Three-dimensional structure of rhesus rotavirus by cryoelectron microscopy and image reconstruction

The structure of rhesus rotavirus was examined by cryoelectron microscopy and image analysis. Three-dimensional reconstructions of infectious virions were computed at 26- and 37-A resolution from electron micrographs recorded at two different levels of defocus. The major features revealed by the reconstructions are (a) both outer and inner capsids are constructed with T = 13l icosahedral lattice symmetry; (b) 60 spikelike projections, attributed to VP4, extend at least 100 A from the outer capsid surface; (c) the outer capsid, attributed primarily to VP7, has a smoothly rippled surface at a mean radius of 377 A and is perforated by 132 aqueous holes ranging from 40-65 A in diameter; (d) the inner capsid has a "bristled" outer surface composed of 260 trimeric-shaped columns of density, attributed to VP6, which merge with a smooth, spherical shell of density at a lower, mean radius of 299 A, and which is perforated by holes in register with those in the outer capsid; (e) a "core" region contains a third, nonspherical shell of density at a mean radius of 225 A that encapsidates the double-stranded RNA genome; and (f) the space between the outer and inner capsids forms an open aqueous network that may provide pathways for the diffusion of ions and small regulatory molecules as well as the extrusion of RNA. The assignment of different viral structural proteins to specific features of the reconstruction has been tentatively made on the basis of excluded volume estimates and previous biochemical characterizations of rotavirus.     

Three-dimensional structure of the rotavirus haemagglutinin VP4 by cryo-electron microscopy and difference map analysis.

The three-dimensional structure of the rotavirus spike haemagglutinin viral protein 4 (VP4) has been determined to a resolution of 26 A by cryo-electron microscopy and difference analysis of intact virions and smooth (spikeless) particles. Native and spikeless virions were mixed prior to cryo-preservation so that both structures could be determined from the same micrograph, thereby minimizing systematic errors. This mixing strategy was crucial for difference map analysis since VP4 only accounts for approximately 1% of the virion mass. The VP4 spike is multi-domained and has a radial length of approximately 200 A with approximately 110 A projecting from the surface of the virus. Interactions between VP4 and cell surface receptors are facilitated by the bi-lobed head, which allows multi-site interactions, as well as the uniform distribution of the VP4 heads at maximum radius. The bi-lobed head is attached to a square-shaped body formed by two rods that have a slight left-handed helical twist. These rods merge with an angled, rod-like domain connected to a globular base approximately 85 A in diameter. The anchoring base displays pseudo 6-fold symmetry. This surprising finding may represent a novel folding motif in which a single polypeptide of VP4 contributes similar but non-equivalent domains to form the arms of the hexameric base. The VP4 spike penetrates the virion surface approximately 90 A and interacts with both outer (VP7) and inner (VP6) capsid proteins. The extensive VP4-VP7 and VP4-VP6 interactions imply a scaffolding function in which VP4 may participate in maintaining precise geometric register between the inner and outer capsids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Three-dimensional biofilm structure quantification

Quantitative parameters describing biofilm physical structure have been extracted from three-dimensional confocal laser scanning microscopy images and used to compare biofilm structures, monitor biofilm development, and quantify environmental factors affecting biofilm structure. Researchers have previously used biovolume, volume to surface ratio, roughness coefficient, and mean and maximum thicknesses to compare biofilm structures. The selection of these parameters is dependent on the availability of software to perform calculations. We believe it is necessary to develop more comprehensive parameters to describe heterogeneous biofilm morphology in three dimensions. This research presents parameters describing three-dimensional biofilm heterogeneity, size, and morphology of biomass calculated from confocal laser scanning microscopy images. This study extends previous work which extracted quantitative parameters regarding morphological features from two-dimensional biofilm images to three-dimensional biofilm images. We describe two types of parameters: (1) textural parameters showing microscale heterogeneity of biofilms and (2) volumetric parameters describing size and morphology of biomass. The three-dimensional features presented are average (ADD) and maximum diffusion distances (MDD), fractal dimension, average run lengths (in X, Y and Z directions), aspect ratio, textural entropy, energy and homogeneity. We discuss the meaning of each parameter and present the calculations in detail. The developed algorithms, including automatic thresholding, are implemented in software as MATLAB programs which will be available at site prior to publication of the paper.     

Three-dimensional structure of the anaphase-promoting complex

The anaphase-promoting complex (APC) is a cell cycle-regulated ubiquitin-protein ligase, composed of at least 11 subunits, that controls progression through mitosis and G1. Using cryo-electron microscopy and angular reconstitution, we have obtained a three-dimensional model of the human APC at a resolution of 24 A. The APC has a complex asymmetric structure 140 A x 140 A x 135 A in size, in which an outer protein wall surrounds a large inner cavity. We discuss the possibility that this cavity represents a reaction chamber in which ubiquitination reactions take place, analogous to the inner cavities formed by other protein machines such as the 26S proteasome and chaperone complexes. This cage hypothesis could help to explain the great subunit complexity of the APC.     

Three-dimensional structure of the gamma-secretase complex

Gamma-secretase belongs to an atypical class of aspartic proteases that hydrolyzes peptide bonds within the transmembrane domain of substrates, including amyloid-beta precursor protein and Notch. gamma-Secretase is comprised of presenilin, nicastrin, APH-1, and PEN-2 which form a large multimeric membrane protein complex, the three-dimensional structure of which is unknown. To gain insight into the structure of this complex enzyme, we purified functional gamma-secretase complex reconstituted in Sf9 cells and analyzed it using negative stain electron microscopy and 3D reconstruction techniques. Analysis of 2341 negatively stained particle images resulted in the three-dimensional representation of gamma-secretase at a resolution of 48 angstroms. The structure occupies a volume of 560 x 320 x 240 angstroms and resembles a flat heart comprised of two oppositely faced, dimpled domains. A low density space containing multiple pores resides between the domains. Some of the dimples in the putative transmembrane region may house the catalytic site. The large dimensions are consistent with the observation that gamma-secretase activity resides within a high molecular weight complex.     

Three-dimensional structure of human follicle-stimulating hormone

The crystal structure of a betaThr26Ala mutant of human follicle-stimulating hormone (hFSH) has been determined to 3.0 A resolution. The hFSH mutant was expressed in baculovirus-infected Hi5 insect cells and purified by affinity chromatography, using a betahFSH-specific monoclonal antibody. The betaThr26Ala mutation results in elimination of the betaAsn24 glycosylation site, yielding protein more suitable for crystallization without affecting the receptor binding and signal transduction activity of the glycohormone. The crystal structure has two independent hFSH molecules in the asymmetric unit and a solvent content of about 80%. The alpha- and betasubunits of hFSH have similar folds, consisting of central cystine-knot motifs from which three beta-hairpins extend. The two subunits associate very tightly in a head-to-tail arrangement, forming an elongated, slightly curved structure, similar to that of human chorionic gonadotropin (hCG). The hFSH heterodimers differ only in the conformations of the amino and carboxy termini and the second loop of the beta-subunit (L2beta). Detailed comparison of the structures of hFSH and hCG reveals several differences in the beta-subunits that may be important with respect to receptor binding specificity or signal transduction. These differences include conformational changes and/or differential distributions of polar or charged residues in loops L3beta (hFSH residues 62-73), the cystine noose, or determinant loop (residues 87-94), and the carboxy-terminal loop (residues 94-104). An additional interesting feature of the hFSH structure is an extensive hydrophobic patch in the area formed by loops alphaL1, alphaL3, and betaL2. Glycosylation at alphaAsn52 is well known to be required for full signal transduction activity and heterodimer stability. The structure reveals an intersubunit hydrogen bonding interaction between this carbohydrate and betaTyr58, an indication of a mechanism by which the carbohydrate may stabilize the heterodimer.     

Three-dimensional solution structure of apo-neocarzinostatin.

Two and three-dimensional solution nuclear magnetic resonance studies of the 11K apoprotein from natural antitumor agent neocarzinostatin (NCS) were extended to elucidation of the high-resolution structure by the use of restrained molecular dynamics computations. The refined structures attained convergency upon three steps of iterative calculations, in which more distance restraints were extracted from experimental data, and the existing distance bounds were optimized on the basis of computed structures. The solution structures of apo-NCS contain seven antiparallel beta-strands, which form two closely located beta-sheets and a short beta-segment. This protein lacks any alpha-helical component. The alignment of the seven beta-strands gives rise to a beta-barrel with an elongated diameter in one direction. The global structure of apo-NCS resembles that of the Ig-fold domain found in immunoglobulins and other structurally related beta-proteins. Residues responsible for side-chain packing and the possible salt-bridge formation important for protein folding were identified. Neocarzinostatin and the analogous proteins are known to exert their biological activity through the interaction of DNA with a chromophoric molecule, which is non-covalently bound to the apo-proteins. This molecular chromophore-binding site in apo-NCS is made of a cavity consisting of residues from the four-beta-stranded sheet and the short beta-segment. Although the solution structures of apo-NCS are similar to that of the analogous apoauromomycin in the crystalline state, difference in the shape of the binding cavities between the two was found. This study provides a structural basis for characterization of the specific recognition and molecular mechanism of the antitumor NCS chromophore binding to its host protein.     

Three-dimensional solution structure of Acanthamoeba profilin-I

We have determined a medium resolution three-dimensional solution structure of Acanthamoeba profilin-I by multidimensional nuclear magnetic resonance spectroscopy. This 13-kD actin binding protein consists of a five stranded antiparallel beta sheet flanked by NH2- and COOH-terminal helices on one face and by a third helix and a two stranded beta sheet on the other face. Data from actin-profilin cross- linking experiments and the localization of conserved residues between profilins in different phyla indicate that actin binding occurs on the molecular face occupied by the terminal helices. The other face of the molecule contains the residues that differ between Acanthamoeba profilins-I and II and may be important in determining the difference in polyphosphoinositide binding between these isoforms. This suggests that lipids and actin bind to different faces of the molecule.     

Three-dimensional structure of a membrane-microtubule complex

The unicellular algae Distigma proteus contain a group of aligned microtubules associated with their cell membrane. The association is maintained in isolated membrane fragments. The membrane-microtubule complex also includes a crystalline array of membrane particles. The major peptide component of this array was identified by labeling whole cells with radioiodine. The entire complex of membrane, particles, and microtubules is sufficiently well ordered to permit reconstruction from electron micrographs by Fourier techniques. A three-dimensional model of the membrane array at a nominal resolution of 2.5 nm has been calculated. Some similarities were apparent between lattice spacings in the membrane array and in microtubules. Analysis of these lattice correlations suggests a way in which the array of membrane particles may serve as scaffolding for microtubule attachment.     

Three-dimensional structure analysis of PROSITE patterns

Pattern matches for each of the sequence patterns in PROSITE, a database of sequence patterns, were searched in all protein sequences in the Brookhaven Protein Data Bank (PDB). The three-dimensional structures of the pattern matches for the 20 patterns with the largest numbers of hits were analysed. We found that the true positives have a common three-dimensional structure for each pattern; the structures of false positives, found for six of the 20 patterns, were clearly different from those of the true positives. The results suggest that the true pattern matches each have a characteristic common three-dimensional structure, which could be used to create a template to define a three-dimensional functional pattern.     

Three-dimensional structure of acylphosphatase. Refinement and structure analysis.

We report here the complete determination of the solution structure of acylphosphatase, a small enzyme that catalyses the hydrolysis of organic acylphosphates, as determined by distance geometry methods based on nuclear magnetic resonance information. A non-standard strategy for the distance geometry calculations was used and is described here some detail. The five best structures were then refined by restrained energy minimization and molecular dynamics in order to explore the conformational space consistent with the experimental data. We address the question of whether the solution structure of acylphosphatase follows the general principles of protein structure, i.e. those learned from analysing crystal structures. Static and dynamic features are discussed in detail. An uncommon beta-alpha-beta motif, so far found only in procarboxypeptidase B and in an RNA-binding protein, is present in acylphosphatase.     

Three-dimensional structure of bacteriophage T4 baseplate

The baseplate of bacteriophage T4 is a multiprotein molecular machine that controls host cell recognition, attachment, tail sheath contraction and viral DNA ejection. We report here the three-dimensional structure of the baseplate-tail tube complex determined to a resolution of 12 A by cryoelectron microscopy. The baseplate has a six-fold symmetric, dome-like structure approximately 520 A in diameter and approximately 270 A long, assembled around a central hub. A 940 A-long and 96 A-diameter tail tube, coaxial with the hub, is connected to the top of the baseplate. At the center of the dome is a needle-like structure that was previously identified as a cell puncturing device. We have identified the locations of six proteins with known atomic structures, and established the position and shape of several other baseplate proteins. The baseplate structure suggests a mechanism of baseplate triggering and structural transition during the initial stages of T4 infection.     

Three-dimensional structure of rat acid phosphatase.

The crystal structure of recombinant rat prostatic acid phosphatase was determined to 3 A resolution with protein crystallographic methods. The enzyme subunit is built up of two domains, an alpha/beta domain consisting of a seven-stranded mixed beta-sheet with helices on both sides of the sheet and a smaller alpha domain. Two disulfide bridges between residues 129-340 and 315-319 were found. Electron density at two of the glycosylation sites for parts of the carbohydrate moieties was observed. The dimer of acid phosphatase is formed through two-fold interactions of edge strand 3 from one subunit with strand 3 from the second subunit, thus extending the beta-sheet from seven to 14 strands. Other subunit-subunit interactions involve conserved residues from loops between helices and beta-strands. The fold of the alpha/beta domain is similar to the fold observed in phosphoglycerate mutase. The active site is at the carboxy end of the parallel strands of the alpha/beta domain. There is a strong residual electron density at the phosphate binding site which probably represents a bound chloride ion. Biochemical properties and results from site-directed mutagenesis experiments of acid phosphatase are correlated to the three-dimensional structure.     

Three-dimensional structure of a bacterial oxalate transporter

The major facilitator superfamily (MFS) represents one of the largest classes of evolutionarily related membrane transporter proteins. Here we present the three-dimensional structure at 6.5 A resolution of a bacterial member of this superfamily, OxlT. The structure, derived from an electron crystallographic analysis of two-dimensional crystals, reveals that the 12 helices in the OxlT molecule are arranged around a central cavity, which is widest at the center of the membrane. The helices divide naturally into three groups: a peripheral set comprising helices 3, 6, 9 and 12; a second set comprising helices 2, 5, 8 and 11 that faces the central substrate transport pathway across most of the length of the membrane; and a third set comprising helices 1, 4, 7 and 10 that participate in the pathway either on the cytoplasmic side (4 and 10) or on the periplasmic side (1 and 7). Overall, the architecture of the protein is remarkably symmetric, providing a compelling molecular explanation for the ability of such transporters to carry out bi-directional substrate transport.     

Three-dimensional structure of kynureninase from Pseudomonas fluorescens

Kynureninase [E.C. 3.7.1.3] is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes the hydrolytic cleavage of l-kynurenine to anthranilic acid and l-alanine. Sequence alignment with other PLP-dependent enzymes indicated that kynureninase is in subgroup IVa of the aminotransferases, along with nifS, CsdB, and serine-pyruvate aminotransferase, which suggests that kynureninase has an aminotransferase fold. Crystals of Pseudomonas fluorescens kynureninase were obtained, and the structure was solved by molecular replacement using the CsdB coordinates combined with multiple isomorphous heavy atom replacement. The coordinates were deposited in the PDB (ID code 1QZ9). The structure, refined to an R factor of 15.5% to 1.85 A resolution, is dimeric and has the aminotransferase fold. The structure also confirms the prediction from sequence alignment that Lys-227 is the PLP-binding residue in P. fluorescens kynureninase. The conserved Asp-201, expected for an aminotransferase fold, is located near the PLP nitrogen, but Asp-132 is also strictly conserved and at a similar distance from the pyridinium nitrogen. Mutagenesis of both conserved aspartic acids shows that both contribute equally to PLP binding, but Asp-201 has a greater role in catalysis. The structure shows that Tyr-226 donates a hydrogen bond to the phosphate of PLP. Unusual among PLP-dependent enzymes, Trp-256, which is also strictly conserved in kynureninases from bacteria to humans, donates a hydrogen bond to the phosphate through the indole N1-hydrogen.