Structure of yeast hexokinase. 3. Low resolution structure of a second crystal form showing a different quaternary structure, heterologous interaction of subunits and substrate binding

A 7 Å resolution electron density map of a second crystal form (called BII) of yeast hexokinase B has been obtained. This crystal form, unlike the first crystal form (BI), binds nucleotide and sugar substrates. While the overall tertiary structure of each subunit appears to be largely the same in both crystal forms, the quaternary structure of the dimer is completely different in the two crystals. The two subunits in the crystallographic asymmetric unit of form BII are related by a molecular screw axis; that is, the two subunits are related by a 160 ° rotation and a 13 Å translation of one subunit relative to the other along the symmetry axis resulting in non-equivalent environments for the two chemically identical subunits. A deep cleft divides each subunit into two domains or lobes of roughly equal size. The helical regions which are clearly visible as rods of electron density in this map constitute at least 40 to 50% of the polypeptide chain and 70 to 80% of one of the lobes. At this resolution the molecule does not appear to be homologous in detail to other kinases such as phosphoglycerate kinase and adenylate kinase. Sugar substrates and inhibitors bind deeply in the cleft which separates the two lobes and produce substantial alterations in the protein structure.     

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.     

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.     

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.     

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.     

Structure of rabbit muscle aldolase at low resolution

X-ray diffraction data were measured by x-ray diffractometry to 5-A resolution for both the monoclinic form of rabbit skeletal muscle aldolase (EC 4.1.2.13) and a platinum derivative. The heavy atom difference patterson was solved at 6-A resolution yielding eight distinct heavy atom sites. Choice was made of the enantiomorph and protein phases were calculated on the basis of single isomorphous replacement differences. The electron density map calculated from these phases was averaged according to the non-crystallographic molecular symmetry. Rotational symmetry analysis of native patterson and site symmetry analysis of refined heavy atom positions are consistent with the aldolase tetramer possessing a very high degree of 222 internal symmetry. The subunits in the tetramer are positioned in a tetrahedral configuration displaying a slight square planar deformation. Each subunit is roughly ellipsoidal in shape with the major axis nearly parallel to a local 2-fold axis. Prominent at the surface of each subunit were structural features resembling alpha helices. Each subunit contributes to its boundary surface at least six helices which are arranged in a barrel-like manner and possessing a right handed twist with respect to each other. Density associated with binding of substrate on the enzyme was located on the surface of each subunit. Cooperative aspects of the conformational changes produced upon substrate binding are discussed.     

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.     

Structure of oxidized thioredoxin to 4 with 5 A resolution

The structure of the oxidized form of Escherichia coli thioredoxin, space group C2, has been determined from X-ray crystallographic data, to a resolution of 4.5 Å using two heavy-atom derivatives, platinum diaminedichloride and 3-pyridyl mercuric chloride. The electron density maps show the molecular shape and the packing of the thioredoxin molecules as well as the positions of the cupric ions necessary for crystallization of thioredoxin. The shape of the thioredoxin molecule is ellipsoidal with approximate dimensions 25 Å × 34 Å × 35 Å. The two thioredoxin molecules in the asymmetric unit appear very similar. They are related by a translation vector with components (0, 0.1, 0.5) along the axis of the unit cell and not by a 2-fold rotation axis. Each of the two molecules in the asymmetric unit belongs to separate infinite layers of molecules parallel to the xy plane. The basic unit in these layers is a dimer formed by interaction of two thioredoxin molecules across the crystallographic 2-fold axis. The structural role of the cupric ions in the crystal lattice is to bridge these dimers within the layers.     

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.     

Studies of asymmetry in the three-dimensional structure of lobster D-glyceraldehyde-3-phosphate dehydrogenase.

An improved electron density map of lobster holo-D-glyceraldehyde-3-phosphate dehydrogenase has been computed to 2.9 A resolution based on two heavy atom isomorphous derivatives. This has been averaged only over the Q molecular 2-fold axis, which is known to be exact in the human holoenzyme. The map showed possible asymmetry between the subunits in which the active centers are closely related across the R axis (that is, between the red and green or between the yellow and blue subunits). A difference map between the electron density of citrate and sulfate-soaked crystals gave further evidence for possible asymmetry. The major differences of electron density between R axis-related subunits appear around the active center and suggest the following interpretations. 1. The conformation of the adenine about the glycosidic bond is the more frequently observed anti with a C-2' endo conformation for the ribose ring in the red and yellow subunits, but is probably syn with a C-3' endo conformation in the green and blue subunits.2. The adenine ribose has its 3'-hydroxyl group hydrogen-bonded to a main chain carbonyl group in the red and yellow subunits but not in the green and blue subunits, as a consequence of the differing ribose conformations. 3. Cysteine-149 is more closely associated with histidine-176 in the green and blue subunits, and appears nearer the nicotinamide in the red and yellow subunits.     

Structure of glycolate oxidase from spinach at a resolution of 5.5 Å

The crystal structure of glycolate oxidase from spinach has been determined to 5.5 Å resolution, using two isomorphous heavy-atom derivatives and their anomalous contributions. In the electron density map the boundaries of the octameric molecules are clearly seen. The subunit molecular weight is 37,000. Two protomers are in very close contact around one of the crystallographic 2-fold axes. Four such dimers are in contact around the 4-fold axis, so that the glycolate oxidase molecules are arranged as octamers with 422 symmetry in the crystal lattice. The roughly spherical octameric molecules have a diameter of approximately 100 Å. These octamers are arranged in a network, such that large solvent channels, approximately 60Å in diameter, pass right through the crystal lattice.The secondary structure of two-thirds of the subunit density has been interpreted in terms of eight consecutive β strand-α-helix units forming a cylinder very similar to the structure of triose phosphate isomerase. This interpretation is based on the very characteristic arrangement of the eight helices which form such a cylinder. The binding site of a substrate analogue, thioglycolate, has been localized in a deep cleft of the subunit at one end of the βα-barrel close to its axis.     

Structure determination of crystalline lobster D-glyceraldehyde-3-phosphate dehydrogenase

Single crystal X-ray data were collected on film for the holoenzyme of lobster d-glyceraldehyde-3-phosphate dehydrogenase to 3·0 Å resolution. Films of potassium tetraiodomercurate, K₂HgI₄, comprising a complete low resolution set, with some additional high resolution terms, were given to us by Drs H. C. Watson and L. J. Banaszak. A 3·0 Å high resolution data set was collected of a p-chloromercuri-phenylsulfonate derivative. All these films were processed on a computer controlled Optronics film scanner. The K₂HgI₄ derivative difference Patterson was initially interpreted in terms of four single sites, one for each polypeptide chain, consistent with the previously determined molecular 222 symmetry. Single isomorphous replacement phases were then sufficient to identify other heavy atom sites. Least-squares refined parameters were used to give multiple isomorphous replacement phases at low resolution, and single isomorphous replacement phases at high resolution. The resultant electron density map was oriented along the molecular 2-fold axes and then averaged over all four equivalent subunits. This process produced a much improved electron density map, which could easily be interpreted in terms of a single polypeptide chain per subunit consistent with the known amino acid sequence. The use of non-crystallographic symmetry to improve the electron density map is equivalent to the molecular replacement method. A comparison is also made with other dehydrogenases.     

The low-resolution structure of human muscle aldolase

The three-dimensional structure of human muscle aldolase has been solved at 5 A resolution with the use of two isomorphous heavy atom derivatives. The enzyme's four subunits are arranged about three mutually perpendicular intersecting twofold axes to form a compact spherical molecule. The subunit boundaries are clearly defined but a possible domain structure is not apparent in this preliminary electron density map.     

Studies on coenzyme binding to glyceraldehyde-3-phosphate dehydrogenase.

Tyrosyl-transfer RNA synthetase from Bacillus stearothermophilus has been crystallized as hexagonal plates, $\text{P3}{}_1\text{21, a = b = 64.6}\text{A}\text{?}\text{, c = 238.8}\text{A}\text{?}$, with the dimeric molecule (molecular weight, 90,000) occupying two crystallographic asymmetric units (Reid et al., 1973). Three heavy-atom derivatives have been identified and X-ray diffraction measurements have been made to 2.7 Å resolution, using the oscillation method. The three heavy-atom derivatives were methyl mercury (two sites, half occupied, 3 Å apart), uranyl acetate (single fully occupied site) and chloroplatinite PtCl₄^2? (three sites of differing occupancy). The results were used to compute an electron density map at 2.7 Å resolution, which shows the monomer as a unit of about 60 Å × 60 Å × 40 Å. The maximum dimension of the dimer is about 130 Å. Most of the polypeptide chain has been traced uniquely. It includes five α-helices more than 12 Å long and several shorter helices. A six-stranded pleated-sheet structure lies in the centre of each subunit. The catalytic site of the enzyme is believed to be adjacent to the mercury-binding group.     

Alpha-carbon coordinates for bovine Cu,Zn superoxide dismutase.

Preliminary α-carbon and metal coordinates are given for a subunit of the bovine erythrocyte Cu⁺⁺, Zn⁺⁺ superoxide dismutase. The coordinates are averaged from 5 sets of measurements made on electron density maps at 3Å resolution: one set from an averaged map and one set from each of the 4 crystallographically independent subunits (2 dimeric molecules) in the asymmetric unit of the crystal. The 3-dimensional structure of the polypeptide backbone is illustrated and briefly described.     

Three-dimensional structure of glutathione reductase at 2 Å resolution

An electron density map of the FAD-containing enzyme glutathione reductase from human erythrocytes was produced at 2 Å resolution using the multi-isomorphous-replacement method. The chemically determined amino acid sequence could be fitted unambiguously to this map. The enzyme has a molecular weight of 104,800 and consists of two identical subunits. Each of them can be subdivided into four domains and a flexible segment of 18 residues at the N-terminus. A subunit contains 11 α-helices comprising 31% of all residues and 32% β-structure in five pleated sheets. An intersubunit disulfide bridge, which is not expected for an intracellular enzyme, was detected in the crystal. The heavy atom binding sites, the subunit interface, and the intermolecular contacts in the crystal are discussed.     

X-ray analysis of the disk of tobacco mosaic virus protein. 3. A low resolution electron density map

The structure of the disk of tobacco mosaic virus protein at low resolution has been determined by X-ray crystal analysis. Signs for the three principal projections were found by isomorphous replacement, using a mercury derivative. The heavy-atom positions were located by interpretation of difference Patterson maps on the basis of the non-crystallographic 17-fold rotational symmetry of the disk, and of the packing of the disks determined in the preceding paper (Finch et al., 1974).The electron density in the corresponding three projections was computed to a resolution of 6 Å. From the projections in the [100] and [010] directions, which are at right-angles to the 17-fold rotation axis, a three-dimensional electron density map has been calculated making use of the non-crystallographic symmetry. The procedure is similar to the three-dimensional image reconstruction technique used in electron microscopy.The map indicates that the subunits in the two rings face the same way and, hence, that the disk is polar. There are differences between the subunits in the two rings at high radius, which are presumably a consequence of the pairing interaction responsible for stabilizing the two-layer polar structure. The map has been compared with a low-resolution map of the intact virus, and certain common features can be identified, notably the site of the nucleic acid.     

The polypeptide chain fold in tyrosine phenol-lyase, a pyridoxal-5'-phosphate-dependent enzyme.

The tyrosine phenol lyase (EC 4.1.99.2) from Citrobacter intermedius has been crystallised in the apo form by vapour diffusion. The space group is P2(1)2(1)2. The unit cell has dimensions a = 76.0 A, b = 138.3 A, c = 93.5 A and it contains two subunits of the tetrameric molecule in the asymmetric unit. Diffraction data for the native enzyme and two heavy atom derivatives have been collected with synchrotron radiation and an image plate scanner. The structure has been solved at 2.7 A resolution by isomorphous replacement with subsequent modification of the phases by averaging the density around the non-crystallographic symmetry axis. The electron density maps clearly show the relative orientation of the subunits and most of the trace of the polypeptide chain. Each subunit consists of two domains. The topology of the large domain appears to be similar to that of the aminotransferases.     

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.     

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.