Protein and virus crystal growth on international microgravity laboratory-2.

Two T = 1 and one T = 3 plant viruses, along with a protein, were crystallized in microgravity during the International Microgravity Laboratory-2 (IML-2) mission in July of 1994. The method used was liquid-liquid diffusion in the European Space Agency's Advanced Protein Crystallization Facility (APCF). Distinctive alterations in the habits of Turnip Yellow Mosaic Virus (TYMV) crystals and hexagonal canavalin crystals were observed. Crystals of cubic Satellite Tobacco Mosaic Virus (STMV) more than 30 times the volume of crystals grown in the laboratory were produced in microgravity. X-ray diffraction analysis demonstrated that both crystal forms of canavalin and the cubic STMV crystals diffracted to significantly higher resolution and had superior diffraction properties as judged by relative Wilson plots. It is postulated that the establishment of quasi-stable depletion zones around crystals growing in microgravity are responsible for self-regulated and more ordered growth.     

Preliminary crystallographic analysis of class 3 rat liver aldehyde dehydrogenase

NAD-linked aldehyde dehydrogenases (A1DH) (EC 1.2.1.3) catalyze the irreversible oxidation of a wide variety of aldehydes to their respective carboxylic acids. Crystals of a class 3 AIDH (from an Escherichia coli expression system) suitable for X-ray analysis have been obtained. These crystals, which can be grown to a size of 0.8 x 0.3 x 0.2 mm, diffract to 2.5 A resolution. Analysis of the diffraction pattern indicates that the crystals belong to the monoclinic space group P21, with cell parameters a = 65.11 A, b = 170.67 A, c = 47.15 A, and beta = 110.5 degrees. Assuming one dimer per asymmetric unit, the value Vm is calculated to be 2.45 and the solvent content of the crystal is estimated to be 50%. A self-rotation function study produced significant rotation peaks (58% of the origin) on the kappa = 180 section at psi = 90 degrees and phi = 71 degrees and 341 degrees, indicating that the pseudo-dimer axis is (or is very nearly) perpendicular to the b-axis.     

Protein crystal growth on board Shenzhou 3: a concerted effort improves crystal diffraction quality and facilitates structure determination

The crystallization of 16 proteins was carried out using 60 wells on board Shenzhou 3 in 2002. Although the mission was only 7 days, careful and concerted planning at all stages made it possible to obtain crystals of improved quality compared to their ground controls for some of the proteins. Significantly improved resolutions were obtained from diffracted crystals of 4 proteins. A complete data set from a space crystal of the PEP carboxykinase yielded significantly higher resolution (1.46A vs. 1.87A), I/sigma (22.4 vs. 15.5), and a lower average temperature factor (29.2A(2) vs. 42.9A(2)) than the best ground-based control crystal. The 3-D structure of the enzyme is well improved with significant ligand density. It has been postulated that the reduced convection and absence of macromolecule sedimentation under microgravity have advantages/benefits for protein crystal growth. Improvements in experimental design for protein crystal growth in microgravity are ongoing.     

Preliminary crystallographic study of glucose-6-phosphate dehydrogenase from rat liver

Crystals of D-glucose-6-phosphate: NADP+ oxidoreductase were obtained with the hanging drop, vapor diffusion and batch methods from ammonium sulfate-containing solutions. X-ray diffraction photographs indicate that the crystals belong to the orthorhombic space groups I222 or I2(1)2(1)2(1) with unit cell dimensions of a = 66.0 A, b = 140.8 A and c = 177.8 A. These data, together with results from sodium dodecyl sulfate/polyacrylamide gel electrophoresis and crystal density experiments, indicate that there is one 116,000 Mr dimer per asymmetric unit. The crystals diffract to at least 2.2 A and are suitable for X-ray crystallographic structure determination.     

Lessons from crystals grown in the Advanced Protein Crystallisation Facility for conventional crystallisation applied to structural biology

The crystallographic quality of protein crystals that were grown in microgravity has been compared to that of crystals that were grown in parallel on earth gravity under otherwise identical conditions. A goal of this comparison was to assess if a more accurate 3D-structure can be derived from crystallographic analysis of the former crystals. Therefore, the properties of crystals prepared with the Advanced Protein Crystallisation Facility (APCF) on earth and in orbit during the last decade were evaluated. A statistical analysis reveals that about half of the crystals produced under microgravity had a superior X-ray diffraction limit with respect of terrestrial controls. Eleven protein structures could be determined at previously unachieved resolutions using crystals obtained in the APCF. Microgravity induced features of the most relevant structures are reported. A second goal of this study was to identify the cause of the crystal quality enhancement useful for structure determination. No correlations between the effect of microgravity and other system-dependent parameters, such as isoelectric point or crystal solvent content, were found except the reduced convection during the crystallisation process. Thus, crystal growth under diffusive regime appears to be the key parameter explaining the beneficial effect of microgravity on crystal quality. The mimicry of these effects on earth in gels or in capillary tubes is discussed and the practical consequences for structural biology highlighted.     

On the isolation, characterisation, and crystallisation of a human Bence-Jones protein of kappa type

The isolation and purification of the human Bence-Jones protein Rei is described. Physico-chemical data are given characterising the protein. It is shown that the protein could be crystallised. It is intended to use the crystals for single crystal X-ray diffraction studies.     

Crystallization and preliminary X-ray investigation reveals that tumor necrosis factor is a compact trimer furnished with 3-fold symmetry

Crystals of recombinant human tumor necrosis factor produced by Escherichia coli have been obtained under different conditions. Crystals suitable for X-ray studies are produced by a vapor diffusion technique using sodium phosphate as both precipitant and buffer at pH 6.5. The crystals belong to the cubic space group, P2(1)3 with unit cell dimensions a = b = c = 95.7 A (1 A = 0.1 nm). Preliminary photography reveals that the crystals are moderately stable to X-rays and diffract to at least 3 A resolution. The diffraction data for native crystals have been collected on a diffractometer at 3 A resolution. Another crystal form, which appeared in a solution containing sodium phosphate at pH 8.0, has the trigonal space group P3 with unit cell dimensions a = b = 63.8 A and c = 54.4 A, and produces measurable reflections to a resolution of 3 A. Hexagonal crystals also have been obtained by the use of polyethylene glycol as precipitant in the range pH 7.6 to 8.0; however, the crystals are fragile and unstable to X-rays. Conservation of 3-fold symmetry in the different crystal forms obtained could reflect the ability of tumor necrosis factor molecules to form trimers in solution and probably the nature of binding of the molecules to cellular receptors.     

Structure of mistletoe lectin I from Viscum album in complex with the phytohormone zeatin

The crystal structure of mistletoe lectin I (ML-I) isolated from the European mistletoe Viscum album in complex with the most active phytohormone zeatin has been analyzed and refined to 2.54 A resolution. X-ray suitable crystals of ML-I were obtained by the counter-diffusion method using the Gel-Tube R crystallization kit (GT-R) onboard the Russian Service Module on the international space station ISS. High quality hexagonal bipyramidal crystals were grown during 3 months under microgravity conditions. Selected crystals were soaked in a saturated solution of zeatin and subsequently diffraction data were collected applying synchrotron radiation. A distinct F(o)-F(c) electron density has been found inside a binding pocket located in subunit B of ML-I and has been interpreted as a single zeatin molecule. The structure was refined to investigate the zeatin-ML-I interactions in detail. The results demonstrate the ability of mistletoe to protect itself from the host transpiration regulation by absorbing the most active host plant hormones as part of a defense mechanism.     

Crystal structure of a light-harvesting protein C-phycocyanin from Spirulina platensis

The crystal structure of C-phycocyanin, a light-harvesting phycobiliprotein from cyanobacteria (blue-green algae) Spirulina platensis has been solved by molecular replacement technique. The crystals belong to space group P2(1) with cell parameters a = 107.20, b = 115.40, c = 183.04 A; beta = 90.2 degrees. The structure has been refined to a crystallographic R factor of 19.2% (R(free) = 23.9%) using the X-ray diffraction data extending up to 2.2 A resolution. The asymmetric unit of the crystal cell consists of two (alphabeta)6-hexamers, each hexamer being the functional unit in the native antenna rod of cyanobacteria. The molecular structure resembles that of other reported C-phycocyanins. However, the unique form of aggregation of two (alphabeta)6-hexamers in the crystal asymmetric unit, suggests additional pathways of energy transfer in lateral direction between the adjacent hexamers involving beta155 phycocyanobilin chromophores.     

The molecular and crystal structure of dextrans: a combined electron and X-ray diffraction study. II. A low temperature, hydrated polymorph

The crystal and molecular structure of a dextran hydrate has been determined through combined electron and X-ray diffraction analysis, aided by stereochemical model refinement. A total of 65 hk0 electron diffraction intensities were measured on frozen single crystals held at the temperature of liquid nitrogen, to a resolution limit of 1.6 A. The X-ray intensities were measured from powder patterns recorded from collections of the single crystals. The structure crystallizes in a monoclinic unit cell with parameters a = 25.71 A, b = 10.21 A, c (chain axis) = 7.76 A and beta = 91.3 degrees. The space group is P2(1) with b axis unique. The unit cell contains six chains and eight water molecules, with three chains of the same polarity and four water molecules constituting the asymmetric unit. Along the chain direction the asymmetric unit is a dimer residue; however, the individual glucopyranose residues are very nearly related by a molecular 2-fold screw axis. The conformation of the chain is very similar to that in the anhydrous structure, but the chain packing differs in the two structures in that the rotational positions of the chains about the helix axes (the chain setting angles) are considerably different. The chains still pack in the form of sheets that are separated by water molecules. The difference in the chain setting angles between the anhydrous and hydrate structures corresponds to the angle between like unit cell axes observed in the diffraction diagrams recorded from hybrid crystals containing both polymorphs. Despite some beam damage effects, the structure was determined to a satisfactory degree of agreement, with the residuals R'(electron diffraction) = 0.258 and R(X-ray) = 0.127.     

Preparation of single crystals of a yolk lipoprotein

Single crystals of a yolk lipoprotein from two species of lamprey oocytes have been prepared and shown to belong to the monoclinic space group C2. The unit cell dimensions combined with molecular weight determinations support the idea that the lipoprotein is a symmetrical dimer centered on a crystallographic 2-fold symmetry axis. The lipid content is estimated to be about 15% (w/w) and the crystals are suitable for structural studies by single crystal X-ray analysis. The X-ray results are correlated with those obtained by electron diffraction.     

Crystal structure and morphology of poly(12-dodecalactone)

Poly(12-dodecalactone) (PDDL) crystals in the form of chain-folded lamellae were prepared by isothermal crystallization from a 1-hexanol solution. The lozenge-shaped crystals with and without spiral growth have been studied by transmission electron microscopy and atomic force microscopy. Wide-angle X-ray diffraction data, obtained from PDDL lamellae sedimented to form oriented mats and annealed solvent-cast film, were supplemented with morphological and structural data from electron microscopy. PDDL crystallizes as an orthorhombic form with a P2(1)2(1)2(1) space group and lattice constants of a = 0.746 +/- 0.001 nm, b = 0.500 +/- 0.001 nm, and c (chain axis) = 3.281 +/- 0.003 nm. There are two chains per unit cell, which existed in an antiparallel arrangement. The fiber repeat distance is appropriate for an all-trans backbone conformation for the straight stems. Molecular packing of this structure has been studied in detail, taking into account both diffraction data and energy calculations. The setting angles, with respect to the a axis, were +/-43 degrees for the corner and center chains according to intensity measurements and structure factor calculations. The optimized shift along the crystallographic c axis is 0.1c (0.328 nm). A final model was obtained to yield R = 0.180 with X-ray diffraction data and R = 0.162 with electron diffraction data. A brief comparison is also made with related polymer structures.     

The second space experiment of protein crystallization with domestic facilities

The second experiment of protein crystallization was performed on domestic re-entry satelliteFSW-2 in 1994-07. The results are superior to the ones of the first mission in 1992: 9 of 10 different proteins were crystallized in space, and 70% of the total 48 samples yielded single crystals. Besides hen egg-white lysozyme which grew high-quality crystals on the first mission, an acidic phospholipase A2(aPLA2) from snake venom and hemoglobin from Anser Indicus produced good-quality crystals suitable for X-ray diffraction analyses. The positive effect of microgravity on protein crystal growth is verified again at this time.     

Crystallization and preliminary X-ray data of bromoperoxidase from Streptomyces aureofaciens ATCC 10762

Bromoperoxidase from Streptomyces aureofaciens ATCC 10762, a non-haem haloperoxidase, has been crystallized using the hanging drop method. Preliminary X-ray diffraction studies show that the crystals belong to the cubic space group P2(1)3 with a = 123.4 A. The asymmetric unit contains a dimer of Mr = 60,200. The crystals diffract to at least 2.3 A resolution and are suitable for crystallographic structure analysis.     

Crystal and molecular structure of cyclo(L-prolyl-glycyl)3. A cyclic hexapeptide with a cis peptide bond

The crystal structure of cyclo(L-Pro-Gly)3 was solved using X-ray crystallographic techniques. The backbone of the peptide is asymmetric and is made up of five trans peptide units and one cis peptide. There is a hydrogen bonded water bridge that links the carbonyl oxygens, O1 and O4. The molecules exist as dimers in the crystal lattice. The two molecules of the dimer are related by crystallographic twofold symmetry and are linked by two N-H ... O hydrogen bonds. The crystals are trigonal, space group P3(2)12 with a = 11.379(3), c = 32.93(1) and z = 6. The structure was solved by multisolution methods and refined by least squares technique to an R of 0.083.     

Crystallization of yeast orotidine 5'-monophosphate decarboxylase complexed with 1-(5'-phospho-beta-D-ribofuranosyl) barbituric acid

Using an incomplete factorial experimental design, we have identified conditions for crystallization of yeast orotidine 5'-monophosphate decarboxylase (ODCase) in an unliganded state and complexed separately to two inhibitors: 6-azauridine 5'-monophosphate (aza-UMP) and 1-(5'-phospho-beta-D-ribofuranosyl) barbituric acid (BMP). Crystals of X-ray diffraction quality have been obtained of yeast ODCase complexed with BMP, a putative transition state analog inhibitor (Ki = 8.8 x 10(-12) M). ODCase:BMP complex crystals with a hexagonal rod habit were grown from a solution initially containing 12 mg/ml ODCase (205 microM dimer) plus 450 microM BMP by microdialysis at 4 degrees C against a mother liquor which consisted of 0.1 M Na-PIPES-acetate (pH 6.4), 37.5 microM BMP, 5 mM mercaptoethanol, 1% polyethylene glycol 400, and 2.3 M ammonium sulfate. Crystals were analyzed using precession photography and were assigned to trigonal space group R32 with unit cell dimensions a = b = 115 A, c = 385 A. The crystal density is 1.245 g/cm3 indicating the presence of two ODCase: BMP complex dimers (118 kDa each) per asymmetric unit with a packing density of 2.08 A3/Da and 41% solvent content. The morphological habit of crystals of the ODCase:BMP complex changed when the initial ammonium sulfate concentration was increased in 0.05 M steps from 2.3 to 2.45 M. All of these crystals diffracted to at least 3.0 A resolution over a period of several weeks at room temperature and are isomorphous.     

Crystallization and preliminary X-ray diffraction studies of a flavoprotein, FP390, from a luminescent bacterium, Photobacterium phosphoreum.

A flavoprotein, FP390, obtained from a luminescent bacterium, Photobacterium phosphoreum, in the purification of luciferase has been crystallized by the vapor-diffusion procedure. Crystals obtained from polyethylene glycol 4000 solutions, whose X-ray photographs show powder diffraction patterns, were unsuitable for further crystallographic work. However, tetragonal crystals grown from potassium phosphate solution well diffracted X-rays beyond 3 A resolution. The space group of this crystal is P4(1)22 or P4(3)22 with unit-cell dimensions of a = b = 76.8 and c = 241 A. Assuming two or three molecules in an asymmetric unit, the value for the crystal volume per unit molecular mass, Vm, is calculated as 3.3 or 2.2 A3/Da, respectively. A total of 13,555 independent reflections for the native crystal was collected up to 3 A resolution using a Weissenberg camera attached to the synchrotron radiation source, the merging R factor being 0.077 for 79,335 measurements.     

Crystallization of a type III chloramphenicol acetyl transferase

A type III variant of chloramphenicol acetyl transferase was purified from Escherichia coli and crystallized in the presence of cobaltic hexamine chloride and 2-methyl-2, 4-pentandiol. Two crystal forms were obtained, one of which proved to be suitable for high-resolution X-ray diffraction studies. The space group of this form is R32 with aR = 74.5 A, alpha R = 92.5 degrees, with a monomer (Mr 25,000) in the asymmetric unit. The crystals diffract to 1.7 A resolution. The crystal symmetry has led to a re-evaluation of the oligomeric symmetry of the enzyme and the proposal that it is a trimer rather than a tetramer, the quaternary structure predicted previously from studies of the association of hybrid s subunits.     

Assembly of Acanthamoeba myosin-II minifilaments. Model of anti-parallel dimers based on EM and X-ray diffraction of 2D and 3D crystals

Current models suggest that the first step in the assembly of Acanthamoeba myosin-II is anti-parallel dimerization of the coiled-coil tails with an overlap of 15 nm. Sedimentation equilibrium experiments showed that a construct containing the last 15 heptads and the non-helical tailpiece of the myosin-II tail (15T) forms dimers. To examine the structure of the 15T dimer, we grew 3D and 2D crystals suitable for X-ray diffraction and electron image analysis, respectively. For both conditions, crystals formed in related space and plane groups with similar unit cells (a=87.7 A, b=64.8 A, c=114.9 A, beta=108.0 degrees). Inspection of the X-ray diffraction pattern and molecular replacement analysis revealed the orientation of the coiled-coils in the unit cell. A 3D density map at 15A in-plane resolution derived from a tilt series of electron micrographs established the solvent content of the 3D crystals (75%, v/v), placed the coiled-coil molecules at the approximate translation in the unit cell, and revealed the symmetry relationships between molecules. On the basis of the low-resolution 3D structure, biochemical constraints, and X-ray diffraction data, we propose a model for myosin interactions in the anti-parallel dimer of coiled-coils that guide the first step of myosin-II assembly.     

Preliminary crystallographic study of a pseudoazurin from methylotrophic bacterium, Methylobacterium extorquens AM1

Single crystals of pseudoazurin, one of the blue copper proteins produced by methylotrophic bacterium Methylobacterium extorquens AM1, have been obtained by the method of vapor diffusion with ammonium sulfate as a precipitant at pH 8.0. Crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit cell dimensions of a = 52.619(4) A, b = 63.280(6) A, c = 35.133(4) A. The asymmetric unit includes one molecule of pseudoazurin (Vm = 2.18 A3/dalton). The crystals are so stable against X-ray irradiation that diffraction intensities of the native crystal up to 1.68 A resolution could be collected from only one crystal. Among the many heavy-metal reagents examined, uranyl acetate gave an effective isomorphous derivative.