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PubMed2003

A simplified counter diffusion method combined with a 1D simulation program for optimizing crystallization conditions.

We developed a new protein crystallization method has been developed using a simplified counter-diffusion method for optimizing crystallization condition. It is composed of only a single capillary, the gel in the silicon tube and the screw-top test tube, which are readily available in the laboratory. The one capillary can continuously scan a wide range of crystallization conditions (combination of the concentrations of the precipitant and the protein) unless crystallization occurs, which means that it corresponds to many drops in the vapor-diffusion method. The amount of the precipitant and the protein solutions can be much less than in conventional methods. In this study, lysozyme and alpha-amylase were used as model proteins for demonstrating the efficiency of this method. In addition, one-dimensional (1-D) simulations of the crystal growth were performed based on the 1-D diffusion model. The optimized conditions can be applied to the initial crystallization conditions for both other counter-diffusion methods with the Granada Crystallization Box (GCB) and for the vapor-diffusion method after some modification.

Capillary Action↗
PubMed2003

Impurity effects on lysozyme crystal growth.

Atomic force microscopy (AFM) and X-ray diffraction experiments have been combined to study the correlation between impurity incorporation, crystal surface morphology and crystal quality. Hen egg-white lysozyme has been used as a model protein, and covalently bound lysozyme dimer as a model impurity. AFM observation of the [101] crystal face revealed that the crystal surface clearly became rough when 5% impurity was added, and the steps disappeared as the impurity concentration increased to 10%. The crystal quality was evaluated by four factors: maximum resolution limit, / , Rmerge, and overall B factor. In every index, the crystal quality tended to degrade as the impurity concentration increased. The B-factor dropped significantly at 5% impurity; at the same time the step roughening was observed. This strongly suggested that the impurity incorporation affected the step growth mechanism and degraded the crystal quality.

Crystallization↗
PubMed2005

Crystallization and preliminary crystallographic analysis of calcium-binding protein-2 from Entamoeba histolytica and its complexes with strontium and the IQ1 motif of myosin V.

Calcium plays a pivotal role in the pathogenesis of amoebiasis, a major disease caused by Entamoeba histolytica. Two domains with four canonical EF-hand-containing calcium-binding proteins (CaBPs) have been identified from E. histolytica. Even though they have very high sequence similarity, these bind to different target proteins in a Ca2+-dependent manner, leading to different functional pathways. Calcium-binding protein-2 (EhCaBP2) crystals were grown using MPD as a precipitant. The crystals belong to space group P2(1), with unit-cell parameters a = 111.74, b = 68.83, c = 113.25 A, beta = 116.7 degrees. EhCaBP2 also crystallized in complex with strontium (replacing calcium) at similar conditions. The crystals belong to space group P2(1), with unit-cell parameters a = 69.18, b = 112.03, c = 93.42 A, beta = 92.8 degrees. Preliminary data for EhCaBP2 crystals in complex with an IQ motif are also reported. This complex was crystallized with MPD and ethanol as precipitating agents. These crystals belong to space group P2(1), with unit-cell parameters a = 60.5, b = 69.86, c = 86.5 A, beta = 97.9 degrees.

Amino Acid Motifs↗
PubMed2005

Crystallization and initial X-ray diffraction studies of scaffolding protein (gp7) of bacteriophage phi29.

The Bacillus subtilis bacteriophage phi29 scaffolding protein (gp7) has been crystallized by the hanging-drop vapour-diffusion method at 293 K. Two new distinct crystal forms that both differed from a previously crystallized and solved scaffolding protein were grown under the same conditions. Form I belongs to the primitive tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 77.13, c = 37.12 A. Form II crystals exhibit an orthorhombic crystal form, with space group C222 and unit-cell parameters a = 107.50, b = 107. 80, c = 37.34 A. Complete data sets have been collected to 1.78 and 1.80 A for forms I and II, respectively, at 100 K using Cu Kalpha X-rays from a rotating-anode generator. Calculation of a VM value of 2.46 A3 Da(-1) for form I suggests the presence of one molecule in the asymmetric unit, corresponding to a solvent content of 50.90%, whereas form II has a VM of 4.80 A3 Da(-1) with a solvent content of 48.76% and two molecules in the asymmetric unit. The structures of both crystal forms are being determined by the molecular-replacement method using the coordinates of the published crystal structure of gp7.

Bacillus Phages↗
PubMed2005

Expression, purification, crystallization and preliminary X-ray analysis of a nucleoside kinase from the hyperthermophile Methanocaldococcus jannaschii.

Methanocaldococcus jannaschii nucleoside kinase (MjNK) is an ATP-dependent non-allosteric phosphotransferase that shows high catalytic activity for guanosine, inosine and cytidine. MjNK is a member of the phosphofructokinase B family, but participates in the biosynthesis of nucleoside monophosphates rather than in glycolysis. MjNK was crystallized as the apoenzyme as well as in complex with an ATP analogue and Mg2+. The latter crystal form was also soaked with fructose-6-phosphate. Synchrotron-radiation data were collected to 1.70 A for the apoenzyme crystals and 1.93 A for the complex crystals. All crystals exhibit orthorhombic symmetry; however, the apoenzyme crystals contain one monomer per asymmetric unit whereas the complex crystals contain a dimer.

Adenosine Triphosphate↗
PubMed2005

Precipitation diagram and optimization of crystallization conditions at low ionic strength for deglycosylated dye-decolorizing peroxidase from a basidiomycete.

The growth of suitably sized protein crystals is essential for protein structure determination by X-ray crystallography. In general, crystals are grown using a trial-and-error method. However, these methods have been modified with the advent of microlitre dispensing-robot technology and of protocols that rapidly screen for crystal nucleation conditions. The use of one such automatic dispenser for mixing protein drops (1.3-2.0 microl in volume) of known concentration and pH with precipitating solutions (ejecting 2.0 microl droplets) containing salt is described here. The results of the experiments are relevant to a crystallization approach based on a two-step procedure: screening for the crystal nucleation step employing robotics followed by optimization of the crystallization conditions using incomplete factorial experimental design. Large crystals have successfully been obtained using quantities as small as 3.52 mg protein.

Basidiomycota↗
PubMed2005

Crystallization of a 2:2 complex of granulocyte-colony stimulating factor (GCSF) with the ligand-binding region of the GCSF receptor.

The granulocyte-colony stimulating factor (GCSF) receptor receives signals for regulating the maturation, proliferation and differentiation of the precursor cells of neutrophilic granulocytes. The signalling complex composed of two GCSFs (GCSF, 19 kDa) and two GCSF receptors (GCSFR, 34 kDa) consisting of an Ig-like domain and a cytokine-receptor homologous (CRH) domain was crystallized. A crystal of the complex was grown in 1.0 M sodium formate and 0.1 M sodium acetate pH 4.6 and belongs to space group P4(1)2(1)2 (or its enantiomorph P4(3)2(1)2), with unit-cell parameters a = b = 110.1, c = 331.8 A. Unfortunately, this crystal form did not diffract beyond 5 A resolution. Since the heterogeneity of GCSF receptor appeared to prevent the growth of good-quality crystals, the GCSF receptor was fractionated by anion-exchange chromatography. Crystals of the GCSF-fractionated GCSF receptor complex were grown as a new crystal form in 0.2 M ammonium phosphate. This new crystal form diffracted to beyond 3.0 A resolution and belonged to space group P3(1)21 (or its enantiomorph P3(2)21), with unit-cell parameters a = b = 134.8, c = 105.7 A.

Chromatography, Ion Exchange↗
PubMed2006

Structure of human salivary alpha-amylase crystallized in a C-centered monoclinic space group.

Human salivary alpha-amylase (HSA) is a major secretory protein component of saliva and has important biological functions, including the initial digestion of starch. HSA acts as a monomer and mediates the hydrolysis of alpha-1,4-glucosidic linkages in oligosaccharides. To date, all published crystal structures of HSA have been crystallized as monomers in space group P2(1)2(1)2(1). Here, the serendipitous purification, crystallization and ultimate structure determination of a HSA non-crystallographic symmetry (NCS) dimer, while attempting to purify human carbonic anhydrase VI (HCA VI) from saliva using an affinity resin for alpha-class carbonic anhydrases, is presented. On further investigation, it was shown that HSA could only be copurified using the affinity resin in the presence of HCA VI which is glycosylated and not the non-glycosylated HCA II. The identification of the HSA crystals was carried out by peptide mapping and mass spectrometry. HSA was shown to have crystallized as an NCS dimer in space group C2, with unit-cell parameters a = 150.9, b = 72.3, c = 91.3 A, beta = 102.8 degrees. The NCS dimer crystal structure is reported to 3.0 A resolution, with a refined Rcryst of 0.228. The structure is compared with the previously reported P2(1)2(1)2(1) monomer structures and the crystal packing and dimer interface are discussed.

Crystallization↗
PubMed2006

Crystallization of the class IV adenylyl cyclase from Yersinia pestis.

The class IV adenylyl cyclase from Yersinia pestis has been cloned and crystallized in both a triclinic and an orthorhombic form. An amino-terminal His-tagged construct, from which the tag was removed by thrombin, crystallized in a triclinic form diffracting to 1.9 A, with one dimer per asymmetric unit and unit-cell parameters a = 33.5, b = 35.5, c = 71.8 A, alpha = 88.7, beta = 82.5, gamma = 65.5 degrees. Several mutants of this construct crystallized but diffracted poorly. A non-His-tagged native construct (179 amino acids, MW = 20.5 kDa) was purified by conventional chromatography and crystallized in space group P2(1)2(1)2(1). These crystals have unit-cell parameters a = 56.8, b = 118.6, c = 144.5 A, diffract to 3 A and probably have two dimers per asymmetric unit and VM = 3.0 A3 Da(-1). Both crystal forms appear to require pH below 5, complicating attempts to incorporate nucleotide ligands into the structure. The native construct has been produced as a selenomethionine derivative and crystallized for phasing and structure determination.

Adenylyl Cyclases↗
PubMed2003

Three-dimensional crystallization of the Escherichia coli glycerol-3-phosphate transporter: a member of the major facilitator superfamily.

Here we report the successful three-dimensional crystallization of GlpT, the glycerol-3-phosphate transporter from Escherichia coli inner membrane. GlpT possesses 12 transmembrane alpha-helices and is a member of the major facilitator superfamily. It mediates the exchange of glycerol-3-phosphate for inorganic phosphate across the membrane. Approximately 20 phospholipid molecules per protein, identified as negatively charged phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, were required for the monodispersity of purified GlpT. Analytical size-exclusion chromatography proved to be efficient in identifying detergents for GlpT monodispersity. Nine such detergents were later used for GlpT crystallization. Screening for crystal nucleation was carried out with a variety of polyethylene glycols as the precipitant over a wide pH range. Subsequent identification of a rigid protein core by limited proteolysis and mass spectroscopy resulted in better-ordered crystals. These crystals exhibited order to 3.7 A resolution in two dimensions. However, the stacking in the third dimension was partially disordered. This stacking problem was overcome by using a detergent mixture and manipulating the ionic interactions in the crystallization solution. The resulting GlpT crystals diffracted isotropically to 3.3 A resolution and were suitable for structure determination by X-ray crystallography.

Chromatography, Affinity↗
PubMed2004

Circular permutation as a tool to reduce surface entropy triggers crystallization of the signal recognition particle receptor beta subunit.

The production of diffraction-quality crystals remains a difficult obstacle on the road to high-resolution structural characterization of proteins. This is primarily a result of the empirical nature of the process. Although crystallization is not predictable, factors inhibiting it are well established. First, crystal formation is always entropically unfavorable. Reducing the entropic cost of crystallizing a given protein is thus desirable. It is common practice to map boundaries and remove unstructured regions surrounding the folded protein domain. However, a problem arises when flexible regions are not at the boundaries but within a domain. Such regions cannot be deleted without adding new restraints to the domain. We encountered this problem during an attempt to crystallize the beta subunit of the eukaryotic signal recognition particle (SRbeta), bearing a long and flexible internal loop. Native SRbeta did not crystallize. However, after circularly permuting the protein by connecting the spatially close N and C termini with a short heptapeptide linker GGGSGGG and removing 26 highly flexible loop residues within the domain, we obtained diffraction-quality crystals. This protein-engineering method is simple and should be applicable to other proteins, especially because N and C termini of protein domains are often close in space. The success of this method profits from prior knowledge of the domain fold, which is becoming increasingly common in today's postgenomic era.

Crystallization↗
PubMed2006

Atomic-resolution crystal structure of Borrelia burgdorferi outer surface protein A via surface engineering.

Outer surface protein A (OspA) from Borrelia burgdorferi has an unusual dumbbell-shaped structure in which two globular domains are connected with a "single-layer" beta-sheet (SLB). The protein is highly soluble, and it has been recalcitrant to crystallization. Only OspA complexes with Fab fragments have been successfully crystallized. OspA contains a large number of Lys and Glu residues, and these "high entropy" residues may disfavor crystal packing because some of them would need to be immobilized in forming a crystal lattice. We rationally designed a total of 13 surface mutations in which Lys and Glu residues were replaced with Ala or Ser. We successfully crystallized the mutant OspA without a bound Fab fragment and extended structure analysis to a 1.15 Angstroms resolution. The new high-resolution structure revealed a unique backbone hydration pattern of the SLB segment in which water molecules fill the "weak spots" on both faces of the antiparallel beta-sheet. These well-defined water molecules provide additional structural links between adjacent beta-strands, and thus they may be important for maintaining the rigidity of the SLB that inherently lacks tight packing afforded by a hydrophobic core. The structure also revealed new information on the side-chain dynamics and on a solvent-accessible cavity in the core of the C-terminal globular domain. This work demonstrates the utility of extensive surface mutation in crystallizing recalcitrant proteins and dramatically improving the resolution of crystal structures, and provides new insights into the stabilization mechanism of OspA.

Amino Acid Substitution↗
PubMed2006

Identifying protein construct variants with increased crystallization propensity--a case study.

This study describes an efficient multiparallel automated workflow of cloning, expression, purification, and crystallization of a large set of construct variants for isolated protein domains aimed at structure determination by X-ray crystallography. This methodology is applied to MAPKAP kinase 2, a key enzyme in the inflammation pathway and thus an attractive drug target. The study reveals a distinct subset of truncation variants with improved crystallization properties. These constructs distinguish themselves by increased solubility and stability during a parallel automated multistep purification process including removal of the recombinant tag. High-throughput protein melting point analysis characterizes this subset of constructs as particularly thermostable. Both parallel purification screening and melting point determination clearly identify residue 364 as the optimal C terminus for the kinase domain. Moreover, all three constructs that ultimately crystallized feature this C terminus. At the N terminus, only three amino acids differentiate a noncrystallizing from a crystallizing construct. This study addresses the very common issues associated with difficult to crystallize proteins, those of solubility and stability, and the crucial importance of particular residues in the formation of crystal contacts. A methodology is suggested that includes biophysical measurements to efficiently identify and produce construct variants of isolated protein domains which exhibit higher crystallization propensity.

Cloning, Molecular↗
PubMed2001

A comparison of salts for the crystallization of macromolecules.

Thirty-one proteins and viruses that we knew from our own experience could be crystallized, or had been reported to have been crystallized by others, were investigated. In this experiment, each protein or virus was subjected to a crystallization screen of 12 different salts, each titrated to pH 7.2 beforehand, at concentrations ranging from 20% saturation to 90% saturation. Eight macromolecules failed to crystallize at all from any salt and were omitted from consideration. From the remaining 23 proteins, each salt was scored according to how many proteins and viruses it successfully crystallized. Among several results, one was particularly striking. Sodium malonate clearly was much more successful than any other salt, resulting in the crystallization of 19 of the 23 macromolecules, almost twice as effective as the next most successful salt, which was a draw between sodium acetate, sodium tartrate, sodium formate, and ammonium sulfate (11 of 22). The high success rate of sodium malonate in producing crystals was even more impressive when an overall unique success rate with individual macromolecules was considered.

Ammonium Sulfate↗
PubMed1995

Calcium oxalate crystal deposition in epithelioid histiocytes of granulomatous lymphadenitis: analysis by light and electronmicroscopy.

In this study of 55 cases of granulomatous lymphadenitis of various aetiologies, both haematoxylin and eosin stained and unstained sections were examined by light and polarizing light microscopy for crystals within epithelioid histiocytes. This investigation was prompted by a case of granulomatous lymphadenitis in which the identification of ovoid birefringent structures within epithelioid histiocytes led to an initial false suggestion of foreign body reaction. Identical single, small, ovoid or biconvex, intensely birefringent crystals, invisible by ordinary light microscopy, were found within the cytoplasm of mononuclear epithelioid histiocytes in 37 cases. There was only minor crystal loss with routine H & E staining. The sections were also examined by scanning electronmicroscopy in secondary and backscattered electron imaging modes and 27 cases were found to contain biconvex crystals which were shown on X-ray energy dispersive spectroscopic microanalysis to be consistent with calcium oxalate. There was a good quantitative correlation between light and scanning electron microscopy. Tissue from the index case was also examined by transmission electronmicroscopy, and electron dense crystals were identified. We confirm the previous findings of ovoid oxalate crystals in a variety of granulomatous conditions including Mycobacterial infection, sarcoidosis and Crohn's disease. Ovoid crystals of calcium oxalate, a common finding in granulomatous lymphadenitis, appear to be endogenously derived, and should be more widely recognized in order to avoid misdiagnosis of foreign body reaction.

Calcium Oxalate↗
PubMed1978

Kappa-type light chain crystal storage histiocytosis.

An autopsy case of systemic histiocytosis with excessive deposition of kappa-type light chain crystals was reported in a 58 year-old man who had consistently showed kappa-type light chain paraproteinemia, Bence Jones proteinuria and hypogammaglobulinemia for about 10 years until his death. However, no bony destruction was found by repeated X-ray examinations. At autopsy, extensive hyperplasia of crystal-storing histiocytes was observed in the bone marrow, spleen, liver, lymph nodes, interstitial tissues of visceral organs and loose connective tissues. In the bone marrow and some other tissues, mild proliferation of plasmocytoid cells containing small crystals were found. Histochemically the crystals positively stained with various methods for amino acids and proteins, especially with Weigerts' method for fibrin. Ultrastructurally intralysosomal crystal deposition was confirmed in the storage histiocytes and derivation of the crystals from Golgi's sacculi in the plasmocytoid cells was suggested. Biochemically the crystals were regarded as mainly consisting of dimers of a variable half of light chain immunoglobulin and immunochemically and immunohistochemically reacted to anti-kappa type light chain serum. Such a generalized storage histiocytosis may be secondarily induced by immunoglobulin synthesized in plasmocytoid cells.

Crystallization↗
PubMed2005

Study of a rat model for calcium oxalate crystal formation without severe renal damage in selected conditions.

BACKGROUND: Although nephrotoxic in high doses, ethylene glycol (EG) has been used with ammonium chloride (NH(4)Cl) or vitamin D(3) to study calcium oxalate stone formation in rat models. In the present study we used EG alone or with NH(4)Cl to study hyperoxaluria, crystaluria, and crystal attachment to renal epithelial cells in rats with minimal renal damage. METHODS: Six-week-old male Sprague-Dawley (SD) rats were given food and special drinking water. In experiment 1 the drinking water contained 1.0% NH(4)Cl plus four different concentrations of EG (0.8%, 0.4%, 0.2%, 0.1%). In experiment 2 the drinking water contained EG alone (0.8%, 0.4%, 0.2%, 0.1%). Urine was collected for 24 h before the rats were sacrificed. In experiment 1 the rats were sacrificed 5-13 days after starting the special water. In experiment 2 the rats were sacrificed 7-21 days after starting the special water. Bladder urine was also obtained. Blood and urine were tested for calcium, phosphorus, and creatinine. In addition, urine was tested for pH, oxalate and N-acetyl-beta-D glucosaminidase (NAG). Kidney sections were stained with hematoxylin-eosin, von Kossa and Pizzolato stain. Crystal morphology was determined using polarizing microscopy, and composition was determined using high-resolution X-ray powder diffraction. RESULTS: Experiment 1: Aggravation of renal function, an increase in urinary oxalate and NAG excretion, and crystals observed in the kidneys all correlated with EG concentration and length of drinking time. In bladder urine, calcium oxalate monohydrate (COM) crystals exceeded calcium oxalate dihydrate (COD) crystals. Experiment 2: Renal function remained unchanged. Oxalate excretion increased and NAG increased slightly. Crystals occurred only in the papillary tip region. Crystals in bladder urine were mostly COD. CONCLUSION: In the current rat model, calcium oxalate crystaluria could be induced without severe renal damage in selected cases. Either and/or both COM and COD might form and interact with kidney epithelium. We propose different experimental conditions to study the various phases of calcium oxalate stone formation in young male SD rats.

Acetylglucosaminidase↗
PubMed2004

The role of glycoproteins in calcium oxalate crystal development.

OBJECTIVE: To assess the effects of a glycoprotein (mucine) on calcium oxalate crystal development in different conditions and situations, to clarify some of its possible effects. MATERIALS AND METHODS: Crystallization was assessed using a batch system in presence of mucine suspensions, by kinetic-turbidimetric measurements, and using a flow system in the presence of retained agglomerates of mucine, evaluating the precipitated calcium oxalate. RESULTS: In batch conditions low mucine concentrations (<15 mg/L) inhibited calcium oxalate nucleation and higher concentrations (<250 mg/L) inhibited calcium phosphate nucleation, whereas at high concentrations there was also promotion. The presence of an aggregate of mucine in the flow system provoked calcium oxalate monohydrate crystallization at 0.691 microg/h per mg of mucine. In flow conditions pyrophosphate at 11.5 micromol/L caused a decrease of 84% in the calcium oxalate crystallized on mucine, 1.32 mmol/L of citrate a decrease of a 83%, 20 mg/L of pentosan polysulphate a decrease by 80%, and 7.58 micromol/L phytate totally prevented the crystallization of calcium oxalate on mucine. CONCLUSION: All substances inhibiting calcium oxalate crystallization with the capacity to interact with calcium ions also have crystallization promoting properties when they are at sufficiently high concentrations, because of their capacity to form agglomerates or the insolubility of their calcium salts.

Calcium Oxalate↗

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