PubMed Health⌕ Search

Biomedical subjects

W Keegstra

Publications and source records attributed to W Keegstra.

11 recordsLinked to original sources
PubMed1989

Crystallization properties and structure of Panulirus interruptus haemocyanin.

Electron-microscopic studies revealed that two types of subunits of Panulirus interruptus haemocyanin crystallize in different ways. Homohexamers of subunit a give close-packed two-dimensional crystals whereas homohexamers of subunit c form open two-dimensional arrays. We applied computer-image analysis to these arrays and studied the differences in crystallization properties by combining the electron-microscopic data with amino acid sequence information and the X-ray diffraction model of subunit a.

Amino Acid Sequence↗
PubMed1986

Two-dimensional crystallization experiments.

Our experience in the growth of two-dimensional crystals of different proteins is presented. Polyethylene glycol was used to produce two-dimensional arrays of haemocyanin from O. vulgaris and of cholera toxin. The arrays showed a hexagonal close-packed structure of only randomly oriented molecules. The increase in protein concentration probably occurred too quickly to allow complete crystallization. Different two-dimensional arrays of hexameric haemocyanin molecules (from P. interruptus) were obtained by microdialysis through the specimen supporting film. A comparison was made with X-ray data. Two-dimensional tetrameric arrays of molecules, possibly rhodopsin, were seen in samples of bovine retinal rod outer segments in the presence of ammonium sulphate. Two-dimensional crystals of complex I (from bovine mitochondria) were prepared by dialysis in the presence of ammonium sulphate. A three-dimensional reconstruction was made from two tilt-series by computer filtration using the direct SIRT procedure. Finally, the possibility of computer crystallization using correlation techniques in combination with correspondence analysis is discussed.

Ammonium Sulfate↗
PubMed1985

Two-dimensional crystallization of bovine rhodopsin.

Bovine rhodopsin has been clustered into two-dimensional crystals in highly purified native rod disk membranes and studied with negative staining and transmission electron microscopy. The lattice is P2(1) with dimensions of 8.3 X 7.9 nm and interaxis angles of 86 +/- 3 degrees. 110 images of ordered areas were digitized and aligned with computer-correlation methods to calculate an average image with diffraction to the fourth order. The images were computer-filtered and reconstructed to approx. 2 nm resolution. When crystals appeared they covered 20-40% of the surface of the preparation and, since rhodopsin is at least 95% of the protein, there is no doubt that the crystals were due to rhodopsin. There appear to be two rhodopsin dimers per unit cell. Each rhodopsin molecules takes up about 7.5 nm2 of membrane area and is estimated to be associated with about 12 lipids on each side of the membrane. The membrane area found for bovine rhodopsin supports the rhodopsin origin of rarely seen but more highly ordered two-dimensional crystals found in detergent-treated frog rod membranes (Corless, J.M., McCaslin, D.R. and Scott, B.L. (1982) Proc. Natl. Acad. Sci. USA 79, 1116-1120). Furthermore, the rhodopsin membrane area is close to that of bacteriorhodopsin and is consistent with a seven transmembrane helix structure proposed for rhodopsin (for references see Dratz, E.A. and Hargrave, D.A. (1983) Trends Biochem. Sci. 8, 128-131). Crystallization was accomplished by lowering the pH to 5.5 near the isoelectric point of rhodopsin, raising the salt concentration of 2 M (NH4)2SO4, adding 5% glucose and 0.02% Hibitane (Ayerst), a cationic amphipathic antiseptic that favored crystal growth.

Animals↗
PubMed1977

Mapping and length measurements of restriction enzyme fragments by electron microscopy.

1. We have mapped by electron microscopy the DNA-fragments formed by the action of the restriction endonuclease from Arthrobacter luteus of phi X 174 replicative form DNA. These fragments were separated by polyacrylamide gel electrophoresis and hybridized to phiX 174 single stranded DNA. The partial duplex molecules were inspected in the electron microscope. In this way the relative order of eleven fragments ranging in size from approximately 100 to 1000 nucleotide pairs has been established and compared with that deduced from reciprocal digestion studies. 2. The measured lengths of the fragments agreed well with the lengths found by gel electrophoresis. 3. The purity of the isolated fragments was checked. Most of the contaminating fragments derive from nearest neighbours in the preparative polyacrylamide gels.

Arthrobacter↗
PubMedDate not supplied

Computer image analysis of two-dimensional crystals of beef heart NADH: ubiquinone oxidoreductase fragments. I. Comparison of crystal structures in various negative stains.

We investigated the structure of two-dimensional crystals from bovine heart mitochondrial NADH: ubiquinone oxidoreductase. A detailed description of uranyl acetate-stained crystals demonstrated that they are composed of fragments in a spatial arrangement according to space group P4212 [J. Brink, S. Hovmöller, C.I. Ragan, M.W.J. Cleeter, E.J. Boekema and E.F.J. van Bruggen, European J. Biochem. 166 (1987) 287]. To gain more structural information on the crystal structure and to assess the effects of various negative stains on the structure preservation and appearance, we examined stained crystals by means of electron microscopy and image analysis. The space group P4212 appeared to be present for several stains tested, i.e. ammonium molybdate, uranyl acetate, uranyl nitrate and uranyl sulphate. Use of phosphotungstic acid and silicotungstate resulted in a reduction of symmetry to pseudo-P4212 or p4. Use of sodium tungstate led to a considerable loss of resolution to 3.8 nm at best, whereas otherwise 1.5 to 1.9 nm could be demonstrated. The lattice vectors were not affected by the stains; they were determined as a = b = 14.9 +/- 0.25 nm with gamma = 89.8 degrees +/- 0.6 degrees. Image analysis showed the presence of similar structures with the molybdate and uranyl compounds. Differences were observed in the case of the tungstate type of stains. Furthermore, the analysis revealed the complete absence of the four small pores of 2.0 nm diameter in the unit cell. This effect was observed irrespective of the type of stain and supporting film, and could be ascribed only to the glow-discharge treatment of the supporting film. The observed difference must be caused by changed interactions between the protein, stain and supporting film. Application of correspondence analysis and clustering algorithms to the various reconstructed images of the crystals showed that they could be separated into several clusters. Each of these clusters corresponded on the average to only one type of stain, whereas a further division according to the specific uranyl compounds was observed. This study therefore shows that under identical preparation conditions subtle differences between individual stains can be detected.

Animals↗