Expression,crystallization and preliminary X-ray diffraction study of FtsY,the docking protein of the signal recognition particle of E. coli

FtsY is the docking protein or SRα homologue in E. coli. It is involved in targeting secretory proteins to the cytoplasmic membrane by interacting with the signal recognition particle, controlled by guanosine 5′-triphosphate. Two different constructs have been used in crystallization studies: the full-length protein and a truncated fragment with a his-tag at the C terminus. Only the second construct resulted in crystals suitable for x-ray diffraction. The crystals belong to the monoclinic space group P2₁ with cell dimensions a = 32.20 Å, b = 79.57 Å, c = 59.21 Å, and β = 94.45, and contain one molecule per asymmetric unit. At cryogenic temperatures the crystals diffract to a resolution limit of 2.5 Å by using a rotating anode, and beyond 1.8 Å by using synchrotron radiation. Proteins 28:285–288, 1997. © 1997 Wiley-Liss Inc.     

Crystallization and preliminary crystallographic analysis of ribonuclease H from Thermus thermophilus HB8

Ribonuclease H from an extreme thermophile, Thermus thermophilus HB8, has been crystallized from solutions at low ionic strength. The crystals belong to the hexagonal space group P6 ₁22 (or P6 ₅22), with unit cell parameters a = b = 44.7 Å, c = 314.7 Å. They contain one 18,000 Mr molecule per asymmetric unit and diffract to 2.8 Å resolution. © 1993 Wiley-Liss, Inc.     

Crystallization and preliminary crystallographic analysis of the N-terminal actin binding domain of human fimbrin

We have crystallized the N-terminal actin binding domain (ABD1) of human fimbrin, a representative member of the largest class of actin crosslinking proteins. Diffraction from these crystals is consistent with the orthorhombic space group P2₁2₁2₁ (a = 50.03 Å, b = 61.24 Å, c = 102.30 Å). These crystals contain one molecule in the asymmetric unit and diffract to at least 1.9 Å resolution. The crystal structure of ABD1 will be the first structure of an actin crosslinking domain. Proteins 28:452–453, 1997. © 1997 Wiley-Liss, Inc.     

Time resolved spectroscopy of the tryptophyl fluorescence of the E. coli LAC repressor.

A new crystal form of a mitogenic lectin from pea seeds ($\text{Pisum sativum}$) has been obtained which is suitable for high resolution structural work. The crystals are orthorhombic, space group P2₁2₁2₁, with unit cell dimensions: $\text{a}$ = 64.2Å, $\text{b}$ = 72. 7Å, $\text{c}$ = 108. 3Å. The asymmetric unit contains one protein molecule.     

Growth and analysis of crystal forms of toxic shock syndrome toxin 1

Native toxic shock syndrom toxin 1 (TSST-1) purified from Staphylococcus aurius has been crystallized in four different forms. The highest resolution data (2.05 Å) was collected from orthorhombic crystals belonging to the space group C222₁. The unit cell dimension are a = 108.7 Å, b = 177.5 Å, c = 97.6 Å. Rotation function analysis of this from indicates that there is trimer of toxin molecules in the asymmetric unit with a local 3-fold axis parallel to the crystallographic c axis. Crystals of a double mutant of TSST-1 have been grown which has a single molecule in the asymmetric unit and diffract to 1.9 Å. The space group is P2₁ with unit cell parameters of a = 44.4 Å, b = 34.0 Å, c = 55.2 Å, β = 93.0°. © 1993 Wiley-Liss, Inc.     

Crystallization of intact monoclonal antibodies

Attempts were made to crystallize four monoclonal antibodies, one IgG_2ak and three IgG_1k. Using a PEG 3350 screen combined with detergents, and developed from our experiments with an IgG_2ak antibody specific for canine lymphoma cells,^1,2 crystals have now been obtained of two of these four immunoglobulins, an antiphenytoin and an antiphenobarbital antibody. A complex between the antiphenobarbital antibody and its drug antigen crystallized as well. The antibody for phenytoin has, to this point, produced only clustered microcrystals, marginally suitable for X-ray analysis. Single crystals of the IgG_1k antibody against phenobarbital, however, were characterized by X-ray diffraction to be primitive monoclinic, with unit cell dimensions a = 67 Å, b = 193 Å, c = 74 Å, and β = 110°. These crystals have an entire IgG_1k molecule as the asymmetric unit and they diffract to at least 3.2 Å resolution. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary x-ray diffraction studies of a monoclonal antibody fab fragment against foot-and-mouth disease virus and of its complex with the main antigenic site peptide

The Fab fragment of the neutralizing monoclonal antibody SD6 elicited against foot-and-mouth disease virus (FMDV) C-SBcl and its complex with a peptide, corresponding to the major antigenic site of FMDV (VPl residues 136–150, YTASARGDLAHLTTT), have been crystallized using the hanging drop vapor diffusion techniques. For the isolated Fab, crystals diffracting to 2.5 Å resolution were obtained at room temperature using ammonium sulfate as precipitant. These crystals are monoclinic, space group C2, and unit cell parameters a = 109.53 Å, b = 89.12 Å, c = 64.04 Å, and β = 112.9° and contain one Fab molecule per asymmetric unit. Crystals from the complex diffract, at least, to 2.8 Å resolution and were obtained, at room temperature, using PEG as precipitant. These crystals are monoclinic, space group P2, and unit cell parameters a = 56.11 Å, b = 60.67 Å, c = 143.45 Å, and β = 95.4°, Density packing considerations indicate that there are two Fab molecules in the asymmetric unit. © 1994 John Wiley & Sons, Inc.     

Crystallization and preliminary X-ray diffraction studies of peroxidase from a fungus Arthromyces ramosus

Peroxidase (donor: H₂O₂ oxi-doreductase [EC 1.11.1.7]) was purified from the culture broth of the hyphomycete Arthromyces ramosus in the early log phase to show a single band on SDS-PAGE. The crystals of A. ramosus peroxidase (ARP) were formed by salting out with ammonium sulfate at room temperature and pH 7.5. The repeated seeding technique was employed to grow the crystals to the size large enough for X-ray diffraction study. The crystals were characterized as tetragonal, space group P4₂2₁2, with unit cell dimensions of a = b = 74.5 Å, c = 117.6 Å. The asymmetric unit contains one molecule of peroxidase. They diffract X-rays to at least 2.0 Å resolution and are stable to X-rays. © 1993 Wiley-Liss, Inc.     

Crystallization and preliminary cryogenic X-ray diffraction analyses of protein L-isoaspartyl O-methyltransferase from human fetal brain

Recombinant human fetal brain protein L-isoaspartyl O-methyltransferase, EC 2.1.1.77, was crystallized in PEG 8000 with adenosine homocysteine by a macroseeding technique. The space group was P2₁ with a = 47.4 Å, b = 53.9 Å, c = 48.7 Å and β = 116.4° for cryofrozen crystals at 90 K. The crystals diffracted to 2.1 Å and have one molecule per asymmetric unit. Proteins 28:457–460, 1997. © 1997 Wiley-Liss, Inc.     

Purification,crystallization, and preliminary X-ray studies on the rhizome lectin from stinging nettle and its complex with NN′N″-triacetylchitotriose

Single crystals were grown from affinity-purified stinging nettle lectin and from its complex with the specific trisaccharide NN′N″ -triacetylchitotriose by vapor diffusion at room temperature. The lectin crystallizes in space group P2₁2₁2₁ with unit cell dimensions a = 54.3 (1) Å, b = 62.2 (1) Å, and c = 92.4 (2) Å, and diffracts to 3.0 Å resolution. The asymmetric unit contains three lectin monomers. The crystals of the lectin-trisaccharide complex have space group P2₁2₁2₁ with cell constants a = 37.69 (4) Å, b = 48.97 (6) Å, and c = 57.32 (4) Å. These crystals diffract to at least 2.0 Å resolution and the asymmetric unit contains one lectin monomer. A three-dimensional X-ray structure determination is on its way. © 1993 Wiley-Liss, Inc.     

Crystallization and preliminary X-Ray analysis of recombinant staphylokinase

Diffraction quality crystals of recombinant staphylokinase (STAR) have been grown by the hanging drop vapor diffusion technique from a solution containing MgCl₂, Tris buffer (pH 8.5), and polyethylene glycol 4000. The crystals belong to the monoclinic space group C2 with unit cell dimensions a = 60.6 Å, b = 43.7 Å, c = 54.3 Å, and β = 115.6°. Å complete native data set to 1.8 Å resolution has been collected using synchrotron radiation. Proteins 27:160–161 © 1997 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray diffraction studies of leishmanolysin,the major surface metalloproteinase from Leishmania major

The membrane-bound GPI-anchored zinc metalloproteinase leishmanolysin purified from Leishmania major promastigotes has been crystallized in its mature form. Two crystal forms of leishmanolysin have been grown by the vapor diffusion method using 2-methyl-2,4-pentanediol as the precipitant. Macroseeding techniques were employed to produce large single crystals. Protein microhet-erogeneity in molecular size and charge was incorporated into both crystal forms. The tetragonal crystal form belongs to the space group P4₁2₁2 or the enantiomorph P4₃2₁2, has unit cell parameters of a = b = 63.6 Å, c = 251.4 Å, and contains one molecule per asymmetric unit. The second crystal form is monoclinic, space group C2, with unit cell dimensions a = 107.2 Å, b = 90.6 Å, c = 70.6 Å, β = 110.6°, and also contains one molecule per asymmetric unit. Both crystal forms diffract X-rays beyond 2.6 Å resolution and are suitable for X-ray analysis. Native diffraction data sets have been collected and the structure determination of leishmanolysin using a combination of the isomorphous replacement and the molecular replacement methods is in progress. © 1995 Wiley-Liss, Inc.     

Preliminary crystallographic analysis of an enzyme involved in erythromycin biosynthesis: Cytochrome P450eryF

Cytochrome P450eryF was overexpressed in Escherichia coli and purified in high yield. Crystals of the protein in the presence of the substrate, 6-deoxyerythronolide B, have been obtained by the hanging drop vapor diffusion method, using polyethylene glycol 4000 as a precipitant. The crystals belong to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions of a = 54.16 Å, b = 79.67 Å, and c = 99.48 Å and one molecule per asymmetric unit. A complete native data set has been collected to a resolution of 2.1 Å, and anomalous dispersion difference Patterson maps have revealed the location of the single heme iron atom. © 1994 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray analysis of nonglycosylated tissue inhibitor of metalloproteinases-1, N30QN78Q TIMP-1

A nonglycosylated (N₃₀QN₇₈Q) form of the human tissue inhibitor of metalloproteinases, TIMP-1, has been prepared and crystallized in a form suitable for X-ray diffraction analysis. Small single crystals have been grown using sodium tartrate as a precipitant. The crystals are in space group P2₁, with cell dimensions a = 35.28, b = 53.95, c = 48.56, and β = 96.0°. There is a single molecule of TIMP-1 in the asymmetric unit. The crystals diffract to at least 2.3 Å resolution. Complete data have been collected to 2.9 Å and a search for heavymetal derivatives is in progress. © 1993 Wiley-Liss, Inc.     

Crystallization and preliminary structural studies of a chorismate mutase catalytic antibody complexed with a transition state analog

The Fab′ fragment of a catalytic antibody with chorismate mutase activity has been crystallized as a complex with the transition-state analog hapten. The complex was crystallized by the vapor diffusion method using ammonium sulfate as the precipitant. The crystals belong to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions a = 37.1 Å, b = 63.3 Å, c = 178.5 Å, and there is one Fab' molecule per asymmetric unit. The crystals diffract X-rays to at least 3.0 Å and are suitable for X-ray crystallographic studies. © 1994 John Wiley & Sons, Inc.     

Crystallization and preliminary X-ray investigation of the recombinant Trypanosoma brucei rhodesiense calmodulin

Bipyramidal crystals of the recombinant calmodulin from Trypanosoma brucei rhodesiense were obtained by vapor diffusion against 55% (v/v) 2-methyl-2,4-pentanediol in 0.05 M cacodylate buffer, pH 5.6. When few nucleation events occurred, crystals grew to 0.25 × 0.25 × 1.20 mm. The space group of the crystal is I4₁22, with unit cell dimensions a = b = 56.88 Å, c = 230.11 Å, α = β = γ = 90°, z = 16. The molecular mass and volume of the unit cell suggest that there is one molecule in the asymmetric unit. The I/σ(I) ratio for data at 3.0 Å resolution was 3.67, indicating that the final structure can be refined at higher resolution. Molecular replacement methods and the PC-refinement technique have not yet yielded the structure under a variety of search conditions. We are currently investigating the multiple isomorphous replacement approach to determine this crystal structure. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray crystallographic studies of ketosteroid isomerase from Pseudomonas putida biotype B

The Δ⁵-3-ketosteroid isomerase from Pseudomonas putida biotype B has been crystallized. The crystals belong to the space group P2₁2₁2₁ with unit cell dimensions of a = 36.48 Å, b = 74.30 Å, c = 96.02 Å, and contain one homodimer per asymmetric unit. Native diffraction data to 2.19 Å resolution have been obtained from one crystal at room temperature indicating that the crystals are quite suitable for structure determination by multiple isomorphous replacement.     

Crystallization of coupling factor 1 (CF1) from spinach chloroplast.

Spinach chloroplast coupling factor (CF₁) was crystal-lized at 20°C from 0.05 M TRIS-PO₄, containing 4 mM ATP, 15mM KCl, 1.0 mM EDTA and 1.80 M (NH₄)₂SO₄, at pH 7.8. Some unit cell parameters were determined by electron microscopy and by X-ray diffraction. The cube shaped crystals have a tetragonal lattice, a = b = 135 Å, c = 280 Å with eight molecules per unit cell; possible space group P422 or P4₂2₁2, hence half a molecule in the asymmetric unit. Crystals grown at pH 7.5 in the absence of ATP have an orthorhombic lattice, a = 125 Å, b = 145 Å, c = 169 Å (C222₁), eight molecules per unit cell.     

Crystals of an antibody FV fragment against an integral membrane protein diffracting to 1.28 Å resolution

The F_v fragment of a monoclonal antibody, 7E2 (IgG₁, κ, murine), which is directed against the integral membrane protein cytochrome c oxidase (EC 1.9.3.1) from Paracoccus denitrificans, was cloned and produced in Escherichia coli. Crystals suitable for highresolution X-ray analysis were obtained by microdialysis under low salt conditions. The crystals belong to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions of a = 51.51 Å, b = 56.15 Å, c = 99.86 Å (1 Å = 0.1 nm) and contain one F _v fragment per asymmetric unit. Using synchrotron radiation diffraction data were collected up to 1.28 Å resolution. This high resolution is very unusual for a heterodimeric protein. The crystals should open the way for refining not only the atomic positions, but also for obtaining information about internal dynamics. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary crystallographic analysis of an amylopullulanase from the hyperthermophilic archaeon Pyrococcus woesei

The thermostable amylopullulanase from Pyrococcus woesei was crystallized. Crystals, suitable for a crystallographic analysis up to a size of 0.6 mm in their longest dimension, have been obtained by the vapor diffusion method in a solution containing polyethyleneglycol 4000 (PEG 4000), isopropanol, and Tris/Cl^? buffer pH 7.5. Crystals grown under these conditions form hexagonal rods and diffract to a maximum resolution of 3 Å. The crystals belong to the trigonal lattice type with the spacegroup P3₁21 or P3₂21, respectively, have the cell dimensions a = b = 96.8 Å, c = 196.2 Å, α = β = 90°,γ = 120°. The crystals have a theoretical packing density of 2.7 ų/Da, assuming one molecule with a molecular weight of 88.8 kDa in the asymmetric unit. Furthermore the self-rotation analysis of the dataset revealed only crystallographic symmetries. The merged native data of two crystals resulted in a 88% complete dataset. © 1995 Wiley-Liss, Inc.