Lipid-layer crystallization and preliminary three-dimensional structural analysis of AcrA, the periplasmic component of a bacterial multidrug efflux pump.

The multidrug efflux complex AcrAB-TolC confers intrinsic drug resistance in Escherichia coli by pumping antibiotics out of the cell. We determined a low-resolution (20 A) structure of AcrA, the periplasmic component, by electron crystallography. Expressed with a His-tag at its carboxyl-terminus, the protein bound to lipid layers containing the nickel-chelating phospholipid DOGS-NTA. Under the lipid layers, AcrA crystallized in layer group P2(1)22, with a unit cell size of 157 by 95 A and a thickness of about 100 A. The four asymmetric units in the unit cell are organized into what appears to be two rings, each with a central opening of 30 A in diameter. Within each ring, the density can be interpreted as following a pseudo-helical path, approximately 210 A long. This length matches that of monomeric AcrA in solution, previously estimated by light scattering and hydrodynamic measurements. On one side the density has a tubular shape, with a thickness of about 25 A, while on the other side the densities of the upper and lower parts of the pseudo-helical path are fused into a shield.

Membrane-bound 3D structures reveal the intrinsic flexibility of annexin VI.

Several quasi-ordered arrays and three two-dimensional crystal forms of annexin VI were obtained on artificial lipid monolayers. Three-dimensional reconstructions of the crystal forms exhibit marked differences in the orientations of the two lobes, revealing flexibility of the linker between the two lobes of annexin VI. Evidence is presented that the lobes may bind the monolayer in a parallel orientation, or an antiparallel orientation, in which the second lobe is turned away from the monolayer. It is hypothesized that annexin VI may also adopt several conformations in vivo, underlying different functional roles.

Crystal structure of bovine annexin VI in a calcium-bound state.

The crystal structure of a calcium-bound form of bovine annexin VI has been determined with X-ray diffraction data to 2.9 A by molecular replacement. Six Ca2+ ions were found, five in AB loops, one in a DE loop. Two loops (II-AB, which binds calcium, and V-AB, which does not) have conformations that differ significantly from those in calcium-free, human recombinant annexin VI. There are only small differences between the calci- and the apo-annexin VI in the rest of the molecule. Calcium by itself does not promote a major conformational change.

Electron crystallography of macromolecular periodic arrays on phospholipid monolayers.

Electron crystallography has the potential of yielding structural information equivalent to x-ray diffraction. The major difficulty has been preparing specimens with the required structural order and size for diffraction and imaging in the electron microscope. 2D crystallization on phospholipid monolayers is capable of fulfilling both of these requirements. Crystals can form as a result of specific interactions with a protein's ligand or an analog, suitably linked to a lipid tail; or on a surface of complementary head-group charge. With such choices, the availability of a suitable lipid is limited only by synthetic chemistry. Ultimately, it is the quality and regularity of the protein-protein interactions that determine the crystalline order, as it is with any protein crystal. In the case of streptavidin, the monolayer crystal diffracts beyond 2.5 A. A 3 A projection map reconstructed from electron diffraction amplitudes and phases from images shows density which can be interpreted as beta-sheets and clusters of side chains. It remains to be shown that the monolayer crystals are flat and diffract as well at high tilt angle as untilted. Technological issues such as charging must be resolved. With parallel advances in data collection and processing, electron crystallography of monolayer macromolecular crystals will eventually take its place beside x-ray crystallography and NMR as a routine and efficient structural technique.

The crystal structure of annexin VI indicates relative rotation of the two lobes upon membrane binding.

The crystal structure of bovine liver annexin VI has been determined to low resolution by molecular replacement. The first lobe (domains 1-4) is rotated about 90 degrees relative to the second lobe (domains 5-8). Since the same crystal form (P4(3), 68 X 68 X 205 A) grew from (NH4)2SO4, polyethylene glycol, and sodium acetate with and without added calcium, this probably reflects the structure in solution. When bound to a lipid monolayer both lobes of annexin VI are coplanar. This implies a significant change in conformation upon binding to membranes.

Visualization of beta-sheets and side-chain clusters in two-dimensional periodic arrays of streptavidin on phospholipid monolayers by electron crystallography.

The biotin-binding protein streptavidin was crystallized as two-dimensional periodic arrays on biotinylated phospholipid monolayers. Electron diffraction patterns and images of the arrays embedded in vitreous ice were recorded to near-atomic resolution. Amplitudes and phases of structure factors were computed and combined to produce a 3 A projection density map. The reliability of the map was verified by comparing it to the available x-ray atomic model of the molecule. Projection densities from beta-strands and some amino acid side chains were identified from the electron cryomicroscopy map. These results demonstrate the first near-atomic image of this type of protein periodic array by electron crystallography, which has a great potential to aid in the structural characterization of molecular arrays engineered on a monolayer for various basic or biotechnological applications.

Electron cryomicroscopy of Bacillus stearothermophilus 50 S ribosomal subunits crystallized on phospholipid monolayers.

50 S ribosomal subunits from Bacillus stearothermophilus have been crystallized as 2-dimensional periodic arrays on phospholipid monolayer films at the water-air interface. These crystals were preserved in vitreous ice and imaged with 100 keV electrons under low dose and low temperature conditions. The unit cell parameters of the crystals are a = 371.3(+/- 3.8) A, b = 152.3(+/- 1.6) A, gamma = 96.3(+/- 1.0) degrees. Some of the image arrays of these crystals have twofold rotational symmetry with a phase residual of less than 25 degrees. The mean figure of merit of the merged structure factors from these image arrays out to 20 A resolution is higher than 0.87. The 2-dimensional projection map shows a level of detail not seen in previous structural studies of the 50 S ribosome subunit. Some of these features may be related to the current 3-dimensional model of the subunit. This analysis illustrates the potential of using the electron crystallographic approach for determining the 3-dimensional structure of the 50 S ribosomal subunit crystallized on a monolayer surface. In addition, the structural information retrieved by electron crystallography might be useful for phasing X-ray data towards an atomic resolution model of the ribosome.

Preliminary electron crystallographic analysis of ice-embedded tropomyosin crystals.

Electron images and diffraction patterns of ice-embedded tropomyosin crystalline sheets have been recorded at 100 and 400 kV. Optical diffractograms from the images indicated an elongated, centered unit cell with a = 799.2 +/- 10.6 A, b = 55.1 +/- 3.5 A. Evaluation of the phases in the computed Fourier transforms up to 7 A resolution revealed the presence of symmetry axes consistent with two-dimensional space group cmm. Electron diffraction patterns show diffuse arcs and discrete sampling at a resolution of 5.1 A, arising from the alpha-helical coiled-coil features of the molecule. These results demonstrate that tropomyosin thin sheets are highly ordered and suggest that retrieval of its high-resolution three-dimensional structure may be feasible by electron crystallography.

Differential effects of ryanodine and tetracaine on charge movement and calcium transients in frog skeletal muscle.

1. Charge movement and myoplasmic calcium transients were simultaneously recorded from frog skeletal muscle fibres by using the double-seal Vaseline-gap technique. Calcium transients were monitored with the fluorescent indicator Rhod-2. 2. Ryanodine modified the kinetics and the total amount of charge moved during depolarizing pulses (Q(on)), while it did not significantly modify the charge after repolarization (Q(off)). The extracellular application of 100 microM-ryanodine elicited a temporary initial increase of the delayed component of charge movement (Q gamma) and the calcium transient. Both phenomena were later blocked with the same temporal course and to the same extent. 3. The blockade of Q gamma and the calcium transient was also observed with ryanodine concentrations of 1-10 microM. For membrane potentials positive to -10mV, the Qon measured was larger in the presence of ryanodine; Qoff was not modified. 4. Tetracaine (400-500 microM) blocked a similar delayed component of Qon, identified as Q gamma, as well as the calcium transient monitored simultaneously. This effect was observed in the first minutes after the addition of tetracaine to the extracellular solution. 5. Tetracaine blocked a faster initial component of Qon for voltages positive to -10 mV, corresponding to the voltage range of activation of the calcium current. At these same membrane potentials, Qoff was also reduced to a similar extent to Qon. 6. Ryanodine and tetracaine showed different effects on calcium current. Whereas the slow calcium current was not modified upon the addition of ryanodine, it was completely blocked in the presence of tetracaine. The blockade of the slow calcium current made evident the fast calcium current. The effects of tetracaine on the charge movement, the calcium transient and the slow calcium current were reversible. 7. These results suggest that ryanodine and tetracaine may act at different sites. Ryanodine exerts its effect on the sarcoplasmic reticulum ryanodine receptor, blocking calcium release and Q gamma, while tetracaine at these concentrations may affect the release channel and the dihydropyridine receptor, causing a blockade of the charge movement, calcium transient and calcium current.

Repetitive stimulation increases the activation rate of skeletal muscle Ca2+ currents.

Voltage clamp experiments were conducted in frog skeletal muscle using repetitive stimulation protocols. The activation rate of Ca2+ currents increased by prepulses to depolarizing potentials or by stimulating the fiber with a frequency of 1.7 Hz at 0 mV. The effect was observable with Ca2+ or Ba2+ ions, and was clearly voltage-dependent. Physiologically, it is relevant that such activation rate increase can take place during a train of action potentials.

Skeletal muscle Ca2+ channels.

Ca2+ channels are widely distributed among different cell types. We shall describe in this paper kinetic properties of voltage-dependent slow Ca2+ channels in mammalian and frog skeletal muscle fibres. In addition, recent data on a fast-activated Ca2+ channel will be presented. Finally, the possible physiological role of the channel will be considered.