Determination and restrained least-squares refinement of the structures of ribonuclease Sa and its complex with 3'-guanylic acid at 1.8 Å resolution

Complex of ribonuclease from Streptomyces aureofaciens with 2'-GMP at 1.7 Å resolution

Acta Crystallographica Section D Biological Crystallography, 1993

The crystal structure of a complex of ribonuclease from Streptomyces aureofaciens (RNase Sa) with guanosine-2'-monophosphate (2'-GMP) has been refined against synchrotron data recorded from a single crystal using radiation from beamline X31 at EMBL, Hamburg, and an imaging plate scanner. The crystals are in space group P212~2~ with cell dimensions a-64.7, b = 78.8 and c = 39.1 A. The structure has two enzyme molecules in the asymmetric unit, complexed with 2'-GMP inhibitor with occupancies of 1 and ] (different to the 3'-GMP complex crystal structure where only one of the two independent RNase Sa molecules binds nucleotide), 492 associated water molecules and one sulfate ion, and was refined using all data between 10.0 and 1.7 A to a final crystallographic R factor of 13.25%. Binding of the base to the enzyme confirms the basis for the guanine specificity but the structural results still do not provide direct evidence of the identity and role of the particular residues involved in the catalytic process. New native RNase Sa data to 1.8 were recorded to provide a reference set measured under comparable experimental conditions. The crystals are in the same space group and have the same lattice as those of the 2'-GMP complex. The native structure with 423 water molecules was refined in a similar manner to the complex to a final R factor of 13.87%. 1.77 A resolution data were independently measured on a 2"-GMP complex crystal at UCLA using an R-AXIS II image plate scanner mounted on

Ribonuclease from Streptomyces aureofaciens at Atomic Resolution

Acta Crystallographica Section D Biological Crystallography, 1996

2. Introduction Crystals of ribonuclease from Streptomyces aureofaciens diffract to atomic resolution at room temperature. Using synchrotron radiation and an imaging-plate scanner, X-ray data have been recorded to 1.20/~ resolution from a crystal of native enzyme and to 1.15/~ from a crystal of a complex with guanosine-2'-monophosphate. Refinement with anisotropic atomic temperature factors resulted in increased accuracy of the structure. The R factors for the two structures are 10.6 and 10.9%. The estimated r.m.s, error in the coordinates is 0.05/~,, less than half that obtained in the previous analysis at 1.7 .~ resolution. For the well ordered part of the main chain the error falls to below 0.02 A as estimated from inversion of the least-squares matrix. The two independent molecules in the asymmetric unit allowed detailed analysis of peptide planarity and some torsion angles. The high accuracy of the analysis revealed density for a partially occupied anion in the nucleotide binding site of molecule A in the native structure which was not seen at lower resolution. The anisotropic model allowed correction of the identity of the residue at position 72 from cysteine to threonine. Cys72SG had been modelled in previous analyses with two conformations. The solvent structure was modelled by means of an automated procedure employing a set of objective criteria. The solvent structure for models refined using different programs with isotropic and anisotropic description of thermal motion is compared.

Crystal Structures of Ribonuclease F1 of Fusarium moniliforme in Its Free Form and in Complex with 2′GMP

Journal of Molecular Biology, 1993

RNase Fl, a guanine-specific ribonuclease from Ji1usariu1n moniliforme, was crystallized in two different forms, in the absence of an inhibitor and in the presence of 2'GMP. The crystal structure of the RNase Fl free fo rm was solved by the molecular replacement method, using the coordinates of the RNase Tl complex with 2'GMP, and was refined to a final R-factor of 18 •7 %, using the data extended to 1• 3 A resol ution. For the crystal structure of the RNase Fl complex with 2'GMP, the solution of the molecular replacement method was obtained on the basis of the coordinates of the RN ase Fl free form, and was refined to a final R-factor of 16-8%, using the data up to 2 A resolution. The two crystal structures of the RNase Fl free form and the complex with 2'GMP are very similar to each other as reflected by a small root mean-square displacement (r.m.s.d.) value of 0•43 A for all ca atoms. The main differences between the two structures are associated with binding of 2'GMP in the substrate recognition site in the loop between Tyr42 and Glu46. A structural comparison bet\veen RNase Fl and RNase Tl shows a substantial similarity between all the ca atoms, as evidenced by a r.m.s.d. value of l •4 A. The loop from residues 32 to 38 was strikingly different between these two enzymes, in both its conformation and its hydrogen bonding schemes. The side-chain of a catalytically active residue, His92, is shifted away from the catalytic site in RNase Fl by l •3 A and 0•85 A with respect to the corresponding positions in the RNase Tl free form and in the RNase Tl complex with 2'GMP, respectively. In the RNase Fl complex, the guanine base of 2'GMP has a syn conformation about the glycosyl bond, and the fu ranose ring assumes a 3'-exo pucker, which is different from that found in the complex with RNase Tl. In the catalytic site of the RNase Fl complex with 2'GMP, one water molecule was observed, which bridges the phosphate oxygen atoms of 2'GMP and the side-chains of the catalytically important residues, His92 and Arg77, through hydrogen bonds. A water molecule occupying the same position was found in the RNase Fl free form. The significance of this water molecule in the hydrolytic reaction is discussed.

The ultrahigh resolution crystal structure of ribonuclease A containing an isoaspartyl residue: hydration and sterochemical analysis

Journal of Molecular Biology, 2000

Crystals of the deamidated form of bovine pancreatic ribonuclease which contains an isoaspartyl residue in position 67 diffract to 0.87 A Ê at 100 K. We have re®ned the crystallographic model using anisotropic displacement parameters for all atoms to a conventional crystallographic residual R 0.101 for all observed re¯ections in the resolution range 61.0-0.87 A Ê . The ratio observations/parameters is 7.2 for the ®nal model. This structure represents one of the highest resolution protein structures to date and interestingly, it is the only example containing more than one molecule in the asymmetric unit with a resolution better than 1.0 A Ê . The non-crystallographic symmetry has been used as a validation check of the geometrical parameters and it has allowed an estimate for an upper limit of errors associated with this high resolution model. In the present structure it was possible to obtain a more accurate picture of the active site whose electron density was not clearly interpretable in the previous 1.9 A Ê resolution structure. In particular, the P1 site is alternatively occupied either by a sulphate anion or by a water molecule network. Most of hydrogen atoms were visible in the electron density maps, including those involved in C a ÐH a Á Á ÁO interactions. Analysis of protein-solvent interactions has revealed the occurrence of an extensive cluster of water molecules, predominantly arranged in pentagonal fused rings and surrounding hydrophobic moiety of side-chains. Finally, in spite of the limited sample of residues, we have detected a clear dependence of backbone N ÐC a ÐC angle on residue conformation. This correlation can be fruitfully used as a valuable tool in protein structure validation.

The complex between ribonuclease T1 and 3'GMP suggests geometry of enzymic reaction path. An X-ray study

European Journal of Biochemistry, 1993

The crystal structure of the complex between ribonuclease T1 and 3'GMP suggests that (a) a substrate GpN is bound to the active site of ribonuclease T1 in a conformation that actively supports the catalytic process, (b) the reaction occurs in an in-line process, (c) His40 NEH' activates 02'-H, (d) Glu58 carboxylate acts as base and His92 NEH' as acid in a general acid-base catalysis.

Structure of ribonuclease A derivative II at 2.1-A resolution

The Journal of biological chemistry, 1994

The crystal structure of bovine pancreatic ribonuclease A derivative II, a covalent derivative obtained by reaction of 6-chloropurine 9-beta-D-ribofuranosyl 5'-monophosphate with the alpha-amino group of Lys-1, has been determined and refined at 2.1-A resolution with an agreement factor R = 0.166 for 6254 reflections in the resolution shell 8.0 to 2.1 A. Crystals are orthorhombic and belong to space group C222(1) with unit cell parameters a = 75.73 A, b = 57.85 A, and c = 53.26 A. This crystal packing had never been reported before for pancreatic ribonuclease nor its complexes. The structure found is in accordance with the location of p2, B3, and R3 subsites at the N-terminal region of the protein and provides an explanation of the catalytic behavior observed for this derivative. In particular, differences in kinetic parameters and in the pKa value of His-119 between derivative II and native ribonuclease A can be interpreted on the basis of the position of the phosphate moiety w...

The refined structure of ribonuclease-A at 1.45 � resolution

Journal of Chemical Crystallography, 1984

Features of the refined X-ray crystal structure of bovine pancreatic ribonuclease-A at 1.45 Å resolution are described. The positions of the protein atoms have been determined within the range 0.004–0.05 Å, and of solvent atoms, assumed to be oxygens, within the range 0.08–0.13 Å. The present model contains 127 solvent molecules, taken to be water, and a sulfate anion located in the active site. Mean square atomic displacement parameters,U iso, refined for each atom, give an indication of the mobility of different parts of the structure. Main-chainU iso values tend to be less than side-chain values, having an average value of 0.15 Å2 compared to 0.25 Å2. Both main-chain and side-chain averageU iso values tend to increase with distance from the center of gravity of the molecule. Side-chain averageU iso values also tend to increase with the number of atoms in the side-chain, with different distributions for ring and chain type residues. Side-chain conformations have been analyzed and found on the whole to follow commonly observed distributions. A notable exception to this is the active-site residue His-119 which occupies two distinct sites. Apart from two small clusters of eight and seven atoms respectively, the solvent molecules are distributed in quite small numbers on the protein surface. The solvent clusters occur in the active-site region and, together with the sulfate anion, appear to stabilize residues in this region. Sixty-three solvent atoms have only one identified hydrogen bond contact. Of the rest, 36 form two, 22 form three, and 6 form four hydrogen bonds. There is a marked tendency for the mean square displacement parameter,U iso, for the solvent atoms to be lower for atoms with many hydrogen bond contacts than for those with fewer contacts.

The refined structure of ribonuclease-A at 1.45 Å resolution

Journal of Chemical Crystallography, 1984

Determination and restrained least-squares refinement of the structures of ribonuclease Sa and its complex with 3'-guanylic acid at 1.8 Å resolution (original) (raw)

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