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PubMed1989

Structure of a second crystal form of Bence-Jones protein Loc: strikingly different domain associations in two crystal forms of a single protein.

We have determined the structure of the immunoglobulin light-chain dimer Loc in a second crystal form that was grown from distilled water. The crystal structure was determined to 2.8-A resolution; the R factor is 0.22. The two variable domains are related by local 2-fold axes and form an antigen binding "pocket". The variable domain-variable domain interaction observed in this crystal form differs from the one exhibited by the protein when crystallized from ammonium sulfate in which the two variable domains formed a protrusion (Chang et al., 1985). The structure attained in the distilled water crystals is similar to, but not identical with, the one observed for the Mcg light-chain dimer in crystals grown from ammonium sulfate. Thus, two strikingly different structures were attained by this multisubunit protein in crystals grown under two different, commonly used, crystallization techniques. The quaternary interactions exhibited by the protein in the two crystal forms are sufficiently different to suggest fundamentally different interpretations of the structural basis for the function of this protein. This observation may have general implications regarding the use of single crystallographic determinations for detailed identification of structural and functional relationships. On the other hand, proteins whose structures can be altered by manipulation of crystallization conditions may provide useful systems for study of fundamental structural chemistry.

Bence Jones Protein↗
PubMed1998

Basic calcium phosphate crystal-induced collagenase production: role of intracellular crystal dissolution.

OBJECTIVE: To investigate the potential therapeutic effects of inhibiting intracellular dissolution of basic calcium phosphate (BCP) crystals in tissue culture by raising lysosomal pH using bafilomycin A1, a specific vacuolar pump inhibitor. DESIGN: 45Ca-labeled crystals were used to demonstrate intracellular crystal dissolution in human foreskin fibroblasts (HF). Mitogenesis was evaluated using [3H]thymidine incorporation assays and cell counts. Northern blot and Western blot were used to study collagenase [matrix metalloproteinase-1 (MMP1)] mRNA accumulation and protein secretion, respectively. RESULTS: Bafilomycin A1 inhibited intracellular dissolution of BCP crystals and caused a concentration-dependent inhibition of BCP crystal-induced mitogenesis. Doses of bafilomycin A1 which inhibited intracellular crystal dissolution and mitogenesis had no effect on BCP crystal-induced MMP1 mRNA accumulation or protein secretion. CONCLUSION: Raising lysosomal pH to inhibit intracellular BCP crystal dissolution attenuates the proliferative response to BCP crystals in HF but does not prevent metalloprotease synthesis and secretion. The therapeutic potential of lysosomotropic agents for preventing joint destruction in BCP crystal deposition disease is limited.

Anti-Bacterial Agents↗
PubMed2001

Development of artificial seed crystal for crystallization of calcium phosphate.

An artifical seed crystal material consisting of calcium silicate hydrate (5CaO x 6SiO2 x 5H2O : tobermorite crystals) applicable for phosphorus removal by crystallization was developed. Card-house shaped tobermorite crystals were developed on the seed material where orthophosphate crystallized as a calcium phosphate. The seed material can be manufactured by mixing siliceous and calcareous raw materials, pelletizing and subsequent autoclaving. Laboratory experiments were conducted to apply the new developed seed crystal material in the phosphorus recovery from sludge sidestreams of a wastewater treatment plant. In this crystallization process, the performance the carbon dioxide degassingprocess usually carried out when applying crystallization was not necessary, the hydroxyapatite was able to crystallize at a pH of 8.0 to 8.5 without precipitation of calcium carbonates. In the treatment of a sidestream with orthophosphate concentrations of 50 mgl(-1) and COD concentrations between 200 to 400 mgl(-1), phosphorus removal efficiencies ranging from 75 to 85% were observed. The seed crystal material was collected after the laboratory experiments and the chemical estimation and the germination test for agricultural reuse were performed. As a result, it was shown that the hydroxyapatite precipitated on the seed material had a 100% fusibility to soil and had characteristics to be a good nutrient source as a fertilizer for plants.

Calcium Phosphates↗
PubMed2004

Crystal packing in two pH-dependent crystal forms of rhamnogalacturonan acetylesterase.

The glycoprotein rhamnogalacturonan acetylesterase from Aspergillus aculeatus has been crystallized in two crystal forms, an orthorhombic and a trigonal crystal form. In the orthorhombic crystal form, the covalently bound carbohydrate at one of the two N-glycosylation sites is involved in crystal contacts. The orthorhombic crystal form was obtained at pH 5.0 and the trigonal crystal form at pH 4.5. In one case, the two crystal forms were found in the same drop at pH 4.7. The differences in crystal packing in the two crystal forms can be explained by the pH-dependent variation in the protonation state of the glutamic acid residues on the protein surface.

Acetylesterase↗
PubMed2005

A nanovolume crystallization robot that creates its crystallization screens on-the-fly.

Protein crystallization generally consists of an initial screen followed by optimization of promising conditions. Whereas the initial screen typically uses a standard set of pre-made crystallization cocktails, optimization requires new cocktails with small perturbations of the original composition. Highly parallel synchronous crystallization robots are ideal for initial screening, but they depend on pre-made crystallization cocktails. Asynchronous crystallization robots can create crystallization cocktails from stock solutions, but in practice this ability is rarely exploited. Instead, large-scale operations typically use a general liquid-handling robot to create optimization screens, whereas academics mostly rely on manual optimization. Here, the use of an asynchronous crystallization robot to create customized crystallization cocktails and set up nanovolume crystallization experiments without a compromise in speed or drop quality is described. This approach avoids the complex integration of hardware, software and dataflow between two robots and saves cost and space. As a proof of principle, a commercial crystal screen has been reproduced with the robot and shows that results are virtually identical to using the actual commercial screen.

Combinatorial Chemistry Techniques↗
PubMed1999

Crystallization within agarose gel in microgravity improves the quality of thaumatin crystals.

To prevent crystals from moving in orbit and sedimenting upon their return to earth, the model protein thaumatin was crystallized in agarose gel in the Advanced Protein Crystallization Facility during the eight-day Space Shuttle mission STS-95 (November 1998). The quality of tetragonal crystals grown in microgravity was compared with that of controls prepared in parallel in the laboratory. On the basis of their diffraction properties, microgravity crystals were more ordered than crystals grown in gel on earth (the latter being, on average, better than reference crystals obtained in solution on earth). It is concluded that protein crystallization within a gel in microgravity may yield crystals of superior quality by combining the advantages of both environments. A possible explanation for the positive effect of microgravity on protein crystallization in gels involving the better quality of the nucleus is discussed.

Crystallization↗
PubMed2005

Supercooled liquid-like solvent in trypsin crystals: implications for crystal annealing and temperature-controlled X-ray radiation damage studies.

The study of temperature-dependent physical changes in flash-cooled macromolecular crystals is pertinent to cryocrystallography and related issues such as crystal annealing, X-ray radiation damage and kinetic crystallography. In this context, the unit-cell volume of flash-cooled trigonal and orthorhombic trypsin crystals has been monitored upon warming from 100 to 200 K and subsequent re-cooling to 100 K. Crystals of both forms were obtained under the same crystallization conditions, yet they differ in solvent content and channel size. An abrupt non-reversible unit-cell volume decrease is observed at 185 K in orthorhombic and at 195 K in trigonal crystals as the temperature is increased; this result is consistent with ultra-viscous solvent leaving the crystals. Concomitant appearance of ice rings in the diffraction patterns suggests that the transported solvent forms crystalline ice. These results demonstrate that solvent in flash-cooled protein crystals is liquid-like near its crystallization temperature, as has been proposed, yet controversially discussed, for the case of pure water. The use of mineral oil prevents the unit-cell volume decrease in trigonal but not in orthorhombic crystals. The observation of liquid-like solvent has implications in the development of annealing protocols and points a way to the rational design of temperature-controlled crystallographic studies that aim either at studying specific radiation damage or at trapping enzymatic intermediate states.

Animals↗
PubMed1984

Preparation of hydroxyapatite crystals and their behavior as seeds for crystal growth.

Samples of crystalline hydroxyapatite [Ca5OH(PO4)3 HA] were prepared by precipitation from aqueous media under a variety of experimental conditions (temperature, concentration of reagents, rates of addition of the reagents, and seeding). The resultant products showed a wide range of particle sizes, i.e., specific surface areas, from 6.39 to 50.1 m2/g. In these preparations, relatively large crystals were obtained with low rates of addition of the reagents or by seeding the precipitating medium. Small differences in super-saturation of the reaction medium can markedly affect the particle sizes and crystalline habits of the resulting products, possibly by altering the processes of nucleation and subsequent crystal growth. When these crystalline materials were used as seeds to study the crystal growth of HA, it was confirmed that the precipitation rate of calcium apatite on seed crystals is highly dependent on the surface areas available for growth, rather than on the particle sizes and amounts of the seed crystals. Small differences in the kinetic runs were observed between the various seed crystals, which can be attributed to differences in the surface properties of these crystals. Additionally, transmission electron microscopy of the seed crystals revealed that some projections form, possibly on the basal planes of the crystals, during crystal growth. Since the growth rate of these projections was greater than the mean growth rate calculated on the basis of changes in solution composition and total surface area, it appears that the kinetics of the growth process is determined, to some extent, by the geometry of the seeds.

Calcium Phosphates↗
PubMed1999

Oxalate: from crystal formation to crystal retention.

Idiopathic calcium oxalate stone formation is a multifactorial disease. It is therefore unlikely that a single underlying condition will be responsible for entire spectrum of the disease; however, it appears that one important factor in the pathogenesis is an abnormality in oxalate metabolism. Whatever the cause, two critical parameters for stone formation are crystal formation and crystal retention in the renal tubules. Although crystal formination and role of oxalate in crystal formation have been evaluated extensively, it is only recently that crystal retention has been addressed. Previous studies from our laboratories demonstrated that oxalate exposure to renal epithelial cells in culture resulted in initiation of a program of events including DNA synthesis and cell death. The present studies evaluated effects of oxalate on cell proliferation and damage to distal tubular (Madin-Darby canine kidney cells) and proximal (LLC-PK1 cells) cells. Effects of oxalate exposure on calcium oxalate monohydrate (COM) crystal adherence to these cells were also evaluated. Results presented herein demonstrate that proximal tubular cells are more sensitive to oxalate than distal tubular cells. Furthermore, oxalate exposure to proximal tubular cells resulted in reinitiation of DNA synthesis, whereas no such effect was observed in distal tubular cells. Higher levels of oxalate (> 1 mM) resulted in cell loss of both proximal and distal tubular cells, as observed by crystal violet staining. Despite these differences, oxalate exposure to both proximal and distal tubular cells resulted in increased COM crystal adherence. Thus, oxalate exposure may promote crystal adherence to renal epithelial cells either secondarily to cell death and proliferation or by a yet unidentified mechanism. These studies provide the first direct evidence for the role of oxalate in promoting COM crystal retention by the urothelium.

Animals↗
PubMed1995

Calcium pyrophosphate dihydrate crystal deposition disease: a review of the literature and a light and electron microscopic study of a case of the temporomandibular joint with numerous intracellular crystals in the chondrocytes.

The pathogenesis of calcium pyrophosphate dihydrate (CPPD) crystal deposition disease of synovial joints is still unclear, although overproduction of extracellular pyrophosphate (PPi) is thought to play a key role. We studied the light and electron microscopic appearances of a case of CPPD crystal deposition disease of the temporomandibular joint (TMJ) in search of new clues for its pathogenesis. Light microscopic examination of CPPD-containing material from the joint space revealed cartilaginous nodules with various degrees of crystallization. Transmission electron microscopic examination revealed numerous extra- as well as intracellular crystals and crystal shaped spaces in the chondrocytes. Other striking ultrastructural features of the chondrocytes included the presence of many mitochondria, frequently containing crystalline material, and the presence of highly dilated rough endoplasmic reticulum and large glycogen islands. The presence of intramitochondrial crystals may hypothetically imply a derangement in mitochondrial adenosine triphosphate or PPi metabolism. The finding of intracellular CPPD crystals in chondrocytes points to the existence of an intracellular pathway of CPPD crystal formation in CPPD crystal deposition disease of the TMJ and possibly in CPPD crystal deposition disease in general.

Cartilage, Articular↗
PubMed2005

Nonspherical colloidal crystals fabricated by the thermal pressing of colloidal crystal chips.

Nonspherical colloids and their ordered arrays may be more attractive in applications such as photonic crystals than their spherical counterparts because of their lower symmetries, although such structures are difficult to achieve. In this letter, we describe the fabrication and characterization of colloidal crystals constructed from nonspherical polyhedrons. We fabricated such nonspherical colloidal crystals by pressing spherical polymer colloidal crystal chips at a temperature slightly lower than the glass-transition temperature (T(g)) of these polymer colloids. During this process, the polymer microspheres were distinctively transformed into polyhedrons according to their crystal structures, whereas the long-range order of the 3D lattice was essentially preserved. Because a working temperature lower than T(g) effectively prevented the colloidal crystals from fusing into films, the spherical colloidal crystals were transformed greatly under pressure, which lead to obvious change in the optical properties of colloidal crystals. Besides their special symmetry and optical properties, these nonspherical colloidal crystals can be used as templates for 2D or 3D structures of special symmetry, such as 2D nano-networks. We anticipate that this fabrication technique for nonspherical colloidal crystals can also be extended to nonspherical porous materials.

Journal Article↗
PubMed2002

Modified photoreactivity due to mixed crystal formation. I. Three mixed crystals between isostructural cobaloxime complexes.

Three crystals of 2-cyanoethyl cobaloxime complexes with 3-chloropyridine, 3-bromopyridine and 3-methylpyridine as axial base ligands are isostructural to one another. Three mixed crystals were formed between the pairs: (3-bromopyridine)(2-cyanoethyl)cobaloxime-(2-cyanoethyl)(3-methylpyridine)cobaloxime(0.45/0.55);(3-chloropyridine)(2-cyanoethyl)cobaloxime-(2-cyanoethyl)(3bromopyridine)cobaloxime (0.61/0.39); (3-chloropyridine)(2-cyanoethyl)cobaloxime-(2-cyanoethyl)(3-methylpyridine)cobaloxime (0.44/0.56). The X-ray crystal structure analysis revealed that the mixed compounds are also isostructural to the component crystals. It was found from the change in IR spectra that the 2-cyanoethyl groups in the three mixed crystals isomerized to 1-cyanoethyl groups on exposure to a xenon lamp, as observed for the 2-cyanoethyl groups in the component crystals. Rate constants for the three mixed and three component crystals, measured under the same conditions, are approximately the same, as the reaction cavities for the 2-cyanoethyl groups in the six crystals have almost the same size. For the mixed crystal between 3-chloropyridine and 3-methylpyridine complexes, the isomerization proceeded with retention of the single-crystal form. The conformation and configuration of the 1-cyanoethyl group that was produced were well explained by the shape of the reaction cavity before irradiation.

Journal Article↗
PubMed2005

Comparison of the crystallization and crystal packing of two Fab single-site mutant protein L complexes.

Protein L from Peptostreptococcus magnus (PpL) is a multidomain protein composed of four or five immunoglobulin-binding domains that target the kappa light chain of a large repertoire of human and murine antibodies. Thus, a single domain of this protein can be used to aid the crystallization of Fab, free or complexed to their antigen when it is not possible to obtain crystals without it. Each wild-type PpL domain has two light-chain binding sites that target the same region of the light chain and can thus bring together two Fab-antigen complexes within the crystal lattice. In this context the small PpL domain is sandwiched between two Fab and cannot participate in crystal contacts, thus mutants are unlikely to increase the chances of crystallizing a particular complex. However, it is possible to design mutants that can bind at only one site by making use of the crystal structures obtained so far. Such mutants will have a free surface that can participate in crystal contacts and that can be modified to improve its crystal contact-forming properties. Here, a comparison of two single-site mutants that differ at three different positions is reported. In both mutants two different tryptophan residues participate in crystal-packing interactions, suggesting that this residue may be particularly interesting for enhancing crystal-contact formation.

Amino Acid Substitution↗
PubMed2005

A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization.

Crystallization trials at the Division of Structural Biology in Oxford are now almost exclusively carried out using a high-throughput workflow implemented in the Oxford Protein Production Facility. Initial crystallization screening is based on nanolitre-scale sitting-drop vapour-diffusion experiments (typically 100 nl of protein plus 100 nl of reservoir solution per droplet) which use standard crystallization screening kits and 96-well crystallization plates. For 294 K crystallization trials the barcoded crystallization plates are entered into an automated storage system with a fully integrated imaging system. These plates are imaged in accordance with a pre-programmed schedule and the resulting digital data for each droplet are harvested into a laboratory information-management system (LIMS), scored by crystal recognition software and displayed for user analysis via a web-based interface. Currently, storage for trials at 277 K is not automated and for imaging the crystallization plates are fed by hand into an imaging system from which the data enter the LIMS. The workflow includes two procedures for nanolitre-scale optimization of crystallization conditions: (i) a protocol for variation of pH, reservoir dilution and protein:reservoir ratio and (ii) an additive screen. Experience based on 592 crystallization projects is reported.

Animals↗
PubMed1990

Observations on the structural features and characteristics of biological apatite crystals. 1. Observation on the shape and arrangement of the cross sectioned enamel crystals.

In a series of studies to investigate the structural features of biological crystals, using transmission electron microscope, the observations have been made on the shape and arrangement of the cross-sectioned crystal obtained from cross sections through the body of adult human enamel rod, in the surface layer, and in the middle and deep layers. The shapes of the cross sectioned crystals in the surface layer of the enamel are generally different from those in the middle and deep layers. In the surface layer of the enamel, the cross sectioned crystals show a considerable variation in shape and size, and have an irregular shape. The crystals are densely packed. Each crystal is adjacent to the neighboring one, and usually, the arrangement of crystals have an appearance rather like a "stone wall". In contrast, the cross sectioned enamel crystals in the middle and deep layer, are somewhat small than those in the surface layer, and have greater gaps between the crystals. In the middle and deep layers of the enamel, it is rare for the shapes of the crystals to be irregular, therefore, they retain their original shape, most of which are long and hexagonal.

Dental Enamel↗
PubMed2005

Supercritical fluid crystallization of griseofulvin: crystal habit modification with a selective growth inhibitor.

Poly (sebacic anhydride) (PSA) was used as a growth inhibitor to selectively modify habit of griseofulvin crystals formed via the Precipitation with a compressed-fluid antisolvent (PCA) process. PSA and griseofulvin were coprecipitated within a PCA injector, which provided efficient mixing between the solution and compressed antisolvent process streams. Griseofulvin crystal habit was modified from acicular to bipyramidal when the mass ratio of PSA/griseofulvin in the solution feed stream was <or=1:1. The habit modification was attributed to the preferential adsorption of PSA to the fastest growing crystal face of the acicular crystal form, which inhibited growth. Scanning electron microscopy (SEM) was used to characterize the griseofulvin and PSA particles, and gave results consistent with a selective growth inhibition mechanism. SEM micrographs showed regions on griseofulvin crystals where PSA microparticles had preferentially adsorbed. X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) analysis of the griseofulvin crystals indicated no changes in the crystalline form after the habit modification. Powder compressibility decreased from 49 +/- 3% to 28 +/- 7% with the modification in crystal habit. No change in the physical stability of the processed powder was observed after being stored at 25 degrees C/60% RH and 40 degrees C/70% RH for 23 days. Despite the change in crystal habit, griseofulvin crystals achieved 100% dissolution within 60 min in a simulated gastric fluid.

Anhydrides↗
PubMed2000

Crystallization and structure determination of a hepatitis delta virus ribozyme: use of the RNA-binding protein U1A as a crystallization module.

Well-ordered crystals of a genomic hepatitis delta virus (HDV) ribozyme, a large, globular RNA, were obtained employing a new crystallization method. A high-affinity binding site for the spliceosomal protein U1A was engineered into a segment of the catalytic RNA that is dispensable for catalysis. Because molecular surfaces of proteins are more chemically varied than those of RNA, the presence of the protein moiety was expected to facilitate crystallization and improve crystal order. The HDV ribozyme-U1A complex crystallized readily, and its structure was solved using standard techniques for heavy-atom derivatization of protein crystals. Over 1200 A(2) of the solvent-accessible surface area of the complex are involved in crystal contacts. As protein-protein interactions comprise 85% of this buried area, these crystals appear to be held together predominantly by the protein component of the complex. Our crystallization method should be useful for the structure determination of other biochemically important RNAs for which protein partners do not exist or are experimentally intractable. The refined model of the complex (R-free=27.9% for all reflections between 20.0 and 2.3 A) reveals an RNA with a deep active site cleft. Well-ordered metal ions are not observed crystallographically in this cavity. Biochemical results of previous workers had suggested an important role in catalysis for cytosine 75. The pyrimidine base of this residue is buried at the bottom of the active site in an environment that could raise its pK(a) value. We propose that this highly conserved cytosine may be the general base that catalyzes the transesterification.

Binding Sites↗
PubMedDate not supplied

Three-dimensional membrane crystals in amphibian cone outer segments. 1. Light-dependent crystal formation in frog retinas.

When frog retinas are exposed to light, a series of three-dimensional crystals develop within the outer segment disk system of cones but not rods. The crystals involve components that span cytoplasmic, disk membrane, and intradisk domains of the outer segment. The crystalline membrane domains are directly continuous with adjacent, noncrystalline lamellar regions. In axial extent, the crystals may involve as few as 1 or 2 disks or as many as 30 disks. However, within each disk, only one crystalline domain typically is observed. Within a crystal, the membranes are more planar in shape and more uniform in axial spacing then adjacent, noncrystalline lamellar regions. Furthermore, as crystalline domains expand laterally, one observes increased axial spacing disorder in noncrystalline lamellar regions, along with an increase in the width of the intradisk compartment. Thus, crystals appear to grow laterally by depleting adjacent lamellar regions of components that influence the normal membrane pair separation and axial spacing of cone outer segment disks. In isolated retinas, the crystalline domains appear to be randomly distributed along the length of the outer segment and show no preference for association with either the closed or open margins of the disk. After 45 min in the light, the crystals occupy approximately 10% of the cone outer segment volume. On the basis of comparative structural, biochemical, and physiological data, cone outer segment crystals may represent a cocrystal between bleached, phosphorylated opsin (providing transmembrane and intradisk elements) and the cytoplasmic protein, arrestin (providing trans-cytoplasmic elements). Thus, crystal formation may provide one mechanism of light adaptation within the cone outer segment. The spontaneous, bleaching-induced formation of these crystals in situ offers the possibility that cocrystals of cone outer segment components can be prepared in vitro for higher resolution crystallographic analyses.

Animals↗