Crystallization of a complex between ribonuclease T1 and 2'-guanylic acid

Ribonuclease T1 was crystallized under various conditions. Form I crystals were produced by microdialysis against 53% (v/v) 2-methyl-2,4-pentanediol in 0.01 M sodium acetate, 0.05% 2'-guanylic acid (2'GMP) and 0.02% NaN3 (pH 6.2-7.2). These crystals are tetragonal, space group P41212 and contain two molecules per asymmetric unit; cell dimensions are a = b = 5.86 nm, c = 13.28 nm. Form IIa and form IIb crystals were obtained by microdialysis from a buffer of 0.01-0.05 M sodium acetate, 0.25-0.5% 2'GMP, 0.02% NaN3 and 2-5 mM calcium acetate (pH 4.0-4.4) in the presence of 50-75% (v/v) 2-methyl-2,4-pentanediol. These crystals are orthorhombic, space group P212121, and contain one molecule per asymmetric unit; cell dimensions are a = 4.66 nm, b = 5.02 nm, c = 4.04 nm (form I) and alpha = 4.44 nm, b = 5.00 nm, c = 4.03 nm (form II). Using high-performance liquid chromatography, it could be shown for all crystal forms that 2'-GMP is bound in the crystals. The molecular ratio between RNase T1 and 2'GMP was 0.9 for form II crystals and thus agreed with a 1:1 enzyme-nucleotide complex. Heavy-atom derivatives were produced with lead acetate for form IIa crystals and with uranyl acetate for from IIb crystals. Three-dimensional X-ray analysis of the RNase-T1 x 2'GMP complex is under way.     

Three-dimensional structure of ribonuclease Ms*3'-guanylic acid complex at 2.5 A resolution.

The crystal structure of ribonuclease Ms*3'-guanylic acid complex has been determined by molecular replacement methods based on the known structure of ribonuclease T1. The pattern of hydrogen-bonds between the enzyme and the guanine base is similar to that discovered by Arni et al. [( 1988) J. Biol. Chem. 263, 15358-15368] in the crystal structure of ribonuclease T1*2'-guanylic acid complex. As for the possible general base in the trans-phosphorylation step of the catalysis, 0 epsilon 1 of Glu57 is within the hydrogen-bond distance (2.7 A) of the 2'-0 of the nucleotide while N epsilon 2 of His39 is significantly more distant (3.4 A) from the 2'-0.     

Refined X-ray structure of the low pH form of ribonuclease T1-2'-guanylic acid complex at 1.9 A resolution

The three-dimensional X-ray structure of the RNase T1[EC 3.1.27.3]-2'GMP complex crystallized at low pH value (4.0) was determined, and refined to 1.9 A resolution to give a final R value of 0.203. The refined model includes 781 protein atoms, 24 inhibitor atoms, and 43 solvent molecules. The imidazole rings of His27 and His40 interact with the carboxyl side chains of Glu82 and Glu58, respectively, whereas that of His92 is in contact with the main chain carbonyl oxygen of Ala75. In the complex, the ribose ring of the 2'GMP molecule adopts a C2'-endo puckering, and the exocyclic conformation is gauche(-)-gauche(+). The glycosyl torsion angle is in the syn range with an intramolecular hydrogen bond between N3 and O5', and the 2'-phosphate orientation is trans-gauche(-). The guanine base of the inhibitor is tightly bound to the base recognition site with five hydrogen bonds (N1--Glu46O epsilon 2, N2---Asn98O,O6---Asn44N, and N7 ---Asn43N delta 2/Asn43N) and is sandwiched between the phenolic ring portions of Tyr42 and Tyr45 by stacking interactions. The 2'-phosphate group interacts with Arg77N eta 2, Glu58O episilon 2, and Tyr 38O eta but not with any of the histidine residues. Arg77N eta 2 also interacts with Tyr38O eta. There is no interaction between the ribose moiety of the inhibitor and the enzyme.     

Crystallization of ribonuclease St

The crystals of ribonuclease St, the extracellular ribonuclease from Streptomyces erythreus, have been obtained from (NH₄)₂SO₄ solution with acetate buffer (pH 4.1). The crystals belong to a monoclinic space group C2 with dimensions $\text{a = 88.4}\text{A}\text{?}\text{, b = 33.0}\text{A}\text{?}\text{, c = 69.0}\text{A}\text{?}\text{, β = 98.4 °}$. There are two protein molecules per asymmetric unit. The crystals diffract beyond 2.0 Å resolution.     

Crystallization of mouse pancreatic ribonuclease

Mouse pancreatic ribonuclease has been crystallized in a form suitable for X-ray structure determination. The crystals grown from solutions of 2-methyl-2,4-pentanediol diffract to high resolution and belong to the hexagonal space group P6(1) (P6(5)) with unit cells dimensions a = b = 64.44 A, c = 53.91 A, y = 120 degrees and V = 1.94 x 10(5) A3 (1 A = 0.1 nm). There are six molecules per unit cell (1 molecule/asymmetric unit), and Vm = 2.3 A3/dalton.     

Formation of a new 5'-guanylic acid helix in neutral solution

5′-GMP forms in neutral solution a regular, ordered structure, presumably helical. The ordered structure is different from that previously reported at pH 5 and is not associated with gel formation. The transition is more cooperative than that of the pH 5 helix, but has a lower T_m.     

Crystallization of a new class of microbial ribonuclease from Rhizopus niveus

Crystals of ribonuclease Rh, a new class of microbial ribonuclease from Rhizopus niveus, were obtained from polyethylene glycol 8000 solution by a vapour diffusion technique in the hanging drop mode. Two crystal forms, type I and type II, were obtained from the same droplet solution. Both forms belong to the space group P2(1)2(1)2(1), but their cell dimensions are markedly different: a = 68.3 A, b = 73.0 A, c = 50.0 A for type I and a = 67.5 A, b = 72.3 A, c = 44.2 A for type II. The type I crystals diffract beyond 2.0 A resolution and are suitable for X-ray structure analysis at high resolution.     

A 15N-NMR study on ribonuclease T1-guanylic acid complex

Ribonuclease T1 is highly specific for the guanylic acid residue in polyribonucleotides. To clarify the origin of the substrate specificity, the interaction sites of guanylic acid with ribonuclease T1 were investigated by the use of 15N-NMR. 95% 15N-enriched guanosine-3'-phosphate was prepared and mixed with purified ribonuclease T1. 15N-NMR spectra of the mixtures at different concentrations were obtained and compared with that of the 15N-enriched substrate alone. Upon complex formation, a 15N signal assigned to the amino group nitrogen at position 2 of guanine shifted and was significantly broadened, suggesting a strong interaction with the enzyme through the amino group. This observation is consistent with the results of studies on the substrate specificity of chemical modification. Nuclear Overhauser effects of signals assigned to N-7 and N-3 were also changed, but not shift was observed. The observations do not support the occurrence of protonation at N-7 upon complex formation, which was previously proposed.     

Crystallization and preliminary X-ray investigation of a complex between a Fab fragment and its antigen, cyclosporin

Preliminary crystallographic data are given for a complex between the cyclic undecapeptide cyclosporin and the Fab fragment of an anti-cyclosporin monoclonal antibody. Crystals of the complex are orthorhombic with space group P2(1)2(1)2(1) and diffract to 2.7 A resolution. The unit cell dimensions are a = 52.6 A, b = 70.2 A and c = 118.4 A. A native data set to 2.7 A resolution has been collected.     

Crystallization and preliminary characterization of mitogillin, a ribosomal ribonuclease from Aspergillus restrictus.

Mitogillin is a ribonuclease secreted by the fungus Aspergillus restrictus. The substrate for mitogillin is a short, universally conserved, sequence in ribosomal RNA. Cleavage of this sequence inactivates protein synthesis. Mitogillin was crystallized by a two-chamber vapor/liquid diffusion method using ethanol as the precipitant. This method has wider potential in the use of volatile organic solvents as precipitants. Crystals of mitogillin diffract X-rays to lattice d-spacings of at least 1.6 A, and belong to the monoclinic space group P2(1), with a = 50.4 A, b = 82.4 A, c = 38.2 A and beta = 99.8 degrees.     

Crystal structure disposition of thymidylic acid tetramer in complex with ribonuclease A.

The crystal structure of ribonuclease A with bound thymidylic acid tetramer is reported at 2.5-A resolution. The diffusion of the tetramer into native orthorhombic crystals of the ribonuclease allows for the formation of a structurally stable complex where the single-stranded nucleic acid enters and leaves the enzyme's catalytic region in a persistent 5'-3' direction. The binding of the tetramer to the enzyme's surface is facilitated and mediated by electrostatic interactions between basic protein residues and nucleotide phosphates. Two pyrimidine nucleotides are bound to the enzyme's active site in a manner similar to that observed for other complexes between ribonuclease A and nucleic acid oligomers.     

Crystallization and preliminary X-ray study of a complex between d(ATGCAT) and actinomycin D

Crystals of a 2:1 complex between the self-complementary DNA hexamer d(ATGCAT) and the antitumor drug actinomycin D have been grown from solutions of polyethylene glycol 400. The crystals are orthorhombic with space group P2(1)2(1)2(1) and a = 95.6, b = 42.7, and c = 40.8 A. A Patterson map calculated from preliminary diffractometer data as well as packing considerations suggest a model in which the actinomycin D is intercalated into a double-stranded DNA hexamer. There are four such complexes in the asymmetric unit.