Two-dimensional Crystalline Arrays Research Articles

Three-dimensional structural analysis of tetanus toxin by electron crystallography

Two-dimensional crystalline arrays of native tetanus toxin have been formed at the interface between a solution of the toxin and a phospholipid monolayer containing a ganglioside. Electron crystallographic analysis has been used to study these periodic arrays. The arrays obey the symmetry of plane group p12 1, with a = 126 A ̊ and b = 84 A ̊ , and a thickness of 90 Å (1 Å = 0.1 nm). The three-dimensional structure of tetanus toxin in negative stain is reconstructed to a nominal resolution of 14 Å from multiple tilt images. The molecule presents an asymmetric three-lobed structure and could interact with the monolayer in two possible orientations.

Crystallization of the Ca 2+-ATPase of skeletal muscle sarcoplasmic reticulum Inhibition by myotoxin a

Decavanadate produces extensive ordered arrays of Ca 2+-ATPase molecules on sarcoplasmic reticulum (SR) vesicle surfaces [(1984) J. Bioenerg. Biomembranes 16, 491–505] and the basic unit of these crystalline structures seems to be a dimer of Ca 2+-ATPase [(]983) J. Ultrastruct. Res. 24, 454–464; (1984) J. Mol. Biol. 174, 193–204]. Myotoxin a, isolated from the venom of the prairie rattlesnake Crotalus viridis viridis, is a muscle-degenerating polypeptide and its primary site of interaction is the SR membrane, where it uncouples Ca 2+-translocation from Ca 2+-dependent ATP hydrolysis [(1986) Arch. Biochem. Biophys. 246, 90–97]. The effect of myotoxin a on decavanadate-induced two-dimensional Ca 2+-ATPase crystals of SR membranes has been investigated. The toxin inhibits the formation of two-dimensional SR-membrane crystals and disrupts previously formed crystals in a time- and concentration-dependent manner, which parallels the uncoupling of ATP hydrolysis from Ca 2+ translocation. Two-dimensional crystalline arrays of the SR membrane have a typical diffraction pattern which, after myotoxin a treatment, displays a progressive loss of order. Decavanadate is an uncompetitive inhibitor of the Ca 2+-ATPase enzyme-myotoxin a complex. The present results suggest that a Ca 2+-ATPase dimer is required for coupling Ca 2+ translocation to Ca 2+-dependent ATP hydrolysis.

Effect of succinylation on images of negatively stained arrays of mitochondrial outer membrane channels

The voltage-dependent anion-selective channels of the outer membrane of Neurospora mitochondria occur in two-dimensional crystalline arrays. Electron microscopic images of negatively stained arrays have been compared for normal membranes and membranes pretreated with succinic anhydride, which changes the functional characteristics of the channel. Succinic anhydride does not alter the lattice parameters or the long-range order in the arrays. Also, it has no significant effect on correlation averages of channel arrays embedded in uranyl acetate. Thus, functional changes induced in the channel by succinic anhydride are probably not due to large-scale conformational changes. The distribution of the anionic stain phosphotungstate on the mitochondrial channel arrays is significantly altered by succinic anhydride pretreatment. There are loci on the channels of reduced phosphotungstate accumulation following succinylation. Since phosphotungstate staectively stains positively charged amino acids, it is proposed that these loci may represent clusters of functionally important, exposed basic amino acids.

Interaction of myotoxin a with the Ca 2+-ATPase of skeletal muscle sarcoplasmic reticulum

Myotoxin a is a muscle-damaging toxin isolated from the venom of Crotalus viridis viridis. Its interaction with the Ca 2+-ATPase of sarcoplasmic reticulum (SR) vesicles purified from rabbit skeletal muscle was investigated. Myotoxin a inhibited Ca 2+ loading and stimulated Ca 2+-dependent ATPase without affecting unidirectional Ca 2+ efflux. Its action was dose, time, and temperature dependent. Myotoxin a partially blocked the binding of specific anti-(rabbit SR Ca 2+-ATPase) antibodies. It is concluded that myotoxin a attaches to the SR Ca 2+-ATPase and uncouples Ca 2+ uptake from Ca 2+-dependent ATP hydrolysis. Myotoxin a also prevented the formation of decavanadate-induced two-dimensional crystalline arrays of the SR Ca2 2+-ATPase.

Structure of purple membrane from halobacterium halobium: recording, measurement and evaluation of electron micrographs at 3.5 Å resolution

Electron micrographs of the purple membrane have been recorded using liquid nitrogen and liquid helium cooling on three cryoelectron microscopes. The best micrographs show optical diffraction spots, arising from the two-dimensional crystal, out to resolutions of around 6 Å. Large areas of several of these micrographs have been analysed using a procedure which determines the strength of the very weak high resolution Fourier components of the image of the crystal. The procedure consists of reciprocal space filtering followed by real space correlation analysis to characterise image distortions, removal of the distortions by interpolation, and finally extraction of the amplitudes and phases of the Fourier components from the distortion-corrected image of the crystal. These raw image amplitudes and phases are then used, together with previously measured amplitude and phase information, to refine the beam tilt and crystal tilt, phase origin and amount of defocus and astigmatism of the image. The phases can then be corrected for the effects of the contrast transfer function, beam tilt and phase origin. The amplitudes of all the spots which are expected to be strong from their known electron diffraction intensity are observed to be significantly above the background noise level, and the independent phases from different images, and from symmetry-related directions in the same image, show excellent agreement out to a resolution of 3.5 Å. Although only images from untilted or slightly tilted ( < 5°) crystals have been analysed using the procedure described in this paper, a simple additional step enables analysis of images at any tilt angle, providing a complete practical method for high resolution analysis of images of two-dimensional crystalline arrays.

Morphological observations on the formation and stability of the crystalline arrays in the plasma membrane of Saccharomyces cerevisiae

Two-dimensional crystalline arrays of freeze-fracture particles are known to occur in abundant quantities in the plasma membrane of stationary state yeast cells. Although these crystalline arrays are seen only infrequently in cells during mid-exponential growth, we now observe that formation of crystalline arrays can be induced in such cells by a “metabolic starvation” protocol. Surprisingly, starvation-induced formation of crystalline patches can be prevented by inhibition of new protein synthesis during the starvation period. The size and quantity of crystalline arrays can be increased by removal of the cell wall prior to starvation. Induction of crystalline arrays in protoplasts has made it possible to investigate the surface morphology of the crystalline particles in isolated membranes as well as at the extracellular surface of intact protoplasts. The stability of isolated crystalline arrays to several detergents has been investigated and conditions have been found that result in improved morphological purity of the isolated crystalline patches.

Crystallization of the Ca2+-ATPase of sarcoplasmic reticulum by calcium and lanthanide ions.

Two-dimensional crystalline arrays of Ca2+-ATPase molecules develop in sarcoplasmic reticulum vesicles exposed to Ca2+ or lanthanide ions. The Ca2+- or lanthanide-induced crystals are presumed to represent the E1 conformation of the Ca2+-ATPase, and their crystal form is clearly different from the earlier described E2 crystals induced by Na3VO4 in the presence of ethylene glycol bis(beta aminoethyl ether)-N,N,N',N'-tetraacetic acid (Taylor, K. A., Dux, L., and Martonosi, A. (1984) J. Mol. Biol. 174, 193-204). Analysis of the crystalline arrays by negative staining or freeze-fracture electron microscopy reveals obliquely oriented rows of particles corresponding to individual Ca2+-ATPase molecules. Computer analysis of the negatively stained lanthanide-induced crystalline Ca2+-ATPase arrays shows that the molecules are arranged in a P1 lattice. The pear-shaped profiles of Ca2+-ATPase molecules seen in projection in the density maps are similar to those seen in vanadate-induced crystals. The space group and unit cell dimensions of the E1 crystals are consistent with Ca2+-ATPase monomers as structural units, while the vanadate-induced E2 crystals form by lateral aggregation of chains of Ca2+-ATPase dimers. The transition between the E1 and E2 conformations may involve a shift in the monomer-oligomer equilibrium of the Ca2+-ATPase. The formation of E1 crystals by PrCl3 is promoted by inside negative membrane potential, presumably through stabilization of the E1 conformation of the enzyme. Cleavage of the Ca2+-ATPase by trypsin into two major fragments (A and B) did not interfere with the Ca2+- or the Pr3+-induced crystallization.

Crystallization of intramembrane particles in rabbit sarcoplasmic reticulum vesicles by vanadate.

Sarcoplasmic reticulum (SR) membranes isolated from rabbit skeletal muscle appear in freeze-fracture as 0.15-0.2 micron vesicles. The concave fracture surface (P-face) contains a dense population of 8.5 nm particles that were previously identified as the Ca2+-transport ATPase. The convex surface (E-face) is mostly smooth, displaying an occasional particle but no complementary arrays of pits. Incubation of the vesicles at 4 degrees C in calcium-free solutions containing 5 mM Na3VO4 induces the formation of two-dimensional crystalline arrays of the Ca2+, Mg2+-ATPase, accompanied by structural changes visible by freeze-etch electron microscopy. Most vesicles elongate into tubules 60-80 nm in diameter and the 8.5 nm intramembrane particles of the P-face become regularly organized into parallel ridges. The ridges are coiled around the tubules in right-handed helices, oriented at 50-60 degrees angle to the long axis of the tubules. The particles repeat along the rows at about 5.5 nm and the rows repeat at 10.5-11.0. Occasionally the ridges seem to break up into 8.5 nm particles. Parallel furrows are visible on the (convex) E-face of the tubules. In high resolution replicas, the furrows are resolved into rows of pits that are complementary images of the ridges. Deep etching and rotary shadowing reveal oblique crests on the protoplasmic surface, consisting of dimeric particles close to 8.5 X 5.5 nm in size, in which each monomer can frequently be resolved into two structural domains. These data suggest that vanadate induces a conformational change in the Ca2+-transport ATPase, with crystallization of the intramembrane particles.

Phospholipase-induced crystallization of channels in mitochondrial outer membranes.

When outer membranes from Neurospora crassa mitochondria are treated with low levels of phospholipase A2 under continuous dialysis, two-dimensional crystalline arrays of the pore protein component of these membranes are formed.

Effect of Na3VO4 and membrane potential on the structure of sarcoplasmic reticulum membrane.

Two-dimensional crystalline arrays of Ca2+-ATPase molecules develop after treatment of sarcoplasmic reticulum vesicles with Na3VO4 in a Ca2+-free medium. The influence of membrane potential upon the rate of crystallization was studied by ion substitution using oxonol VI and 3,3'-diethyl-2,2'-thiadicarbocyanine (Di-S-C2(5] to monitor inside positive or inside negative membrane potentials, respectively. Positive transmembrane potential accelerates the rate of crystallization of Ca2+-ATPase, while negative potential disrupts preformed Ca2+-ATPase crystals, suggesting an influence of transmembrane potential upon the conformation of Ca2+-ATPase.

Three-stage melting of an annelide-type copper complex. A new type of organized phase: Tegma crystals

The solid—solid phase transition of an annlide type copper complex are described. As the temperature is raised, three different constituents of the complex successively melt. In the temperature range 77–95°C, the polyethylene oxide chains and the paraffinic tail are both in a very mobile state while the copper complex sub-units still form a two-dimensional crystalline array.

The regulation of ATPase-ATPase interactions in sarcoplasmic reticulum membrane. I. The effects of Ca2+, ATP, and inorganic phosphate.

Two-dimensional crystalline arrays of Ca2+-ATPase molecules develop after treatment of sarcoplasmic reticulum vesicles with Na3VO4 in calcium-free medium (Dux, L., and Martonosi, A. (1983) J. Biol. Chem. 258, 2599-2603). The formation of Ca2+-ATPase crystals is inhibited by Ca2+ (2 microM), or ATP (5 mM), but not by ADP, 5'-adenylylimidodiphosphate, or adenylylmethylenediphosphonate. ATPase crystals did not form at 37 degrees C and exposure of preformed crystals to 37 degrees C for 1 h caused the disappearance of crystal lattice. Inorganic orthophosphate (1 mM at pH 6.0) promoted the formation of a distinct crystal form of Ca2+-ATPase, which was different from that produced by Na3VO4. These observations indicate that Ca2+, ATP, inorganic phosphate, pH, and temperature influence the interactions between ATPase molecules in the sarcoplasmic reticulum membrane.

Membrane crystals of Ca2+-ATPase in sarcoplasmic reticulum of normal and dystrophic muscle.

Two-dimensional crystalline arrays of the Ca2+ transport ATPase develop after treatment of sarcoplasmic reticulum vesicles with Na3VO4. The dimensions of the crystal lattice are similar in sarcoplasmic reticulum membranes isolated from normal and genetically dystrophic human, mouse, and chicken muscles. These observations indicate similar requirements for ATPase-ATPase interactions in sarcoplasmic reticulum of normal and dystrophic muscles and lessen the likelihood of a molecular defect of the Ca2+ pump in the various forms of genetic muscular dystrophies.

Ca2+-ATPase membrane crystals in sarcoplasmic reticulum. The effect of trypsin digestion.

Vanadate induces the formation of two-dimensional crystalline arrays of Ca2+-ATPase molecules in sarcoplasmic reticulum. The Ca2+-ATPase membrane crystals are evenly distributed among the terminal cisternae and longitudinal tubules of sarcoplasmic reticulum, but very few crystals were observed in the T tubules. Tryptic cleavage of the Ca2+ transport ATPase into two major fragments (A and B) did not interfere with the vanadate-induced formation of membrane crystals. The ability of Ca2+-ATPase to crystallize was lost after further cleavage of the A fragment into the A1 and A2 subfragments that is known to be accompanied by loss of Ca2+ uptake. Vanadate (0.1-5 mM) inhibited the secondary cleavage of Ca2+-ATPase by trypsin suggesting that the susceptibility of the tryptic cleavage sites is influenced either by the conformation of the enzyme or by the formation of ATPase crystals.

X-ray diffraction analysis of matrix porin, an integral membrane protein from Escherichia coli outer membranes

An integral membrane protein forming channels across Escherichia coli outer membranes, porin, has been crystallized using a polyethylene glycol or salt generated two-phase system. Monodispersity and homogeneity of proteindetergent complexes were found to be prerequisites for reproducible formation of crystals amenable to X-ray structural analysis. By varying pH, detergent and buffer type, large crystals of three different habits can be obtained, two of which are discussed in this paper. The tetragonal form (space group P4 2; unit cell dimensions, a = b = 155 A ̊ , c = 172 A ̊ ) is suitable for X-ray analysis. Low temperature induces a change of the space group to P4 222, with a single trimer in the asymmetric unit. This crystal form diffracts to a resolution beyond 2.9 Å. The hexagonal crystal form (space group P6 322; unit cell dimensions, a = b = 93 A ̊ , c = 220 A ̊ ) is limited in resolution to 4.5 Å, but reveals a packing arrangement very similar to that in two-dimensional membrane-like crystalline arrays.

Structure of the S-layer of Sulfolobus acidocaldarius

The outermost component of many bacterial cell envelopes is a two-dimensional crystalline array of protein molecules, termed the S-layer1. Despite considerable effort to investigate its structure and composition, its precise function remains uncertain. We report here the first determination of the three-dimensional structure of an S-layer, that from the thermophilic bacterium, Sulfolobus acidocaldarius. Our map shows a complex and strongly interconnected structure, penetrated by numerous channels which appear to provide little barrier to anything but rather large molecules. The external surface is fairly smooth, but the cellular side is sculpted with large cavities and protruding ‘pedestals’. The protein substructure consists of three types of globular domain, connected by narrow bridges. We suggest that these may be flexible ‘hinges’, allowing the S-layer to form a curved surface, and that the multidomain structure allows the S-layer to maintain strong connectivity as it grows with the bacterium.

Artefact in 20Å-Resolution Electron Images of Negatively Stained Twodimensional Membrane Protein Crystals

A number of computing systems devoted to the averaging of electron images of two-dimensional macromolecular crystalline arrays have facilitated the visualization of negatively-stained biological structures. Either by simulation of optical filtering techniques or, in more refined treatments, by cross-correlation averaging, an idealized representation of the repeating asymmetric structure unit is constructed, eliminating image distortions due to radiation damage, stain irregularities and, in the latter approach, imperfections and distortions in the unit cell repeat. In these analyses it is generally assumed that the electron scattering from the thin negativelystained object is well-approximated by a phase object model. Even when absorption effects are considered (i.e. “amplitude contrast“), the expansion of the transmission function, q(x,y)=exp (iσɸ (x,y)), does not exceed the first (kinematical) term. Furthermore, in reconstruction of electron images, kinematical phases are applied to diffraction amplitudes and obey the constraints of the plane group symmetry.

Electron microscopy of human hepatitis B virus cores by negative staining-carbon film technique.

The ultrastructure of the nucleocapsid component of human hepatitis B virus (core particle) was studied by negative staining-carbon film technique. Using this method an improved image of core particles was obtained in respect of resolution and contrast. Two-dimensional crystalline arrays of core particles were formed in vitro. Under these arrays the distance between the particle centers was 28.3 nm, corresponding to the capsid diameter, when analyzed through optical diffraction patterns. Positively stained images of these arrays revealed that core particles contain an electron-dense center of nucleoid-like area about 21 nm in diameter. The capsid surface rarely exhibited small capsomeres, ie, small spheres or ring-like structures measuring 4.0-4.2 nm. From the dimension of these structures and the analysis by Markham's rotational technique, it was suggested that each of these capsomeres is an individual subunit (monomer) and 180 of these subunits build up the core particle capsid according to the icosahedral symmetry (T = 3), but not clustering into distinct morphological features.

Structure of NADH:Q oxidoreductase from bovine heart mitochondria studied by electron microscopy

Two-dimensional crystalline arrays of NADH:Q oxidoreductase preparations have been obtained by microdiffusion of protein dissolved in detergent against a 15 mM sodium acetate buffer of pH 5.5 containing 10% ( w v ) ammonium sulphate. Electron microscopy was used to study the structure of negatively stained crystals. Computer-reconstructed images were obtained by the Fourier peak filtering method. The crystals have p4 symmetry and a square unit cell with dimensions of 15.2 ± 0.5 nm. The four asymmetric units in the unit cell form a single tetrameric molecule with a dimension in the third direction of 8.2 nm. It is concluded on the basis of the estimated molecular mass that each tetramer cannot contain more than only one FMN molecule. This implies that the tetramers possibly are only a part of Complex I, since there is much evidence that one functional enzyme molecule of Complex I contains two FMN molecules.

Inter- and intrahexonal connections between adenovirus hexon polypeptides in the two-dimensional crystalline array

High-resolution micrographs revealed the presence of linkers between adenovirus hexon capsomers organized into two-dimensional, closely packed arrays. Examination of electron micrographs directly and by Markham's rotational integration technique permitted the establishment of the arrangement of the interhexonal connective elements within small, distinct parts of the crystalline array. Every hexon is joined to its six nearest neighbors by six times two parallel connections. Intrahexonal connections were found also among the three polypeptide subunits constituting one complete hexon.