Prolonged incubation of membrane fragments containing homogeneous (Na+ + K+)-ATPase with Mg2+, K+ and VO; at 4°C resulted i_n formation of two-dimensional crystals of this enzyme with unit cell parameters: a = 66 A, b = 118 A, y = 108". The crystals correspond to the two-sided plane group ~21. By combining tilted electron microyopic views of the crystals, a three-dimensional structure of (Na+ -t K+)-ATPase was calculated at -20 A resolution. The unit cell is formed by two (@)-promoters which are in contact in their central parts. The structure was compared with chemical modification and immunochemical data; the arrangement of intra-and extramembrane domains was proposed.
SynopsisThe protein actinoxanthin (isolated from Actinomyces gZobisporus-molecular weight, 10,300; 107 amino acid residues) crystallizes in space group P212121 with cell dimensions: a = 30.9 A, b = 48.8 A, c = 64.1 A, and 2 = 4. The three-dimensional structure of actinoxanthin was determined by the x-ray multiple isomorphous replacement method at 2.5-A resolution. The molecule is kidney-shaped and has a well-defined cavity. Its characteristic features are the absence of a-helices and the presence of enhanced content of antiparallel @-structure (-55%). A cylinder-shaped formation of seven antiparallel @-strands comprises the main part of the protein structure. The established P-supersecondary structure is characterized by a three-dimensional topology similar to that of immunoglobulin domains, superoxide dismutase subunits, and azurin and plastocyanin proteins.
Two-dimensional crystals of photosynthetic reaction centers from CMarqkus auranfiacus were obtained from protein-lipid-detergent micelles by detergent dialysis. The size of crystals was up to 2 e. Some of them were multi~yer~ crystals. However, other crystal forms were also observed. Preliminary image processing analysis showed that crystals of one crystal form referred to two-sided piane group p2 and had the following unit cell parameters: a= 17,6 nm, b= I8,O nm, y=84". The contour map of the crystal stain-excluding region was calculated by the Fourier-filtering procedure at about 2 nm resolution.Reaction center; Two~imensionai crystal; Electron microscopy; Image processing 1" INTRODUCTIONPrimary photochemical reactions in bacterial photosynthesis are known to occur in integral membrane protein systems, i.e. reaction centers (RC). Although the detailed mechanisms of the functioning of these muitimolecular systems are still obscure, the studies of the structural organization of the photosynthetic systems at all levels are important. The membrane organization of the bacteria RC is under extensive study. The structure of the RC molecules of purple bacteria Rhodopseudoareas viridis [ 1,2] and R~odops~d~~onus sphaeroids [3,4] were determined at atomic resolution by X-ray analysis. The spatial structure of the photosynthetic RC of green bacteria C~loro~ex~s aurant~ac~s in the membrane is as yet unknown. Staehelin with coworkers analyzed a general morphology of Cklorofrexus aurantiacus bacteria 151. Recently primary structures of the L-and ~-subunits of C~~oro~~s RC were established, that allowed modeling the topography of the polypeptide chains in the membrane [S, 71.The structural organization of membrane proteins can be studied by electron microscopy of two-dimensional crystals followed by computer image reconstruction techniques [8], The present work was aimed at twodimensional crystallization of Ckloroflexus RC and 2, MATERIALS AND METHODSIsolation and purification of RC, determination of spectral and other chemical parameters were carried out as described in [4,5]. Lecithin from soybean (Sigma) was solubilized by IauryI~methyl-amine N-oxide (LDAO). For reconstitution experiments, solubilized lecithin was added to the RC sample (10 mg/ml of protein in 50 mM of Tris-HCl, pH 9.0, &l@?o LDAO) at various proportions. Then detergent was removed by dialysis under various conditions (temperature, dialysis rate, ionic strength, ion composition of dialysis media) 181.Electron microscopy studies, optical diffraction and image processing were done as described in [IO].
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