Crystallographic studies and primary structure of the antitumor monoclonal CC49 Fab′

The Fab′ of CC49, a murine monoclonal antibody directed against the human tumor-associated antigen TAG-72 has been crystallized. The crystals are monoclinic, space group P2₁ with cell parameters a = 115.6 Å, b = 116.4 Å, and c = 70.3 Å; β = 97.8°. The size of the unit cell is compatible with four Fab′ molecules in the asymmetric unit. The Fab molecules are related by two approximately perpendicular pseudo-2-fold axes. One pseudo-2-fold axis is parallel to the crystallographic 2-fold axis and was found by inspection of the Harker section of the native Patterson map; the other was found by a self rotation function. The primary structures of the variable regions of the CC49 antibody light and heavy chains have been determined and are compared with those of the related antitumor antibody B72.3. © 1993 Wiley-Liss, Inc.     

Characterization of crystals of a cytochrome oxidase (nitrite reductase) from Pseudomonas aeruginosa by x-ray diffraction and electron microscopy

Cytochrome oxidase from Pseudomonas aeruginosa has been crystallized from 2 m-ammonium sulfate. The crystals occur principally as thin diamond-shaped plates of space group P2₁2₁2 with unit cell dimensions of 92 Å × 115 Å × 76 Å. Determination of the density of glutaraldehyde-fixed, water-equilibrated crystals (1.167 g/cm³), coupled with the unit cell volume (804,000 ų), indicates that there is one subunit (~63,000 Mr) per asymmetric unit. X-ray diffraction data which were limited to 12 Å resolution due to small crystal size were obtained for the hk0 and 0kl zones using precession photography. Amplitude and phase data for the hk0, 0kl, and h0l zones were obtained from computer-based Fourier analysis of appropriate micrographs recorded from negatively stained microplates and thin sections of larger crystals using minimal beam electron microscopy. For crystals embedded in the presence of tannic acid it was possible to achieve 20 Å resolution which is comparable to the resolution achieved with negative staining of thin crystalline arrays. In addition, unstained electron diffraction on glutaraldehyde-fixed, glucose-stabilized plates was recorded to a resolution of 9 Å. The three-dimensional packing of the cytochrome oxidase dimer in the unit cell has been deduced from computer reconstructed images of the three principal projections along the crystallographic axes. The cytochrome oxidase dimer is located in the unit cell with the dimer axis coincident with a crystallographic 2-fold axis; thus within the resolution of the present data in projection (9 Å) the two subunits are identical, in agreement with biochemical evidence. The crystals have been prepared with the enzyme in the fully oxidized state and upon reduction a progressive cracking of the crystals is observed, possibly due to a conformational change dependent on the oxidation state of the heme iron.     

Structure of Panulirus interruptus hemocyanin at 5 Å resolution

The hemocyanin from the spiny lobster Panulirus interruptus, a hexamer with a molecular weight of approximately 540,000, was crystallized in space group P2₁ with two molecules in the unit cell and cell dimensions $\text{a = 119.8}\text{A}\text{?}\text{, b = 193.1}\text{A}\text{?}\text{, c = 122.2}\text{A}\text{?}\text{and}\text{β = 118.1 °}$. With screened precession photographs a three-dimensional set of reflections was collected up to 10 Å resolution. Both the conventional and the fast rotation function programs were applied and gave results that were in excellent agreement with each other. The hemocyanin hexamer has 32 point group symmetry. Its 3-fold molecular axis runs approximately parallel to the crystallographic 2-fold screw axis.X-ray diffraction data to 5 Å resolution were collected by the oscillation method. Rotation function studies with data between 7 and 5 Å resolution confirmed the 10 Å studies and, furthermore, showed that the rotation axes relating subunits within one hexameric molecule can be distinguished from the rotation axes relating subunits belonging to different hexamers in the unit cell. The local 3-fold axis in the hexamer makes an angle of about 6 ° with the crystallographic 2-fold screw axis.For a mercury and a platinum derivative three-dimensional data sets were collected to 5 Å by the oscillation method. The difference Patterson of the platinum derivative could be solved. The eventual number of heavy-atom sites was 36 for the platinum derivative and 70 for the mercury derivative. From the well-occupied sites the point-group symmetry of the molecule could be established accurately. In addition, the centre of the hexamer could be located within 0.2 Å.Protein phases were obtained from isomorphous as well as anomalous differences. A “best” electron density map calculated with these phases showed the shape of the hexameric molecule as well as the boundaries of the six subunits. Correlation coefficients between the densities of the subunits showed little variation, suggesting a random distribution of the different subunit types (Van Eerd & Folkerts, 1981) over the six positions in the hexamer.The subunits are positioned at the corner of an antiprism. When viewed along the 3-fold axis the hexamer is roughly hexagonal in shape, with a diameter of approximately 120 Å. Viewed along one of the 2-fold axes the molecule is of rectangular shape with dimensions 95 Å × 120 Å. The subunit can be described as an ellipsoid of irregular shape with axes of 80 Å, 55 Å and 48 Å. Each subunit makes extensive contacts with three other subunits in the hexamer and, possibly, a much weaker contact with a fourth subunit.     

Twinning in crystals of human skeletal muscle d-glyceraldehyde-3-phosphate dehydrogenase

The coenzyme-bound form of human skeletal muscle d-glyceraldehyde-3-phosphate dehydrogenase has been shown to crystallize in the space group C2 and not C222₁ as previously reported. The unit cell contains two tetrameric molecules with the dimer of molecular weight 72,000 as the crystallographic asymmetric unit. The recorded X-ray intensity distribution clearly indicates the presence of non-crystallographic 2-fold axes perpendicular to the crystallographic 2-fold axis showing that the subunits are arranged with near perfect 222 symmetry.Isomorphous derivatives of the enzyme have been prepared and the heavy atom positions defined in complete agreement with the C2 space group assignment. Further confirmation that the space group is C2 and not C222₁ comes from the 3.5 Å resolution electron density map of the human enzyme, which appears almost identical to that of the lobster holo-enzyme where no such space group ambiguity exists.     

Growth and analysis of crystal forms of toxic shock syndrome toxin 1

Native toxic shock syndrom toxin 1 (TSST-1) purified from Staphylococcus aurius has been crystallized in four different forms. The highest resolution data (2.05 Å) was collected from orthorhombic crystals belonging to the space group C222₁. The unit cell dimension are a = 108.7 Å, b = 177.5 Å, c = 97.6 Å. Rotation function analysis of this from indicates that there is trimer of toxin molecules in the asymmetric unit with a local 3-fold axis parallel to the crystallographic c axis. Crystals of a double mutant of TSST-1 have been grown which has a single molecule in the asymmetric unit and diffract to 1.9 Å. The space group is P2₁ with unit cell parameters of a = 44.4 Å, b = 34.0 Å, c = 55.2 Å, β = 93.0°. © 1993 Wiley-Liss, Inc.     

Non-crystallographic symmetry in the crystal dimer of wheat germ agglutinin

Three isomorphous heavy atom derivatives of wheat germ agglutinin crystals, KAu(CN)₂, K₂Pt(NH₃)₂(NO)₂ and mersalyl, have been examined at high resolution. Heavy atom sites were located from difference Patterson maps in three dimensions at 2.15 Å resolution for the gold and platinum derivatives and with less certainty in the centrosymmetric [010] projection for the mersalyl derivative. These sites are distributed in the crystallographic asymmetric unit such that one half of them can be related to the other half by a 180 ° rotation about an axis parallel to a, and an additional translation of about 6.35 Å along that axis. It is suggested that the two subunits of the wheat germ agglutinin dimer, which represent the asymmetric unit of the C2 unit cell, are related by the same symmetry axis, causing heterologous subunit contacts due to the 6.35 Å translation of one relative to the other subunit.     

A preliminary crystallographic study of isocitrate dehydrogenase from Azotobacter vinelandii

Large single crystals of isocitrate dehydrogenase from Azotobacter vinelandii have been grown by vapor diffusion from ammonium sulfate and phosphate solutions. The crystals are tetragonal, space group P4₂2₁2 with cell dimensions $\text{a = 122.1}\text{A}\text{?}$, $\text{c = 163.9}\text{a}\text{?}$. There are two molecules of 80,000 molecular weight per asymmetric unit. Native data to 5.5 Å resolution have been collected on a diffractometer. A rotation function using data between 10 Å and 6 Å resolution indicates three possible orientations of the non-crystallographic 2-fold axis relating the two molecules.     

Structure of yeast hexokinase. II. A 6 angstrom resolution electron density map showing molecular shape and heterologous interaction of subunits

An electron density map of yeast hexokinase has been calculated at 6 Å resolution using six heavy atom derivatives. The map shows each of the enzyme's two 51,000 molecular weight subunits to consist of two separate lobes connected by a narrow bridge of density. Furthermore, these two subunits are related to each other in the asymmetric unit of the crystal by a quasi-2-fold rather than a true 2-fold axis. That is, they are related by a rotation of 180 ° plus a relative translation of 3.6 Å along the symmetry axis. This gives rise to a heterologous subunit interaction and a possibility of non-identical structure and function for these chemically identical subunits. The molecule is quite asymmetric, having dimensions of 150 Å × 45 Å × 55 Å. Each subunit is about 80 Å × 40 Å × 50 Å.A portion of an electron density map at 3 Å resolution has been also calculated, based on phases from two heavy atom derivatives. Polypeptide backbone and side chains are visible in this map.     

The crystal structure of phosphorylase beta at 6 A resolution

The determination of the crystal structure of phosphorylase b in the presence of IMP at 6 Å resolution is described. The structure determination is based on two heavy-atom isomorphous derivatives and their anomalous contributions. The molecular boundary is clearly distinguishable in the electron density map, except in the region of subunit-subunit contact about the crystallographic dyad axis, which is the symmetry axis of the dimer. The dimer molecule is roughly ellipsoidal in shape with dimensions 63 Å × 63 Å × 116 Å. There is a pronounced cavity on the enzyme surface but it is not yet known if this is a substrate binding site.     

Low resolution crystal structure of hagfish insulin

Insulin from the Atlantic hagfish, Myxine glutinosa, crystallizes in space group P4₁2₁2 with a monomer in the asymmetric unit. The application of the Rossmann &; Blow (1962) rotation function, utilizing the known 2-zinc pig insulin crystal structure, has established the existence of an insulin dimer containing a crystallographic 2-fold axis. The position of the hagfish insulin molecule in the unit cell has been determined and a set of calculated phases derived. These are compared to phases found from isomorphous replacement studies. A 6 Å resolution electron density map has been calculated which shows the A and B chains are folded in a similar way to pig insulin and that the monomers are similarly organized into dimers.     

Molecular Symmetry of the ClpP Component of the ATP-dependent Clp Protease,anEscherichia coliHomolog of 20 S Proteasome

The ClpP component Clp protease fromEscherichia colihas been crystallized and examined by X-ray crystallography and self-rotation function calculations. The crystal belongs to the monoclinic space groupP2₁with unit cell dimensions ofa=196.9 Å,b=104.3 Å,c=162.4 Å and β=98.3°. The X-ray diffraction pattern extends at least to 2.5 Å Bragg spacing when exposed to CuKα X-rays. Self-rotation function analyses indicate that the ClpP oligomer has 72-point group symmetry. This symmetry suggests that the ClpP oligomer is a tetradecamer, (ClpP)₁₄, consisting of two heptamers, (ClpP)₇stacked on top of each other in a head-to-head fashion. The measurement of crystal density indicates that two independent copies of the ClpP oligomers are present in the asymmetric unit, giving a crystal volume per protein mass (V_M) of 2.73 ų/Da and a solvent content of 54.9% (v/v). Self-rotation function calculations are consistent with the presence of two ClpP tetradecamers in the asymmetric unit. The Patterson function suggests that a translation ofx=0.5 andy=0.5 relates a pair of ClpP oligomers in one asymmetric unit to another pair in the other asymmetric unit. And the two independent tetradecamers in one asymmetric unit are related by a relative rotation of about 18° around the 7-fold axis.     

Low resolution structure of adenylate kinase

A low resolution model of adenylate kinase has been derived from a 6 Å electron density map. The molecular shape can be described approximately as an oblate ellipsoid with dimensions 40 Å × 40 Å × 30 Å. The molecule is composed of two globular units separated by a 10 Å deep cleft. In contrast to the bigger unit, the smaller globule appears to contain a high amount of α-helical structure. The location of the active centre is discussed.The crystals used for X-ray diffraction analysis belong to one of the enantiomorphic trigonal space groups P3₁21 or P3₂21, with one molecule in the asymmetric unit. The phase determination was based on four isomorphous heavy atom derivatives. Frequent transitions between different crystal forms complicate the analysis.     

Crystallization of a scRIP—gelonin isolated from plant seeds Gelonium multiforum

Single crystals of the protein gelonin isolated from the seeds of Gelonium multiforum have been grown at room temperature by vapor diffusion method. The crystals are monclinic with a = 49.4 Å, b = 44.9 Å, c = 137.4 Å, and β = 98.3°. The space group is P2₁, with two molecules in the asymmetric unit which are related by a noncrystallographic 2-fold axis along ψ =13° and ? =88°. The crystals diffract X-rays to high resolution, making it possible to obtain an accurate structure of this single chain ribosome inactivating protein. © 1994 Wiley-Liss, Inc.     

The three-dimensional structure of mitochondrial aspartate aminotransferase at 4.5 A resolution

An X-ray crystallographic study at 4.5 Å resolution has been carried out with triclinic crystals of chicken mitochondrial aspartate aminotransferase.In the electron density map, the enzyme is clearly visible as an isologous α₂-dimer (105 Å × 60 Å × 50 Å) in which the subunits are associated about a molecular 2-fold axis. Each subunit of dimensions 70 Å × 50 Å × 40 Å contains at least seven helices, one of which is about 50 Å long.Difference maps have revealed the positions of the pyridoxyl and the phosphate moieties of the coenzyme as well as the general substrate binding area. The active sites are on opposite sides of the dimer, about 30 Å apart and close to the intersubunit boundary, so that probably both subunits contribute to each active site. An isolated chain segment, passing in front of the active site and ending in contact with the neighbouring subunit is interpreted as one of the chain termini.     

Crystallography of hemerythrin

X-ray crystallographic studies of hemerythrin from Golfingia gouldii show that the molecules pack in a tetragonal cell with two sets of molecules in an apparently face-centred array seen in projection along the 4-fold axis, but displaced relatively to each other by approximately $\text{c}\text{10}$ in the axial direction. Both sets of molecules lie on 4-fold rotation axes, so that the subunits of each octameric molecule are related in pairs by a molecular 4-fold rotation axis. The two subunits of each pair are probably related by non-crystallographic 2-fold rotation axes perpendicular to the 4-fold axes and lying at 10 ° and 55 ° to the a and b axes. At low resolution the subunits are apparently arranged approximately in the form of a square prism. Along each 4-fold crystallographic axis there are two hemerythrin molecules, nearly equidistant and having similar but not identical orientations.     

Helical channels in crystals of gramicidin A and of a cesium--gramicidin A complex: an x-ray diffraction study.

X-ray crystallographic studies of gramicidin A crystallized from methanol (P2₁) and ethanol (P2₁2₁2₁), and of a Cs⁺ gramicidin A complex crystallized from methanol (P222₁, P2₁2₁2 or P2₁2₁2₁) are reported here. The asymmetric unit consists of two molecules of gramicidin A in the native crystals and four molecules in the cesium complex crystal. Patterson analyses show that gramicidin A in these crystals forms a cylindrical helical channel. In the two types of native gramicidin crystals, the diameter of this channel is about 5 å and its length is about 32 å. Cesium ions are bound inside this channel in crystals of the cesium-gramicidin A complex. The channel in this complex is considerably shorter (26 Å) and wider (6·8Å) than in the native forms. The Patterson maps of these three crystal forms are compatible with either the single-stranded β-helix (Urry, 1971) or the double-stranded parallel or anti-parallel, β-helix (Veatch et al., 1974).     

A Regular 1:1 Complex of Two Allosteric States in the Single Crystal of l-Lactate Dehydrogenase from Bifidobacterium longum

Single crystals of the regular 1:1 complex of T and R-state tetramers of l -lactate dehydrogenase from Bifidobacterium longum have been grown from polyethylene glycol 6000 solution in the presence of NADH, fructose 1,6-bisphosphate and oxamate. The crystals belong to space group F222 with unit cell dimensions of a = 148·4 Å, b = 295·9 Å and c = 71·0 Å. The crystals are suitable for X-ray analysis and diffract to beyond 2·5 Å spacing. A crystallographic study showed that the asymmetric unit contains two subunits and that one of them belongs to the T-state tetramer and the other to the R-state tetramer.     

The high-resolution crystal structure of a parallel intermolecular DNA G-4 quadruplex/drug complex employing syn glycosyl linkages

We have determined the X-ray structure of the complex between the DNA quadruplex d(5′-GGGG-3′)₄ and daunomycin, as a potential model for studying drug–telomere interactions. The structure was solved at 1.08 Å by direct methods in space group I4. The asymmetric unit comprises a linear arrangement of one d(GGGG) strand, four daunomycin molecules, a second d(GGGG) strand facing in the opposite direction to the first, and Na and Mg cations. The crystallographic 4-fold axis generates the biological unit, which is a 12-layered structure comprising two sets of four guanine layers, with four layers each of four daunomycins stacked between the 5′ faces of the two quadruplexes. The daunomycin layers fall into two groups which are novel in their mode of self assembly. The only contacts between daunomycin molecules within any one of these layers are van der Waals interactions, however there is substantial π–π stacking between successive daunomycin layers and also with adjacent guanine layers. The structure differs significantly from all other parallel d(TGGGGT)₄ quadruplexes in that the 5′ guanine adopts the unusual syn glycosyl linkage, refuting the widespread belief that such conformations should all be anti. In contrast to the related d(TGGGGT)/daunomycin complex, there are no ligand–quadruplex groove insertion interactions.     

Crystallization and X-ray analysis of a single fab binding domain from protein L of Peptostreptococcus magnus

Protein L is a multi domain cell wall constituent of certain strains of Peptostreptococcus magnus which binds to the variable domain of immunoglobulin κ-light chains. A single immunoglobulin-binding domain of M_r = 9000 from this protein has been isolated and crystallized. The crystals are of space group P4₂2₁2, with cell dimensions a = b = 66.9 Å, c = 68.3 Å, and diffract to at least 2.2 Å resolution. The asymmetric unit of the crystal contains two molecules of the protein L domain, related by a noncrystallographic 2-fold axis, as revealed by a self-rotation function calculated with native diffraction data. © 1995 Wiley-Liss, Inc.     

The crystal structure of penicillopepsin at 6Åresolution

A 6Åresolution electron density map of crystals of penicillopepsin, an acid protease from Penicillium janthinellum, has been computed from multiple isomorphous replacement phases determined from two heavy metal derivatives, K₂PtCl₆ and UO₂Cl₂. The mean figure of merit of the map is 0.939. The boundaries of the molecules, of which there are four per unit cell, are readily discernible. The molecule is highly asymmetric with approximate dimensions 60Å× 40Å× 30Å. The molecule consists of two distinct lobes separated by a deep cleft, which is probably the extended substrate binding site.