Lyle H. Jensen

A crystallographic model for azurin at 3 Å resolution

Abstract The structure of the blue copper protein azurin (Mr 14,000) from Pseudomonas aeruginosa has been determined from a 3.0 A resolution electron density map computed with phases based on a uranyl derivative to 3 A resolution and a platinum derivative to 3.7 A. Interpretation of the somewhat noisy map was based on comparison of the density of the four molecules in the asymmetric unit with their averaged density. The polypeptide chain folds into an eight-strand β barrel with an additional flap containing a short helix. The copper atom is bound at one end and on the inside of the barrel, probably to a cysteine, a methionine, and two histidine residues.

The structure of rubredoxin at 1.2 Å resolution

Structural details of the model of Clostridium pasteurianum rubredoxin are presented, based on the refined model at 1.2 A resolution. The molecule contains no extensive regions of pleated-sheet or helical structure. Regular secondary structure consists primarily of residues 3 to 7, 11 to 13 and 48 to 52 in a small region of pleated-sheet; and residues 14 to 18, 19 to 23, 29 to 33 and 45 to 49 in 310 helical corners. Interbond angles in the helical corners average as much as 10 ° greater than normally accepted values and a number of the peptide groups deviate significantly from planarity. Rubredoxin has a pronounced asymmetry in the distribution of charged groups on its surface. This would lead to highly favored molecular orientations when the protein interacts with other charged molecules. Bond lengths in the iron-sulfur complex range from 2.24 a to 2.33 A, and bond angles range from 104 ° to 114 °.

Crystallographic refinement of rubredoxin at 1·2 Å resolution

The model for rubredoxin from Clostridium pasteurianum has been refined by least-squares against a 1·2 A resolution data set. The conventional R value is 0·128 for the 10,936 reflections with I >2 σ(I) and 10 A> d >1· A. The precision of the model is much improved over the earlier refinement (Watenpaugh et al. , 1973), the mean standard deviation in the C α −C β bond lengths from the present refinment being ∼0·1 A. The Fe−S bond lengths in the FeS 4 complex range from 2·24 A to 2·33 A with an average value of 2·29 A. Standard deviations in the individual Fe−S bonds are ∼0·02 A. The B parameters, which measure the distribution of atoms about their mean positions, range from 7 A 2 to values greater than 50 A 2 . Their magnitudes are found to correlate with both inter- and intramolecular structural features, the larger B values occurring where the atoms would be expected to be less rigidly bound. The relatively large B values found for rubredoxin and characteristic of crystalline proteins generally imply relatively large amplitudes of atomic motion and pliable, readily deformable molecules. Such basic molecular properties are likely to play an important role in the mechanisms by which the molecules function. In the best determined regions of the rubredoxin molecule, hydrogen atoms are visible in the difference map.

Structure of rubredoxin: An X-ray study to 2.5 Å resolution

Abstract The non-heme iron protein, rubredoxin (molecular weight 6300) crystallizes as rhombs which possess the symmetry of the space group R 3. The parameters of the unit cell referred to hexagonal axes are a = b = 64.5 A and c = 32.7 A with nine molecules per unit cell. The structure was first solved at 3 A resolution with data from the native protein and a single derivative (mercuri-iodide) by using the effects of anomalous scattering. The map was readily interpretable in terms of what was known about the structure. The resulting model was confirmed by a 3 A electron density map based on phases determined using a second derivative alone (uranyl derivative). Data for the native protein and the HgI 4 2− and UO 2 2+ derivatives have been extended to include all planes with spacings greater than 2.5 A. The electron density map based on these data shows that the molecule consists of an irregularly folded polypeptide chain which contains an appreciable amount of anti-parallel pleated sheet structure but no alpha helix. The iron atom is bound to four cysteine sulfur atoms to form an apparently tetrahedral complex.

Refined crystal structure of ferredoxin II from Desulfovibrio gigas at 1·7 Å☆

The crystal structure of ferredoxin II from Desulfovibrio gigas has been determined using phasing from anomalous scattering data at a resolution of 1.7 A and refined to an R-factor of 0.157. The molecule has an overall chain fold similar to that of the other bacterial ferredoxins of known structure. The molecule contains a single 3Fe-4S cluster with geometry indistinguishable from the 4Fe-4S clusters, and a disulfide bond near the site corresponding to the position of the second cluster of two-cluster ferredoxins. The cluster is bound by cysteine residues 8, 14 and 50. The side-chain of cysteine 11 extends away from the cluster, but could rotate to become the fourth cysteine ligand in the four-iron form of the molecule given a local adjustment of the polypeptide chain. This residue is modified, however, by what appears to be a methanethiol group. There are a total of eight NH . . . S bonds to the inorganic and cysteine sulfur atoms of the Fe-S cluster. There is an additional residue found that is not reported for the chemical sequence: according to the electron density a valine residue should be inserted after residue 55.

Stereochemistry of nucleic acid constituents: I. Refinement of the structure of cytidylic acid b‡

The crystal structure of cytidylic acid b, ‡ cytidine-3′-phosphate, has been refined by the method of full matrix least-squares to an R factor of 0·045, R = Σ | F o | − Σ | F c |/ Σ | F o | where | F o | and | F c | are respectively the observed and calculated diffraction amplitudes. The nucleotide is found to exist as a zwitter ion. The cytosine ring is slightly nonplanar, with N (3) protonated. The ribose ring is puckered with C (2′) -endo, and displaced (on the same side of C (5′) ) about 0·6 A from the plane of the remaining ring atoms. These latter appear to deviate slightly from a plane. Because the base in nucleic acids need not necessarily be planar and because C (1′) can be significantly displaced from the least-squares plane of the base, we suggest that the definition of the torsion angle Φ CN defined by Donohue & Trueblood (1960) , be modified as follows. The torsion angle Φ CN of the bond C (1′) −N is the angle formed by the projection of C (1′) −O (1′) relative to the projection of N (1′) −C (6) (in pyrimidine ring) and N (9) −C (8) (in purine ring) when viewed along C (1′) −N. This angle is taken as zero when O (1′) is anti-planar to C (2) of the pyrimidine or C (4) of the purine ring, and positive angles are taken as those measured in a clockwise direction when viewing from C (1′) to N. For this structure Φ CN = −42·1° and the conformation anti . All 14 hydrogen atoms in the structure appear in a ΔF synthesis and their parameters have also been refined. Several of the hydrogen isotropic thermal parameters have an anomalously low value. The standard deviation of the atomic co-ordinates from the least-squares refinement are P = 0·0011 A, O = 0·0036 A, N = 0·0042 A, C = 0·005 A and H = 0·06 A.

Stereochemistry of nucleic acid constituents: II. A comparative study

Stereochemical results for some nucleic acid constituents are presented in the light of recent studies. In nucleotides, the ring nitrogen atom, N(3) in pyrimidines and N(1) in purines, is protonated. Similarities in certain bond lengths and bond angles occur between protonated cytosine and thymine and between protonated adenine and guanine. In the pyrimidine and purine bases, the substituent atoms may be markedly displaced on either side of the plane determined by the ring atoms. Moreover, the ring atoms may deviate significantly from a plane. Probable values for the molecular dimensions of β- D -ribose and 2-deoxy-β- D -erythro pentose are given for the four possible conformations of the furanose ring; C(3′)-endo, C(2′)-endo, C(3′)-exo and C(2′)-exo. The P−O bond lengths in the phosphate group are dependent on the nature and number of substituents.

Water structure in a protein crystal: Rubredoxin at 1.2 Å resolution

Abstract The model for rubredoxin based on X-ray diffraction data has been extensively refined with a 1.2 A resolution data set. Water oxygen atoms were deleted from the model if B exceeded 50 A 2 and occupancy was less than 0.3 eA −3 . The final water model consists of 127 sites with B values ranging from 15 to 6 0 A 2 and occupancies from unity down to 0.3, the most tightly bound water oxygen atoms being hydrogen bonded to two or more main-chain nitrogen or oxygen atoms. The water forms extensive hydrogen bond networks bridging the crevices on the molecular surfaces and between adjacent molecules. The minimum distances of the water sites from the protein surface are distributed about two distinct maxima, the major one at 2.5 to 3 A and a minor one at 4 to 4.5 A. Beyond 5 to 6 A from the protein surface, the discrete water merges into the aqueous continuum.

A structural model of rubredoxin from Desulfovibrio vulgaris at 2 A resolution.

The structure of the iron-sulfur protein rubredoxin from Desulfovibrio vulgaris has been determined from X-ray diffraction data by the molecular replacement method. The starting model was derived from the one for Clostridium pasteurianum rubredoxin on the basis of atoms common to the two rubredoxins according to the chemical sequence. Partial refinement has been carried out by difference Fourier methods with 350 of the 389 atoms in the molecule, the conventional R factor being 0·31 for the 2 A resolution data set. Positions have been found for most of the remaining atoms. The two rubredoxin structures differ mainly in groups on the surface of the molecule, no major differences in conformation or internal hydrogen bonding being apparent at this point.

The structure of a copper complex of the growth factor glycyl-L-histidyl-L-lysine at 1.1 Å resolution

Abstract Interest in the crystal structure of a copper complex of the growth factor, glycyl-L-histidyl-L-lysine has been stimulated by the tripeptides ability to facilitate copper uptake in cultured hepatoma cells and by the copper complexs tendency to induce angiogenesis. The coordination polyhedron is a distorted square pyramid, CuN3O2, with the four basal ligating atoms bonded to the copper at about 2.00 A and the apical ligating atom at 2.49 A. One tripeptide furnishes three of the basal atoms, the glycine amino nitrogen atom, the peptide nitrogen atom of the histidine, and the imine nitrogen atom of the imidazole. A second tripeptide is involved via its terminal carboxyl oxygen atom while the fifth copper ligand is a carboxyl oxygen atom of a third tripeptide. The carboxyl oxygen atoms form bridges between copper centers and thus the system is polymeric in the solid state. The crystal structure can be used to propose a model for the first step in the transport of copper into cells via a copper-tripeptide complex.