of 14.4 (4)°. In the crystal structure, mol­ecules are linked into centrosymmetric dimers by inter­molecular C—H⋯O hydrogen bonds. The monoclinic crystal under investigation shows pseudo-merohedral twinning with twin fractions of 0.63 and 0.37.

The asymmetric unit of the title salt C8H22N4 2+·2C7H5O2 −, comprises two independent pairs of half a 2,2′-(piperazine-1,4-di­yl)diethanaminium dication plus a benzoate anion. The dications are symmetrical and lie across crystallographic centres of inversion. The crystal structure was refined as a two-component pseudo-merohedral twin using the twin law 001 0-10 100 [he domain fractions are 0.8645 (8) and 0.1355 (8)]. The anions and cations are linked by N—H⋯O hydrogen bonds and weak N—H⋯O inter­molecular inter­actions to form infinite two-dimensional networks parallel to [101]. The conformation adopted by the cation in the crystal structure is very similar to that adopted by the same cation in the structures of the 2-hy­droxy­benzoate [Cukrowski et al. (2012 ▶). Acta Cryst, E68, o2387], the nitrate and the tetra­hydrogen penta­borate salts.

The enanti­opure title compound, C4H10NO2 +·Cl−·H2O, forms a two-dimensional network by inter­molecular hydrogen bonding parallel to (010). Non-merohedral twinning with a twofold rotation about the reciprocal c* axis as twin operation was taken into account during intensity integration and structure refinement. This twinning leads to alternative orientations of the stacked hydrogen-bonded layers.