Structural analysis of the bacteriophage T3 head-to-tail connector

The connector protein of bacteriophage T3, p8, has been overexpressed in Escherichia coli. Purification of the oligomers built by several copies of p8 reveals a mixed population of dodecamers and tridecamers. The percentages of these two types of oligomers differ in every culture growth, indicating that assembly of this protein depends upon the conditions of the expression system. Those cultures that generated a majority of dodecamers allowed, after purification of the connectors, the two-dimensional crystallization of the dodecamers in a tetragonal arrangement, while the tridecamers did not form crystals. The processing and averaging of several images of frozen-hydrated crystals and their internal phase comparison shows that the crystals are arranged in a P42(1)2 space group, with cell unit dimensions of 165 x 165 A. The three-dimensional reconstruction generated with images of crystals ranging from 0 degrees to 60 degrees tilt reveals a wide domain surrounded by 12 protrusions and a narrow domain that serves to interact with the tail of the bacteriophage. A channel runs along the connector wide enough to allow the translocation of a double-stranded DNA molecule into the prohead. The general structure of the T3 connector is very similar to those obtained for other nonrelated bacteriophages and strongly suggests that the shape of this important viral structure is intimately related to its function.     

Three-dimensional reconstruction of the connector of bacteriophage phi 29 at 1.8 nm resolution

The three-dimensional reconstruction of the connector of bacteriophage phi 29 has been obtained from tilt series of negatively stained tetragonal ordered aggregates under low-dose conditions and up to a resolution of (1/1.8) nm-1. These connectors are built up as dodecamers of only one structural polypeptide (p10). Two connectors form the crystal unit cell, each one facing in the opposite direction with respect to the plane of the crystal and partially overlapping. The main features of the two connectors that build the unit cell were essentially the same, although they were negatively stained in slightly different ways, probably due to their situations with respect to the carbon-coated support grid. The main features of the phi 29 connector structure revealed by this three-dimensional reconstruction are: the existence of two clearly defined domains, one with a diameter of around 14 nm and the other narrower (diameter approximately equal to 7.5 nm); an inner hole running all along the structure (around 7 to 8 nm in height) with a cylindrical profile and an average diameter of 4 nm; a general 6-fold symmetry along the whole structure and a 12-fold one in the wider domain; a clockwise twist of the more contrasted regions of both domains from the narrower towards the wider domain (the direction of DNA encapsidation). These features are compatible with an active role for the connector in the process of DNA packaging.     

The bacteriophage phi29 head-tail connector imaged at high resolution with the atomic force microscope in buffer solution.

The surfaces of two- and three-dimensional phi29 connector crystals were imaged in buffer solution by atomic force microscopy (AFM). Both topographies show a rectangular unit cell with dimensions of 16.5 nm x 16.5 nm. High resolution images of connectors from the two-dimensional crystal surface show two connectors per unit cell confirming the p42(1)2 symmetry. The height of the connector was estimated to be at least 7.6 nm, a value close to that found in previous studies using different techniques. The 12 subunits of the wide connector domain were clearly resolved and showed a right-handed vorticity. The channel running along the connector had a diameter of 3.7 nm in the wide domain, while it was 1.7 nm in the narrow domain end, thus suggesting a tronco-conical channel shape. Moreover, the narrow connector end appears to be rather flexible. When the force applied to the stylus was between 50 and 100 pN, the connector end was fully extended. At forces of approximately 150 pN, these ends were pushed towards the crystal surface. The complementation of the AFM data with the three-dimensional reconstruction obtained from electron microscopy not only confirmed the model proposed, but also offers new insights that may help to explain the role of the connector in DNA packing.     

Structure of the connector of bacteriophage T7 at 8A resolution: structural homologies of a basic component of a DNA translocating machinery

The three-dimensional structure of the bacteriophage T7 head-to-tail connector has been obtained at 8A resolution using cryo-electron microscopy and single-particle analysis from purified recombinant connectors. The general morphology of the T7 connector is that of a 12-folded toroidal homopolymer with a channel that runs along the longitudinal axis of the particle. The structure of the T7 connector reveals many structural similarities with the connectors from other bacteriophages. Docking of the atomic structure of the varphi29 connector into the three-dimensional reconstruction of T7 connector reveals that the narrow, distal region of the two oligomers are almost identical. This region of the varphi29 connector has been suggested to be involved in DNA translocation, and is composed of an alpha-beta-alpha-beta-beta-alpha motif. A search for alpha-helices in the same region of the T7 three-dimensional map has located three alpha-helices in approximately the same position as those of the varphi29 connector. A comparison of the predicted secondary structure of several bacteriophage connectors, including among others T7, varphi29, P22 and SPP1, reveals that, despite the lack of sequence homology, they seem to contain the same alpha-beta-alpha-beta-beta-alpha motif as that present in the varphi29 connector. These results allow us to suggest a common architecture related to a basic component of the DNA translocating machinery for several viruses.     

Three-dimensional structure of bovine NADH:ubiquinone oxidoreductase of the mitochondrial respiratory chain

We have studied the structure of bovine heart mitochondrial NADH:ubiquinone (Q) oxidoreductase (EC 1.6.99.3) by image analysis of electron micrographs. A three-dimensional reconstruction was calculated from a tilt-series of a two-dimensional crystal of the molecule. Our interpretation of the position of the molecule in the unit cell of the crystal is supported by additional (low-resolution) analysis of images of single molecules. The three-dimensional reconstruction was calculated with the aid of an iterative real-space reconstruction algorithm. The various projections used as input to the algorithm were obtained by averaging the images of the tilted crystal through a Fourier-space peak-filtering procedure. The reconstructed unit cell measures 15.2 X 15.2 nm in the plane of the two-dimensional crystal and has a height of 10-11 nm. The unit cell contains one molecule consisting of four large subunits. At the present resolution of about 1.3 nm in the untilted projection, these four monomers are seen as two dimers related by a two-fold axis. Two views of the single particles have been recognized; they are the top and side view of the building block of the crystal. After computer image alignment and correspondence analysis, clusters of similar particles have been averaged. In the averages an uneven stain distribution is seen around the molecules, which may result from preferential staining of hydrophilic parts of the molecule. The molecular mass of the whole molecule was determined from scanning transmission electron microscopy measurements as (1.6 +/- 0.2) X 10(6) daltons.     

Three-dimensional structure of the regular surface layer (HPI layer) of Deinococcus radiodurans

The low-resolution structure of the regular surface layer of Deinococcus radiodurans has been determined from negatively stained specimens by three-dimensional electron microscopy. The layer has P6 symmetry, a lattice constant of 18 nm and a thickness of 6.5 nm. Three-dimensional reconstruction was performed by a hybrid real space/Fourier space approach that incorporates partial compensation of lattice distortions: The model obtained is discussed in the light of independent information about the surface structure of this layer, derived from metal shadowing and surface relief reconstruction. While agreement is quite satisfactory for the apparently more rigid inner surface, the outer surface shows severe flattening effects. The structure of the HPI layer is compared with other bacterial surface layers using a classification scheme that is outlined in the Appendix.     

Single particle reconstruction of membrane proteins: A tool for understanding the 3D structure of disease-related macromolecules

Membrane proteins play important roles in cell functions such as neurotransmission, muscle contraction, and hormone secretion, but their structures are mostly undetermined. Several techniques have been developed to elucidate the structure of macromolecules; X-ray or electron crystallography, nuclear magnetic resonance spectroscopy, and high-resolution electron microscopy. Electron microscopy-based single particle reconstruction, a computer-aided structure determination method, reconstructs a three-dimensional (3D) structure from projections of monodispersed protein. A large number of particle images are picked up from EM films, aligned and classified to generate two-dimensional (2D) averages, and, using the Euler angle of each 2D average, reconstructed into a 3D structure. This method is challenging due to the necessity for close collaboration between classical biochemistry and innovative information technology, including parallel computing. However, recent progress in electron microscopy, mathematical algorithms, and computational ability has greatly increased the subjects that are considered to be primarily addressable using single particle reconstruction. Membrane proteins are one of these targets to which the single particle reconstruction is successfully applied for understanding of their structures. In this paper, we will introduce recently reconstructed channel-related proteins and discuss the applicability of this technique in understanding molecular structures and their roles in pathology.     

Helical structure of unidirectionally shadowed metal replicas of cyanide hydratase from Gloeocercospora sorghi

The helical filaments of the cyanide hydratase from Gloeocercospora sorghi have been reconstructed in three dimensions from freeze dried, unidirectionally shadowed specimens using iterative real-space helical reconstruction. The average power spectrum of all selected images has three clear reflections on different layer lines. The reconstruction is complicated by the fact that three possible indexing schemes are possible and reconstructions using the starting symmetries based on each of these indexing schemes converge on three-dimensional volumes which appear plausible. Because only one side is visible in shadowed specimens, it is necessary to examine the phases from a single filament by cryo-electron microscopy in order to make an unequivocal assignment of the symmetry. Because of the novel nature of the reconstruction method used here, conventional cryo-EM methods were also used to determine a second reconstruction, allowing us to make comparisons between the two. The filament is shown to have a left-handed one-start helix with D(1) symmetry, 5.46 dimers per turn and a pitch of 7.15nm. The reconstruction suggests the presence of an interaction across the groove not previously seen in nitrilase helical fibres.     

The three-dimensional structure of a DNA translocating machine at 10 A resolution.

BACKGROUND: Head-tail connectors are viral substructures that are very important in the viral morphogenetic cycle, having roles in the formation of the precursor capsid (prohead), DNA packaging, tail binding to the mature head and in the infection process. Structural information on the connector would, therefore, help us to understand how this structure is related to a multiplicity of functions. RESULTS: Recombinant bacteriophage phi29 connectors have been crystallized in two-dimensional aggregates. An average projection image and a three-dimensional map have been obtained at 8 A and 10 A resolution, respectively, from untilted and tilted images of vitrified specimens of the two-dimensional crystals. The average projection image reveals a central mass surrounding a channel with 12 appendages protruding from the central mass. The three-dimensional map reveals a wide domain surrounded by 12 appendages that interact with the prohead vertex, and a narrow domain that interacts with the bacteriophage tail. At the junction of the two domains, 12 smaller appendages are visualized. A channel runs along the axis of the connector structure and is sufficiently wide to allow a double-stranded DNA molecule to pass through. CONCLUSIONS: The propeller-like structure of the phi29 connector strengthens the notion of the connector rotating during DNA packaging. The groove formed by the two lanes of large and small appendages may act as a rail to prevent the liberation of the connector from the prohead vertex during rotation.     

The three-dimensional structure of frozen-hydrated Nudaurelia capensis β Virus, a T = 4 insect Virus

The three-dimensional structure of Nudaurelia capensis beta virus (N beta V) was reconstructed to 3.2-nm resolution from images of frozen-hydrated virions. The distinctly icosahedral capsid (approximately 40-nm diameter) contains 240 copies of a single 61-kDa protein subunit arranged with T = 4 lattice symmetry. The outer surface of unstained virions compares remarkably well with that previously observed in negatively stained specimens. Inspection of the density map, volume estimates, and model building experiments indicate that each subunit consists of two distinct domains. The large domain (approximately 40 kDa) has a cylindrical shape, approximately 4-nm diameter by approximately 4-nm high, and associates with two large domains of neighboring subunits to form a Y-shaped trimeric aggregate in the outer capsid surface. Four trimers make up each of the 20 planar faces of the capsid. Small domains (approximately 21 kDa) presumably associate at lower radii (approximately 13-16.5 nm) to form a contiguous, non-spherical shell. A T = 4 model, constructed from 80 trimers of the common beta-barrel core motif (approximately 20 kDa) found in many of the smaller T = 3 and pseudo T = 3 viruses, fits the dimensions and features seen in the N beta V reconstruction, suggesting that the contiguous shell of N beta V may be formed by intersubunit contacts between small domains having that motif. The small (approximately 1800 kDa), ssRNA genome is loosely packed inside the capsid with a low average density.     

Three-dimensional structure of unstained, frozen-hydrated extended tails of bacteriophage T4

Unsupported, unstained frozen-hydrated extended tails of bacteriophage T4 have been studied by cryo-electron microscopy. Their three-dimensional structure has been reconstructed after correlation and averaging of the information from different particles. While the reconstructions of hydrated tails show all the features found by conventional electron microscopy, they are characterized by an open structure. Individual subunits constituting the axial repeat cannot be outlined unambiguously, as the density connectivity is sensitive to the phase-contrast transfer function effects. In order to minimize these effects, we found that the best data set for three-dimensional reconstruction is composed of layer-lines corrected for the phase-contrast transfer function and an uncorrected equator.     

Three-dimensional structure of the surface protein layer (MW layer) of Bacillus brevis 47

The three-dimensional (3D) structure of one surface protein layer from Bacillus brevis 47, the middle wall (MW) layer, has been reconstructed from tilted-view electron micrographs after correlation averaging to a resolution of 2 nm. The MW layer has p6 symmetry with a center-to-center spacing of 18.3 nm and a minimum thickness of 5.5 nm. The reconstruction reveals a distinct domain structure: the heavier domain of six monomers jointly forms a massive core centered at the sixfold symmetry axis, and lighter domains interconnect adjacent unit cells. In addition, the larger domains collectively form a pore by making contact with each other towards the inner surface, while the smaller domains establish a second connectivity towards the outer surface of the S layer. The MW layer of B. brevis resembles the S layer of Acetogenium kivui in various aspects: they have very similar lattice parameters and highly reminiscent 3D structures; the pores penetrate through the whole core and appear to determine the porosity of the S layers.     

Structure of two-dimensional crystals of membrane-bound Na,K-ATPase as analyzed by correlation averaging

The structure of two-dimensional crystals of membrane-bound Na,K-ATPase from rabbit kidney has been analyzed with a correlation averaging procedure. Two principally different crystal forms are observed with p1 and p21 symmetry, respectively. In the p1 form the averaged projection structure shows a triangular shaped protein domain interpreted as a protomer (alpha beta-unit) of Na,K-ATPase. In the p21-form the stain-deficient area is extended toward a twofold symmetry axis. The results are in good agreement with a previous analysis where Fourier methods were applied to well ordered crystals of pig kidney Na,K-ATPase and illustrate that the correlation averaging procedure can be used for the analysis of membrane crystals of Na,K-ATPase showing curved lattice lines.     

Gap junctions. Structural changes after uncoupling procedures

The freeze-fracture appearance of rat stomach and liver gap junctions changes after uncoupling procedures such as inhibition of the metabolism of perfusion with hypertonic sucrose. In control stomach, either fixed immediately or kept for 1 h in a well-oxygenated Tyrode's solution at 37 degrees C, most gap junctions between mucous cells contain particles irregularly packed at an average center-to-center spacing of 10.3-10.5 nm. After 1-h treatment with 2,4-dinitrophenol (DNP), at the same temperature and oxygenation, most particles aggregate hexagonally at an average spacing of approximately 8.5 nm. Similar changes are seen in hypoxic specimens. In control liver, fixed by perfusion, most junctional particles are irregularly packed at an average center-to-center spacing of approximately 10 mm. Small areas of fairly regular hexagonal packing are occasionally seen, where the average particle spacing is 9.2-9.5 nm. In hypoxic liver, the junctional particles form regular hexagonal packings in which the average center-to-center particle spacing is approximately 8.5 nm. In liver perfused with hypertonic sucrose-calcium solutions, following EDTA solutions, most junctions are pulled apart. The separated junctional membranes, expected to be highly impermeable, contain particles regularly and tightly packed as in hypoxic or DNP-treated junctions. Preliminary measurements indicate also a possible change in particle diameter, from approximately 8.6 nm (control) to approximately 7.7 nm (treated). The structural changes are similar to those previously reported in crayfish and may reflect conformational changes in particle subunits resulting in functional uncoupling.     

Three-dimensional structure of the regularly constructed surface layer from Synechocystis sp. strain CLII

The isolated, outermost cell wall layer from Synechocystis sp. strain CLII is described using electron microscopy and Fourier reconstruction to study the three-dimensional structure of the proteins within the layer to a resolution of ca. 3 nm. This surface layer forms regular hexagonal arrays (a = b = 15.2 nm). The two-dimensional space group is p6. The monomer proteins form hexamers arranged around a central hollow cylinder. The linkers between the hexamers are of the delta type and are located approximately in the central section between the top and bottom of the protein layer.     

冷冻电镜单颗粒三维重构密度掩模的自动生成方法研究

冷冻电镜单颗粒三维重构技术是用来解析生物大分子三维结构的常用方法.然而目前在单颗粒三维重构过程中,溶剂平滑操作还存在一定缺陷:没有一款主流的单颗粒三维重构程序能够自动寻找掩模(mask)三维密度图,使得三维重构过程难免受到噪音统计学模型计算偏差的干扰.为解决这一问题,本研究借鉴X射线晶体学中解析优化相位所广泛采用的溶剂平滑方法,采用高斯滤波、坎尼边缘检测、最小误差阈值处理等方法处理重构所得三维密度图,优化溶剂平滑操作,发展在单颗粒三维重构过程中自动寻找mask三维密度图的方法.运用三维密度图傅里叶壳层相关系数(fourier shell correlation,FSC)曲线图、模拟颗粒数据重构角度误差散点图等指标评估此方法的效果.结果表明,自动寻找mask密度图的方法能够较好地找到涵盖分子结构信号区域的mask密度图,较为明显提高三维重构所得密度图分辨率.     

Two-dimensional crystallization of herpes simplex virus type 1 single-stranded DNA-binding protein, ICP8, on a lipid monolayer

Herpes simplex virus type 1 single-stranded DNA-binding protein (ICP8) has been crystallized on a positively charged lipid monolayer. The crystals belong to the planar group p2 with a=39 nm, b=23.2 nm and gamma=87.2 degrees. The projected map of ICP8 crystals calculated at a resolution of 3.9 nm shows four ICP8 monomers per unit cell with the crystals formed by a parallel arrangement of 16.2 nm helical ICP8 filaments. This novel filamentous form has not been reported before. The ICP8 monomers show different appearances in projection, suggesting that they may adopt different orientations, probably reflecting the strong intermolecular and lipid-filament interactions in the crystal. When the 23 nm diameter filaments formed by ICP8 in solution at low temperature in the presence of magnesium were generated and then layered on the phospholipid monolayer, highly ordered arrays of an 8.5 nm filament with a shallow 31.2 nm pitch were observed and reconstruction revealed a double-helical structure.     

Three-dimensional reconstruction of bacteriophage phi 29 neck particles at 2 X 2 nm resolution

The three-dimensional structure of the head-to-tail connecting region of bacteriophage phi 29 has been studied by analysing two-dimensional, hexagonal ordered aggregates of negatively stained viral necks to a resolution of 2 X 2 nm. These necks are composed of two proteins, p10 and p11; p10 being the connector protein. A 12-folded and a 6-folded axially symmetric domain are present in the specimen. The 12-folded domain is the larger part of the structure; it consists of 12 subunits associated in pairs. These subunits appear to be more closely paired towards the centre, where only six subunits are resolved forming the 6-folded domain. The pairs of subunits present an important twist between the 12-folded and the 6-folded areas. A conical hole is formed at the centre of the structure. This hole is more open at the 12-folded domain than at the level of the possible zone of interaction between p10 and p11, where it is almost closed. Protein p11 is very poorly represented in the reconstruction, probably due to lack of staining. The structure described for the phi 29 neck has many of the attributes expected for an active device involved in bacteriophage DNA encapsidation.     

Electron microscopical investigation of citrate lyase single molecules

Electron micrographs of citrate lyase from Rhodopseudomonas gelatinosa and Klebsiella aerogenes reveal two characteristic molecular forms. The "basket" form and the "star" form were subjected to two-dimensional image reconstruction using a technique involving averaging of superposed single molecular images after rotational correlation. A three-dimensional image reconstruction shows that the images of these forms can be interconverted by rotation and that they therefore represent different views of the same structure.