Electron microscopy of subnanometer surface features on metal-decorated protein crystals.

Crystals of heavy riboflavin synthase from Bacillus subtilis were freeze-etched and vacuum-coated at normal incidence with 0.1 to 0.4 nm of gold and silver, respectively. This decoration technique was applied to probe the protein surface for preferential nucleation sites. Image processing of the electron micrographs revealed two particular decoration sites for silver and a different one for gold. According to X-ray crystallography, the riboflavin synthase molecules are spherical and smooth except for a surface corrugation of less than 1 nm, which can not be depicted by heavy-metal shadowing. Thus the decoration sites represent sites of specific physical-chemical interactions between the condensing metal and the protein. The decoration pattern correctly reflects the icosahedral symmetry of the almost spherical protein molecules. Owing to the molecule's symmetry, the position of these topochemical sites with respect to the symmetry axes can be localized within 5A. The packing of the molecules in the crystal can be directly observed on shadowed replicas. Only decoration, however, makes it possible to observe the exact orientation of the molecules within the crystal planes and to derive the true lattice constant along the 6-fold screw axis. This proves decoration to be a technique suitable for studying crystal packing and the molecular symmetry of protein complexes at high resolution. The technique can be applied to crystals that are not large enough or insufficiently ordered for X-ray crystallography.

Basal-body-associated disks are additional structural elements of the flagellar apparatus isolated from Wolinella succinogenes.

The intact flagella of Wolinella succinogenes, a gram-negative, anaerobic bacterium with a single polar flagellum, were obtained by an improved procedure, introduced recently by Aizawa et al. (S.-J. Aizawa, G. E. Dean, C. J. Jones, R. M. Macnab, and S. Yamaguchi, J. Bacteriol. 161:836-849, 1985) for the flagellum of Salmonella typhimurium. Disks with a diameter of 130 +/- 30 nm, which were attached to the basal body of the isolated intact flagella, could be identified by electron microscopy as additional structural elements of the bacterial flagellar apparatus. In freeze-dried and metal-shadowed samples, two rings of the basal body were detected on one side and a terminal knob was located on the other side of the disks. Suspension of the flagellar apparatus in acidic solution dissociated the flagellar filaments, yielding hook-basal body complexes with and without the associated disks. If whole cells were subjected to low pH, double disks of the same diameter and with a central hole of about 13 nm could be isolated. Similar parallel disks could be seen also in negatively stained whole cells. When uranyl acetate was used for negative staining of the intact flagella, concentric rings were detected on the disks, similar to the concentric membrane rings found by Coulton and Murray (J. W. Coulton and R. G. E. Murray, J. Bacteriol. 136:1037-1049, 1978) on platelike arrays of proteins in outer membrane preparations of Aquaspirillum serpens. Because the disks of W. succinogenes can be isolated together with the flagellar hook-basal body complex, they appear to be basal-body-rather than secondary membrane-associated structures. It is possible that these disks are the bearing or stator of this rotary device.

Three-dimensional structure of the regular surface glycoprotein layer of Halobacterium volcanii from the Dead Sea.

A three-dimensional reconstruction from electron micrographs of negatively stained cell envelopes of Halobacterium volcanii has revealed the structure of the surface glycoprotein to a resolution of 2 nm. The glycoprotein is arranged on a p6 lattice with a lattice constant of 16.8 nm. It forms 4.5 nm high, dome-shaped, morphological complexes with a narrow pore at the apex opening into a ;funnel' towards the cell membrane. The polarity of the structure was derived from freeze-etching experiments and ;edge' views. Six radial protrusions emanate from each morphological complex and join around the 3-fold axis to provide lateral connectivity. Using the primary structure of the surface glycoprotein of the closely related species Halobacterium halobium (Lechner and Sumper, 1987) and the cell envelope profile from a previous X-ray analysis of the same species (Blaurock et al., 1976) we have integrated our reconstruction into a model of halobacterial cell envelope.

Bacterial surface proteins. Some structural, functional and evolutionary aspects.

The structure of several eubacterial and archaebacterial surface (glyco)proteins as determined by three-dimensional electron microscopy is described. Particular emphasis is placed on surface proteins which interact with membranes. Some structure-function relationships deduced from the structural information, such as shape maintenance and molecular recognition phenomena, are discussed.

Three-dimensional structure of the surface protein of Desulfurococcus mobilis.

The spherical cells of the thermophilic, sulfur-dependent archaebacterium Desulfurococcus mobilis are completely covered with a relatively poorly ordered, tetragonally arrayed surface protein. The structure of this surface protein was examined by using three-dimensional electron microscopy. The protein lattice forms an open meshwork composed of cross-shaped morphological units, which are released when glycerol is added. These subunits make contact at the distal ends of their four arms. The p4 symmetry requires that each of these morphological subunits represents a tetramer. The strong interaction of the monomers within the crosses and the relatively weak interaction of the intersecting arms of the crosses within the lattice structure suggest that the tetramers are assembled before their incorporation into the lattice.

The cell envelope of Thermoproteus tenax: three-dimensional structure of the surface layer and its role in shape maintenance.

The sulphur-dependent archaebacterium Thermoproteus tenax has a cylindrical cell shape variable in length, but constant in diameter. Its whole surface is covered by a regular protein layer (S-layer). The lattice has p6 symmetry and a lattice constant of 32.8 nm. The three-dimensional reconstruction from a tilt series of isolated and negatively stained S-layer shows a complex mass distribution of the protein: a prominent, pillar-shaped protrusion is located at the 6-fold crystallographic axis with radiating arms connecting neighbouring hexamers in the vicinity of the 3-fold axis. The base vectors of the S-layer lattice have a preferred orientation with respect to the longitudinal axis of the cell. The layer can be seen as a helical structure consisting of a right-handed, two-stranded helix, with the individual chains running parallel. Supposing that new S-layer protein is inserted at lattice faults (wedge disclinations) near the poles, growing of the layer would then proceed by moving a disclination at the end of the helix. The constant shape of the cell, as well as the particular structure of the layer, strongly suggest that this S-layer has a shape-maintaining function.

The structure of the photoreceptor unit of Rhodopseudomonas viridis.

The thylakoid membrane of Rhodopseudomonas viridis contains extensive, regular arrays of photoreceptor complexes arranged on a hexagonal lattice with a repeat distance of 130 A. Single membrane sheets were obtained by mild treatment of the thylakoid fraction with the detergent Triton X-100. Heavy metal shadowing and electron microscopy of isolated thylakoids indicated a strong asymmetry of the membrane, showing a smooth plasmic and a rough exoplasmic side. Fourier processing of rotary-shadowed specimens showed the different surface relief on both sides of the membrane. Structural units on both sides were roughly circular and showed 6-fold symmetry at a resolution close to 20 A. The structural unit was characterised by a central core that seemed to extend through the membrane, protruding on the exoplasmic side. The core was surrounded by a ring showing 12 subunits on the plasmic side. Rotary-shadowed as well as negatively-stained membranes indicated a handedness of the structure. Treatment of thylakoid vesicles with higher detergent concentrations yielded a fraction of particles showing the same features as Fourier maps of the structural units. The isolated particles therefore appeared to represent structurally intact units of photosynthesis.