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1.
Structure of the Azotobacter vinelandii surface layer.   总被引:7,自引:6,他引:1       下载免费PDF全文
Electron microscopy of the Azotobacter vinelandii tetragonal surface array, negatively stained with ammonium molybdate in the presence of 1 mM calcium chloride, showed an apparent repeat frequency of 12 to 13 nm. Image processing showed dominant tetrad units alternating with low-contrast cruciform structures formed at the junction of slender linkers extending from corner macromolecules of four adjoining dominant units. The actual unit cell showed p4 symmetry, and a = b = 18.4 nm. Distilled water extraction of the surface array released a multimeric form of the single 60,000 molecular-weight protein (S protein) which constitutes the surface layer. The molecular weight of the multimer was estimated at 255,000 by gel filtration, indicating a tetrameric structure of four identical subunits and suggesting that this multimer was the morphological subunit of the S layer. Tetrameric S protein exhibited low intrinsic stability once released from the outer membrane, dissociating into monomers when incubated in a variety of buffers including those which served as the base for defined media used to cultivate A. vinelandii. The tetramer could not be stabilized in these buffers at any temperature between 4 and 30 degrees C, but the addition of 2 to 5 mM Ca2+ or Mg2+ completely prevented its dissociation into monomers. Circular dichroism measurements indicated that the secondary structure of the tetramer was dominated by aperiodic and beta-sheet conformations, and the addition of Ca2+ did not produce any gross changes in this structure. Only the tetrameric form of S protein was able to reassemble in vitro in the presence of divalent cations onto the surface of cells stripped of their native S layer.  相似文献   

2.
Sections and freeze-fractured preparations showed an S layer on the surface of Pseudomonas-like strain EU2. Polyacrylamide gel electrophoresis of cell envelopes extracted with 1% sodium dodecyl sulfate (SDS) at room temperature showed three proteins (45K, 55K, and 110K). The 55K protein was identified as the S-layer protein. Incubation in 1.5 M guanidine hydrochloride removed the S layer from cell envelopes and dissociated the structure into subunits. The soluble 55K protein reassembled into planar sheets upon removal of the guanidine hydrochloride by dialysis. Electron microscopy and image processing indicated that these sheets had p4 symmetry in projection with a lattice constant of 13.2 +/- 0.1 nm (corresponding to 9.3 nm between adjacent fourfold axes). In some instances these reassemblies appeared to form small three-dimensional crystals which gave particularly clear views of the structure in projection because of the superimposition of information from a number of layers. A model is proposed with molecules having rounded lobes connected by a narrower linker region and joining at the lobes to form the fourfold axes of the array. The pattern superficially resembles those of other bacterial S layers, such as those of Aeromonas salmonicida, Aeromonas hydrophila, and Azotobacter vinelandii. Extraction of cell envelopes with 1% SDS at 50 degrees C released the 110K protein from the envelopes and removed an amorphous backing layer from the S layer. The 45K protein displayed heat-modifiable migration in SDS-polyacrylamide gel electrophoresis and was insoluble in SDS at 50 degrees C or in high concentrations of guanidine hydrochloride, suggesting that it was associated with the peptidoglycan.  相似文献   

3.
The three-dimensional structure of the protein which forms the S layer of Aquaspirillum serpens strain VHA has been determined by electron microscopy. Structures have been reconstructed to a resolution of about 1.6 nm for single-layered specimens and about 4 nm for two-layered specimens. The structure, which has hexagonal symmetry, consists of a core in the shape of a cup, with six projections arising from the rim of the cup to join adjacent subunits at the threefold symmetry axes. The model is consistent with edge views of the S layer which have been obtained in this and other work. It is now clear from this work and from three-dimensional reconstructions of other bacterial S layers that a wide diversity exists in the morphology of surface layers.  相似文献   

4.
Cell-free culture supernatants of a lipopolysaccharide (LPS) O-polysaccharide-deficient, single-insertion transposon mutant of the tetragonal surface protein array (S layer)-containing fish pathogen Aeromonas salmonicida were examined by electron microscopy. Negative staining showed that the S layer was released as sheets of tetragonal material, indicating that although surface retention of assembled S layer requires the presence of wild-type LPS oligosaccharides, initial assembly of S-layer subunits into sheets does not require the presence of O-polysaccharide chains. The three-dimensional structure of the S layer was reconstructed from tilted micrographs of the released sheets. Horizontal sections through this reconstruction showed that the released sheets were composed of two identical S layers that were perfectly in register. The reconstructed layer had a lattice constant of 12.5 nm. At a resolution of 1.6 nm, the layer consisted of a major tetragon at one fourfold axis of symmetry and a minor tetragon at the second fourfold axis of symmetry. The core, composed of four of the major domains, contained a large depression and was located toward the inside of the layer. The minor tetragon provided connectivity within the layer and was located toward the outer surface of the layer. Projections through the double layer gave a type I (closed) pattern (M. Stewart, T. J. Beveridge, and T. J. Trust, J. Bacteriol. 166:120-127, 1986), yet projections through the single layer indicated that the type II (open) pattern was present. This open pattern was indistinguishable from that seen in S layer released from the surfaces of wild-type cells.  相似文献   

5.
The 2D crystallization method using a liquid–liquid interface has been applied to theRhodococcus20S proteasome. Two types of ordered arrays were obtained, both large enough for high-resolution analysis. The first one is a hexagonal close-packed array, whereas the second one has fourfold symmetry. By image analysis based on a real space correlation averaging technique, the close-packed array was found to be hexagonally packed but the molecules had complete rotational freedom. The fourfold array is, however, a true crystal with p4 symmetry. Lattice constants area=b= 20.0 nm and the unit cell of this crystal contains two proteasomes. The diffraction pattern computed from the original picture shows the spots up to (4.5) that correspond to 3.1 nm resolution. After applying an unbending procedure, the diffraction pattern shows spots extending to 1.8 nm resolution.  相似文献   

6.
The regular surface layer (S-layer) of Clostridium aceticum has been isolated and the three-dimensional structure determined to a resolution of 2.0 nm from tilt series of negatively stained preparations. It has tetragonal symmetry with a lattice constant of 12 nm and a thickness of 6 nm; there are probably 4 protein monomers per unit cell. A large proportion of the protein is concentrated in massive "cores" at the major four-fold axes which are situated towards the inner surface of the layer. From these cores, delicate arms extend towards the minor four-fold axes, where secondary connectivity is established near the exterior surface. When viewed from the outside, each of the cores appears to have a large central depression, rather than a true "pore". Since this general pattern of mass distribution is shared by the hexagonal S-layer of Clostridium thermohydrosulfuricum, some consideration has been given to the possible evolutionary steps leading to changes in symmetry. From modelling experiments, it is evident that the change from four-fold to six-fold symmetry in this instance could be accomplished simply by the loss of a structural "domain" from the protomer.  相似文献   

7.
The three-dimensional structures of the crystalline surface layers of two species of Eubacteria have been determined by electron microscopy and computerized image processing. The S-layer of Eubacterium sp. ES4C has tetragonal symmetry, with a unit cell spacing of 10.6 nm and a thickness of 9.5 nm, while that of Eubacterium sp. AHN 990 has hexagonal symmetry a = b = 15.7 nm and a thickness of 13 nm. The resolutions in the reconstructions were 2.5 nm and 1.8 nm, respectively. The reconstruction of the S-layer of strain ES4C reveals a distinct domain structure: a major tetramer, arms connecting adjacent unit cells, and a minor tetramer. The S-layer of strain AHN 990, on the other hand, has a rather complex arrangement, centred around the six-fold axis.  相似文献   

8.
The cell envelope of the hyperthermophilic sulphur-reducing archaebacterium Pyrobaculum organotrophum H10 was found to be composed of two distinct hexagonally arranged crystalline protein arrays. Electron microscopic analysis of freeze-etched cells and isolated envelopes in conjunction with image processing showed that the inner layer (lattice centre-to-centre spacing 27.9 nm) is essentially identical to the protein array of Pyrobaculum islandicum GEO3, a complex, rigid structure implicated in the maintenance of cell shape. The outer layer has clear p6 symmetry and a lattice spacing of 20.6 nm. Its three-dimensional structure was reconstructed from a negative stain tilt series of an intact double-layered envelope using Fourier filtration to separate the desired information from the other lattices present. The outer layer is a unique, porous network of blocklike dimers disposed around six-fold axes, and exhibits minimal asymmetry between its inner and outer faces. It appears to be rather loosely associated with the outer surface of the inner layer. In most H10 envelopes, the inner layer is orientated with one base vector exactly perpendicular to the long axis of the cell, so that the cylindrical portion is composed of a series of parallel cell-girdling hoops of hexameric morphological units. All the other known Pyrobaculum strains were found to have a GEO3-type envelope structure, consisting of a single rigid protein array and a fibrous capsule. Although H10 does not possess a capsule, fibrils appear to be sandwiched between the two protein layers.  相似文献   

9.
Aquaspirillum sinuosum cell walls bear two paracrystalline, proteinaceous surface layers (S layers). Each shows a different symmetry: the inner layer is closely apposed to the outer membrane and is a tetragonal array (90 degrees axes; 5-nm units; repeat frequency 8 nm); the outer layer is a hexagonal array on the external surface (14-nm units; repeat frequency 18 nm) and, although the units have a six-pointed stellate form, the linkage between units is not resolved. The outer layer consists of a major 130-kDa protein and a 180-kDa minor component; these co-extract, co-assemble, and are inseparable by hydroxylapatite chromatography or by recrystallization. The solubilizing effects of reagents suggest stabilization by hydrogen bonding and Ca2+. The two outer layer proteins are serologically related and show partial identity by peptide mapping. Periodic acid--Schiff staining of the 180-kDa band suggests that this may be a glycosylated form of the 130-kDa component. The inner layer components form a doublet of 75- and 80-kDa polypeptides with extreme resistance to extraction. Close apposition to the outer membrane, resistance to chaotropes, aqueous insolubility, and behaviour in charge-shift electrophoresis suggest hydrophobic interaction between subunits and an integral association with the outer membrane.  相似文献   

10.
Two-dimensional crystals of alpha-latrotoxin from the venom of black widow spider (Latrodectus mactans tredecimguttatus) were studied by the negative staining electron microscopy. Two-dimensional crystals were obtained by adsorption of the protein solution with a high Mg2+ concentration on carbon-coated electron microscopy grids. The crystals were about 0.4 mkm in size, had the unit cell parameters: a = b = 15.55 nm, gamma = 90 degrees, p4 plane group symmetry. The contour map of a stain-excluding region of such crystals was calculated by the Fourier-filtering procedure at about 4 nm resolution. The calculation of molecular weight of the unit cell, with the symmetry p4 taken into account, showed that alpha-latrotoxin particles, revealed by negative staining, consisted of 4 or 8 protomers.  相似文献   

11.
The regular surface layer of a strain of a Comamonas-like organism was examined by electron microscopy. The surface layer protein was easily extracted from the cell surface by a 2.5 M solution of lithium chloride. The protein subunit has a molecular size of 32,000 daltons, but usually forms a large aggregate of more than 1,200,000 daltons. In the extract it formed a regular array of p4 symmetry and was observed to be intimately associated with fragments of lipopolysaccharide. The size of a subunit determined by the negative staining method and the image processing method measured 5.2 × 6.4 nm (width and length), was arranged in a cobblestone-like pattern, and was located in a lattice space measuring 13.0 nm square.  相似文献   

12.
The conformation of the chondroitin-4-sulfate calcium salt was investigated by X-ray analysis. The following results were obtained. 1, The repeat length per disaccharide was 0.913 nm: 2, The molecular chain had three-fold screw symmetry: 3, The shape of the unit cell was a trigonal prism with dimensions a=b=1.28 nm, c=2.74 nm, and gamma=120 degrees: 4, The number of disaccharide residues in the unit cell was six. Two molecular chains were packed in the unit cell.  相似文献   

13.
The two-dimensional (2D) structure of the regularly structured surface layer (S-layer) of the gram-negative eubacterium Lampropedia hyalina has been determined at the molecular level to a nominal resolution of 2.1 nm by transmission electron microscopy and digital image processing. The inner, or “perforate,” layer consists of dimeric block-shaped units located at two-fold symmetry axes. These morphological dimers associate around three-fold symmetry axes to form a continuous layer with p6 symmetry and a lattice constant of 14.6 ± 0.4 nm. Scanning transmission electron microscopy (STEM) yields a mass-per-area (MPA) value for the perforate layer of 3.5 kDa/nm2. The outer, or “punctate,” layer is composed of long, roughly cylindrical units centered on six-fold symmetry axes, which are connected by six fine linking arms joining at the three-fold symmetry axes to create a hexagonal layer with a lattice constant of 25.6 ± 0.5 nm. The MPA of the “composite”-i.e., perforate plus punctate—layer is 10.2 kDa/nm2.  相似文献   

14.
The cell walls of a number of filamentous, gliding cyanobacteria of the genus Oscillatoria were examined by transmission electron microscopy of ultrathin sections, of freeze-etched replicas, and of whole cells crushed between glass slides and negatively stained. All three techniques revealed the presence of a highly ordered array of parallel fibrils, seen in transverse sections to be situated between the peptidoglycan and the outer membrane. Approximately 200 individual fibrils, each 25 to 30 nm in width, form a parallel, helical array that completely surrounds each cyanobacterial filament, running at an angle of 25 to 30° to its long axis. This highly regular arrangement of the fibrillar layer may imply some underlying symmetry responsible for its organization. A possible source of such symmetry would be the peptidoglycan, and some form of interaction between this layer and the fibrils might provide the necessary scaffolding for the fibrillar array. In crushed, negatively stained samples of fresh cells, individual fibrils were seen outside the filament, released from the cell wall. These released fibrils were of the same width as those observed in situ but were in short lengths, mostly of 100 to 200 nm, and were invariably bent, sometimes even into U shapes, implying great flexibility. Negative staining of released fibrils showed no evidence that they were hollow tubes but did give some indication of a substructure, implying that they were composed of many subunits. The function of this fibrillar array is unknown, although its position in the cell wall, as well as the correspondence between the angle of the fibrils with respect to the long axis of the filament and the rotation of the filament during gliding, may imply an involvement in gliding motility.  相似文献   

15.
The hexameric central subunit (Mr = 360,000) of the multi-subunit complex transcarboxylase has been crystallized by bulk dialysis against 250 mM-sodium acetate (pH 5.5). The crystals are cubic, a = 193.1 A, space group P4(1)32 or enantiomorph. The number of molecules per unit cell is four and was deduced from the density of the crystals (1.10 g cm-3) and the mother liquor (1.01 g cm-3) and the specific volume of the protein calculated from molecular dimensions obtained from electron microscopy studies. Four molecules per cell requires the central subunits to lie on 3-fold axes, which are perpendicular to 2-fold rotation axes, so that the molecules satisfy 32 symmetry giving one subunit as the asymmetric unit. Of the four possible models that have been considered for the quaternary structure of transcarboxylase, only that with antiparallel subunits, two sets of isologous binding sites and D3 symmetry is in agreement with the symmetry requirements of the cubic crystals.  相似文献   

16.
The three-dimensional structure of the regular surface layer of Sporosarcina ureae has been determined to a resolution of 1.7 nm by electron microscopy and image reconstruction. The S-layer has p4 symmetry, a lattice constant of 12.9 nm, and a minimum thickness of 6.6 nm. The reconstruction reveals a distinct domain structure: a massive core, arms connecting adjacent unit cells, and spurs which make contact at the subsidiary fourfold symmetry axes. In the z-direction the domains appear to be arranged in three planes, creating two entirely different surface reliefs. The S-layer has a complex pattern of pores and gaps that are 2 to 3 nm wide. In addition, the secondary-structure composition has been determined by infrared spectroscopy: about 35% of the polypeptide appears to have a beta-structure conformation.  相似文献   

17.
Characterization of a dynamic S layer on Bacillus thuringiensis.   总被引:5,自引:3,他引:2       下载免费PDF全文
The surfaces of three Bacillus thuringiensis strains possess an S layer composed of linear arrays of small particles arranged with p2 symmetry and with a = 8.5 nm, b = 7.2 nm, and gamma = 73 degrees. Platinum shadows of whole cells and S-layer fragments revealed the outer surface of the array to be smooth and the inner surface to be corrugated. Treatment with 2 M guanidine hydrochloride at pH 2.5 to 4 best removed the S layer for chemical characterization; it was a relatively hydrophilic 91.4-kilodalton protein with a pI of 5, no detectable carbohydrate, cysteine, methionine or tryptophan, and 21.2% nonpolar residues. No N-terminal homology with other S-layer proteins was evident. Antibody labeling experiments confirmed that the amount of S layer was proportional to the growth phase in broth cultures. Late-exponential- and stationary-growth-phase cells typically sloughed off fragments of S layer, and this may be the result of wall turnover. Indigenous autolytic activity in isolated walls rapidly digested the wall fabric, liberating soluble S-layer protein. At the same time, proteases frequently reduced the molecular weight of the 91.4-kilodalton protein, but these polypeptides could still be identified as S-layer components by immunoblotting. As cultures were serially subcultured, the frequency of appearance of the S layer diminished, and it was eventually lost. The dynamic nature of this S layer makes it atypical of most previously identified S layers and made it unusually difficult to characterize.  相似文献   

18.
The Caliciviridae is a family of nonenveloped, icosahedral, positive-sense single-stranded RNA viruses. This family of viruses consists of both animal and human pathogens. Adapting human caliciviruses to cell culture has not been successful, whereas some animal caliciviruses, including San Miguel sea lion virus, have been successfully propagated in vitro. Here we report the crystallization of San Miguel sea lion virus serotype 4 (SMSV4) and the preliminary X-ray crystallographic analysis of the crystals. SMSV4 have been crystallized using the hanging-drop method. These crystals diffracted to approximately 3A resolution using a synchrotron radiation source. A single crystal under cryo-conditions yielded a complete set of diffraction data. Data processing of the diffraction patterns showed that SMSV crystals belong to I23 space group with cell dimensions a=b=c=457 A. The crystallographic asymmetric unit includes five icosahedral asymmetric units, each consisting of three capsid protein subunits. In the space group I23, given the icosahedral symmetry and the size of the virus particle, the location of the particle is constrained to be at the point where the crystallographic 2- and 3-fold axes intersect. The orientation of the virus particle in the unit cell was ascertained by self-rotation function calculations.  相似文献   

19.
Abstract Analysis by electron microscopy of cells of Thermus thermophilus revealed the presence of a crystalline layer on the cell surface with the characteristic appearance of an S-layer. The layer is apparently built up by a single protein with a M r of 100 000 in a hexagonal array. The unit cell dimension of the S-layer is 24 ± 2 nm.  相似文献   

20.
Image analysis of electron micrographs of thin-sectioned myosin subfragment-1 (S1) crystals has been used to determine the structure of the myosin head at approximately 25-A resolution. Previous work established that the unit cell of type I crystals of myosin S1 contains eight molecules arranged with orthorhombic space group symmetry P212121 and provided preliminary information on the size and shape of the myosin head (Winkelmann, D. A., H. Mekeel, and I. Rayment. 1985. J. Mol. Biol. 181:487-501). We have applied a systematic method of data collection by electron microscopy to reconstruct the three-dimensional (3D) structure of the S1 crystal lattice. Electron micrographs of thin sections were recorded at angles of up to 50 degrees by tilting the sections about the two orthogonal unit cell axes in sections cut perpendicular to the three major crystallographic axes. The data from six separate tilt series were merged to form a complete data set for 3D reconstruction. This approach has yielded an electron density map of the unit cell of the S1 crystals of sufficient detail. to delineate the molecular envelope of the myosin head. Myosin S1 has a tadpole-shaped molecular envelope that is very similar in appearance to the pear-shaped myosin heads observed by electron microscopy of rotary-shadowed and negatively stained myosin. The molecule is divided into essentially three morphological domains: a large domain on one end of the molecule corresponding to approximately 60% of the total molecular volume, a smaller central domain of approximately 30% of the volume that is separated from the larger domain by a cleft on one side of the molecule, and the smallest domain corresponding to a thin tail-like region containing approximately 10% of the volume. This molecular organization supports models of force generation by myosin which invoke conformational mobility at interdomain junctions within the head.  相似文献   

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