Subunit Packing Research Articles

Formation of single-layer arrays and 6-Angstrom electron imaging of the surface layer protein of Aquaspirillum serpens

Aquaspirillum serpens possesses a surface layer protein which exhibits a regular hexagonal packing of the morphological subunits (HP-protein). Buckmire and Murray concluded that a “backing layer”, later identified to be the outer membrane, was required for HP-protein to form 2-d arrays.We developed a modified procedure of isolation of outer wall fragments. The yield was about 10 times increased, and patches with structured arrays were bigger and more frequent. Large, light arrays of good crystalline order were obtained, with which the optical diffraction patterns always show “handedness” (see Fig. 1); whereas with the dark patches, the optical diffraction patterns appear to be symmetrical. We conclude that the light arrays are real single layer protein arrays.

Reconstructions of tubulin protofilaments: Different appearances of the same structure

We compare the structures of tubulin protofilaments obtained by image reconstruction of tubulin sheets and hoops, and by X-ray diffraction of microtubules. Negatively stained specimens yield up to eight different appearances of protofilaments. They all represent the same intrinsic structure. The effects are explained by the packing of the protein subunits and the stain distribution resulting from it. They cannot be interpreted directly in terms of protein structure or composition; in particular, differences in staining cannot be attributed to microtube-associated proteins (MAPs). None of the reconstructions reproduce the X-ray structure faithfully. The discrepancies between the reconstructions are seen at resolutions of 4 nm or less. This appears to be the limit of structural fidelity of negatively stained tubulin specimens, even when the nominal resolution of the micrographs is better. Most of the differences may be defined in terms of the diffraction patterns and are therefore genuine. Additional differences become apparent after computing the 3D reconstruction and may be genuine and/or due to the data treatment. Several factors affecting the appearances are discussed.

Gap junction structures. VI. Variation and conservation in connexon conformation and packing

Correlation of structural changes in isolated gap junctions with the mechanism of channel gating is complicated by the effects of isolation procedures and the lack of a direct functional assay. The effect of variations in the isolation procedure are examined by comparison of the structures of gap junctions isolated by different protocols. X-ray diffraction data from over two hundren specimens are compared to provide a basis for identification of invariant aspects of the connexon structure and variable properties related either to functional switching or experimental modifications. We discuss the relationship between subunit tilt, lattice symmetry and packing, and membrane curvature and demonstrate that membrane curvature may be a natural consequence of the structure of the connexons and the patterns of interactions between them.

Structural analysis of the surface-layer protein of spirillum serpens by high-resolution electron microscopy

Spirillum serpens possesses a surface layer protein which exhibits a regular hexagonal packing of the morphological subunits. A morphological model of the structure of the protein has been proposed at a resolution of about 25 Å, in which the morphological unit might be described as having the appearance of a flared-out, hollow cylinder with six ÅspokesÅ at the flared end. In order to understand the detailed association of the macromolecules, it is necessary to do a high resolution structural analysis. Large, single layered arrays of the surface layer protein have been obtained for this purpose by means of extensive heating in high CaCl2, a procedure derived from that of Buckmire and Murray. Low dose, low temperature electron microscopy has been applied to the large arrays.As a first step, the samples were negatively stained with neutralized phosphotungstic acid, and the specimens were imaged at 40,000 magnification by use of a high resolution cold stage on a JE0L 100B. Low dose images were recorded with exposures of 7-9 electrons/Å2. The micrographs obtained (Fig. 1) were examined by use of optical diffraction (Fig. 2) to tell what areas were especially well ordered.

Structure of Satellite tobacco necrosis virus at 3.0 Å resolution

The structure of Satellite tobacco necrosis virus (STNV) has been determined to 3.0 Å resolution by X-ray crystallography. Electron density maps were obtained with phases based on one heavy-atom derivative and several cycles of phase refinement using the 60-fold non-crystallographic symmetry in the particle. A model for one protein subunit was built using a computer graphics display. The subunit is constructed mainly of a β-roll structure forming two β-sheets, each of four antiparallel strands. The N-termini of the subunits form bundles of three α-helices extending into the RNA region of the virus at the 3-fold axis. The topology of the polypeptide chain is the same as, and the conformation clearly similar to, that of the shell domains of the Tomato bushy stunt virus (TBSV) and Southern bean mosaic virus (SBMV) protein subunits. The subunit packing in the T = 1 STNV structure is, however, significantly different from the packing of these T = 3 viruses: parts of some of the structural elements facing the RNA in TBSV and SBMV are utilized for subunit-subunit contacts in STNV. No RNA structure is obvious in the present icosahedrally averaged electron density maps. The protein surface facing the RNA contains mainly hydrophilic residues, especially lysine and arginine.

Crystal structure of cat muscle pyruvate kinase at a resolution of 2.6 Å

The structure of pyruvate kinase (EC 2.7.1.40) has been determined from a 2.6 Å resolution electron density map. This map shows more detail than the previous 3.1 Å map (Stammers & Muirhead, 1977) and has enabled a detailed chain folding to be established for two out of the three domains which make up each of the four identical subunits. A provisional chain folding has been established for the third domain. The results have been briefly reported in a previous paper (Levine et al., 1978). Details of the structure determination and a further discussion of the results are presented in this paper. Domain A (the three domains of pyruvate kinase are referred to as A, B and C) can be described in terms of a cylindrical eight-stranded parallel β sheet and an outer coaxial cylinder of eight α helices. The α helices connect adjacent strands of the β sheet. Domain B is made up of a closed anti-parallel β sheet structure. Domain C is a five-stranded β sheet of which the fourth strand is anti-parallel and the rest parallel. These strands are also interconnected by α helices. Domain A can be dissected into eight consecutive β strand—α helix units starting from the N-terminus. The arrangement of these relative to each other can be most simply described by relating them to eight planes, each at 40 ° to the cylinder axis and symmetrically placed around the cylinder. When unit 2 is aligned with one of these planes then units 1, 3, 4, 5 and 8 are also closely aligned with a plane. This analysis is also applied to triosephosphate isomerase and a strikingly similar arrangement is found. A detailed comparison of the two structures is presented. Although the lack of a chemical sequence makes it difficult to identify the amino acid residues of pyruvate kinase, side-chains are clearly visible in the map and this information is correlated with the results of previous 6 Å substrate soaking experiments and with the structure of triosephosphate isomerase. The similarities and differences are discussed in terms of similarities and differences in the reactions catalysed and also of different subunit packing.

The quaternary structure of the sheaths of defective phages similar to PBS X.

The contractile sheaths of five defective, PBS X-like bacteriophages from Bacillus subtilis and B. licheniformis were investigated by electron microscopy, dodecylsulphate gel electrophoresis and immunodiffusion. Electron microscope images of the extended and contracted sheaths were of similar appearance, although their lengths were different. The surface lattices of both the extended and the contracted sheaths were determined by optical diffraction. This showed that the quaternary structure of the sheaths of all five defective phages originated from identical surface lattices, which could be approximately expressed by the selection rules L = -2n' + 3m and L = 9N' + 17M for the extended and contracted sheaths respectively, in which 6n' = n with n = 0 or an integer multiple of 6. These results indicated that the packing of the protein subunits in these sheaths differed from those of other bacteriophages, for example T4 and millimicron [Amos and Klug, J. Mol. Biol. 99, 51--73 (1975); Admiraal and Mellema, J. Ultrastruct. Res. 56, 48--64 (1976)]. The molecular weight of the main sheath protein of the defective phages, as determined by dodecylsulphate gel electrophoresis, was approximately 50000. This value differed from that for T4, but was similar to that of millimicron [Admiraal and Mellema, J. Ultrastruct. Res. 56, 48--64 (1976); King and Laemmli, J. Mol. Biol, 75, 315--337 (1973)]. The results of immunodiffusion experiments, however, pointed to a chemical difference between the sheath proteins of the defective phages and millimicron, in addition to T4.

A quantitative EM study op the packing and shape of the protein subunits organized in contractile sheaths

Phages for example from the T-even family have a complex structure. They consist of a head in which the DNA is packaged, a contractile sheath and a complex structure with which the phage attaches itself to the membrane of the host cell.We are studying the structural mechanism by which the sheath transmits the DNA in the recipient cell by electron microscopy and image analysis techniques. The contractile sheath of these types of phages consists of a large number of identical protein subunits which are arranged in a regular way.To study the arrangement of the subunits in five PBS X-like defective phages, from B.subtilis and B.licheniformis, negatively stained specimens were imaged by electron microscopy and the images were subjected to optical diffraction and digital analysis. The extended and contracted sheath configuration of the five defective phages were found to be similar and differed from the known cases of T4 and Mu.

Location of tyrosine residues in the disk of tobacco-mosaic-virus protein and comparison of the subunit packing with that of the virus.

The products of iodination of the disk of tobacco mosaic virus coat protein have been investigated in order to locate the reactive amino acid residues in the three-dimensional structure. Reaction occurs mainly with tyrosine-139 and, to a lesser extent, tyrosine-2 and the positions of these modified residues have been determined by X-ray crystallography. Different extents of reaction are found in the two rings of the disk and also, on adding the high salt concentration needed for stabilisation of the crystal during reaction, some conformational changes in the polypeptide chain in the inner part of the disk. Comparison of the relative positions of residues 27 and 139 in the disk and virus shows that little distortion occurs in the outer part of the subunit during the transition between the disk and virus structures.

An Analysis of the Contracted Sheath Structure of Bacteriophage Mu

Polymers of contracted sheath particles of bacteriophage Mu were imaged by the negative-staining technique and the electron images were analyzed by optical and digital methods. By means of the three-dimensional reconstruction technique of DeRosier and Klug [Nature (Lond.) 217, 130--134 (1968)] an averaged density map of the sheath structure at a resolution of about 2.0 nm was derived. The sheath is known to consist of one type of protein with a molecular weight of about 52000 [Admiraal and Mellema, J. Ultrastr. Res. 56, 48--64 (1976)]. The interpretation of the map has given information about packing and shape of the protein subunits. One way to describe the structure is by a set of annular rings with 6-fold symmetry. The height of these rings is about 1.8 nm and successive rings in the structure change by about 33 degrees in azimuth. The protein subunit which occupies more than one ring in the polymer, is elongated. The long axis of the protein subunit is at an angle of about 20 degrees with the plane normal to the polymer axis. These data are discussed in relation to changes in the sheath molecules upon contraction.

Different arrangements of protein subunits and single-stranded circular DNA in the filamentous bacterial viruses fd and Pf1

A single-stranded circular DNA molecule of 6690 ± 450 nucleotides accounts for 5.5 ± 0.3% of the mass of Pf1 virus. The remaining mass is contributed almost entirely by subunits of the major coat protein. A non-integral nucleotide to subunit ratio of 0.87 ± 0.05 is calculated from the DNA content, the average nucleotide mass (309), and the known mass of one protein subunit (4609). There are therefore 7690 ± 680 major coat protein subunits in the virus. The virus length determined by electron microscopy is 1960 ± 70 nm. The data give an average axial distance of 2.55 ± 0.24 Å between protein subunits in dry virus. Since there is an up strand and a down strand of the circular DNA within the virus filament, an axial distance between bases in a given strand of 5.9 ± 0.5 Å is calculated. Available X-ray data show that an axial repeat of 72 Å, or slightly less, would be expected for dry Pf1 virus (0% relative humidity). A structural model in which 27 protein subunits and 24 nucleotides are contained in this repeat would be consistent with our data. The DNA conformation and the subunit packing in Pf1 differ considerably from those in fd, even though both are filamentous viruses containing single-stranded circular DNA. The uncertainties cited are 95% confidence limits.

X-ray small-angle studies of the pyruvate dehydrogenase core complex from Escherichia coli K-12. I. Overall structure of the core complex.

The pyruvate dehydrogenase core complex from E. coli K-12, defined as the multienzyme complex which can be obtained with a unique polypeptide chain composition, has been investigated in solution with the X-ray small-angle technique. The molecular mass of the core complex of 3.78-10(6) daltons verifies the ratio of polypeptide chains of 16:16:16 of the three enzyme components, pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase, present in the complex. In connection with the values obtained for the radius of gyration (156.5A), volume (1.07(7) A3) and amount of solvent associated with the complex (1.03 g/g) a loose packing of subunits in the complex has to be assumed. The maximum diameter of the core complex of 433 A, as determined from the correlation function, corroborates the large extension of the complex. The comparison of experimental and theoretical scattering curves reveals a relatively isometric overall shape of the core complex.

The ultrastructure and development of tubular and crystalline P-protein in the sieve elements of certain papilionaceous legumes

The ultrastructure of the primary sieve elements of several papilionaceous legumes was studied using hypocotyl and young internode segments fixed in glutaraldehyde followed by osmium tetroxide. In particular, the study sought to determine whether the crystalline “flagellar inclusions” characteristic of these species are developmentally related to the P-protein bodies present in the phloem of these and other legumes and of angiosperms generally. The crystalline inclusions consist of a central body terminated at one or both ends by a gradually tapering tail. The central body is usually spindle shaped in longitudinal section and square in cross section. In all species examined, the inclusion is first seen as a small, thin crystal in the cytoplasm of young sieve elements. The crystal enlarges and acquires tails as the sieve element develops. In certain species, exemplified byDesmodium canadense, numerous tubules are formed in the cytoplasm near the crystal and appear to be concerned in its growth. The observations on the structure and interactions of these two components, tubules and crystalline inclusions, suggest that both represent forms of P-protein: the tubules are continuous with the crystal and are striated like the crystal near the tubule-crystal junction, suggesting that they are adding onto the crystal body; the tubules closely resemble the P-protein tubules described in the literature in that they measure 157 A in diameter, accumulate in spindle-shaped bundles, and disperse into striated fibrils late in the ontogeny of the sieve element; and finally, the crystal also disperses into fine filaments. The crystalline inclusion therefore probably represents still another aggregation state of P-protein, one that is characteristic of papilionaceous legumes. The different stages of crystal aggregation and the diverse forms of P-protein now known are discussed briefly in relation to the control of macromolecular assembly and subunit packing.

Fine structure of trichocyst fibrils of the dinoflagellate Peridinum westii

Electron micrographs of cells of the dinoflagellate Peridinium westii showed an intracellular accumulation of cross-striated fibrils (trichocysts) during cellular aging and degeneration. Negative staining of isolated fibrils revealed an axial repeating period of 650–700 A with distinct intraperiod bands, and a lateral packing of filamentous subunits. The amino acid profile of the isolated fibrils was not similar to that of collagen.

Electro-optic evidence for the control of the structure of bacteriophage f1 by a minor coat protein

The structures of the filamentous bacteriophage f1 and its gene 3 amber mutant R4 have been compared using electric birefringence, electric dichroism, and ultrasonic vibration. The electro-optic experiments showed that the phage particles can be oriented in an electric field. The birefringence and dichroism as a function of field strength are not the same for the A-protein mutant and for the wild-type particle. Studies using ultrasonic vibration to fragment the bacteriophage show that the stabilities of the f1 and R4 particles differ. We have interpreted the structural differences between the particles as reflecting alterations in the arrangement of the subunits of the major capsid protein, the B-protein; further, the observed suppressor dependence indicates that the subunit packing is controlled by the A-protein. Models for the mechanism of A-protein function are discussed.

Symmetry in virus architecture

Virus particles (virions) fall into three main morphological groups characterized by (1) helical symmetry, (2) cubic symmetry, and (3) other symmetries. In this paper geometrical aspects of particles have been discussed in the light of recent evidence. Especial attention has been given to the limitations of packing of morphological subunits (capsomeres) in the shells (capsids) of virions with cubic symmetry. Four series of packing arrangements of hexagonal and pentagonal capsomeres in capsids with 5:3:2 symmetry have been considered. Of these, the series of potentially icosahedral capsids where capsomere numbers are given by 10( n − 1) 2 + 2 has so far been encountered most often; it is believed that this arrangement is most likely because it involves least strain. The discussion has been extended to consider briefly- function in terms of the structure of the virion. Finally, the validity of some morphological criteria for classification is discussed.