Crystals of glutamine synthetase from Escherichia coli.

Further details are given of crystals of glutamine synthetase prepared from Escherichia coli. Crystals of two kinds have been observed: (1) rhombic dodecahedra which correspond to the morphology of the crystals studied by Eisenberg et al. (1971) (and which were found by them to contain dodecamers), and (2) rhombohedra, reported here. Cell dimensions and packing considerations led to the consideration of two possible structures for the rhombohedral crystals. These we have called the “T = 7 structure” and the “B.C.C. structure”. The T = 7 structure would be related to that derived by Eisenberg and would contain dodecamers, but is inconsistent with our X-ray intensity data. The B.C.C. structure is considered more probable. It is built of cubic octomers or square tetramers. Electron micrographs of our glutamine synthetase preparations show a wide variety of aggregates, including dodecamers and tetramers. The unit cell dimensions of our crystals are $\text{a = 140 ± 2}\text{A}\text{̊}$, and $\text{c = 148 ± 2}\text{A}\text{̊}$. The Laue symmetry group is $\text{3}\text{̄}$m P3₁.     

Hydrophobic channels in crystals of an α-aminoisobutyric acid pentapeptide

The crystal structure of the pentapeptide p-toluene-sulfonyl-(α-aminoisobutyryl)₅-methyl ester (Tosyl-(Aib)₅-OMe) has been determined in the space group P$\text{I}$. Pentapeptide molecules are folded in the 3₁₀ helical conformation and packed together, so as to yield a hydrophobic channel with a minimim diameter of 5.2 Å. The channel contains crystallographically disordered material. This structure provides a model for channel formation by hydrophobic peptide aggregates and should prove useful in studies of alamethicin, suzukacillin and related Aib containing membrane channels. Triclinic (P$\text{I}$) crystals of the pentapeptide are obtained in the presence of LiClO₄ in aqueous methanol, whereas crystallization from methanol alone yields crystals in the space group Pbca. The conformations of the peptide in the two crystal forms are very similar and only the molecular packing is dramatically different.     

Tyrocidine A: crystal growth and preliminary x-ray diffraction data.

Tyrocidine A was crystallized from 2-methyl-2,4-pentanediol and water, or methanol, to yield crystals that are large enough for X-ray diffraction studies. Four crystals were examined; three were in equilibrium with mother liquor and the fourth was air-dried. They belong to the rhombohedral space group R32. Parameters of the hexagonal cell of the fully solvated crystals vary slightly and are approximately $\text{a = 34}\text{A}\text{?}\text{and}\text{c = 50}\text{A}\text{?}$. The asymmetric unit consists of one molecule of tyrocidine and several molecules of 2-methyl-2,4-pentanediol. Air-dried crystals appear to contain about half the number of solvent molecules. Three-dimensional X-ray diffraction data showing a maximum resolution of $\text{s = (1.7}\text{A}\text{?}\text{)}{}^{\mathrm{?1}}$ have been recorded.     

Letter: Crystallization and preliminary x-ray diffraction studies on tyrosyl-transfer RNA synthetase from Bacillus stearothermophilus

Conditions have been established for the crystallization of tyrosyl-transfer RNA synthetase from Bacillus stearothermophilus at room temperature. The crystals are extremely well-ordered, exhibiting diffraction spots out to at least 2.7 Å, and can be grown to a convenient size for X-ray crystallographic analysis. The crystals are trigonal with a space group P3₁21, the unit cell having dimensions of $\text{a = 64.4}\text{A}\text{?}$ and $\text{c = 238}\text{A}\text{?}$; the crystallographic asymmetric unit is probably one subunit of the dimeric (2 × 45,000, mol. wt) enzyme. The enzyme crystals are extremely stable and exhibit good resistance to radiation damage. This amino-acyl-tRNA synthetase appears to be amenable to complete structure determination by X-ray crystallography.     

Characterization and preliminary crystallographic data on the VL-related fragment of the human kI Bence Jones protein Wat

A “naturally occurring” human κI V_L dimer, designated Wat, has been isolated and crystallized. Protein Wat consists of two non-covalently bound monomers, each having a molecular weight of ~ 11,500. The monomer subunit is composed of an entire variable region light chain (V_L) domain closely homologous to that of the κI Bence Jones protein Roy (Hilschmann &; Craig, 1965) as evidenced from amino acid composition, tryptic peptide map, and sequence analysis. Immunochemical studies substantiated that protein Wat is of the κ chain subgroup κI and lacks the isotypic and allotypic antigenic determinants associated with the κ constant region light chain domain. Two types of crystals of V_L dimer Wat were obtained from ammonium sulfate or polyethylene glycol solutions. The type I crystals have unit cell dimensions of $\text{a = b = 82.6}\text{A}\text{?}\text{, c = 60.3}\text{A}\text{?}$, and the space group is hexagonal P6₂ or P6₄. The asymmetric unit consists of one V_L dimer; the fractional volume of unit cell occupied by solvent is 0.51. The unit cell dimensions of the type II crystals are $\text{a = b = 1,08.3}\text{A}\text{?}\text{, c = 108.8}\text{A}\text{?}$; the space group is hexagonal P6₁22 or P6₅22. Three variable domains constitute the asymmetric unit of the type II crystals; the fractional value of the solvent (0.52) is compatible with the value obtained for the type I crystals.     

Purification and preliminary crystallographic studies on azurin and cytochrome c' from Alcaligenes denitrificans and Alcaligenes sp. NCIB 11015.

Purification and crystallisation procedures are reported for azurin and cytochrome c′ from Alcaligenes denitrificans and Alcaligenes sp. NCIB 11015. The azurin crystals from A. denitrificans are suitable for high-resolution X-ray structure analysis. They are orthorhombic, space group C222₁ (with marked tetragonal pseudo-symmetry), cell dimensions $\text{a = 75.0}\text{A}\text{?}\text{, b = 74.1}\text{A}\text{?}\text{, c = 99.5}\text{A}\text{?}$, with two molecules per asymmetric unit. The cytochrome c′ crystals from both species are hexagonal, space group P6₁22 (or P6₅22), cell dimensions $\text{a = b = 54.7}\text{A}\text{?}\text{, c ~ 185}\text{A}\text{?}$, γ = 120 °, with one subunit (molecular weight 14,000) in the asymmetric unit.     

The transverse location of tryptophan residues in the purple membranes of Halobacterium halobium studied by fluorescence quenching and energy transfer

Fluorescence quenching by a series of spin-labelled fatty acids is used to map the transverse disposition of tryptophan residues in bacteriorhodopsin (the sole protein in the purple membranes of Halobacterium halobium). A new method of data analysis is employed which takes into account differences in the uptake of the quenchers into the membrane. Energy transfer from tryptophan to a set of $\text{n-(9-}\text{anthroyloxy)}$ fatty acids is used as a second technique to confirm the transverse map of tryptophan residues revealed by the quenching experiments. The relative efficiencies of quenching and energy transfer obtained experimentally are compared with those predicted on the basis of current models of bacteriorhodopsin structure. Most of the tryptophan fluorescence is located near the surface of the purple membrane. When the retinal chromophore of bacteriorhodopsin is removed, tryptophan residues deep in the membrane become fluorescent. These results indicate that the deeper residues transfer their energy to retinal in the native membrane. The retinal moiety is therefore located deep within the membrane rather than at the membrane surface.     

Crystallographic analysis of the phytoagglutinin from Abrus precatorius by X-ray diffraction and electron microscopy

The lectin from the seeds of Abrus precatorius has been crystallized and the crystals subjected to study by X-ray diffraction and electron microscopy. Three closely related crystal forms were obtained, of orthorhombic space group P2₁2₁2₁ with $\text{a = 138}\text{A}\text{?}$, $\text{b = 142}\text{A}\text{?}$, and $\text{c = 173}\text{A}\text{?}$, of tetragonal space group P4₁2₁2 with $\text{a = b = 136}\text{A}\text{?}$, $\text{c = 176}\text{A}\text{?}$, and a twinned intermediate of the first two. From electron microscopy and two-dimensional spatial filtering of electron micrographs of the crystals, the molecule appears to consist of four similar domains grouped in a roughly planar diamond-shaped arrangement having a local intramolecular dyad axis. The average diameter of the Abrus lectin molecule is 50 to 60 Å and the individual domains appear to have a diameter of about 25 Å.     

A preliminary crystallographic study of β-glucuronidase from rat preputial gland

Large single crystals of rat preputial gland β-glucuronidase have been grown by dialysis against 2-methyl-2,4-pentanediol. The crystals are tetragonal, space group P4₁2₁2 or P4₃2₁2 with cell dimensions $\text{a = 103.5}\text{A}\text{?}\text{and}\text{c = 279.8}\text{A}\text{?}$. Each asymmetric unit contains one half of a tetramer composed of subunits of 69,000 molecular weight.     

Protein rotation and chromophore orientation in reconstituted bacteriorhodopsin vesicles

Bacteriorhodopsin has been reconstituted into lipid vesicles with dipalmitoyl and dimyristoyls phosphatidylcholine. Circular dichroism (CD) measurements show that the proteins are in a monomeric state above the main lipid phase transition temperature (T_c), 41 and 23°C for dipalmitoyl and dimyristoyl phosphatidylcholine, respectively. Below T_c, the CD spectrum is the same as that found for the purple membrane. The latter result implies that the orientation of the chromophore at these temperatures is most likely the same as in the purple membrane ($\text{70° ± 5°}$ from the normal to the membrane plane).Transient dichroism measurements show that below T_c the proteins are immobile, while above this temperature protein rotation around an axis normal to the plane of the membrane is occurring. In addition, from the data the angle of the chromophore for the rotating proteins with respect to the rotational diffusion axis can be calculated. This angle is found to be $\text{30° ± 3°}$ and $\text{29° ± 4°}$ in dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, respectively. This is considerably smaller than the value of $\text{70° ± 5°}$ for the natural biomembrane. A reversible reorientation of the chromophore above and below the respective main T_c transition temperature could explain the change of angle observed provided that all the molecules rotate above T_c.     

Preliminary crystallographic study of macromomycin: The apoprotein of the antitumor antibiotic auromomycin

Macromomycin (M_r 12,000) is the apoprotein of the antitumor drug auromomycin, which inhibits DNA synthesis by causing single-strand breaks in DNA. Two orthorhombic crystal forms of macromomycin have been observed. The platelike crystals of one form belong to space group P2₁2₁2₁, with cell dimensions $\text{a = 48·92}\text{A}\text{?}\text{, b = 54·71}\text{A}\text{?}\text{, c = 103·31}\text{A}\text{?}\text{, Z = 8}$. The crystals of the second form are needle-shaped, and belong to space group P2₁2₁2, with cell dimensions $\text{a = 46·1}\text{A}\text{?}\text{, b = 54·4}\text{A}\text{?}\text{, c = 41·2}\text{A}\text{?}\text{, Z = 4}$. At this point in time, the platelike crystals appear the most suitable for continued crystallographic studies.     

Crystallization of alfalfa mosaic virus coat protein as a T = 1 aggregate

Reassembled alfalfa mosaic virus coat protein was partially digested with trypsin to remove the first 26 amino acids (Bol et al., 1974). These particles are empty icosahedral protein shells built with 60 alfalfa mosaic virus protein subunits. This aggregate has been crystallized in two different crystal forms, one of which diffracts X-rays to at least 3.4 Å resolution. The type I crystals (space group $\text{P6}{}_3\text{, a = 200}\text{A}\text{?}\text{, c = 314}\text{A}\text{?}$) contain two particles per cell separated by 195 Å with each sitting on a 3-fold axis. The type II crystals contain three particles per cell in space group P3₁or P3₂ ($\text{a = 201}\text{A}\text{?}\text{, c = 485}\text{A}\text{?}$). Other T = 1 viral particles have very similar diameters.     

Crystallographic characterization of a principal non-toxic lectin from seeds of Ricinus communis

Large hexagonal crystals of ricinus lectin, present as a major component in the seeds of Ricinus communis, have been obtained at 4 °C in the presence of polyethylene glycol 6000 by vapor diffusion against media containing acetic acid. The crystals are of space group P622, with hexagonal unit cell parameters $\text{a = b = 166}\text{A}\text{?}\text{and}\text{c = 341}\text{A}\text{?}$. The asymmetric unit contains two molecules of molecular weight 125,000. The crystals are extremely unstable, both to environmental changes and to X-irradiation, and have a solvent content of approximately 55% by volume.     

Crystallization of human α-lactalbumin

Crystals of human milk α-lactalbumin have been grown from ammonium sulfate solutions in the presence of calcium ions at 35 °C. They belong to space group P2₁2₁2, with unit cell dimensions: $\text{a = 33.6}\text{A}\text{?}\text{, b = 69.9}\text{A}\text{?}\text{, c = 47.3}\text{A}\text{?}$ and have a single molecule in the asymmetric unit. The crystals diffract X-rays strongly to Bragg spacings of 2 Å and appear suitable for X-ray structural analysis.     

Preliminary crystallographic data on buffalo beta-lactoglobulin.

Lactoglobulin isolated from Italian buffalo milk has been crystallized in two different forms. Crystals of type I have been grown at pH 3.5 and belong to space group P6₃; the unit cell dimensions are $\text{a = b = 67}\text{A}\text{?}\text{, c = 142}\text{A}\text{?}$, and accordingly the asymmetric unit contains one dimer of molecular weight 36,000. Type II crystals, grown at pH 8.5, have only one monomer per asymmetric unit; the space group is P3₁21 (or enantiomorph) and the unit cell edges are: $\text{a = b = 54.4}\text{A}\text{?}\text{, c = 113.2}\text{A}\text{?}$. The crystals of the trigonal form show low radiation damage and are suitable for a structural investigation.     

A preliminary crystallographic study of isocitrate dehydrogenase from Azotobacter vinelandii

Large single crystals of isocitrate dehydrogenase from Azotobacter vinelandii have been grown by vapor diffusion from ammonium sulfate and phosphate solutions. The crystals are tetragonal, space group P4₂2₁2 with cell dimensions $\text{a = 122.1}\text{A}\text{?}$, $\text{c = 163.9}\text{a}\text{?}$. There are two molecules of 80,000 molecular weight per asymmetric unit. Native data to 5.5 Å resolution have been collected on a diffractometer. A rotation function using data between 10 Å and 6 Å resolution indicates three possible orientations of the non-crystallographic 2-fold axis relating the two molecules.     

Heterogeneity of spacer lengths in circles of amplified ribosomal DNA of two insect species, Dytiscus marginalis and Acheta domesticus.

Crystals of subtilopeptidase A have been studied by means of X-ray diffraction. They are orthorhombic, space group P2₁2₁2₁ with $\text{a = 76 73}\text{A}\text{?}\text{, b = 55.35}\text{A}\text{?}\text{and}\text{c = 53.00}\text{A}\text{?}$. There is one molecule per asymmetric unit and the solvent content is 42%. The crystals diffract very well and appear suitable for detailed study of the three-dimensional structure of the enzyme.     

Crystallization of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli

Crystals of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli have been grown out of ammonium sulfate by the hanging drop method of vapor diffusion. The crystals belong to the hexagonal space group P6₁22 or P6₅22, with $\text{a = 124}\text{A}\text{?}\text{and}\text{c = 381}\text{A}\text{?}$, and diffract to 3.8 Å resolution.     

Preliminary crystallographic data for adenylosuccinate synthetase from rabbit skeletal muscle

Single crystals of adenylosuccinate synthetase, an essential component of purine biosynthesis, extracted from rabbit skeletal muscle were prepared as suitable specimens for X-ray structure analysis. The crystal belongs to the space group P4₃2₁2 or P4₁2₁2 with unit cell dimensions $\text{a = b = 71.2}\text{A}\text{?}\text{, c = 194.8}\text{A}\text{?}$. The asymmetric unit contains one protein molecule of 54,000 molecular weight.     

Crystallization and preliminary crystallographic data of rabbit uteroglobin

Uteroglobin is a steroid-binding protein of 15,800 molecular weight, composed of two chemically identical subunits which, in the oxidized form, are covalently linked by disulphide bridges. Large crystals have been grown from ammonium sulphate solutions by vapour diffusion as well as by equilibrium dialysis. The crystals are very stable under X-rays, diffract to at least 2.2 Å resolution and belong to space group P2₁. The unit cell, with dimensions $\text{a = 43.3}\text{A}\text{?}\text{, b = 31.1}\text{A}\text{?}\text{, c = 34.5}\text{A}\text{?}\text{, and}\text{β = 90.7 °}$, contains two dimeric molecules. The crystals exhibit a prominent pseudo symmetry corresponding to space group P2₁2₁2 which indicates that the two subunits should be structurally nearly identical.