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PubMed2004

A new crystal form of XT6 enables a significant improvement of its diffraction quality and resolution.

Xylanases (1,4-beta-D-xylan xylanhydrolases; EC 3.2.1.8) hydrolyze the 1,4-beta-D-xylopyranosyl linkage of xylans. The detailed structural characterization of these enzymes is of interest for the elucidation of their catalytic mechanism and for their rational modification toward improved stability and specificity. An extracellular xylanase from Geobacillus stearothermophilus T-6 (XT6) has recently been cloned, overexpressed, purified and biochemically characterized. Previous crystallographic efforts resulted in a hexagonal crystal form, which subsequently proved to be of limited use for structural analysis, mainly because of its relatively poor diffraction quality and resolution. A systematic search for more suitable crystals of XT6 recently resulted in a new crystal form of this enzyme with significantly improved diffraction characteristics. The new crystals belong to a C-centred monoclinic crystal system (space group C2), with unit-cell parameters a = 121.5, b = 61.7, c = 89.1 A, beta = 119.7 degrees. These crystals diffract X-rays to better than 1.5 A resolution, showing a very clear diffraction pattern of relatively high quality. The crystals are mechanically strong and exhibit excellent radiation-stability when frozen under cold nitrogen gas. A full diffraction data set to 1.45 A resolution (94.1% completeness, R(merge) = 7.0%) has been collected from flash-frozen crystals of the native enzyme at 95 K using synchrotron radiation. Crystals of the E159A/E265A catalytic double mutant of XT6 were found to be isomorphous to those of native XT6. They were used for a full measurement of 1.8 A resolution diffraction data at 100 K (90.9% completeness; R(merge) = 5.0%). These data are currently being used for the high-resolution structure determination of XT6 and its mutant for mechanistic interpretations and rational introduction of thermostability.

Bacterial Proteins↗
PubMed2004

Phase transition of triclinic hen egg-white lysozyme crystal associated with sodium binding.

A triclinic crystal of hen egg-white lysozyme obtained from a D2O solution at 313 K was transformed into a new triclinic crystal by slow release of solvent under a temperature-regulated nitrogen-gas stream. The progress of the transition was monitored by X-ray diffraction. The transition started with the appearance of strong diffuse streaks. The diffraction spots gradually fused and faded with the emergence of diffraction from the new lattice; the scattering power of the crystal fell to a resolution of 1.5 A from the initial 0.9 A resolution. At the end of the transition, the diffuse streaks disappeared and the scattering power recovered to 1.1 A resolution. The transformed crystal contained two independent molecules and the solvent content had decreased to 18% from the 32% solvent content of the native crystal. The structure was determined at 1.1 A resolution and compared with the native structure refined at the same resolution. The backbone structures of the two molecules in the transformed crystal were superimposed on the native structure with root-mean-square deviations of 0.71 and 0.96 A. A prominent structural difference was observed in the loop region of residues Ser60-Leu75. In the native crystal, a water molecule located at the centre of this helical loop forms hydrogen bonds to main-chain peptide groups. In the transformed crystal, this water molecule is replaced by a sodium ion with octahedral coordination that involves water molecules and a nitrate ion. The peptide group connecting Arg73 and Asn74 is rotated by 180 degrees so that the CO group of Arg73 can coordinate to the sodium ion. The change in the X-ray diffraction pattern during the phase transition suggests that the transition proceeds at the microcrystal level. A mechanism is proposed for the crystal transformation.

Animals↗
PubMed2006

Development of an automated large-scale protein-crystallization and monitoring system for high-throughput protein-structure analyses.

Protein crystallization remains one of the bottlenecks in crystallographic analysis of macromolecules. An automated large-scale protein-crystallization system named PXS has been developed consisting of the following subsystems, which proceed in parallel under unified control software: dispensing precipitants and protein solutions, sealing crystallization plates, carrying robot, incubators, observation system and image-storage server. A sitting-drop crystallization plate specialized for PXS has also been designed and developed. PXS can set up 7680 drops for vapour diffusion per hour, which includes time for replenishing supplies such as disposable tips and crystallization plates. Images of the crystallization drops are automatically recorded according to a preprogrammed schedule and can be viewed by users remotely using web-based browser software. A number of protein crystals were successfully produced and several protein structures could be determined directly from crystals grown by PXS. In other cases, X-ray quality crystals were obtained by further optimization by manual screening based on the conditions found by PXS.

Automation↗
PubMed1998

Crystallization and preliminary X-ray studies of allophycocyanin from red alga Porphyra yezoensis.

Allophycocyanin from red alga Porphyra yezoensis has been crystallized in three crystal forms. Form 1 crystals with space group P4132 or P4332 and cell parameters a = 286 A, alpha = 90 degrees were obtained by using isopropanol as precipitant. These crystals did not diffract beyond 5.8 A and could not be used in structure analysis. Form 2 crystals were obtained when crystallization conditions were slightly changed by adding 0.2 M magnesium chloride. The space group of form 2 was not determined because it was difficult to get large single crystals. Form 3 crystals were obtained by using ammonium sulfate as precipitant. The space group of form 3 is R32 with cell dimensions a = b = 105.3, c = 189.4 A and one alpha beta unit in the asymmetric unit. These crystals diffract up to 2.06 A resolution and are suitable for structure determination by molecular-replacement methods.

Crystallization↗
PubMed1998

Macromolecular crystal annealing: overcoming increased mosaicity associated with cryocrystallography.

Although cryogenic data collection has become the method of choice for macromolecular crystallography, the flash-cooling step can dramatically increase the mosaicity of some crystals. Macromolecular crystal annealing significantly reduces the mosaicity of flash-cooled crystals without affecting molecular structure. The process, which cycles a flash-cooled crystal to ambient temperature and back to cryogenic temperature, is simple, quick and requires no special equipment. The annealing process has been applied to crystals of several different macromolecules grown from different precipitants and using a variety of cryoprotectants. The protocol for macromolecular crystal annealing also has been applied to restore diffraction from flash-cooled crystals that were mishandled during transfer to or from cryogenic storage. These results will be discussed in relation to crystal mosaicity and effects of radiation damage in flash-cooled crystals.

Crystallization↗
PubMed1999

Crystallization and preliminary X-ray diffraction studies of phospholipase D from Streptomyces antibioticus.

Phospholipase D (E.C. 3.1.4.4) from Streptomyces antibioticus has been crystallized in six crystal forms using the hanging-drop vapour-diffusion method. The type III and V crystals belong to monoclinic and hexagonal systems, respectively. All of the other crystal forms, types I, II, IV and VI, belong to orthorhombic space group P212121. Of these four types, the type VI crystals are suitable for X-ray structure determination. Crystal data for type VI crystals are: a = 50.1, b = 98.7, c = 107.6 A, V = 532100 A3, Z = 4 and Vm = 2.47 A3 Da-1. Type VI crystals diffract to at least 2.3 A resolution. A total of 11295 independent reflections to 3 A resolution have been collected from a type VI crystal using a conventional X-ray source, and its structural analysis is currently being conducted using isomorphous replacement methods.

Crystallization↗
PubMed1999

Crystallogenesis studies on yeast aspartyl-tRNA synthetase: use of phase diagram to improve crystal quality.

Aspartyl-tRNA synthetase (AspRS) extracted from yeast is heterogeneous owing to proteolysis of its positively charged N-terminus; its crystals are of poor quality. To overcome this drawback, a rational strategy was developed to grow crystals of sufficient quality for structure determination. The strategy is based on improvement of the protein homogeneity and optimization of crystallization, taking advantage of predictions from crystal-growth theories. An active mutant lacking the first 70 residues was produced and initial crystallization conditions searched. The shape and habit of initial crystals were improved by establishing a phase diagram of protein versus crystallizing-agent concentrations. Growth of large well faceted crystals takes place at low supersaturations near the isochronic supersolubility curve. Further refinement led to reproducible growth of two crystalline forms of bipyramidal (I) or prismatic (II) habit. Both diffract X-rays better than crystals previously obtained with native AspRS. Complete data sets were collected at 3 A resolution for form I (space group P41212) and form II (space group P3221) and molecular-replacement solutions were found in both space groups.

Aspartate-tRNA Ligase↗
PubMed1999

Crystallization and preliminary X-ray analysis of human rhinovirus serotype 2 (HRV2).

Human rhinoviruses, the major cause of mild recurrent infections of the upper respiratory tract, are small icosahedral particles. Over 100 different serotypes have been identified. The majority (91 serotypes) use intercellular adhesion molecule 1 as the cell-attachment site; ten serotypes (the minor group) bind to members of the low-density lipoprotein receptor. Three different crystal forms of the minor-group human rhinovirus serotype 2 (HRV2) were obtained by the hanging-drop vapour-diffusion technique using ammonium sulfate and sodium/potassium phosphate as precipitants. Monoclinic crystals, space group P2(1), diffracted at least to 2.8 A resolution, and two complete virus particles were located in the crystal asymmetric unit. A second type of crystals had a compact cubic like morphology and diffracted beyond 2.5 A resolution. These crystals belong to a primitive orthorhombic space group, with unit-cell parameters a = 309.3, b = 353.5, c = 759.6 A, and contain one virus particle in the asymmetric unit. A third type of crystals, with a prismatic shape and belonging to space group I222, was also obtained under similar crystallization conditions. These latter crystals, with unit-cell parameters a = 308.7, b = 352.2, c = 380.5 A, diffracted to high resolution (beyond 1.8 A) and contained 15 protomers per asymmetric unit; this requires that three perpendicular crystal twofold axes coincide with three of the viral particle's dyad axes.

Crystallization↗
PubMed1999

Crystallization of truncated human apolipoprotein A-I in a novel conformation.

The crystallization of recombinant human apolipoprotein A-I (apo A-I), the major protein component of high-density lipoprotein, in a new crystal form is described. The fragment crystallized, residues 44-243 of native apo A-I [apo Delta(1--43)A-I], is very similar to intact native apo A-I in its ability to bind lipid, to be incorporated into high-density lipoproteins and to activate lecithin-cholesterol acyl transferase. Apo Delta(1-43)A-I crystallizes, in the presence of beta-D-octylglucopyranoside, in space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 37. 11, b = 123.62, c = 164.65 A and a diffraction limit of 3.2 A. These form II crystals grow under conditions of significantly lower ionic strength than the original form I crystals (space group P2(1)2(1)2(1), a = 97.47, b = 113.87, c = 196.19 A, diffraction limit 3.0 A). Packing arguments show that the unusual open conformation of apo Delta(1-43)A-I found in the form I crystals cannot be packed into the smaller oddly proportioned form II unit cell. Monomeric apo Delta(1-43)A-I, as either a four-helix bundle ( approximately 75 x 30 x 30 A) or an extended helical rod (approximately 150 x 20 x 20 A), can be packed into the form II unit cell. It is concluded, therefore, that apo Delta(1-43)A-I may have crystallized in one of these distinct conformations in the form II crystals.

Apolipoprotein A-I↗
PubMed2002

Experiments testing the abatement of radiation damage in D-xylose isomerase crystals with cryogenic helium.

Helium is a more efficient cryogen than nitrogen, and for macromolecular data collection at high-flux beamlines will deliver lower temperatures. An open-flow helium cryostat developed at the University of Toledo (the Pinkerton Device) has been used for macromolecular data collection. This device differs from standard commercial He cryostats by having a much narrower aperture providing a high velocity stream of He around the crystal that maximizes convective and conductive heat exchange between the crystal and the cryogen. This paper details a series of experiments conducted at the IMCA-CAT 17ID beamline using one crystal for each experimental condition to examine whether helium at 16 K provided better radiation-damage abatement compared with nitrogen at 100 K. These studies used matched high-quality crystals (0.94 A diffraction resolution) of D-xylose isomerase derived from the commercial material Gensweet SGI. Comparisons show that helium indeed abates the indicators of radiation damage, in this case resulting in longer crystal diffractive lifetimes. The overall trend suggests that crystals maintain order and that high-resolution data are less affected by increased radiation load when crystals are cooled with He rather than N(2). This is probably the result of a lower effective temperature at the crystal with concomitant reduction in free-radical diffusion. Other features, such as an apparent phase transition in macromolecular crystals at lower temperatures, require investigation to broaden the utility of He use.

Aldose-Ketose Isomerases↗
PubMed2005

Systematic replacement of lysine with glutamine and alanine in Escherichia coli malate synthase G: effect on crystallization.

Two proposals recommend substitution of surface lysine residues as a means to improve the quality of protein crystals. In proposal I, substitution of lysine by alanine has been suggested to improve crystallization by reducing the entropic cost of ordering flexible side chains at crystal contacts. In proposal II, substitution of lysine by residues more commonly found in crystal contacts, such as glutamine, has been proposed to improve crystallization. 15 lysine residues on the surface of Escherichia coli malate synthase G, distributed over a variety of secondary structures, were individually mutated to both alanine and glutamine. For 28 variants, detailed studies of the effect on enzymatic activity and crystallization were conducted. This has permitted direct comparison of the relative effects of the two types of mutations. While none of the variants produced crystals suitable for X-ray structural determination, small crystals were obtained in a wide variety of conditions, in support of the general approach. Glutamine substitutions were found to be more effective than alanine in producing crystals, in support of proposal II. Secondary structure at the site of mutation does not appear to play a major role in determining the rate of success.

Alanine↗
PubMed2006

A single point mutation changes the crystallization behavior of Mycoplasma arthritidis-derived mitogen.

Mycoplasma arthritidis-derived mitogen (MAM) functions as a conventional superantigen (SAg). Although recombinant MAM has been crystallized by the hanging-drop vapour-diffusion method, the crystals diffracted poorly to only 5.0 A resolution, with large unit-cell parameters a = 163.8, b = 93.0, c = 210.9 A, beta = 93.7 degrees in the monoclinic space group P2(1). Unit-cell content analysis revealed that as many as 24 molecules could be present in the asymmetric unit. Systematic alanine mutagenesis was applied in order to search for mutants that give crystals of better quality. Two mutants, L50A and K201A, were crystallized under the same conditions as wild-type MAM (MAMwt). Crystals of the L50A mutant are isomorphous with those of MAMwt, while a new crystal form was obtained for the K201 mutant, belonging to the cubic space group P4(1)32 with unit-cell parameters a = b = c = 181.9 A. Diffraction data were collected to 3.6 and 2.8 A resolution from crystals of the MAM L50A and K201A mutants, respectively. Molecular-replacement calculations suggest the presence of two molecules in the asymmetric unit for the MAM K201A mutant crystal, resulting in a VM of 5.0 A Da(-1) and a solvent content of 75%. An interpretable electron-density map for the MAM K201A mutant crystal was produced using the molecular-replacement method.

Antigens↗
PubMed2006

Crystallization and preliminary X-ray diffraction studies on the bicupin YwfC from Bacillus subtilis.

A central tenet of evolutionary biology is that proteins with diverse biochemical functions evolved from a single ancestral protein. A variation on this theme is that the functional repertoire of proteins in a living organism is enhanced by the evolution of single-chain multidomain polypeptides by gene-fusion or gene-duplication events. Proteins with a double-stranded beta-helix (cupin) scaffold perform a diverse range of functions. Bicupins are proteins with two cupin domains. There are four bicupins in Bacillus subtilis, encoded by the genes yvrK, yoaN, yxaG and ywfC. The extensive phylogenetic information on these four proteins makes them a good model system to study the evolution of function. The proteins YvrK and YoaN are oxalate decarboxylases, whereas YxaG is a quercetin dioxygenase. In an effort to aid the functional annotation of YwfC as well as to obtain a complete structure-function data set of bicupins, it was proposed to determine the crystal structure of YwfC. The bicupin YwfC was crystallized in two crystal forms. Preliminary crystallographic studies were performed on the diamond-shaped crystals, which belonged to the tetragonal space group P422. These crystals were grown using the microbatch method at 298 K. Native X-ray diffraction data from these crystals were collected to 2.2 A resolution on a home source. These crystals have unit-cell parameters a = b = 68.7, c = 211.5 A. Assuming the presence of two molecules per asymmetric unit, the V(M) value was 2.3 A3 Da(-1) and the solvent content was approximately 45%. Although the crystals appeared less frequently than the tetragonal form, YwfC also crystallizes in the monoclinic space group P2(1), with unit-cell parameters a = 46.7, b = 106.3, c = 48.7 A, beta = 92.7 degrees.

Amino Acid Sequence↗
PubMed2000

Oxygen binding by alpha(Fe2+)2beta(Ni2+)2 hemoglobin crystals.

Oxygen binding by hemoglobin fixed in the T state either by crystallization or by encapsulation in silica gels is apparently noncooperative. However, cooperativity might be masked by different oxygen affinities of alpha and beta subunits. Metal hybrid hemoglobins, where the noniron metal does not bind oxygen, provide the opportunity to determine the oxygen affinities of alpha and beta hemes separately. Previous studies have characterized the oxygen binding by alpha(Ni2+)2beta(Fe2+)2 crystals. Here, we have determined the three-dimensional (3D) structure and oxygen binding of alpha(Fe2+)2beta(Ni2+)2 crystals grown from polyethylene glycol solutions. Polarized absorption spectra were recorded at different oxygen pressures with light polarized parallel either to the b or c crystal axis by single crystal microspectrophotometry. The oxygen pressures at 50% saturation (p50s) are 95 +/- 3 and 87 +/- 4 Torr along the b and c crystal axes, respectively, and the corresponding Hill coefficients are 0.96 +/- 0.06 and 0.90 +/- 0.03. Analysis of the binding curves, taking into account the different projections of the alpha hemes along the optical directions, indicates that the oxygen affinity of alpha1 hemes is 1.3-fold lower than alpha2 hemes. Inspection of the 3D structure suggests that this inequivalence may arise from packing interactions of the Hb tetramer within the monoclinic crystal lattice. A similar inequivalence was found for the beta subunits of alpha(Ni2+)2beta(Fe2+)2 crystals. The average oxygen affinity of the alpha subunits (p50 = 91 Torr) is about 1.2-fold higher than the beta subunits (p50 = 110 Torr). In the absence of cooperativity, this heterogeneity yields an oxygen binding curve of Hb A with a Hill coefficient of 0.999. Since the binding curves of Hb A crystals exhibit a Hill coefficient very close to unity, these findings indicate that oxygen binding by T-state hemoglobin is noncooperative, in keeping with the Monod, Wyman, and Changeux model.

Crystallization↗
PubMed1998

The comparative potency of cholesterol crystallization-effector proteins in supersaturated model bile systems: association with vesicle transformation.

Various proteins which affect cholesterol crystallization are known to be present in bile, although the relative potency of their action is yet to be established. In this study, we evaluated the comparative potency of nucleating-effector proteins using a recently developed method for quantitative assessment of vesicle transformation in supersaturated model bile systems, to partially characterize mechanisms of their action. Concanavalin A-bound glycoproteins isolated from human gall-bladder bile shortened cholesterol crystallization time by 40% and increased cholesterol growth rate and final crystal mass by 161 and 19%, respectively, when compared to the control. In addition, immunoglobulins isolated from human gall-bladder bile increased cholesterol growth rate by 9%, but showed no significant effect on cholesterol crystallization time and final crystal mass. In contrast, human serum apolipoproteins A-I and B reduced cholesterol growth rate by 26 and 31% and reduced final crystal mass by 12 and 21%, but did not affect cholesterol crystallization time. Gel permeation chromatography revealed that proteins were distributed to both vesicles and bile salt micelles, but that no marked redistribution of lipids was caused by addition of these proteins. Furthermore, no significant difference in crystal structure was observed by video-enhanced contrast microscopy. These results indicate that nucleating-effector substances tested in this study may modulate vesicular cholesterol-holding capacity, thus affecting cholesterol crystallization. Such modulation is based upon the protein-vesicle association which defines the physico-chemical metastability of vesicular cholesterol.

Analysis of Variance↗
PubMed1992

In vitro inhibition of struvite crystal growth by acetohydroxamic acid.

Struvite (MgNH4PO46H2O) crystals were produced by Proteus mirabilis growth in artificial urine, in the presence and absence of the urease inhibitor, acetohydroxamic acid (AHA). In the absence of AHA, struvite crystals assumed an "X-shaped" or dendritic crystal habit due to rapid growth along their 100 axis. When AHA was present, crystal growth, as monitored by phase contrast light microscopy, was greatly slowed, and the crystals assumed an octahedral crystal habit. Scanning electron microscopy revealed that crystals grown in the presence of AHA were pitted on their surface. This pitting was absent in control samples. While most of this inhibition by AHA was due to lowered urease activity, some crystal growth inhibition occurred in struvite produced in the absence of urease activity through NH4OH titration of artificial urine. We conclude that while AHA is primarily a urease inhibitor, it may also disrupt struvite growth and formation directly through interference with the molecular growth processes on crystal surfaces.

Animals↗
PubMed1998

Control of calcium oxalate crystal structure and cell adherence by urinary macromolecules.

Crystal polymorphism is exhibited by calcium oxalates in nephrolithiasis, and we have proposed that a shift in the preferred crystalline form of calcium oxalate (CaOx) from monohydrate (COM) to dihydrate (COD) induced by urinary macromolecules reduces crystal attachment to epithelial cell surfaces, thus potentially inhibiting a critical step in the genesis of kidney stones. We have tested the validity of this hypothesis by studying both the binding of monohydrate and dihydrate crystals to renal tubule cells and the effect of macromolecular urinary solutes on crystal structure. Renal tubule cells grown in culture bound 50% more CaOx monohydrate than dihydrate crystals of comparable size. The effects of macromolecules on the spontaneous nucleation of CaOx were examined in HEPES-buffered saline solutions containing Ca2+ and C2O4(2-) at physiologic concentrations and supersaturation. Many naturally occurring macromolecules known to be inhibitors of crystallization, specifically osteopontin, nephrocalcin and urinary prothrombin fragment 1, were found to favor the formation of calcium oxalate dihydrate in this in vitro system, while other polymers did not affect CaOx crystal structure. Thus, the natural defense against nephrolithiasis may include impeding crystal attachment by an effect of macromolecular inhibitors on the preferred CaOx crystal structure that forms in urine.

Calcium Oxalate↗
PubMed2004

Tamm-Horsfall protein is a critical renal defense factor protecting against calcium oxalate crystal formation.

BACKGROUND: The tubular fluid of the mammalian kidney is often supersaturated with mineral salts, but crystallization rarely occurs under normal conditions. The unique ability of the kidney to avoid harmful crystal formation has long been attributed to the inhibitory activity of the urinary macromolecules, although few in vivo studies have been carried out to examine this hypothesis. Here we examined the role of Tamm-Horsfall protein (THP), the principal urinary protein, in urinary defense against renal calcium crystal formation, using a THP knockout model that we recently developed. METHODS: Wild-type and THP knockout mice were examined for the spontaneous formation of renal calcium crystals using von Kossa staining. The susceptibility of these mice to experimentally induced renal crystal formation was evaluated by administering mice with ethylene glycol, a precursor of oxalate, and vitamin D(3), which increases calcium absorption. Renal calcium crystals were visualized by von Kossa stain, dark field microscopy with polarized light and scanning electron microscopy. RESULTS: Inactivating the THP gene in mouse embryonic stem cells results in spontaneous formation of calcium crystals in adult kidneys. Excessive intake of calcium and oxalate, precursors of the most common type of human renal stones, dramatically increases both the frequency and the severity of renal calcium crystal formation in THP-deficient, but not in wild-type mice. Under high calcium/oxalate conditions, the absence of THP triggers a marked, adaptive induction in renal epithelial cells of osteopontin (OPN), a potent inhibitor of bone mineralization and vascular calcification. Thus, OPN may serve as an inducible inhibitor of calcium crystallization, whereas THP can serve as a constitutive and apparently more effective inhibitor. CONCLUSION: These results provide the first in vivo evidence that THP is a critical urinary defense factor and suggest that its deficiency could be an important contributing factor in human nephrolithiasis, a condition afflicting tens of millions of people in the world annually.

Animals↗