Yeast tRNA(Asp)-aspartyl-tRNA synthetase complex: low resolution crystal structure

Yeast aspartyl-tRNA synthetase, a dimer of molecular weight 125,000, and two molecules of its cognate tRNA (Mr = 24160) cocrystallize in the cubic space group I432 (a = 354 A). The crystal structure was solved to low resolution using neutron and X-ray diffraction data. Neutron single crystal diffraction data were collected in five solvents differing by their D2O content in order to use the contrast variation method to distinguish between the protein and tRNA. The synthetase was first located at 40 A resolution using the 65% D2O neutron data (tRNA matched) tRNA molecules were found at 20 A resolution using both neutron and X-ray data. The resulting model was refined against 10 A resolution X-ray data, using density modification and least-squares refinement of the tRNA positions. The crystal structure solved without a priori phase knowledge, was confirmed later by isomorphous replacement. The molecular model of the complex is in good agreement with results obtained in solution by probing the protected part of the tRNA by chemical reagents.     

Yeast tRNA Asp-Aspartyl-tRNA Synthetase: The Crystalline Complex

Abstract

Aspartyl-tRNA synthetase from yeast, a dimer of molecular weight 125,000 and its cognate tRNA (Mr=24,160) were co-crystallized using ammonium sulfate as precipitant agent. The presence in the crystals of both components in the two-to-one stoichiometric ratio was demonstrated by electrophoresis, biological activity assays and crystallographic data. Crystals belong to the cubic space group 1432 with cell parameter of 354 Å and one complex particle per asymmetric unit.

The solvent content of about 78% is favorable for a low resolution structural investigation. By exchanging H₂O for D₂O in mother liquors, advantage can be taken from contrast variation techniques with neutron radiations. Diffraction data to 20 Å resolution were measured at five different contrasts, two of them being close to the theoretical matching point of RNA and protein in the presence of ammonium sulfate. The experimental extinction of the diffracted signal was observed to be close to 36% D₂O, significantly different from the predicted value of 41%. The phenomenon can be explained by the existence of a large interface region between the two tRNAs and the enzyme. These parts of the molecules are hidden from the solvent and their protons are less easily exchangeable. Accessibility studies toward chemicals of tRNA^Asp in solution and in the presence of synthetase are in agreement with such a model.     

Neutron diffraction studies on crystals of nucleosome cores using contrast variation

Neutron diffraction data have been collected from crystals of intact nucleosome core particles to a resolution of 25 Å. By varying the proportion of D₂O, the scattering of the mother liquor relative to the protein and DNA can be altered. At 39% D₂O, the solvent scattering matches that of the protein and so only the DNA is scattering, and similarly at 65% D₂0 only the protein scatters. Using this approach the neutron scattering of the two components and of the complete particle (0% D₂O) have been measured. The data corresponding to the principal projections are consistent with a model in which 1.8 turns of a DNA superhelix of pitch 27·5 Å and radius 42 Å are wound around a protein core.     

f-Element/crown ether complexes. 1. Synthesis and structure of [Y(OH2)8]Cl3·(15-crown-5)

The crystal and molecular structure of [Y(OH₂)₈]Cl₃·(15-crown-5) has been determined by single- crystal X-ray diffraction. The complex crystallizes in the monoclinic space group P2₁/n with Z = 4. Lattice parameters are a = 9.202(2), b = 17.247(3), c = 15.208(3) Å, and β = 92.39(2)°. The structure was solved by Patterson and Fourier techniques and refined by least-squares to a final conventional R value of 0.081. The Y(III) ion is eight coordinate, bonded to the oxygen atoms of the eight water molecules. Three of the water molecules are hydrogen bonded to crown ether molecules. The three chloride ions participate in hydrogen bonds with the remaining five water molecules. The YO(water) distances range from 2.322(6) to 2.432(7) Å and average 2.37(4) Å. The average O(water)···Cl and O(water)···O(crown) hydrogen bonded separations are 3.08(4) and 2.76(7) Å, respectively.     

Hydrogen-bond network and pH sensitivity in transthyretin: Neutron crystal structure of human transthyretin

Transthyretin (TTR) is a tetrameric protein associated with human amyloidosis. In vitro, the formation of amyloid fibrils by TTR is known to be promoted by low pH. Here we show the neutron structure of TTR, focusing on the hydrogen bonds, protonation states and pH sensitivities. A large crystal was prepared at pD 7.4 for neutron protein crystallography. Neutron diffraction studies were conducted using the IBARAKI Biological Crystal Diffractometer with the time-of-flight method. The neutron structure solved at 2.0 Å resolution revealed the protonation states of His88 and the detailed hydrogen-bond network depending on the protonation states of His88. This hydrogen-bond network is composed of Thr75, Trp79, His88, Ser112, Pro113, Thr118-B and four water molecules, and is involved in both monomer–monomer and dimer–dimer interactions, suggesting that the double protonation of His88 by acidification breaks the hydrogen-bond network and causes the destabilization of the TTR tetramer. In addition, the comparison with X-ray structure at pH 4.0 indicated that the protonation occurred to Asp74, His88 and Glu89 at pH 4.0. Our neutron model provides insights into the molecular stability of TTR related to the hydrogen-bond network, the pH sensitivity and the CH···O weak hydrogen bond.     

Complicated water orientations in the minor groove of the B-DNA decamer d(CCATTAATGG)2 observed by neutron diffraction measurements

It has long been suspected that the structure and function of a DNA duplex can be strongly dependent on its degree of hydration. By neutron diffraction experiments, we have succeeded in determining most of the hydrogen (H) and deuterium (D) atomic positions in the decameric d(CCATTAATGG)₂ duplex. Moreover, the D positions in 27 D₂O molecules have been determined. In particular, the complex water network in the minor groove has been observed in detail. By a combined structural analysis using 2.0 Å resolution X-ray and 3.0 Å resolution neutron data, it is clear that the spine of hydration is built up, not only by a simple hexagonal hydration pattern (as reported in earlier X-ray studies), but also by many other water bridges hydrogen-bonded to the DNA strands. The complexity of the hydration pattern in the minor groove is derived from an extraordinary variety of orientations displayed by the water molecules.     

X‐ray crystal structure of anhydrous chitosan at atomic resolution

We determined the crystal structure of anhydrous chitosan at atomic resolution, using X‐ray fiber diffraction data extending to 1.17 Å resolution. The unit cell [a = 8.129(7) Å, b = 8.347(6) Å, c = 10.311(7) Å, space group P2₁2₁2₁] of anhydrous chitosan contains two chains having one glucosamine residue in the asymmetric unit with the primary hydroxyl group in the gt conformation, that could be directly located in the Fourier omit map. The molecular arrangement of chitosan is very similar to the corner chains of cellulose II implying similar intermolecular hydrogen bonding between O6 and the amine nitrogen atom, and an intramolecular bifurcated hydrogen bond from O3 to O5 and O6. In addition to the classical hydrogen bonds, all the aliphatic hydrogens were involved in one or two weak hydrogen bonds, mostly helping to stabilize cohesion between antiparallel chains. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 361–368, 2016.     

Sizes of two amino acyl-tRNA synthetase complexes from E. coli with their cognate tRNAs.

The complexes of valyl-tRNA synthetase with tRNA_I^Val and arginyl-tRNA synthetase with tRNA_II^Arg from $\text{E. coli}$ were studied by light scattering measurements and analytical ultracentrifugation of concentrations as low as 40 μg/ml. The molecular weights determined from these studies were 260,000 ± 2,000 for the valyl-tRNA synthetase·tRNA complex, and 310,000 ± 1,500 for the arginyl-tRNA synthetase·tRNA complex at pH 7.1. The stoichiometry for the complexes are apparently 2:1 for valyl-tRNA synthetase and tRNA and 4:1 in the case of the arginyl-tRNA synthetase and tRNA. From the angular dependence of the scattered intensity a radius of gyration of 54.5 Å for the complex between valyl-tRNA synthetase and tRNA was found, whereas for the other complex a value of 59.1 Å was found.     

Crystallization and preliminary X-ray diffraction studies of leishmanolysin,the major surface metalloproteinase from Leishmania major

The membrane-bound GPI-anchored zinc metalloproteinase leishmanolysin purified from Leishmania major promastigotes has been crystallized in its mature form. Two crystal forms of leishmanolysin have been grown by the vapor diffusion method using 2-methyl-2,4-pentanediol as the precipitant. Macroseeding techniques were employed to produce large single crystals. Protein microhet-erogeneity in molecular size and charge was incorporated into both crystal forms. The tetragonal crystal form belongs to the space group P4₁2₁2 or the enantiomorph P4₃2₁2, has unit cell parameters of a = b = 63.6 Å, c = 251.4 Å, and contains one molecule per asymmetric unit. The second crystal form is monoclinic, space group C2, with unit cell dimensions a = 107.2 Å, b = 90.6 Å, c = 70.6 Å, β = 110.6°, and also contains one molecule per asymmetric unit. Both crystal forms diffract X-rays beyond 2.6 Å resolution and are suitable for X-ray analysis. Native diffraction data sets have been collected and the structure determination of leishmanolysin using a combination of the isomorphous replacement and the molecular replacement methods is in progress. © 1995 Wiley-Liss, Inc.     

X-ray crystallographic studies of polymorphic forms of yeast phenylalanine transfer RNA

Yeast phenylalanine transfer RNA has been found to crystallize in five different crystal systems involving eight different space groups. The X-ray diffraction characteristics of these forms are described. One of the orthorhombic forms yields a diffraction pattern with higher resolution than either the hexagonal, the cubic or the monoclinic forms. One region of this orthorhombic diffraction pattern is particularly sensitive to X-ray exposure and to changes in the concentration of various solutes. The diffraction pattern from the cubic crystal form extends to a resolution of 3 Å, and there are a number of strong reflections in the 3 to 4 Å region which suggest that double-helical segments of the tRNA molecules are oriented along the 4-fold axes. Some comments are made regarding the nature of the polymorphism in the transfer RNA crystals.     

High-resolution structure of phosphoketolase from Bifidobacterium longum determined by cryo-EM single-particle analysis

In bifidobacteria, phosphoketolase (PKT) plays a key role in the central hexose fermentation pathway called “bifid shunt.” The three-dimensional structure of PKT from Bifidobacterium longum with co-enzyme thiamine diphosphate (ThDpp) was determined at 2.1 Å resolution by cryo-EM single-particle analysis using 196,147 particles to build up the structural model of a PKT octamer related by D₄ symmetry. Although the cryo-EM structure of PKT was almost identical to the X-ray crystal structure previously determined at 2.2 Å resolution, several interesting structural features were observed in the cryo-EM structure. Because this structure was solved at relatively high resolution, it was observed that several amino acid residues adopt multiple conformations. Among them, Q546–D547–H548–N549 (the QN-loop) demonstrate the largest structural change, which seems to be related to the enzymatic function of PKT. The QN-loop is at the entrance to the substrate binding pocket. The minor conformer of the QN-loop is similar to the conformation of the QN-loop in the crystal structure. The major conformer is located further from ThDpp than the minor conformer. Interestingly, the major conformer in the cryo-EM structure of PKT resembles the corresponding loop structure of substrate-bound Escherichia coli transketolase. That is, the minor and major conformers may correspond to “closed” and “open” states for substrate access, respectively. Moreover, because of the high-resolution analysis, many water molecules were observed in the cryo-EM structure of PKT. Structural features of the water molecules in the cryo-EM structure are discussed and compared with water molecules observed in the crystal structure.     

Crystallization and preliminary crystallographic analysis of the N-terminal two domain fragment of vascular cell adhesion molecule-1 (VCAM-1)

A molecular fragment comprising the first two domains of the human vascular cell adhesion molecule-l (VCAM-l) has been crystallized by the vapor diffusion method. Two crystal forms have been examined by X-ray analysis: One crystal form belongs to the space group C2 with two molecules in the asymmetric unit and cell parameters: a = 122.1 Å, b = 48.9 Å, c = 73.4 Å, and β = 117.4°. The other crystal form belongs to the space group P2₁ with one molecule in the asymmetric unit and cell parameters: a = 40.4 Å, b = 45.7 Å, c = 54.7 Å, and β = 100.5°. Diffraction data up to 1.9 Å resolution have been collected for the C2 crystal form. © 1994 Wiley-Liss, Inc.     

Purification,crystallization, and preliminary X-ray diffraction studies of tRNA-guanine transglycosylase from Zymomonas mobilis

The tRNA modifying enzyme tRNA–guanine transglycosylase (Tgt) catalyzes the exchange of guanine in the first position of the anticodon with the queuine precursor 7-aminomethyl-7-deazaguanine. Tgt from Zymomonas mobilis has been purified by crystallization and further recrystallized to obtain single crystals suitable for x-ray diffraction studies. Crystals were grown by vapor diffusion/gel crystallization methods using PEG 8,000 as precipitant. Macroseeding techniques were employed to produce large single crystals. The crystals of Tgt belong to the monoclinic space group C2 with cell constants a = 92.1 Å, b = 65.1 Å, c = 71.9 Å, and β = 97.5°, and contain one molecule per asymmetric unit. A complete diffraction data set from one native crystal has been obtained at 1.85 Å resolution.     

Neutron scattering studies of escherichia coli tyrosyl-trna synthetase and of its interaction with trna tyr

Small-angle neutron scattering studies of Escherichia coli tyrosyl-tRNA synthetase indicate that in solution this enzyme is a dimer of M_r, 91 (±6) × 10³ with a radius of gyration R_G of 37.8 ± 1.1 Å.The increase in the scattering mass of the enzyme upon binding tRNA^Tyr has been followed in 20 mm-imidazole · HCl (pH 7.6), 10 mm-MgCl₂, 0.1 mm-EDTA, 10 mm-2-mercaptoethanol, 150 mm-KCl. A stoichiometry of one bound tRNA per dimeric enzyme molecule was found. The R_G of the complex is equal to 41 ± 1 Å. Titration experiments in 74% ²H₂O, close to the matching point of tRNA, show an R_G of 38.5 ± 1 Å for the enzyme moiety in the complex. From these values, a minimum distance of 49 Å between the centre of mass of the bound tRNA and that of the enzyme was calculated.In low ionic strength conditions (20 mm-imidazole-HCl (pH 7.6), 10 mm-MgCl₂, 0.1 mm-EDTA, 10 mm-2-mercaptoethanol) and at limiting tRNA concentrations with respect to the enzyme, titrations of the enzyme by tRNA^Tyr are characterized by the appearance of aggregates, with a maximum scattered intensity at a stoichiometry of one tRNA per two enzyme molecules. At this point, the measured M_r and R_G values are compatible with a compact 1:2, tRNA: enzyme complex. This complex forms with a remarkably high stability constant: (enzyme:tRNA:enzyme)/(enzyme:tRNA)(enzyme) of 0.1 to 0.3(× 10⁶) m^?1 (at 20 °C). Upon addition of more tRNA, the complex dissociates in favour of the 1:1, enzyme:tRNA complex, which has a higher stability constant (1 to 3 (× 10⁶) m^?1).     

Purification,crystallization, and preliminary X-ray studies of 10-formyltetrahydrofolate synthetase from Clostridia acidici-urici

The monofunctional enzyme 10-formyltetrahydrofolate synthetase (THFS), which is responsible for the recruitment of single carbon units from the formate pool into a variety of folate-dependent biosynthetic pathways, has been subcloned, purified, and crystallized. The crystals belong to space group P2₁, with unit cell dimensions a= 102.4 Å b= 116.5 Å c= 115.8 Å and β = 103.5 Å. The crystal unit cell and diffraction is consistent with an asymmetric unit consisting of the enzyme tetramer, and a specific volume of the unit cell of 2.7 Å₃/Da. The crystals diffract to at least 2.3 Å resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector. © 1997 Wiley-Liss, Inc.     

The crystal and molecular structure of tri-o-ethylamylose (tea 3)

The crystal and molecular structure of a tri-O-ethylamylose polymorph, TEA 3, has been solved by stereochemical conformation and packing analysis, combined with X-ray fibre diffraction analysis. The unit cell is orthorhombic, space group P2₁2₁2₁, with a  15.36 (±0.03) Å, b  12.18 (±0.05) Å, and c (fibre repeat)  15.48 (±0.01) Å. The actual chain conformation is a 4₃ helix with the EtO-6 group in the tg position, as was found in the polymorph TEA 1.     

Crystallization of Thermus thermophilus histidyl-tRNA synthetase and Its complex with tRNAHis

Histidyl-tRNA synthetase (HisRS) has been purified from the extreme thermophile Thermus thermophilus. The protein has been crystallized separately with histidine and with its cognate tRNA^His. Both crystals have been obtained using the vapor diffusion method with ammonium sulphate as precipitant. The crystals of HisRS with histidine belong to the spacegroup P2₁2₁2 with cell parameters a = 171.3 Å, b = 214.7 Å, c = 49.3 Å, α = β = γ = 90°. A complete data set to a resolution of 2.7Å with an R_merge on intensities of 4.1% has been collected on a single frozen crystal. A partial data set collected on a crystal of HisRS in complex with tRNA_His shows that the crystals are tetragonal with cell parameters a = b = 232 Å, c = 559 Å, α = β = γ = 90° and diffract to about 4.5 Å resolution. © 1995 Wiley-Liss, Inc.     

Crystal Structure of a Tightly Bound Inhibitor of Adenosine Transport N6-(4-Nitrobenzyl)-β-D-2′-Deoxyadenosine Methanol Solvate,C17H18N6O5.1/2 CH3OH

Abstract

The molecular structure of N⁶-(4-nitrobenzyl)-β-D-2′-deoxyadenosine (I) has been determined by single crystal X-ray diffraction. A potent inhibitor of adenosine permeation in cultured S49 mouse lymphoma cells, I binds tightly (K_D 2.4 nM) to high affinity membrane sites present on the nucleoside transporter elements of these cells. Compound I crystallizes in the trigonal space group P3₂21 with unit cell dimensions a = b = 8.0009(9)Å, c = 49.174(8)Å, and Z = 6. The structure was solved by direct methods and refined by least-squares to a final R = 0.038. The mean plane of the 4-nitrobenzyl group, an important substituent for potent nucleoside transport inhibition in a series of S6-substituted 6-thioinosine derivatives, is inclined at an angle of 120.6° to the plane of the adenine ring. The torsion angles around the methylene carbon atom of this benzyl group are C(6)-N(6)-C(10)-C(11), 96.6° and N(6)-C(10)-C(11)-C(12), 93.6°. The glycosidic torsion angle, X, is 217.1° which corresponds to the common anti nucleoside conformation. The deoxyribose ring, however, has the unusual C(1′)-exo conformation, with C(1′) displaced 0.608Å from the plane of C(2′), C(3′), C(4′) and O(4′). The conformation about the exocyclic C(4′)-C(5′) bond is gauche⁺.     

Impact of lyoprotectors on protein-protein separation in the solid state: Neutron- and X-ray-scattering investigation

BackgroundPolyhydroxycompounds (PHC) are used as lyoprotectors to minimize aggregation of pharmaceutical proteins during freeze-drying and storage.MethodsLysozyme/PHC mixtures with 1:1 and 1:3 (w/w) ratios are freeze-dried from either H₂O or D₂O solutions. Disaccharides (sucrose and trehalose), monosaccharide (glucose), and sugar alcohol (sorbitol) are used in the study. Small-angle neutron and X-ray scattering (SANS and SAXS) are applied to study protein-protein interaction in the freeze-dried samples.ResultsProtein interaction peak in the freeze-dried mixtures has been detected by both SANS (D₂O-based samples only) and SAXS (both D₂O- and H₂O-based). In the 1:1 mixtures, protein separation distances are similar (center-of-mass distance of approx. 31 Å) between all lyoprotectors studied. Mixtures with a higher content of the disaccharides (1:3 ratio) have a higher separation distance of approx 40 Å. The higher separation could reduce protein-protein contacts and therefore be associated with less favourable aggregation conditions. In the 1:3 mixtures with glucose and sorbitol, complex SANS and SAXS/WAXS patterns are observed. The pattern for the glucose sample indicate two populations of lysozyme molecules, while the origin of multiple SAXS peaks in the lysozyme/sorbitol 1:3 mixture is uncertain.ConclusionsProtein-protein separation distance is determined predominantly by the lyoprotector/protein weight ratio.General significanceUse of SANS and SAXS improves understanding of mechanisms of protein stabilization by sugars in freeze-dried formulations, and provide a tool to verify hypothesis on relationship between protein/protein separation and aggregation propensity in the dried state.