Crystallization and Preliminary X-Ray Analysis of Neuropsin, a Serine Protease Expressed in the Limbic System of Mouse Brain

Neuropsin (M_r25 032) is a serine protease expressed in the limbic system of mouse brain. It has been implicated in various neurological processes including formation of memory and may be important as a drug target in the treatment of epilepsy. The recombinant protein was produced using a baculovirus expression system and was purified. Two crystal forms were obtained by a hanging-drop vapor-diffusion method with polyethylene glycol. Preliminary X-ray crystallographic analysis revealed that crystal form I belongs to triclinic space groupP1 with unit cell dimensionsa= 97.16 Å,b= 97.12 Å,c= 46.75 Å and α = 99.17°, β = 99.77°, γ = 117.35°. Self-rotation function analysis of these data of form I indicates the position of a noncrystallographic threefold axis. There are six molecules in the crystallographic asymmetric unit. Crystal form II also belongs to triclinic space groupP1 but has unit cell dimensions ofa= 38.40 Å,b= 55.16 Å,c= 65.37 Å and α = 95.38°, β = 89.98°, γ = 110.46° with two molecules in the crystallographic asymmetric unit. Form II has a noncrystallographic twofold axis. Intensity data to 3.1 Å resolution for form I and to 2.2 Å resolution for form II have been collected.     

New Crystal Forms of the Motile Major Sperm Protein (MSP) ofAscaris suum

We have obtained two new crystal forms of theAscarismajor sperm protein (MSP) that mediates amoeboid cell motility in nematode sperm. We obtained crystals with C2 symmetry from bacterially expressed α-MSP witha= 216.5 Å,b= 38.6 Å,c= 32.5 Å, γ = 93.1° and also crystals with P2₁symmetry from native β-MSP witha= 63.1 Å,b= 91.7 Å,c= 72.5 Å, γ = 91.3°. A full native data set has been collected for each crystal form using synchrotron radiation. Both crystal forms diffract to 2 Å and are suitable for high-resolution structural investigation.     

Crystallization and Characterization of Pumilio: A Novel RNA Binding Protein

Axis determination in early Drosophila embryos is controlled, in part, by regulation of translation of mRNAs transcribed in maternal cells during oogenesis. The Pumilio protein is essential in posterior determination, binding to hunchback mRNA in complex with Nanos to suppress hunchback translation. In order to understand the structural basis of RNA binding, Nanos recruitment, and translational control, we have crystallized a domain of the Drosophila Pumilio protein that binds RNA. The crystals belong to the space group P6₃ with unit cell dimensions of a = b = 94.5 Å, c = 228.9 Å, α = β = 90°, γ = 120° and diffract to 2.6 Å with synchrotron radiation. We show that the purified protein actively binds RNA and is likely to have a novel RNA binding fold due to a very high content of α-helical secondary structure.     

Purification, crystallization and preliminary X-ray diffraction studies of the bacteriophage φ29 connector particle

The connector or portal particle from double-stranded DNA bacteriophage φ29 has been crystallized. This structure, which connects the head of the virus with the tail and plays a central role in prohead assembly and DNA packaging and translocation, is formed by 12 subunits of the p10 protein and has a molecular weight of 430 kDa. The connector structure was proteolysed with endoproteinase Glu-C from Staphylococcus aureus V8, which removes 13 and 18 amino acids from the amino- and carboxy-terminal regions of the p10 protein, respectively. Two crystal forms were grown from drops containing an alcohol solution and paraffin oil. Crystals of form I are monoclinic, space group C2 with cell dimensions a=416.86 Å, b=227.62 Å, c=236.68 Å and β=96.3° and contain four connector particles per asymmetric unit. Crystals of form II are tetragonal, space group P4₂2₁2 with cell dimensions a=b=170.2 Å, c=156.9 Å and contain half a particle per asymmetric unit. X-ray diffraction data from both native crystal forms have been collected to 6.0 and 3.2 Å respectively, using synchrotron radiation. Crystals of form II are likely to have the same packing arrangement as the two-dimensional crystals analyzed previously by electron microscopy.     

Crystal structures of cyclomaltohexaose (α-cyclodextrin) complexes with p-chlorophenol and p-cresol

Crystal structures of cyclomaltohexaose (α-cyclodextrin) complexes with p-chlorophenol and p-cresol have been determined by single-crystal X-ray diffraction studies. The space group of the α-cyclodextrin–p-chlorophenol complex is P2₁2₁2₁ with unit cell dimensions of a=15.299(3), b=24.795(5), c=13.447(5) Å, and that of the α-cyclodextrin–p-cresol complex is P2₁ with unit cell dimensions of a=7.927(7), b=13.568(7), c=24.54(1) Å, β=90.41(8)°. In spite of the similar structures of guest molecules, both complexes have different inclusion modes and packing structures.     

The X-ray structure analysis of bis-2,2′,N,N′-bipyridyl ketone cobalt(III) nitrate dihydrate

An X-ray structural analysis of bis-2,2′,N,N′-bipyridyl ketone cobalt(III) nitrate dihydrate, CoC₂₂H₂₀N₄O₄⁺· NO₃⁻·2H₂O,M_r=559.38 g/mol, P , a=8.862(2), b=16.195(3), c=8.772(2) Å, α=103.54(2), β=95.74(3), γ=105.07°, V=1164.4(4) ų, Z=2, D_x=1.595 g/cm³, Mo Kα radiation (λ=0.71073 Å), μ=7.8 cm⁻¹ and R=0.079, revealed a Co(III) cation in a slightly distorted octahedral environment. The structure reveals that the ligand di-2-pyridyl ketone (dpk) has undergone a hydration reaction across the ketone double bond and one of the hydrate oxygen atoms coordinated to the metal forming a tridentate chelate. This new Co(dpk-hydrate)₂⁺ complex displays the least distorted geometry yet reported for either 1:1 or 1:2 (metal:ligand) complexes. A geometry optimization using the INDO model Hamiltonian as implemented in the program ZINDO was performed on the title complex with the Co³⁺ modeled as a singlet. The result of the computation corroborates the geometry of the title complex as that expected for Co³⁺.     

Synthesis and crystal structure of the MgCl2(CH3COOC2H5)2· (CH3COOC2H5) adduct

The reaction of α-MgCl₂ with boiling ethyl acetate affords MgCI₂(CH₃COOC₂H₅)₂· (CH₃COOC₂H₅), which is obtained as crystals suitable for X-ray analysis only from the mother liquor. M=315.5, orthorhombic, space group P2₁22₁ (No. 18), a=25.077(3), b=8.616(1), c=7.345(1) Å, V=1587.0(3) ų, Z=4, D_x=1.32 g cm⁻³,λ A(Mo Kα)=0.71069 Å, μ=4.17 cm⁻¹, F(000)=664, T=298 K, observed reflections: 1667, R=0.059 and R_w=0.069. The structure is composed of polymeric chains of MgCl₂(CH₃COOC₂H₅₎₂ and the ethyl acetate molecules occupy a mutually trans position.     

A study of the reactivity and structure of cyclic α,β-unsaturated Fischer-type carbene complexes

Cycloaddition reactions with α,β-unsaturated carbene complexes of the Fischer-type bearing the carbene carbon atom and the double bond incorporated in the same ring are described. Pentacarbonyl(2H-benzopyran-2- ylidene)chromium(0) complexes (2a-c) and pentacarbonyl(4-methoxy-3,3-dimethyl-2-oxacyclopentylidene)- chromium(0) (3) show a rather low reactivity towards 1,3-dipoles and 1,3-dienes. The reactions with diazomethane are regioselective but not chemoselective; compounds 2 and 3 show two sites of attack: the α,β carbon-carbon and the carbon-metal double bond. The crystal and molecular structures of 2a and 3 have been elucidated by single crystal X-ray analysis. Crystals of 2a are monoclinic, space group P2₁/c, a=7.614(3), b=14.033(3), c=12.766(3) Å, β=95.24°, V=1358.3(7) Å Z=4; crystals of 3 are triclinic, space group P , a=6.553(1), b=9.408(1), c=10.620(1) Å α=92.70(1), β=92.30(1), γ=92.12(1)°, V=653.0(1), ų, Z=2. Final agreement indices for 2a and 3 are R=0.034 and 0.033, respectively. Vibrational properties of the Cr(CO)₅ moiety were interpreted by FT-IR and FT-Raman spectroscopy. Electronic spectra and π electron distribution were interpreted by resonance Raman spectroscopy.     

Crystallization and Preliminary X-Ray Analysis of Thermoactinomyces vulgaris R-47 α-Amylase II

Crystals of Thermoactinomyces vulgaris α-amylase II, which is a new type of α-amylase having hydrolysis activities for pullulan and cyclodextrins, have been obtained and diffraction data to 2.9 Å resolution were collected. The crystal belongs to an orthorhombic system with cell dimensions of a = 119.5 Å, b = 120.6 Å, and c = 114.6 Å and a space group of P 2₁2₁2₁. Two or three protein molecules are expected to exist in an asymmetric unit.     

Synthesis and structure of an organosamarium aryloxide complex, (C5 Me5)2 Sm(OC6HMe4-2,3,5,6)

Bis(pentamethylcyclopentadienyl)samarium bis- (tetrahydrofuranate), (C₅Me₅)₂Sm(THF)₂, reacts with 2,3,5,6-tetramethylphenol in toluene to yield (C₅Me₅)₂Sm(OC₆HMe₄-2,3,5,6). The compound crystallizes in the space group P2₁/c with a = 8.725(3) Å, b=18.821(6) Å, c=18.461(6) Å, β= 111.17(2)°, V = 2827(2) ų and D_c=1.340 g cm⁻³ for Z = 4. Molecules of the aryloxide complex are monomeric and exhibit a bent metallocene structure with a nearly linear Sm---O---C(aryloxide) linkage: Sm---O = 2.13(1) Å, O---C = 1.29(2) Å, and Sm---O---C = 172.3(13)°. Reaction of the samarium complex with phenyllithium produces the previously- characterized species (C₅Me₅)₂Sm(C₆H₅)(THF).     

Crystallization and Preliminary X-Ray Diffraction Analysis of the 190-Å-Long Coiled-Coil Dimerization Domain of the Actin-Bundling Protein Cortexillin I fromDictyostelium discoideum

We have crystallized the ≈190-Å-long parallel two-stranded coiled-coil oligomerization domain of the actin-bundling protein cortexillin I fromDictyostelium discoideum. The orthorhombic crystals belong to the space group C222₁with unit cell dimensions ofa= 71.3 Å,b= 127.8 Å, andc= 91.6 Å. As both native and selenomethionine-substituted protein crystals diffract to 3.0 and 2.85 Å resolution, respectively, using synchrotron radiation, they are suitable for the first high-resolution structural analysis of a two-stranded coiled coil comprising more than six heptad repeats. Moreover, because the polypeptide chain fragment contains a recently identified two-heptad-repeat long sequence that is indispensable for the assembly of the cortexillin I coiled-coil oligomerization domain, its high-resolution structure should enable us to extend our knowledge on the molecular mechanisms underlaying coiled-coil formation and to establish the precise manner in which the two “trigger” sequences interact with one another in the dimer.     

Preparation, X-ray structure and solution behavior of [Ag(9-EtGH-N7)2]NO3·H2O (9-EtGH=9-ethylguanine)

The preparation and X-ray structure of [Ag(9-EtGH-N7)₂]NO₃·H₂O(9-EtGH=neutral 9-ethylguanine) is reported. The compound crystallizes in the triclinic system, space group P with a=7.063(6), b=7.153(3), c=11.306(10) Å, α=83.36(6), β=76.66(7), γ=81.44(6)°. The cation is centrosymmetric with Ag(I) coordinated via two N7 positions and Ag---N7 bond lengths of 2.11(1) Å. Applying ¹⁰⁹Ag NMR spectroscopy, complex formation constants for both the 1:1 complex (log β₁=0.6) and the title compound (log β₂=1.6) in Me₂SO have been determined.     

Synthesis and crystal structure of a complex between lanthanum nitrate and a bicyclic polyether

Reaction between lanthanum nitrate hexahydrate and a macrobicyclic polyether in ethanol has yielded a product of overall stoichiometry 3:2. The ligand, 21R, 26S, 29R, 34S-21, 22, 23, 24, 25, 26, 29, 30, 31,32,33, 34-dodecahydro-1,4,7,14,17,20,28,35-octaoxa (2^3,29syn2^18,34syn) (7.7) orthocyclophane, L, provides 8 oxygen donor atoms. Crystals were obtained from MeOH/EtOH (50/50). Crystal structure determination on 9590 observations, R=0.059, has shown the triclinic unit cell, a = 26.562(6), b = 13.486(3), c = 12.154(3) Å, α=63.9(1), β = 100.0(1), γ = 102.0(1)° space group P , V = 3806 Å to contain, as the asymmetric unit, two complex cations (La(NO₃)₂L)⁺ and one complex anion (La- (NO₃)₅MeOH)²⁻. The lanthanum is 11-coordinated in the anion and one of the cations, in which there is one bidentate and one monodentate nitrate anion and 12-coordinated in the other cation. For the monodentate nitrate La---O = 2.448(9) Å, all other nitrate ions are bidentate (La---O = 2.594(9)−2.743(10) Å). Most La---O bonds are shorter in the 11 than in the 12-coordinated cation. There are large differences in La---O bond lengths according to the nature of the carbon atoms to which the oxygen is attached. The methanol molecule forms a hydrogen bond to one oxygen atom of the monodentate nitrate group.     

Synthesis of [Tl4Se16]: a novel tetranuclear Tl polyselenide

(Ph₄P)₄[Tl₄Se₁₆] was prepared hydrothermally in a sealed pyrex tube by the reaction of TlCl, K₂Se₄ and Ph₄PCl in a 1:1:1 molar ratio at 110 °C for one day. The red crystals were obtained in 50% yield. Crystals of (Ph₄P)₄[Tl₄Se₁₆]: triclinic P (No. 2), Z=1, a=12.054(9), b=19.450(10), c=11.799(6) Å, α=104.63(4), β=98.86(6), γ=101.99(6)° and V=2555(3) ų at 23 °C, 2θ_max=40.0°, μ=120.7 cm⁻¹, D_calc=2.23. The structure was solved by direct methods. Number of data collected: 5206. Number of unique data having F_o²>3σ(F_o²): 1723. Final R=0.075 and R_w=0.089. [Tl₄Se₁₆]⁴⁻ consists of four, almost already linearly arranged, tetrahedral thallium centers which are coordinated by two chelating Se₄²⁻, two bridging Se₂²⁻ and four bridging Se²⁻ ligands. [Tl₄Se₁₆]⁴⁻ sits on an inversion center and possesses a central {Tl₂Se₂}²⁺ planar core. The Tl(1)–Tl(1)′ distance in this core is 3.583(6) Å. These two thallium atoms are then each linked to two cyclic Tl(Se₄) fragments via bridging Se₂²⁻ and Se²⁻ ligands forming Tl₂Se(Se₂) five-membered rings.     

Type IIβ Regulatory Subunit of cAMP-Dependent Protein Kinase: Purification Strategies to Optimize Crystallization

To elucidate the structural basis for important differences between types I and II regulatory subunit isoforms (RI and RII) of adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent protein kinase, the full-length RIIβ isoform and five RIIβ deletion mutants were constructed, expressed, purified, and screened for crystallization. Only one of these six proteins yielded diffraction quality crystals. Crystals were grown of the RIIβ deletion mutant (Δ1–111) monomer potentially in complex with two cAMP molecules. X-ray diffraction quality data were obtained only after significant modification to existing purification procedures. Modifications required a Sepharose, not agarose, support for cAMP affinity chromatography followed by rapid, quantitative removal of free cAMP by size-exclusion chromatography under reducing conditions. Data to 2.4 Å resolution were collected at 29°C using synchrotron radiation on a single crystal measuring 0.2 × 0.3 × 1.2 mm³. Data were 99% complete. The hexagonal crystal belonged to space group P6₍₁₎ or P6₍₅₎ with unit cell dimensions a = b = 161.62 Å and c = 39.66 Å.     

Structure and properties of a novel OH bridged dimetal helical complex with 6,6-dimethyl-2,2′:6′,2″:6″,2:6,2-quinquepyridine ligand

A new monohelical OH⁻ bridged dinuclear complex [Zn₂(dmqpy)(OOCCH₃)₂(μ-OH)][ClO₄] · 0.5EtOH, where dmqpy is 6,6-dimethyl-2,2′:6′,2″:6″,2:6,2-quinquepyridine, has been synthesized and characterized by X-ray crystallography: monoclinic, space group P2₁/c, a=13.670(1), b=14.751(1), c=16.782(1) Å, β=96.59(1)°, U=3361.7(4) ų, Z=4, R=0.0601. Two Zn(II) ions are in different coordination modes, one is five-coordinate with a N₃O₂ donor set and the other is N₂O₂ four-coordinate with a distorted tetrahedral geometry, and the zinc ions are bridged by a hydroxyl group. The presence of the OH⁻ bridge is further confirmed by electrospray mass and infrared spectroscopies. The solution properties of the complex were investigated by ¹H NMR spectroscopy. The results of NMR indicate that the complex has higher symmetry in solution than in the solid state.     

Synthesis, structural analysis and anticonvulsant activity of a ternary Cu(II) mononuclear complex containing 1,10-phenanthroline and the leading antiepileptic drug valproic acid

The synthesis and characterization of the binary complex of copper(II) with the antiepileptic drug valproic acid sodium salt (Valp) and the related ternary complex with 1,10-phenanthroline (phen) are reported, as well as the anticonvulsant properties of the latter. The characterization was carried out by means of elemental analyses, infrared (IR), UV–visible (UV–vis) spectrophotometry and Electron Paramagnetic Resonance (EPR). The X-ray crystal structure of the mononuclear complex bis(2-propylpentanoate)(1,10-phenanthroline)copper(II) [Cu(Valp)₂phen] is showed for the first time. It crystallized in C2/c space group with unit cell dimensions of a = 14.939(1) Å, b = 19.280(1) Å, c = 9.726(1) Å, β = 97.27(2)°, V = 2778.8(4) Å ³ and Z = 8. The carboxylates bond in an asymmetric chelating mode and the copper atom adopts a highly distorted octahedral coordination, characterized by the sum of the angles of 365.9° around Cu(II) and its nearest atoms in the CuN₂O₂ + O₂ chromophore instead of the expected 360° for a basal square planar geometry found in most Cu(II) complexes. Molecules assemble three by three through slipped π–π stacking of the aromatic phen with respectively 3.519 and 3.527 Å distances, in a head-to-tail arrangement. Studies of the anticonvulsant properties of this bioligand chelate evidenced its lack of efficacy in preventing MES-induced seizures. Interestingly, complex 4 protected mice against the Minimal Clonic seizures at doses that do not cause Rotorod toxicity, with an ED₅₀ documenting very potent anticonvulsant activity in this model of seizure, a particularly useful pharmacological profile of activity for the treatment of Petit Mal seizures.     

Heterocyclic compounds containing antimony 1. Synthesis, physicochemical properties, crystal and molecular structure of 2-(β-hydroxyethylthio) 1,3,2-oxathiastibolane

The compound (HOCH₂CH₂S) ) (1) has been prepared by the reaction of antimony(III) isopropoxide and 2-mercaptoethanol in a 1:2 molar ratio. Reaction of 1 with MOCH₃ (where M = Li, Na and K) yields bimetallic products of the type, M[(OCH₂CH₂S) )]. All these derivatives have been characterized by elemental analysis, IR, NMR (¹H and ¹³C) spectra and molar conductivity measurements. Crystals of 1 are triclinic, space group P , with a = 6.449(2), b = 10.285(2), c = 13.494(1) Å, α = 78.08(1), β = 75.99(1), γ = 71.54(2)°, V = 815.48 ų, Z = 4, D_calc = 2.239 g cm⁻³, (Mo Kα) λ = 0.7107 Å, μ = 3.55 mm₋₁, F(000) = 528, T = 295 K, final R = 0.0189 for 2344 reflections. One of the two mercaptoethanol moieties in 1 forms a five-membered chelate ring with antimony, Sb(1)---O(11) = 2.023(2) Å and Sb(1)---S(11) = 2.434(1) Å, while the other is bonded through the S atom only, Sb(1)---S(12) = 2.434(1) Å. The angles between these primary bonds with a mean value of 90.2° suggest a basically pyramidal, or pseudo tetrahedral structure if the stereochemically active lone pair is included in the coordination sphere. Two molecules are linked by intermolecular hydrogen bridges. The presence of weak intermolecular secondary bonding, Sb(1)---O(12) = 2.567(3) Å, in the complex indicates that the overall coordination polyhedron is best described in terms of a distorted trigonal bipyramidal arrangement.     

N7 and C8 coordinated purine complexes of [(NH3)5Os]. Crystal structures of 7-[9MeHyp(NH3)5Os]Cl3·H2O and 8-[1,3,7Me3Xan(NH3)5Os]Cl3·2H2O

Pentaammineosmium(III) coordinates to both the N7 and C8 positions of purine rings. The compound 7-[9MeHyp(NH₃)₅Os]Cl₃·H₂O crystallizes in the orthorhombic space group Pnma (No. 62) with the unit cell parameters: a=11.542(2), b=6.9841(8), c=21.960(3) Å and Z=4. The compound 8-[1,3,7Me₃Xan(NH₃)₅Os]Cl₃·2H₂O crystallizes in the monoclinic space group P2₁/c (No. 14) with the unit cell parameters: a=7.1228(X), b=14.613(1), c=19.667(1) Å, β=91.782(9)° and Z=4. The Os---C bond in the latter structure is 2.039(9) Å and the imidazolylidine ligand exerts a slight trans influence seen in the lengthening of the Os---N_ax distance (2.172(8) Å) by about 0.05 Å relative to the average of the equatorial Os---N_eq value of 2.123(8) Å. The spectroscopic, electrochemical and structural properties of these and additional N-bound purine complexes are compared with those of similar N7 and C8 ruthenium(III) species.     

Binuclear complexes of a new hexadentate macrocyclic ligand. The crystal and molecular structure of [LCu2(CH3CO2)2](ClO4)2·5H2O

The 30-membered hexaaza macrocylic ligand, L (L=3,7,11,18,22,26-hexaazatricyclo-[26.2.2.2^13,16]tetratriaconta-1(31),13(33),14,16(34),28(32),29-hexaene), is capable of forming binuclear complexes with the divalent transition metal ions Ni, Cu and Zn. The two metal ions are bound by the two dipropylenetriamine units of the macrocycle. Extra coordination sites on the metal ions can be occupied by exogenous ligands such as acetate, chloride and thiocyanate. The crystal structure of one of the di-copper complexes is described: [LCu₂(CH₃CO₂)₂](ClO₄)₂·5H₂O crystallizes in the monoclinic space group P2₁/c (No. 14), with a=9.369(2), b=17.644(3), c= 27.466(3) Å, β=92.90(1)°, U=4534.7 ų and Z=4. The Cu1···Cu2 separation is 8.40(3) Å. The access for potential exogenous bridging ligands, to the cavity between the copper ions, is somewhat restricted by the two phenyl units of the macrocycle which appear almost parallel in the structure. The redox potential of the couple L(Cu²⁺)₂/L(Cu⁺)₂, recorded by cyclic voltammetry for the chloride adduct, [LCu₂Cl₂]Cl₂·5H₂O, is −0.061 V versus SCE in DMF.