Approximate Unit-cell Parameters Research Articles

Crystallization and preliminary X-ray analysis of the ytxM gene product from Bacillus subtilis.

The ytxM gene product from Bacillus subtilis has been cloned, expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. Multiple-wavelength anomalous dispersive X-ray data have been collected to 2.0 A resolution on a single selenomethionine-incorporated crystal. This crystal belongs to the primitive orthorhombic system, with approximate unit-cell parameters a = 44.3, b = 90.9, c = 136.1 A, alpha = beta = gamma = 90 degrees and two monomers in the asymmetric unit.

Crystallization and preliminary X-ray analysis of substrate complexes of leucine dehydrogenase from Thermoactinomyces intermedius.

Leucine dehydrogenase is an octameric enzyme which belongs to the superfamily of amino-acid dehydrogenases and catalyses the reversible oxidative deamination of leucine to 2-ketoisocaproate, with the corresponding reduction of the cofactor NAD(+). Catalysis by this enzyme is thought to involve a large-scale motion of the enzyme's two domains between an 'open' and 'closed' form, with the latter representing a conformation of the enzyme in which the partners involved in the hydride-transfer reaction are appropriately positioned for catalysis. Whilst a structure for the open form of the enzyme has been determined, the nature of the closed form has yet to be observed. In order to trap a closed form, crystals of the complexes of leucine dehydrogenase from Thermoactinomyces intermedius with 2-ketoisocaproate and with 2-ketoisocaproate and NAD(+) have been obtained by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The crystals of the binary complex with 2-ketoisocaproate belong to space group P2(1)2(1)2(1), with approximate unit-cell parameters a = 106, b = 118, c = 320 A and an octamer in the asymmetric unit, corresponding to a V(M) of 3.1 A(3) Da(-1). The crystals of the non-productive ternary complex belong to space group P6(1) or P6(5), with approximate unit-cell parameters a = b = 117, c = 502 A and an octamer in the asymmetric unit, corresponding to a V(M) of 3.0 A(3) Da(-1). These crystals diffract X-rays on a synchrotron-radiation source to at least 2.8 and 3.3 A resolution, respectively, and are suitable for a full structure determination.

Cloning, purification, crystallization and preliminary crystallographic analysis of Bacillus subtilis LuxS.

LuxS of Bacillus subtilis is a member of a novel family of proteins with a potential role in quorum sensing, controlling important aspects of cellular physiology in a range of microbial species. B. subtilis luxS was cloned, expressed in Escherichia coli, purified and crystallized using the hanging-drop method of vapour diffusion with ammonium sulfate as the precipitant. The crystals belong to one of the enantiomorphic space groups P6(1)22 or P6(5)22, with approximate unit-cell parameters a = b = 63.6, c = 151.5 A and one subunit in the asymmetric unit, corresponding to a packing density of 2.5 A(3) Da(-1). The crystals diffract X-rays to at least 1.55 A resolution on a synchrotron-radiation source. Determination of the structure will provide insights into the key determinants of function of this class of proteins, for which no structures are currently available.

Overexpression, purification, crystallization and data collection of a single-stranded DNA-binding protein from Sulfolobus solfataricus.

Single-stranded DNA-binding proteins are recruited when single-stranded DNA is exposed by disruption of the duplex. Many important biological processes such as DNA replication can only occur when the two strands of the duplex are separated. A defining trait of these proteins is the presence of the so-called OB fold. The single-stranded DNA-binding protein of the crenarchaeote Sulfolobus solfataricus has a number of interesting differences and similarities to both the eubacterial and eukaryotic homologues. It has an extended C-terminal tail with significant sequence identity to a similar region in the eubacterial protein. However, the sequence of the OB fold is much more like the eukaryotic and euryarchaeal proteins. The S. solfataricus protein remains a monomer in the absence of DNA but rapidly polymerizes upon binding - a behaviour not seen in the Escherichia coli protein. The protein has been overexpressed, purified and crystallized. The protein crystallizes in two related forms, both having space group P6(1) (or P6(5)) with approximate unit-cell parameters a = b = 75, c = 69 A, but the crystals are distinguished by their size and morphology. The larger crystals are hexagonal bipyramids and are merohedrally twinned, diffracting to 1.34 A with diffraction observed to 1.2 A. Smaller needle-like crystals diffract to about 2.0 A but are not twinned. Molecular-replacement attempts have failed owing to low identity with available search models. The structure will be determined by multiple-wavelength methods.

Crystallization and preliminary X-ray analysis of the glycerol-3-phosphate 1-acyltransferase from squash (Cucurbita moschata)

Glycerol-3-phosphate 1-acyltransferase (E.C. 2.3.1.15; G3PAT) catalyses the incorporation of an acyl group from either acyl-acyl carrier proteins (acylACPs) or acylCoAs into the sn-1 position of glycerol 3-phosphate to yield 1-acylglycerol 3-phosphate. Crystals of squash G3PAT have been obtained by the hanging-drop method of vapour diffusion using PEG 4000 as the precipitant. These crystals are most likely to belong to space group P2(1)2(1)2(1), with approximate unit-cell parameters a = 61.1, b = 65.1, c = 103.3 A, alpha = beta = gamma = 90 degrees and a monomer in the asymmetric unit. X-ray diffraction data to 1.9 A resolution have been collected in-house using a MAR 345 imaging-plate system.

Purification, crystallization and quaternary structure analysis of a glycerol dehydrogenase S305C mutant from Bacillus stearothermophilus.

Bacillus stearothermophilus glycerol dehydrogenase (GlyDH) is a 39.5 kDa molecular weight metalloenzyme which catalyzes the oxidation of glycerol to dihydroxyacetone with the concomitant reduction of NAD(+) to NADH. Despite its classification as a member of the 'iron-containing' polyol dehydrogenase family, studies on recombinant B. stearothermophilus GlyDH have shown this enzyme to be Zn(2+)-dependent. Crystals of a S305C GlyDH mutant were obtained by the hanging-drop vapour-diffusion method, using ammonium sulfate and PEG 400 as precipitating agents, in the presence and absence of NAD(+). The crystals belong to space group I422, with approximate unit-cell parameters a = b = 105, c = 149 A and one subunit in the asymmetric unit, corresponding to a packing density of 2.6 A(3) Da(-1). The crystals diffract X-rays to at least 1.8 A resolution on a synchrotron-radiation source. Determination of the structure will provide insights into the key determinations of catalytic activity of this class of enzymes, for which no structures are currently available.

Quantitative Phase Analysis using the Whole-Powder-Pattern Decomposition Method. I. Solution from Knowledge of Chemical Compositions

A new procedure for quantitative phase analysis using the whole-powder-pattern decomposition method is proposed. The procedure consists of two steps. In the first, the whole powder patterns of single-component materials are decomposed separately. The refined parameters of integrated intensity, unit cell and profile shape for respective phases are stored in computer data files. In the second step, the whole powder pattern of a mixture sample is fitted, where the parameters refined in the previous step are used to calculate the profile intensity. The integrated intensity parameters are, however, not varied during the least-squares fitting, while the scale factors for the profile intensities of individual phases are adjusted instead. Weight fractions are obtained by solving simultaneous equations, coefficients of which include the scale factors and the mass-absorption coefficients calculated from chemical formulas of respective phases. The procedure can be applied to all mixture samples, including those containing an amorphous material, if single-component samples with known chemical compositions and their approximate unit-cell parameters are provided. The procedure has been tested by using two- to five-component samples, giving average deviations of 1 to 1.5%. Optimum refinement conditions are discussed in connection with the accuracy of the procedure.

Automatic indexing of rotation diffraction patterns

A method is described which assigns indices to a set of single-crystal reflections recorded by the rotation-oscillation technique using a fixed X-ray wavelength. It is assumed that the space group and approximate unit-cell parameters are known. The unknown crystal orientation is determined directly from the observed diffraction pattern of one or several oscillation data records. A local indexing procedure is described which tolerates large initial errors in the parameters controlling the diffraction pattern. These parameters are refined subsequently, thereby satisfying the constraints imposed by the space-group symmetry.

Indexing of powder diffractograms and refinement of unit-cell data: general distributions of differences between observed and calculated quadratic forms

A method is described to search for the most probable assignment of indices to powder diffractograms starting from approximate unit-cell parameters. The difference ΔQi = ΔQoi−ΔQci between observed and calculated quadratic forms of the ith peak is attributed to three contributions: ΔQi* due to imprecise unit-cell constants, ΔQi′ due to incorrect indexing and ∊i due to experimental error. The points ΔQi plotted as a function of ΔQoi give rise to distributions D′, D* (or under special conditions Dg*) and D∊. The differences between these distributions are evinced by differences in their moments and probability function. At the start the nearly normal distribution Dg* is searched for, by changing the initial reciprocal-cell parameters by small increments δa*, δb*, δc*. Highly probable indices can then be assigned to the Qoi's and the unit-cell constants refined by the least-squares method. At the end the normal distribution D∊ with mean μ = 0 must be obtained. Statistically significant deviations of D∊ from the normal distribution with μ = 0 indicate that some systematic experimental errors affect the data. The method is suitable for applications to electronic computers and is particularly recommended for plotters.

Computer indexing of x-ray and neutron powder diffraction patterns of cubic, tetragonal, orthorhombic and hexagonal structures

A computer programming procedure is described for indexing X-ray or neutron powder diffraction data from cubic, tetragonal, orthorhombic and hexagonal crystals. Corrections can be made for X-ray absorption errors and indices are assigned by comparison of observed and calculated values of sin θ. The input data required are either trial Miller indices for one, two or three reflections or approximate unit cell parameters.