Muscle contraction involves the cyclic interaction of the myosin cross-bridges with the actin filament, which is coupled to steps in the hydrolysis of ATP. While bound to actin each cross-bridge undergoes a conformational change, often referred to as the "power stroke", which moves the actin filament past the myosin filaments; this is associated with the release of the products of ATP hydrolysis and a stronger binding of myosin to actin. The association of a new ATP molecule weakens the binding again, and the attached cross-bridge rapidly dissociates from actin. The nucleotide is then hydrolysed, the conformational change reverses, and the myosin cross-bridge reattaches to actin. X-ray crystallography has determined the structural basis of the power stroke, but it is still not clear why the binding of actin weakens that of the nucleotide and vice versa. Here we describe, by fitting atomic models of actin and the myosin cross-bridge into high-resolution electron cryo-microscopy three-dimensional reconstructions, the molecular basis of this linkage. The closing of the actin-binding cleft when actin binds is structurally coupled to the opening of the nucleotide-binding pocket.

Elucidation of the molecular contacts between actin and myosin is central to understanding the force-generating process in muscle and other cells. Actin, a highly conserved globular protein found in all eukaryotes, polymerizes into filaments (F-actin) for most of its biological functions. Myosins, which are more diverse in sequence, share a conserved globular head of about 900 amino acids in length (subfragment-1 or S1) at the N-terminal end of the molecule. S1 contains all the elements necessary for mechano-chemical force transduction in vitro. Here we report an atomic model for the actomyosin complex produced by combining the atomic X-ray structure of F-actin and chicken myosin S1 with a three-dimensional reconstruction from electron micrographs of frozen-hydrated F-actin decorated with recombinant Dictyostelium myosin S1. The accuracy of the reconstruction shows the position of actin and myosin molecules unambiguously.

Microemulsions were rapidly frozen in liquid propane by using a newly developed mechanical plunging device. Fracturing in vacuo, Ta-W shadowing, and replication of the fracture face permit the discrimination between waterand oil-rich domains by transmission electron microscopy. The experimental procedure is described in detail and the possible influence of freezing artifacts is carefully considered. Images are shown for microemulsions with varying water-to-oil ratio at constant amphiphile concentration, 7 wt %, for the system H20-«-octane-C12E5. The images support the notion of a bicontinuous network at comparable amounts of H20 and n-octane. For the system D20-/i-decane-A0T a water in oil droplet structure at equal volumes of water and oil with 20 wt % of AOT is observed. Dilution of this solution with oil leads to smaller droplet number densities at about constant droplet size. Also for the system H20-n-dodecane-DDAB water in oil droplet structures are observed. For the classical microemulsion system H20-n-dodecane-l -hexanol-potassium oleate the freezing behavior seems to indicate the existence of a droplet structure for this system as well. Thus, we find from our freeze fracture electron micrographs general support of results on the microstructure concluded from different techniques in the literature.

We show images of dilute lamellar (La) and anomalous isotropic (L3) phases obtained by freeze fracture electron microscopy. The images of the La phase show stacks of bilayers. In some parts, undulations of the bilayers may be seen. The images from the L3 phase show an apparently bicontinuous network of two aqueous subvolumes separated by a random bilayer network. Saddle point structures with a mean curvature close to zero and a negative Gaussian curvature are observed. The bilayers are separated by several hundred angstroms, and the repeat distances seen on the images agree with those measured previously by SANS. Furthermore, the transition from La to LB through the intermediate state of passage formation seems to be captured. indebted to him for his continuing support. G.P.

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