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PubMed1999

The crystal growth technique--a laboratory evaluation of bond strengths.

An ex vivo study was carried out to determine differences in the bond strengths achieved with brackets placed using a crystal growth technique compared with a conventional acid-etch technique. A solution of 37 per cent phosphoric acid was used for acid-etching and a commercially available polyacrylic acid gel, Crystal-lok for crystal growth. A heavily-filled composite resin was used for all samples to bond brackets to healthy premolar teeth extracted for orthodontic purposes. Polycrystalline ceramic and stainless steel brackets were used and tested to both tensile and shear failure using an Instron Universal Testing machine. The tensile and shear bond strengths were recorded in kgF. In view of difficulties experienced with previous authors using different units to describe their findings, the data were subsequently converted to a range of units in order to facilitate direct comparison. The crystal growth technique produced significantly lower bond strengths than the acid-etch technique for ceramic and stainless steel brackets, both in tensile and shear mode. The tensile bond strength for stainless steel brackets with crystal growth was 2.2 kg compared with 6.01 kg for acid-etch, whilst with ceramic brackets the tensile bond strengths were 3.9 kg for crystal growth and 5.55 kg for acid-etch. The mean shear bond strength for stainless steel brackets with crystal growth was 12.61 kg compared with 21.55 kg for acid-etch, whilst with ceramic brackets the shear bond strengths were 7.93 kg with crystal growth compared with 16.55 kg for acid-tech. These bond strengths were below those previously suggested as clinically acceptable.

Acid Etching, Dental↗
PubMed1989

A novel dialysis procedure for the crystallization of proteins.

Various dialysis methods are commonly employed for the crystallization of proteins. Typical procedures include the use of dialysis bags, dialysis buttons or Zeppezauer microdiffusion cells. The general principle involved is that the protein solution is gradually brought to a point of supersaturation by imposing a gradient of ionic strength or organic solvent concentration across the wall of the dialysis membrane. However, in some cases, the imposition of this gradient across the dialysis membrane can result in the formation of a large number of crystal nucleation sites, thereby giving rise to a reduction in the maximum size of the crystals which can be obtained. A novel 'double-dialysis' procedure which incorporates a second dialysis membrane, thus reducing the rate of equilibration in the crystallization experiment, has been developed in our laboratory. The system has been employed successfully on the delta toxin of Staphylococcus aureus resulting in a useful increase in crystal size. A more quantitative analysis of the technique has been carried out on rat liver malic enzyme. The results of a limited series of crystallization trials with this protein have shown that employment of the 'double-dialysis' technique allows a fine control of the rate of crystal nucleation and therefore provides a mechanism for the controlled growth of large crystals.

Animals↗
PubMed1994

Rapid crystallization of T4 lysozyme by intermolecular disulfide cross-linking.

In an attempt to facilitate crystallization, engineered cysteines were used to promote formation of a 'back-to-back' dimer that occurs in different crystal forms of wild-type and mutant T4 lysozymes. The designed double mutant, N68C/A93C, in which the surface residues Asn68 and Ala93 were replaced by cysteines, formed dimers in solution and crystallized isomorphously to wild-type, but at a much faster rate. Overall, the mutant structure remained very similar to wild-type despite the formation of two intermolecular disulfide bridges. The crystals of cross-linked dimers ahd thermal factors somewhat lower than wild-type, indicating reduced mobility, but did not diffract to noticeably higher resolution. Introduction of the same cross-links was also used to obtain crystals in a different space group of a T4 lysozyme mutant that could not be crystallized previously. The results suggest that the formation of the lysozyme dimer is a critical intermediate in the formation of more than one crystal form and that covalent cross-linking of the intermediate accelerates nucleation and facilitates crystal growth. The disulfide cross-links are located on the 'back' of the molecule and formation of the cross-linked dimer appears to leave the active sites completely unobstructed. Nevertheless, the cross-linked dimer is completely inactive. One explanation for this behavior is that the disulfide bridges prevent hinge-bending motion that may be required for catalysis. Another possibility is that the formation of the dimer increases the overall bulk of the enzyme and prevents its access to the susceptible glycosidic bonds within the cell wall substrate.

Bacteriophage T4↗
PubMed1996

A novel technique for containerless protein crystallization.

Heterogeneous nucleation, which is often detrimental to the production of suitable crystals for X-ray diffraction, can be induced by the contact of a crystallization sample with the walls of its supporting vessel. A novel method for creating a 'containerless' environment for the growth of protein crystals is described. Contact between the container walls and a crystallization drop is eliminated by suspending the drop between two oils of different density: one of higher and the other of lower density than that of water and the common precipitating agents. A number of proteins were crystallized in 2-10 microliters drops using this procedure. It was found that the number of crystals obtained in such suspended drops was reduced significantly compared with the number of crystals obtained in trials where the crystallization drop was situated at the bottom of a vial under a single layer of oil. This method has potential in controlling heterogeneous nucleation.

Carrier Proteins↗
PubMedDate not supplied

Observations on embryonic chick-bone crystals by high resolution transmission electron microscopy.

High resolution transmission electron microscopy of bone mineral at a vary early stage in its deposition in embryonic chick frontal bone demonstrates crystals of 2-2.5 nm thickness and a profile up to 40 nm long. They appear in groups which may show as few as one or two crystals. Associated with the crystals is a less dense amorphous material. Using specimen tilting, the change in crystal appearance indicates that crystals are tablet-shaped (rectangular) rather than rod-shaped. The amorphous material may be tablet-shaped crystals in orientations which are not dense enough to register as crystals in the image. Truly amorphous mineral does not show by bright-field electron microscopy, but preliminary results indicate that the dark-field technique may prove to be useful in imaging amorphous mineral provided that sections can be cut thinly enough and that precautions are taken during specimen preparation to avoid mineral loss. Crystals have been located in various parts of the osteoid zone and were not associated specifically with any one component. Rapid processing using anhydrous techniques must be used to avoid the possibility of artefactually altering the in vivo conditions of the mineral at the time of excision of the specimen.

Animals↗
PubMed2003

Cholesterol crystal morphology in acalculous gallbladder disease.

BACKGROUND: Biliary crystal morphology is best described in patients with gallbladder stones, but most patients undergoing bile collection for microscopy have a clinical diagnosis of acalculous gallbladder disease. We investigated the morphology of biliary crystals in such patients. STUDY: Bile was obtained for polarizing microscopy from fresh cholecystectomy specimens of patients with a clinical diagnosis of acalculous or calculous gallbladder disease. Slides for microscopy were prepared by touch contact with bile in freshly opened gallbladder specimens, and following aspiration of gallbladder bile through a 5-French cannula. RESULTS: Bile was examined from five patients with a clinical diagnosis of acalculous gallbladder disease and five patients with known gallstones. Needle-like cholesterol crystals predominated in most patients without gallstones, whereas plate-like and dot-like crystals were more common in patients with gallstones. All three crystal types were seen in most patients. Crystal morphology was not affected by aspiration of bile through a 5-French cannula. CONCLUSIONS: Birefringent needles and dots should be recognized as cholesterol crystals during bile microscopy. These crystal morphologies may predominate in some patients with a clinical diagnosis of acalculous gallbladder disease.

Adult↗
PubMed1998

The inhibition of calcium oxalate monohydrate crystal growth by maleic acid copolymers.

The crystallization of calcium oxalate monohydrate (COM) was investigated at conditions of constant supersaturation both in the absence and in the presence of synthetic maleic acid copolymers at 37C, 0.15 M NaCl. The dependence of the rates of COM crystallization in the absence of inhibitors was found to be second order at low and first order at higher supersaturations suggesting a surface diffusion controlled mechanism. The presence of all copolymers tested at concentration levels up to 5 ppm retarded the rates of COM crystal growth up to 90%. The decrease of the COM crystal growth rates by the polymers depended on the nature of the comonomer polymerized with maleic acid and the order of inhibition was found to be vinyl acetate > N-vinylpyrrolidone > styrene. Taking into consideration kinetics data published in the literature concerning the inhibition of COM crystal growth, it is suggested that molecular weight also plays a role, with more inhibition at higher molecular weights. The morphology of the COM crystals grown was unaffected yet the crystals growing at lower rates in the presence of the copolymers were larger and their size more uniform. It is concluded that maleic acid copolymers are strong inhibitors of the crystallization of COM, the inhibitory activity being more pronounced in the case of the linear copolymers.

Adsorption↗
PubMed2004

Diffraction in crystalline colloidal-array photonic crystals.

We characterized the diffraction and crystal structure of a crystalline colloidal array (CCA) photonic crystal composed of 270 nm diameter polystyrene spheres which have a nearest neighbor spacing of approximately 540 nm. This CCA diffracts light in first order at approximately 1200 nm and shows strong diffraction in the visible spectral region from higher order planes. We quantitatively examined the relative diffraction intensities of the putative fcc (111), (200), (220), and (311) planes. Comparing these intensities to those calculated theoretically we find that the crystal structure is fcc with significant stacking faults. Essentially, no light transmits at the Bragg angle for the fcc (111) planes even through thin approximately 40 microm thick CCA. However, much of this light is diffusely scattered about the Bragg angle due to crystal imperfections. Significant transmission occurs from thin samples oriented at the Bragg condition for the fcc (200), (220), and (311) planes. We also observe moderately intense two-dimensional diffraction from the first few layers at the crystal surfaces. We also examined the sample thickness dependence of diffraction from CCA photonic crystals prepared from approximately 120 nm polystyrene spheres whose fcc (111) planes diffract in the visible spectral region. These experimental observations, aided by calculations based upon a simple but flexible model of light scattering from an arbitrary collection of colloidal spheres, make clear that fabrication of three-dimensional photonic band gap crystals will be challenged by crystal imperfections.

Biophysics↗
PubMed1998

Crystal growth and crystallography.

Selected topics that may be of interest for both crystal-structure and crystal-growth communities are overviewed. The growth of protein crystals, along with that of some other compounds, is one of the topics, and recent insights into related phenomena are considered as examples of applications of general principles. The relationship between crystal growth shape and structure is reviewed and an attempt to introduce semiquantitative characterization of binding for proteins is made. The concept of kinks for complex structures is briefly discussed. Even at sufficiently low supersaturations, the fluctuation of steps may not be sufficient to implement the Gibbs-Thomson law if the kink density is low enough. Subsurface ordering of liquids and growth of rough interfaces from melts is discussed. Crystals growing in microgravity from solution should be more perfect if they preferentially trap stress-inducing impurities, thus creating an impurity-depleted zone around themselves. Evidently, such a zone is developed only around the crystals growing in the absence of convection. Under terrestrial conditions, the self-purified depleted zone is destroyed by convection, the crystal traps more impurity and grows stressed. The stress relief causes mosaicity. In systems containing stress-inducing but poorly trapped impurities, the crystals grown in the absence of convection should be worse than those of their terrestrial counterparts.

Animals↗
PubMed1992

Crystallization and preliminary data analysis of Flock House virus.

Flock House virus, purified from infected cultured Drosophila cells, crystallizes into three different forms under identical growth conditions. Two crystal forms grow in the trigonal space group R3, both with equivalent cell constants a = 323.6 A, alpha = 61.7 degrees. The difference between the two trigonal crystal forms is 1.1 degrees in the orientation of the virus particle as determined from the rotation function. Early crystal setups grew in one form, while recent crystals grew in the other form. The third space group, which accounts for 5% of the observed crystals and grows with both trigonal forms, is orthorhombic I222 with cell parameters a = 416.7, b = 332.1, c = 351.2 A. The trigonal crystal forms contain one virion per unit cell and the orthorhombic form contains two particles per cell. All three crystal forms diffract X-rays to 2.8 A resolution.

Animals↗
PubMed2000

Preliminary X-ray analysis of a new crystal form of the Escherichia coli KDO8P synthase.

3-Deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the biosynthesis of an essential component of the lipopolysaccharide of all Gram-negative bacteria. The structure and mechanism of KDO8P synthase are being actively studied as this enzyme represents an important target for antibiotic therapy. The structure of the Escherichia coli KDO8P synthase in cubic crystals (space group I23) has recently been determined and the enzyme shown to be a tetramer of identical subunits. However, this information is challenged by biochemical studies, which suggest that the enzyme behaves in solution as a homotrimer. Here, the preparation and preliminary X-ray analysis of monoclinic crystals of KDO8P synthase are reported. The crystals belong to space group P2(1), with unit-cell parameters a approximately 50, b approximately 140, c approximately 74 A, beta approximately 105 degrees. The structure of KDO8P synthase in the monoclinic crystal form was determined by molecular replacement, using as a search model one of the subunits of the enzyme in the cubic crystals. A tetramer of KDO8P synthase with 222 local symmetry is also present in the asymmetric unit of the P2(1) crystals, with a solvent content of 43%. The observation that the same quaternary structure of KDO8P synthase is observed in two different crystal forms belonging to distinct crystal systems (monoclinic and cubic) suggests that a tetramer is the native form of the enzyme.

Aldehyde-Lyases↗
PubMed2000

Characterization of crystal content by ESI-MS and MALDI-MS.

A general approach based on mass spectrometry is described for the rapid identification of the content of macromolecular crystals. The experimental procedure was established using lysozyme crystals and then successfully applied to various systems containing specifically bound molecules not easily detectable by other classical techniques. This procedure can be carried out on crystals containing macromolecules of a different nature, such as proteins, nucleic acids and small organic molecules and their non-covalent complexes, grown under various crystallization conditions including PEGs and salts. It can be applied very early on in the crystallization process - as soon as the crystals can be handled. It allows the biologist to control precisely the sequence integrity and homogeneity of the crystallized proteins (in particular at the C-terminus) as well as to verify whether the protein has crystallized with all its expected partners or ligands (nucleic acid molecules, cofactor or small organic molecules).

Aldehyde Reductase↗
PubMed2001

Identification of many crystal forms of Aspergillus nidulans dehydroquinate synthase.

Extensive crystallization trials of Aspergillus nidulans dehydroquinate synthase, a potential novel target for antimicrobial drugs, in complexes with different ligands have resulted in the identification of nine crystal forms. Crystals of unliganded DHQS, binary complexes with either the substrate analogue, carbaphosphonate or the cofactor NADH, as well as the ternary DHQS-carbaphosphonate-cofactor complex, were obtained. The ternary complex crystallizes from ammonium sulfate and CoCl(2) in space group P2(1)2(1)2, with unit-cell parameters a = 133.8, b = 86.6, c = 74.9 A. The binary carbaphosphonate complex crystallizes from PEG 6000 in space group P2(1)2(1)2(1), with a = 70.0, b = 64.0, c = 197.6 A, and the binary cofactor complex crystallizes from PEG 3350 and sodium potassium tartrate in space group P2(1), with a = 83.7, b = 70.4, c = 144.3 A, beta = 89.2 degrees. DHQS in the absence of ligands crystallizes in space group P2(1), with a = 41.0, b = 68.9, c = 137.7 A, beta = 94.8 degrees. Each of these crystal forms are suitable for high-resolution structure determination. Structures of a range of DHQS-ligand complexes will be of value in the structure-based design of novel antimicrobial drugs.

Aspergillus nidulans↗
PubMed2001

Crystallization and preliminary X-ray analysis of maltose O-acetyltransferase.

Maltose O-acetyltransferase (Mac) is a member of the hexapeptide-repeat family of enzymes, which contains proteins with left-handed parallel beta-helix architecture forming homotrimers. Diffraction data for four well diffracting crystal forms were collected. Crystal form I diffracted beyond 1.53 A resolution but was perfectly merohedrally twinned with an apparent space group P622. Crystal forms II and III (space groups R3 and C2, respectively) could be obtained under very similar conditions by adjusting the buffer pH differently. Crystal forms II and III had several monomers in the asymmetric unit and were difficult to derivatize. However, during soaking with trimethyl lead acetate, the form III crystals dissolved and crystals with a different habit and space group grew in their place (form IV). In three of the crystal forms, a ladder of peaks was visible in the native Patterson maps along the c axis. These peaks were interpreted as corresponding to the vectors between the beta-strands in the turns of the beta-helix. Crystal form IV is suitable for structure determination of Mac exploiting the anomalous scattering of lead.

Acetyltransferases↗
PubMed2002

X-ray scattering studies of Aspergillus flavus urate oxidase: towards a better understanding of PEG effects on the crystallization of large proteins.

The determination of the three-dimensional structures of biological macromolecules by X-ray diffraction generally requires large good-quality crystals, which are often difficult to obtain as crystal nucleation and growth depend upon a great number of physicochemical parameters. In the future, the emergence of structural genomic projects will require new and rapid methods to determine crystallization conditions. Until now, the prediction of crystallization conditions has essentially been based on the knowledge of interparticular interactions in solutions inferred from studies on small soluble proteins in the presence of salts. The present study, by small-angle X-ray scattering, of urate oxidase from Aspergillus flavus, a homotetrameric enzyme of 128kDa, allowed the extension of the results to the crystallization of large proteins in the presence of polyethylene glycol (PEG). The protein crystallization, the nucleation rate and the different morphological crystal shapes obtained were correlated with the second virial coefficient (A(2)), which was found to be in a restricted range at the low end of the 'crystallization slot' proposed by George & Wilson [(1994). Acta Cryst. D50, 361--365].

Aspergillus flavus↗
PubMed2002

Crystal contacts engineering of aspartyl-tRNA synthetase from Thermus thermophilus: effects on crystallizability.

To understand how surface residues in a protein structure influence crystal growth, packing arrangement and crystal quality, crystal surfaces were modified and crystallizability of seven different mutants investigated. The model was aspartyl-tRNA synthetase-1 from Thermus thermophilus, a homodimer (M(r) 122000) with a subunit of 580 amino acids. Engineering concerned modification of amino acids involved in packing contacts in the orthorhombic lattice (P2(1)2121) of the synthetase. Comparison of the crystallization behaviour of the mutants indicates a correlation between disruption/addition of packing interactions and crystallizability of the mutants: disruption or modification of lattice contacts prevents crystallization or leads to crystals of poor quality. In contrast, addition of potential contacts leads to well-shaped crystals of same space group and cell parameters than wild-type crystals.

Aspartate-tRNA Ligase↗
PubMed2002

The impact of Glu-->Ala and Glu-->Asp mutations on the crystallization properties of RhoGDI: the structure of RhoGDI at 1.3 A resolution.

It is hypothesized that surface residues with high conformational entropy, specifically lysines and glutamates, impede protein crystallization. In a previous study using a model system of Rho-specific guanine nucleotide dissociation inhibitor (RhoGDI), it was shown that mutating Lys residues to Ala results in enhanced crystallizability, particularly when clusters of lysines are targeted. It was also shown that one of these mutants formed crystals that yielded diffraction to 2.0 A, a significant improvement on the wild-type protein crystals. In the current paper, an analysis of the impact of surface mutations replacing Glu residues with Ala or Asp on the stability and crystallization properties of RhoGDI is presented. The Glu-->Ala (Asp) mutants are generally more likely to produce crystals of the protein than the wild-type and in one case the resulting crystals yielded a diffraction pattern to 1.2 A resolution. This occurs in spite of the fact that mutating surface Glu residues almost invariably affects the protein's stability, as illustrated by the reduced deltaG between folded and unfolded forms measured by isothermal equilibrium denaturation. The present study strongly supports the notion that rational surface mutagenesis can be an effective tool in overcoming problems stemming from the protein's recalcitrance to crystallization and may also yield dramatic improvements in crystal quality.

Alanine↗
PubMed2003

Comparison of three commercial sparse-matrix crystallization screens.

Sparse-matrix sampling using commercially available crystallization screen kits has become the most popular way of determining the preliminary crystallization conditions for macromolecules. In this study, the efficiency of three commercial screening kits, Crystal Screen and Crystal Screen 2 (Hampton Research), Wizard Screens I and II (Emerald BioStructures) and Personal Structure Screens 1 and 2 (Molecular Dimensions), has been compared using a set of 19 diverse proteins. 18 proteins yielded crystals using at least one crystallization screen. Surprisingly, Crystal Screens and Personal Structure Screens showed dramatically different results, although most of the crystallization formulations are identical as listed by the manufacturers. Higher molecular weight polyethylene glycols and mixed precipitants were found to be the most effective precipitants in this study.

Crystallization↗