Urban Kavéus

The projection structure of α-toxin from Staphylococcus aureus in human platelet membranes as analyzed by electron microscopy and image processing

Abstract Most strains of Staphylococcus aureus produce α-toxin, a 33-kDa membrane active protein which is considered to be an important virulence factor of this bacterium. When α-toxin interacts with membranes an oligomeric form of the toxin can be seen by electron microscopy as characteristic ring structures in the membrane. A two-dimensional study of these annular structures, incorporated in membranes of human platelets, was performed, introducing a partly new method for rotational alignment of individual particles. It is shown that the averaged oligomer consists of six subunits. At neutral pH the outer diameter of the ring is about 75 A. The stain-filled pore or cavity in the center has a diameter of about 25 A. The size of the hexamer is increased if the pH is lowered.

Three-dimensional structure of Na,K-ATPase determined from membrane crystals induced by cobalt-tetrammine-ATP

The three-dimensional structure of Na,K-ATPase has been analyzed with electron microscopy and image processing. The enzyme, purified from pig kidney outer medulla, was arranged in a new form of tetragonal two-dimensional membrane crystals after incubation with cobalt-tetrammine-ATP, a stable MgATP complex analogue. Each continuous protein domain, as delineated by negative stain, consists of two alpha beta-protomers related by a dyad axis. The two rod-like regions are connected by a bridge displaced about 20 A away from the center of the structure toward the lipid bilayer. The domain connecting the two promoters is more constricted and closer to the center of the structure in the Co(NH3)4ATP-induced crystals than in the vanadate-induced p21 crystals. These observations suggest that the difference between previously analyzed dimers of two-dimensional p21 crystals induced with vanadate/magnesium and dimers of p4 crystals induced with Co(NH3)4ATP reflects two different conformational states of the enzyme.

Structure analysis of fibrinogen by electron microscopy and image processing

Human fibrinogen was observed by electron microscopy following rotary shadowing with tungsten. Structure analysis of the molecules was performed by image processing of electron micrographs. A method is described for selection, alignment, and classification of molecules. The widely accepted overall trinodular structure of the protein was observed. The flexibility about the central domain of the molecule was quantitatively analyzed. A Gaussian distribution of this conformational parameter was obtained having an average corresponding to a maximally extended structure. Correspondence analysis applied to the aligned images showed that the degree of folding of the molecule was continuously distributed. The averaged structure of fibrinogen was estimated to be 450 A long. The central domain had a diameter of 50 A and the peripheral domains were 90 A long and 50 A wide. The latter regions had two separated maxima of scattering density.

Coexistence of different forms ofNa,K‐ATPase in two‐dimensional membrane crystals

Two‐dimensional membrane crystals of renal Na,K‐ATPase were analyzed by electron microscopy and image processing. The particular property of the crystals in this work was that they showed unit cell parameters similar to the previously studied p21 crystals but lacked the dyad axis as observed in nominal 0°‐projections. A three‐dimensional reconstruction revealed that structural differences between αβ‐units of the enzyme gave rise to the asymmetry. A high degree of two‐fold rotational symmetry was observed in the middle of the structure while the protein units had different three‐dimensional shapes at levels above and below the central sections. The simultaneous coexistence of different forms of Na,K‐ATPase suggests that the conformational flexibility of the enzyme plays an important role in the pumping process.

The three-dimensional structure of trypsin-treated Staphylococcus aureus α-toxin

Trypsin treatment of staphylococcal alpha-toxin cleaves the molecule into two roughly equally sized parts, which results in inactivation of the toxin. Tetragonal arrays of oligomers, closely resembling the native ones, can however be formed on lipid layers. From tilted views of negatively stained crystals a 3D structure to 23 A resolution has been determined by electron microscopy and image processing. On comparison with the 3D structure of the native alpha-toxin (Olofsson et al., J. Mol. Biol. 214, 299-306, 1990) the subdomains are more separated, confirming the differences found when comparing the projection maps (Olofsson et al., J. Struct. Biol. 106, 199-204, 1991). The tryptic cleavage takes place in a postulated hinge region. The results are consistent with the hypothesis that the conformational change required for inducing the membrane permeabilizing property takes place in this region. Furthermore, we present a refined projection map at approximately 10 A resolution based on the analysis of a large number of crystals using unbending methods.