Crystallization and preliminary X-ray analysis of electron transfer flavoproteins from human and Paracoccus denitrificans.

Mammalian electron transfer flavoprotein (ETF) is a soluble, heterodimeric flavoprotein responsible for the oxidation of at least nine primary matrix flavoprotein dehydrogenases. Crystals have been obtained for the recombinant human electron transfer flavoprotein (ETFhum) by the sitting-drop vapor diffusion technique using polyethylene glycol (PEG) 1500 at pH 7.0 as the precipitating agent. ETFhum crystallizes in the monoclinic space group P2(1), with unit cell parameters a = 47.46 angstrum, b = 104.10 angstrum, c = 63.79 angstrum, and beta = 110.02 degrees. Based on the assumption of one alpha beta dimer per asymmetric unit, the Vm value is 2.69 angstrum 3/Da. A native data set has been collected to 2.1 angstrum resolution. One heavy-atom derivative has also been obtained by soaking a preformed crystal of ETFhum in 2 mM thimerosal solution for 2h at 19 degrees C. Patterson analysis indicates one major site. The analogous electron transfer flavoprotein from Paracoccus denitrificans (ETFpar) has also been crystallized using PEG 8000 at pH 5.5 as the precipitating agent. ETFpar crystallizes in the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters a = 79.98 angstrum, b = 182.90 angstrum, and c = 70.07 angstrum. The Vm value of 2.33 angstrum 3/Da is consistent with two alpha beta dimers per asymmetric unit. A native data set has been collected to 2.5 angstrum resolution.     

Preliminary crystallographic analysis of the ATP-hydrolysing domain of the Escherichia coli DNA gyrase B protein.

The 43 kDa N-terminal ATPase domain of the Escherichia coli DNA gyrase B protein has been purified from an over-expressing strain. This protein has been crystallized in two crystal forms, both in the presence of the non-hydrolysable ATP analogue 5'-adenylyl-beta,gamma-imidodiphosphate. The first crystal form is monoclinic P2(1), with cell dimensions a = 76 A, b = 88 A, c = 82 A, beta = 105.5 degrees, and diffracts to at least 2.7 A resolution using synchrotron radiation. Crystal density measurements suggest that there are two molecules in the asymmetric unit (Vm = 3.08 A3/Da). The second crystal form is orthorhombic C222(1), with cell dimensions a = 89.2 A, b = 143.1 A and c = 79.8 A. The crystals diffract to beyond 3 A and are stable for at least 100 hours when exposed to X-rays from a rotating anode source. The asymmetric unit of this crystal form appears to contain one molecule (Vm = 2.96 A3/Da). Data have already been collected to 5 A resolution from native crystals of this second form, and to 6 A resolution from three heavy-atom derivatives. Electron density maps calculated using phases obtained from these derivatives show features consistent with secondary structural elements, and have allowed the molecular boundary to be determined. Higher resolution native and derivative data are being collected.     

Crystallization and preliminary X-ray study of pig lens aldose reductase

Crystals of pig lens aldose reductase have been grown from polyethylene glycol solutions at pH 6.2 and analysed by X-ray diffraction. Two crystal forms were obtained. The first belongs to space group P1 with unit cell dimensions a = 81.3 A, b = 85.9 A, c = 56.6 A, alpha = 102.3 degrees, beta = 103.3 degrees, gamma = 79.0 degrees, with four molecules in the unit cell related by a 222 non-crystallographic symmetry. The second crystal form is hexagonal. The space group is P6(2)22 with a = b = 101 A, c = 257 A and two molecules in the asymmetric unit. Both forms are suitable for X-ray structure analysis to better than 3 A resolution.     

Crystallization and preliminary diffraction studies of recombinant human granulocyte-stimulating factor (KW2228).

Human granulocyte colony-stimulating factor (hG-CSF) specifically stimulates proliferation of neutrophils. Two crystal forms of a mutant of hG-CSF expressed in Escherichia coli have been obtained using the hanging drop vapour diffusion method. One form is triclinic, space group P1, with cell dimensions a = 37.3 A, b = 46.4 A, c = 47.7 A, alpha = 105.5 degrees, beta = 98.0 degrees and gamma = 109.4 degrees. The other is monoclinic, space group C2, with cell dimensions a = 82.0 A, b = 49.2 A, c = 49.4 A and beta = 113.9 degrees. Both crystal forms diffract beyond 2.0 A and are suitable for X-ray analysis.     

Crystallization and preliminary diffraction studies of hydroxypyruvate reductase (D-glycerate dehydrogenase) from Hyphomicrobium methylovorum.

Two crystal forms of hydroxypyruvate reductase (D-glycerate dehydrogenase) from the methylotrophic bacterium Hyphomicrobium methylovorum have been grown from ammonium sulphate solutions. One crystal form is triclinic, with unit cell parameters a = 60.4 A, b = 60.5 A, c = 66.3 A, alpha = 102.3 degrees, beta = 113.7 degrees and gamma = 102.7 degrees, suggesting that a dimer (monomer M(r) 38,000) occupies the unit cell. This crystal form diffracts to beyond 2.4 A resolution and is suitable for crystallographic structure analysis.     

Crystallization and preliminary diffraction analysis of cholesterol esterase from Candida cylindracea

Cholesterol esterase (EC 3.1.1.13) from the microorganism Candida cylindracea has been crystallized in two forms. Crystals, typically 0.30 x 0.15 x 0.10 mm in size, diffract rotating anode generated x-rays to beyond 3 A are suitable for data collection for an x-ray crystallographic investigation. A monoclinic crystal form in the space group P2(1) was found to have cell dimensions of a = 122.9 A, b = 101.0 A, c = 95.2 A and beta = 108.3 degrees. The asymmetric unit of the cell contains two dimers of 129 kDa each. A second crystal form, in the triclinic space group P1, has cell dimensions of a = 58.6 A, b = 88.7 A, c = 58.6 A, alpha = 93.3 degrees, beta = 113.8 degrees and gamma = 96.0 degrees, and has one dimer per asymmetric unit.     

Supramol--a program for structure analysis of intercalates using molecular simulations: the structure of VOPO4*C6H4O2

A method of structure analysis of intercalates has been developed that uses a combination of molecular simulations with powder diffraction. The program Supramol for the determination of intercalated structures uses crystal energy minimization in conjunction with powder diffraction data.The program solves the multiple minima problem in molecular mechanics, generating initial models systematically and searching for the global energy minimum by comparing the experimental and calculated diffraction patterns. The program is compatible with the Cerius2 modeling environment.Two intercalated crystal structures solved by Supramol are presented in the present paper: vanadyl phosphate intercalated with p-benzoquinone and the high temperature phase of vanadyl phosphate intercalated with dioxane. The structure of vanadyl phosphate intercalated with p-benzochinone is tetragonal, space group I4/ m, the unit cell parameters a=6.21 A, b=6.21 A, c=20.18 A and the density is rho=2.30 g x cm(-3), Z=4. The crystal structure of vanadyl phosphate intercalated with dioxane (high temperature phase) is monoclinic, space group C2/ m, unit cell parameters are: a= b=8.94 A, c=8.22 A, alpha=gamma=90 degrees, beta=106.30 degrees, Z=4, density 2.248 g x cm(-3).     

Crystallization and preliminary crystallographic analysis of carboxypeptidase G2 from Pseudomonas sp. strain RS-16

Carboxypeptidase G2, a zinc metalloenzyme isolated from Pseudomonas sp. strain RS-16, which catalyses the hydrolytic cleavage of reduced and non-reduced folates to pteroates and L-glutamate, has been crystallized from polyethylene glycol (average Mr 4000) by vapour diffusion. The crystal symmetry is monoclinic C2, with unit cell dimensions a = 206 A, b = 82 A, c = 116 A and beta = 118 degrees. The molecular mass and volume of the unit cell suggest that there are two dimers of the enzyme in the asymmetric unit. The crystals diffract to at least 3.0 A and are suitable for X-ray structure analysis.     

Crystallization and preliminary X-ray studies of the Ia component of Clostridium perfringens iota toxin complexed with NADPH.

A recombinant Ia component of Clostridium perfringens iota toxin, which ADP-ribosylates actin, was crystallized by the hanging drop vapor diffusion method using PEG4000 as a precipitating agent. The crystals were obtained in the presence of NADPH, which is similar to a real substrate, NADH, and belongs to the space group P1 (a = 47.9 A, b = 54.5 A, c = 103.1 A, alpha = 99.0 degrees, beta = 93.3 degrees, and gamma = 107.2 degrees ). The Matthews coefficient of native crystal was 2.7, assuming 2 mol/asymmetric unit. Native data were collected at 2.4-A resolution. The results from a heavy-atom search showed that lanthanide ions (samarium, holmium) altered the molecular packing, judging from the unit-cell difference. The crystals also belonged to the space group P1 (a = 47.7 A, b = 53.9 A, c = 54.6 A, alpha = 68.9 degrees, beta = 78.3 degrees, and gamma = 73.7 degrees ), which is consistent with only one molecule per asymmetric unit.     

Crystallization of chicken egg white cystatin, a low molecular weight protein inhibitor of cysteine proteinases, and preliminary X-ray diffraction data

The shorter-chain form of chicken egg white cystatin has been crystallized in 1.6 M-phosphate buffer at pH 4.0 by vapour diffusion. The crystals are of trigonal space group P3121 (or P3221), have cell constants a = b = 47.9 A, c = 87.5 A, alpha = beta = 90 degrees, gamma = 120 degrees, and contain one molecule of 12,191 molecular weight per asymmetric unit. They diffract well to about 2.0 A resolution and are suitable for X-ray crystal structure analysis.     

Crystallization of isoelectrically homogeneous cholera toxin

Past difficulty in growing good crystals of cholera toxin has prevented the study of the crystal structure of this important protein. We have determined that failure of cholera toxin to crystallize well has been due to its heterogeneity. We have now succeeded in overcoming the problem by isolating a single isoelectric variant of this oligomeric protein (one A subunit and five B subunits). Cholera toxin purified by our procedure readily forms large single crystals. The crystal form (space group P2(1), a = 73.0 A, b = 92.2 A, c = 60.6 A, beta = 106.4 degrees, one molecule in the asymmetric unit) has been described previously [Sigler et al. (1977) Science (Washington, D.C.) 197, 1277-1278]. We have recorded data from native crystals of cholera toxin to 3.0-A resolution with our electronic area detectors. With these data, we have found the orientation of a 5-fold symmetry axis within these crystals, perpendicular to the screw dyad of the crystal. We are now determining the crystal structure of cholera toxin by a combination of multiple heavy-atom isomorphous replacement and density modification techniques, making use of rotational 5-fold averaging of the B subunits.     

Crystallization of a ternary complex of lactate dehydrogenase from Bacillus stearothermophilus

Bacillus stearothermophilus lactate dehydrogenase was purified from an overexpressing Escherichia coli cell line. The enzyme has been crystallized in several different forms. All of these crystal forms were grown in the presence of NADH, sodium oxamate and fructose 1,6-bisphosphate. Three crystal forms have been characterized, an orthorhombic P2(1)2(1)2 (type III, a = 86 A, b = 105 A, c = 136 A) and two monoclinic P21 forms (type IV, a = 85 A, b = 118 A, c = 136 A, beta = 96 degrees; type V, a = 112 A, b = 85 A, c = 136 A, beta = 91 degrees). Precession photographs from these crystal forms are very alike, suggesting the molecular packing to be similar in all three forms. The P21 type IV crystals diffract to beyond 2 A spacing and are stable to irradiation with X-rays. A complete medium-resolution (4.7 A) dataset has been collected from a single crystal using synchrotron radiation. Rotation function studies with these data show the two tetramers of the asymmetric unit to be in very similar orientations. Higher-resolution data are being collected.     

Crystallization of diphtheria toxin.

Two new crystal forms (forms III and IV) have been grown of diphtheria toxin (DT), which kills susceptible cells by catalyzing the ADP-ribosylation of elongation factor 2, thereby stopping protein synthesis. Forms III and IV diffract to 2.3 A and 2.7 A resolution, respectively. Both forms belong to space group C2; the unit cell parameters for form III are a = 107.3 A, b = 91.7 A, c = 66.3 A and beta = 94.7 degrees and those for form IV are a = 108.3 A, b = 92.3 A, c = 66.1 A and beta = 90.4 degrees. Both forms have one protein chain per asymmetric unit with the dimeric molecule on a twofold axis of symmetry. Form IV is exceptional among all crystal forms of DT in that it can be grown reproducibly. Thus the form IV crystals should yield a crystallographic structure giving insight into the catalytic, receptor-binding and membrane-insertion properties of DT.     

Human plasminogen kringle 4. Crystallization and preliminary diffraction data of two different crystal forms

Human plasminogen kringle 4 has been crystallized in two different crystal forms: monoclinic, a = 32.78(3), b = 49.17(2), c = 46.27(3) A, beta = 100.67 degrees, space group P2(1), four molecules/unit cell, two molecules/asymmetric unit; orthorhombic, a = 32.09(7), b = 49.14(6), c = 49.47(9) A, space group P2(1)2(1)2, four molecules/unit cell. Both crystal forms have a large protein fraction (66% for monoclinic and 62% for orthorhombic) and diffract x-rays to 2.0 A resolution. A self-rotation function has been calculated with monoclinic data indicating a non-crystallographic 2-fold rotation approximately parallel to a* (peak height of 14.3 x sigma). Cross-rotation function calculations are in progress utilizing the coordinates of the conserved structure of kringle 1 of prothrombin and plasminogen kringle 4.     

Crystal structure of an anti-Lewis alpha Fab determined by molecular replacement methods

The anti-Lewis alpha mouse immunoglobulin CF4C4 (IgGl, k) Fab has been crystallized from 58% saturated ammonium sulfate in space group Pl; unit cell dimensions a = 43.4 A b = 41.7 A, c = 62.0 A, a = 72.7 degrees, beta = 96.6 degrees, gamma = 100.1 degrees. X-ray diffraction data have been measured beyond 3.0 A Bragg spacing. The crystal structure has been determined by molecular replacement methods, using as search models the constant and variable domains of the mouse immunoglobulin McPC603 (IgA, kappa) Fab. The crystallographic residual for the data 5.0 to 4.0 A, is 0.47. The approximate 2-fold axis relating the VL and the VH domains forms an angle of 164 degrees with the 2-fold axis relating the constant domains. The crystal packing is reasonable.     

Crystallization of human leukocyte elastase with its inhibitor Pro44-eglin c

Human leukocyte elastase has been crystallized in complex with recombinant Pro44-eglin c in the orthorhombic space group P2(1)2(1)2(1). The cell constants are a = 126.1 A, b = 127.8 A, c = 69.4 A, alpha = beta = gamma = 90 degrees. The crystals diffract to at least 2.5 A resolution and are suitable for crystallographic structure analysis.     

Crystals of the complex between recombinant N-acetyleglin c and subtilisin. A preliminary characterization

The complex between recombinant N-acetyleglin c and subtilisin has been crystallized into a triclinic cell. The cell constants are a = 38.3 A, b = 41.5 A, c = 56.6 A, alpha = 110.6 degrees, beta = 83.8 degrees, and gamma = 104.7 degrees. The crystals diffract to better than 2.3-A resolution and are large enough for data collection. The crystallization conditions and general crystal data are presented.     

Crystallization and preliminary X-ray crystallographic analysis of Ace: a collagen-binding MSCRAMM from Enterococcus faecalis

Ace is a collagen-binding bacterial cell surface adhesin from Enterococcus faecalis. The collagen-binding domain of Ace (termed Ace40) and its truncated form Ace19 have been crystallized by the vapor-diffusion hanging-drop method. Ace19 was crystallized in two different crystal forms. A complete 1.65 A data set has been collected on the orthorhombic crystal form with unit cell parameters a=38.43 b=48.91 and c=83.73 A. Ace40 was crystallized in the trigonal space group P3(1)21 or P3(2)21 with unit cell parameters a=b=80.24, c=105.91 A; alpha=beta=90 and gamma=120 degrees. A full set of X-ray diffraction data was collected to 2.5 A. Three heavy atom derivative data sets have been successfully obtained for Ace19 crystals and structural analysis is in progress.     

Crystallization and preliminary X-ray analysis of flavocytochrome c(3), the fumarate reductase from Shewanella frigidimarina.

The fumarate reductase (flavocytochrome c(3)) from Shewanella frigidimarina (formerly S. putrefaciens) NCIMB400 has been crystallized in the space group P2(1), with cell dimensions of a = 45.447 A, b = 92.107 A, c = 78.311 A, and beta = 91.038 degrees and one molecule per asymmetric unit. A native data set has been collected to 1.8 A. The gene encoding Fcc(3) from the S. frigidimarina type strain ACAM591 has been cloned and sequenced and the protein crystallized in space group P2(1) with cell dimensions of a = 45.359 A, b = 88.051 A, c = 77.473 A, and beta = 104.499 degrees. Anomalous data have also been collected from the NCIMB400 crystal allowing the heme iron positions to be identified.     

Crystallization and preliminary x-ray diffraction studies of recombinant human interleukin-1 beta

Recombinant human interleukin-1 beta has been crystallized into a tetragonal cell. The unit cell constants are a = b = 54.9 A, c = 76.8 A, and alpha = beta = gamma = 90 degrees. The crystals diffract to better than 1.9 A and are suitable for high resolution data collection. The crystallization conditions and general crystal data are presented.