Two-dimensional averaged images of the dynactin complex revealed by single particle analysis

The dynactin complex interacts with dynein and numerous other proteins to provide for a wide range of subcellular transport functions. A detailed understanding of the structure and subunit organization of dynactin should yield new insights into its function. In the present study, we used single particle analysis to obtain a two-dimensional averaged image of dynactin isolated from chick embryo brains and visualized by negative stain electron microscopy (EM). Each individual image, consisting of the shoulder/sidearm and the rod, closely resembled the previously published quick-freeze deep-etch rotary-shadow electron micrographs. However, the averaged image revealed novel structural features that may have functional significance. The bulky shoulder complex has a triangular shape and is 13 nm wide and 8 nm high. The rod, with an overall length of 40 nm, consists of clearly defined lobes that are apparently grouped into three parts, the pointed-end complex, the middle segment, and the extra lobes at the barbed end. The pointed-end complex reveals the characteristic protrusions and clefts that were previously observed only in the isolated pointed-end complex. In the middle segment, the seven lobes are fitted to the helical symmetry of F-actin. A narrow but prominent gap separates the previously unidentified extra three lobes at the barbed end from the middle segment. The averaged image we obtained contrasts dramatically with the simple Arp1 polymer that was previously reported by single particle analysis of bovine brain dynactin. These apparent structural differences are probably due to the greater stability and integrity of the chick embryo brain dynactin preparation. We propose a new structural model for dynactin, based on our observations.     

Orientation of the pigments in Photosystem II: low-temperature linear-dichroism study of a core particle and of its chlorophyll-protein subunits isolated from Synechococcus sp.

The orientation of the pigments in the Photosystem II core particle isolated from the thermophilic cyanobacterium Synechococcus sp. has been investigated by linear dichroism spectroscopy at 10 K of macroscopically oriented samples. The absorbance (A), linear dichroism (LD) and LD/A spectra are remarkably similar to those previously reported for a core complex isolated from Chlamydomonas reinhardtii (Biochim. Biophys. Acta 850 (1986) 156–161). The spectra of the Synechococcus core particle are compared to the corresponding spectra obtained on its two main constituent chlorophyll-protein complexes CP2-b (photochemically active) and CP2-c (photochemically inactive). The various features seen in the spectra of the core particle appear well segregated into the spectra of one or the other of the two subparticles without significant loss of orientation of the pigments. The orientation of the chlorophyll macrocycles, with the Y and X optical axis preferentially parallel and perpendicular to the plane of largest cross-section of the particle, respectively, is very similar in the two subparticles. CP2-b contains mainly the beta-carotene pool absorbing around 505 and 470 nm, which is oriented close to the membrane plane, while CP2-c contains the beta-carotene pool absorbing around 495 and 465 nm and oriented closer to the normal to the membrane plane. A shoulder at 682 nm in the absorbance and linear dichroism spectra of the core complex is fully segregated in the spectra of CP2-c, thus excluding the possibility that this spectral feature could be assigned to the primary donor of PS II. A negative linear dichroism component peaking around 691 nm (LD 691) in the core particle is mainly segregated in CP2-b together with the photoactive pheophytin acceptor molecule responsible for the 544 nm positive linear dichroism signal (LD 544). While the ratio of the amplitudes LD 691/LD 544 is approximately the same for the core particle and for the CP2-b complex, the amplitude of LD 691 is significantly reduced in CP2-b compared to the core particle.     

Sequence information within the lamB genes in required for proper routing of the bacteriophage lambda receptor protein to the outer membrane of Escherichia coli K-12

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.     

Multivariate statistical analysis of the Pteridophytic diversity of District Battagram,Khyber Pakhtunkhwa,Pakistan

The Pteridophytes represent a great but poorly explored potential as an ecological indicators. Ecological indicator is a useful tool to link empirical results, models and theories with environmental applications. Habitat diversity, precipitation, moisture and rainfall have a high influence on species richness with altitudinal gradients. From 15 different habitats 45 species belonged to 25 genera and 13 families were documented. The leading family were Pteridaceae with 14 taxa, Dryopteridaceae were the second largest family with 7 taxa, followed by Thylepteridaceae with 6 taxa and Aspleniaceae with 5 taxa. Adiantum capillus-veneris and Diplazium esculentum survive in a wide range of habitats while Pellaea calomelanos have a narrow range of habitat. Five plant associations were sorted out through cluster analysis and two ways indicator species analysis. In Bray Curtis ordination the maximum Regression coefficient were recorded for axis 1 (?10.66) with a variance of 0.61. The response data were compositional and have a gradient of 3.2 SD units long. In DCA ordination maximum Eigenvalue and gradient length were recorded for axis 1. Species grouped on the basis of habitat types and habitats having common species. CCA ordination showed that the plants have a linear relationship with environmental variables. The permutation test results on all axis were pseudo-F = 2.4, P = 0.002 indicating the significance value. CCA ordination showed that the environmental variables had a great impact on the association and distribution of majority of the species.     

Pf1 virus particle dynamics

The overall dynamics of the Pf1 filamentous bacteriophage particle in solution are characterized by nmr experiments. The chemical-shift anisotropy powder-pattern lineshapes from both DNA and protein backbone sites of the virus are motionally averaged in the same way, indicating that the entire particle undergoes rapid (< 10⁴ Hz) reorientation about the long axis of the filament when the virus is in solution at high pH. In contrast, the virus particles in samples at low pH are immobile on this time scale.     

The reaction center complex from the green sulfur bacterium Chlorobium tepidum: a structural analysis by scanning transmission electron microscopy.

The three-dimensional (3D) structure of the reaction center (RC) complex isolated from the green sulfur bacterium Chlorobium tepidum was determined from projections of negatively stained preparations by angular reconstitution. The purified complex contained the PscA, PscC, PscB, PscD subunits and the Fenna-Matthews-Olson (FMO) protein. Its mass was found to be 454 kDa by scanning transmission electron microscopy (STEM), indicating the presence of two copies of the PscA subunit, one copy of the PscB and PscD subunits, three FMO proteins and at least one copy of the PscC subunit. An additional mass peak at 183 kDa suggested that FMO trimers copurify with the RC complexes. Images of negatively stained RC complexes were recorded by STEM and aligned and classified by multivariate statistical analysis. Averages of the major classes indicated that different morphologies of the elongated particles (length=19 nm, width=8 nm) resulted from a rotation around the long axis. The 3D map reconstructed from these projections allowed visualization of the RC complex associated with one FMO trimer. A second FMO trimer could be correspondingly accommodated to yield a symmetric complex, a structure observed in a small number of side views and proposed to be the intact form of the RC complex.     

Electron microscopic study of troponin

Troponin and its components or fragments were observed in an electron microscope by the use of the rotary shadowing technique. In freshly prepared troponin with low viscosity, globular particles were mainly observed. The size of the long axis of the particles was 13.2 +/- 1.3 nm and the size perpendicular to the long axis was 9.5 +/- 1.2 nm. The mean axial ratio was 1.4 +/- 0.3. Most of the particles observed in a stored troponin preparation, having a higher viscosity than that of fresh troponin, had a globular head with a thin tail, with the total length of 25.4 +/- 1.4 nm (head-tail type particles). The axial size of the globular portion was 8.3 +/- 1.2 nm and the tail length was 17.1 +/- 1.6 nm. Observation of various particles during the transitional stages indicated that, in the globular particles, the tail region of head-tail type particle was associated along the globular head region. Troponin T was a filamentous particle with 16.9 +/- 1.5 nm length. The 26K fragment of troponin T, which was devoid of the N-terminal 45 residues from troponin T, was a filamentous particle with the length of 14.4 +/- 1.3 nm. Troponin T1, one of two chymotryptic subfragments of troponin T, was a filamentous particle of 11.6 +/- 1.4 nm length. Troponin C.T in the presence of Ca2+ was a particle with a globular head (7 nm in size) and a tail of about 17 nm length. The Fab fragment of anti-troponin T1 formed regular transverse striations along the thin filament of rabbit skeletal muscle with a 38 nm period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antenna organization and evidence for the function of a new antenna pigment species in the green photosynthetic bacterium Chloroflexus aurantiacus

Whole cells and isolated chlorosomes (antenna complex) of the green photosynthetic bacterium Chloroflexus aurantiacus have been studied by absorption spectroscopy (77 K and room temperature), fluorescence spectroscopy, circular dichroism, linear dichroism and electron spin resonance spectroscopy. The chlorosome absorption spectrum has maxima at 450 (contributed by carotenoids and bacteriochlorophyll (BChl) a Soret), 742 (BChl c) and 792 nm (BChl a) with intensity ratios of 20:25. The fluorescence emission spectrum has peaks at 748 and 802 nm when excitation is into either the 742 or 450 nm absorption bands, respectively. Whole cells have fluorescence peaks identical to those in chlorosomes with the addition of a major peak observed at 867 nm. The CD spectrum of isolated chlorosomes has an asymmetric-derivative-shaped CD centered at 739 nm suggestive of exciton interaction at least on the level of dimers. Linear dichroism of oriented chlorosomes shows preferential absorption at 742 nm of light polarized parallel to the long axis of the chlorosome. This implies that the transition dipoles are also oriented more or less parallel to the long axis of the chlorosome. Treatment with ferricyanide results in the appearance of a 2.3 G wide ESR spectrum at g 2.002. Whole cells grown under different light conditions exhibit different fluorescence behavior when absorption is normalized at 742 nm. Cells grown under low light conditions have higher fluorescence intensity at 748 nm and lower intensity at 802 nm than cells grown under high light conditions. These results indicate that the BChl c in chlorosomes is highly organized, and transfers energy from BChl c (742 nm) to a connector of baseplate BChl B792 (BChl a) presumably located in the chlorosome baseplate adjacent to the cytoplasmic membrane.     

Small angle neutron scattering studies of chromatin subunits in solution

Neutron scattering studies have been performed on dilute solutions of the fundamental subunit of chromatin, the nucleosome. The subunits contain approximately 195 base paris (bp) of DNA and histones H2A, H2B, H3, and H4. Measurements of the small angle scattering curves in various H2O/D2O solvents allow the contrast dependence of the radius of gyration of the subunits to be examined and give the mean scattering density of the particle. Further application of contrast variation to the higher angle scatter curves allows the contributions from the shape and internal structure of the subunits to be analyzed separately. From these results, we are able to propose a spherically averaged structure with most of the histones closely packed into a core of radius 3.2 nm surrounded by a loosely packed DNA-rich shell of 2.0 nm thickness resulting in a particle of 5.2 nm average radius. Model calculations for ellipsoids show that the outer shape of the subunit must have an axial ratio between 0.5 and 1.4 but is probably best described by more spherical particle. These results are correlated with the diffraction from chromatin films to provide an explanation for some of the diffraction rings.     

Phycobilisomes of Porphyridium cruentum. I. Isolation

A procedure was developed for the isolation of phycobilisomes from Porphyridium cruentum. The cell homogenate, suspended in phosphate buffer (pH 6.8), was treated with 1% Triton X-100, and its supernatant fraction was centrifuged on a sucrose step gradient. Phycobilisomes were recovered in the 1 M sucrose band. The phycobilisome fraction was identified by the characteristic appearance of the phycobilisomes, and the absorbance of the component pigments: phycoerythrin, R-phycocyanin, and allophycocyanin Isolated phycobilisomes had a prolate shape, with one particle axis longer than the other. Their size varied somewhat with their integrity, but was about 400–500 A (long axis) by 300–320 A (short axis). Phycobilisome recovery was determined at six phosphate buffer concentrations from 0.067 M to 1.0 M. In 0.5 M phosphate, phycobilisome yield (60%) and preservation were optimal. Such a preparation had a phycoerythrin 545 nm/phycocyanin 620 nm ratio of 8.4. Of the detergents tested (Triton X-100, Tween 80, and sodium deoxycholate), Triton X-100 gave the best results Freezing of the cells caused destruction of phycobilisomes.     

Invertebrate myosin filament: subfilament arrangement in the wall of tubular filaments of insect flight muscles

Transverse sections (100 to 140 nm thick) of the flight muscles of the fleshfly Phormia terrae-novae and the housefly Musca domestica were studied. The images of 56 tubular myosin filaments of the fleshfly and 62 filaments of the housefly were digitized and computer processed by rotational averaging. The rotational power spectra of more than 80% of the filaments showed peaks for 6-fold rotational frequency. The average of these images for each species showed a characteristic pattern consisting of 12 subunits arranged in six pairs around the wall of the filament. This pattern was enhanced by rotationally filtering the average images using the 6-fold components of the rotational power spectrum. On tilting individual images, the subunits behaved like rods perpendicular to the plane of the transverse section and they were therefore considered to be subfilaments essentially parallel to the long axis of the filament. The center-to-center spacing between the subfilaments of a pair is 2.8 nm, and the center-to-center spacing between the adjacent subfilaments of neighboring pairs is 4.0 nm. The observation of 12 subfilaments is consistent with a four-stranded helical arrangement of myosin cross-bridges on the surface of the filaments.     

Production of human papillomavirus 6b L1 virus-like particles incorporated with enhanced green fluorescent whole protein in silkworm larvae

Using human papillomavirus (HPV) as a subunit vaccine and its manipulation of surface loops is current trending research. Since the atomic model of L1 protein conformations were deciphered, their manipulations of epitopes bring multivalent vaccines. Here, in the present study, we have manipulated antigenic loops of HPV 6b L1 capsid proteins in the amino acid regions 174 ～ 175 (L1:174EGFP) and 348 ～ 349 (L1:348EGFP) with whole enhanced green fluorescent protein(EGFP), expressed in the silkworm larva using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid technology. The expressed proteins were partially purified using sucrose density-gradient centrifugation and size-exclusion chromatography (SEC). The display of EGFP in virus-like particles (VLPs) was confirmed by immuno-fluorescence microscopy, Western blots and immune-transmission electron microscopy (immuno-TEM). There was higher expression of EGFP incorporated L1:174EGFP than L1:348EGFP. Hydrodynamic diameter of VLPs was corroborated by dynamic light scattering, confirming the size of expected range of around 160 nm and substantiating the incorporation of EGFP. From immuno-TEM, each L1:EGFP VLP formed small particles, suggesting that small particles of L1:EGFP fusion protein were aggregated. Our study illustrates that incorporation of whole protein can efficiently form chimeric VLPs, without hindering the conformation. HPV L1 protein accommodated a whole protein on its antigenic loop as a small particle, but an inserted whole protein was unstable.     

Protein and DNA residue orientations in the filamentous virus Pf1 determined by polarized Raman and polarized FTIR spectroscopy

The Pseudomonas bacteriophage Pf1 is a long ( approximately 2000 nm) and thin ( approximately 6.5 nm) filament consisting of a covalently closed, single-stranded DNA genome of 7349 nucleotides coated by 7350 copies of a 46-residue alpha-helical subunit. The coat subunits are arranged as a superhelix of C(1)()S(5.4)() symmetry (class II). Polarized Raman and polarized FTIR spectroscopy of oriented Pf1 fibers show that the packaged single-stranded DNA genome is ordered specifically with respect to the capsid superhelix. Bases are nonrandomly arranged along the capsid interior, deoxynucleosides are uniformly in the C2'-endo/anti conformation, and the average DNA phosphodioxy group (PO(2)(-)) is oriented so that the line connecting the oxygen atoms (O.O) forms an angle of 71 degrees +/- 5 degrees with the virion axis. Raman and infrared amide band polarizations show that the subunit alpha-helix axis is inclined at an average angle of 16 degrees +/- 4 degrees with respect to the virion axis. The alpha-helical symmetry of the capsid subunit is remarkably rigorous, resulting in splitting of Raman-active helix vibrational modes at 351, 445 and 1026 cm(-)(1) into apparent A-type and E(2)()-type symmetry pairs. The subunit tyrosines (Tyr 25 and Tyr 40) are oriented with phenoxyl rings packed relatively close to parallel to the virion axis. The Tyr 25 and Tyr 40 orientations of Pf1 are surprisingly close to those observed for Tyr 21 and Tyr 24 of the Ff virion (C(5)()S(2)() symmetry, class I), suggesting a preferred tyrosyl side chain conformation in packed alpha-helical subunits, irrespective of capsid symmetry. The polarized Raman spectra also provide information on the orientations of subunit alanine, valine, leucine and isoleucine side chains of the Pf1 virion.     

Observation of Aspherical Particle Rotation in Poiseuille Flow via the Resistance Pulse Technique: I. Application to Human Erythrocytes

The resistance pulse detector (Coulter counter) has been widely applied to the problem of determining the volumes of insulating particles in electrolyte solutions. This technique is based on the simple relationship, ΔR/R = fv/V, between the fractional resistance change ΔR/R and the ratio of particle volume v to pore volume V. The proportionality constant f is a function of particle shape and orientation. Direct observation of the expected resistance anisotropies for aspherical particles is reported here. As predicted by simple hydrodynamic theory each individual resistance pulse has a periodically varying amplitude as it traverses a long pore in the shear field of Poiseuille flow. Characteristics of the particle motion allow improved volume distribution determinations by properly accounting for the shape factor. Application is made to normal human erythrocytes and a gaussian volume distribution with a coefficient of variation ~ 19% is found. The electrical shape effect for erythrocytes is consistent with an oblate ellipsoidal particle with a diameter-to-thickness ratio of 4. Analysis of the data indicates that the convergent entrance flow orients the cells so that they enter the pore with their axis of symmetry perpendicular to the pore axis.     

Molecular structure of flavocytochrome b2 at 2.4 A resolution

The crystal structure of flavocytochrome b2 has been solved at 3.0 A resolution by the method of multiple isomorphous replacement with anomalous scattering. Area detector data from native and two heavy-atom derivative crystals were used. The phases were refined by the B.C. Wang phase-filtering procedure utilizing the 67% (v/v) solvent content of the crystals. A molecular model was built first on a minimap and then on computer graphics from a combination of maps both averaged and not averaged about the molecular symmetry axis. The structure was extended to 2.4 A resolution using film data recorded at a synchrotron and refined by the Hendrickson-Konnert procedure. The molecule, a tetramer of Mr 230,000, is located on a crystallographic 2-fold axis and possesses local 4-fold symmetry. Each subunit is composed of two domains, one binding a heme and the other an FMN prosthetic group. In subunit 1, both the cystochrome and the flavin-binding domain are visible in the electron density map. In subunit 2 the cytochrome domain is disordered. However, in the latter, a molecule of pyruvate, the product of the enzymatic reaction, is bound at the active site. The cytochrome domain consists of residues 1 to 99 and is folded in a fashion similar to the homologous soluble fragment of cytochrome b5. The flavin binding domain contains a parallel beta 8 alpha 8 barrel structure and is composed of residues 100 to 486. The remaining 25 residues form a tail that wraps around the molecular 4-fold axis and is in contact with each remaining subunit. The FMN moiety, which is located at the C-terminal end of the central beta-barrel, is mostly sequestered from solvent; it forms hydrogen bond interactions with main- and side-chain atoms from six of the eight beta-strands. The interaction of Lys349 with atoms N-1 and O-2 of the flavin ring is probably responsible for stabilization of the anionic form of the flavin semiquinone and hydroquinone and enhancing the reactivity of atom N-5 toward sulfite. The binding of pyruvate at the active site in subunit 2 is stabilized by interaction of its carboxylate group with the side-chain atoms of Arg376 and Tyr143. Residues His373 and Tyr254 interact with the keto-oxygen atom and are involved in catalysis. In contrast, four water molecules occupy the substrate-binding site in subunit 1 and Tyr143 forms a hydrogen bond to the ordered heme propionate group. Otherwise the two flavin-binding domains are identical within experimental error.(ABSTRACT TRUNCATED AT 400 WORDS)     

CsdA, a cold-shock RNA helicase from Escherichia coli, is involved in the biogenesis of 50S ribosomal subunit

CsdA, a DEAD-box protein from Escherichia coli, has been proposed to participate in a variety of processes, such as translation initiation, gene regulation after cold-shock, mRNA decay and biogenesis of the small ribosomal subunit. Whether the protein really plays a direct role in these multiple processes is however, not clear. Here, we show that CsdA is involved in the biogenesis of the large rather than the small ribosomal subunit. Deletion of the csdA gene leads to a deficit in free 50S subunits at low temperatures and to the accumulation of a new particle sedimenting around 40S. Analysis of the RNA and protein contents of this particle indicates that it corresponds to a mis-assembled large subunit. Sucrose gradient fractionation shows that in wild-type cells CsdA associates mainly with a pre50S particle. Presumably the RNA helicase activity of CsdA permits a structural rearrangement during 50S biogenesis at low temperature. We showed previously that SrmB, another DEAD-box RNA helicase, is also involved in 50S assembly in E.coli. Our results suggest that CsdA is required at a later step than SrmB. However, over-expression of CsdA corrects the ribosome defect of the srmB-deleted strain, indicating that some functional overlap exists between the two proteins.     

Expression of HBcAg mutant with long internal deletion in Saccharomyces cerevisiae and observation of its self-assembly particles by atomic force microscopy (AFM)

An internally truncated C gene of adr hepatitis B virus core antigen with long internal deletion (aa81–aa116) (ΔHBcAg with 36aa truncation) was expressed in Saccharomyces cerevisiae and the products (ΔrHBcAg) were purified from a crude lysate of the yeast by three steps: Sephrose CL-4B chromatography, sucrose step-gradient ultracentrifugation and CsCl-isopycnic ultracentrifugation. Results of ELISA test and density analysis of CsCl-isopycnic ultracentrifugation indicated that the purified products (ΔrHBcAg protein) with HBeAg antigenicity mainly located at the densities of 1.23 g ml⁻¹. Observation and analysis of the purified ΔrHBcAg products by AFM indicated that the ΔrHBcAg (core) protein produced in S. cerevisiae could self-assemble into three or more size classes of core particles which exhibited a polymorphous distribution of ΔrHBcAg (core) particles. These different size classes of core particles mainly centred on the range whose mean diameter was from 10 nm to 48 nm, especially on the position of 11 nm, 15.6 nm and the range from 27 nm to 41 nm, respectively. Furthermore, the most number of core particles mainly centred on the range whose mean diameter was from 27 nm to 41 nm. These results above indicated that the truncated internal long fragment (aa81–aa116) probably had no effect on self-assembly of the HBcAg core particles which implied the internal length fragment (aa81–aa116) was not the sole domain for self-assembly of HBcAg dimer or the truncated HBcAg protein subunit formed the fresh interactive domain with each other. These initial results above by AFM analysis were very important for further research on the self-assembly, ultrastructure, subunit interaction and core internal deletion mutant (CIDM) function of HBcAg core particles.     

Structural study of modified fibrinogen microcrystals by electron microscopy

The same molecular shape of proteolytically modified fibrinogen has been identified in two different crystalline forms: orthorhombic P2₁2₁2 with a = 44.7(± 1.7) nm, b = 11.8(± 1.0) nm, c = 3.8 nm and monoclinic P2₁ with a = 17.7(± 0.6) nm, b = 16.2(± 1.0) nm, c = 4.8 nm and β = 95 °. The shape of the molecule is more detailed than has been reported. It has the commonly accepted elongated form 44.5(± 1.5) nm long with a 2-fold axis perpendicular to the major axis of the molecule. Each end domain shows a distinctive substructure and has an asymmetry similar to that reported by Williams (1981) and Mosesson et al. (1981) from single molecule observations. The ends are flattened, having overall dimensions of approximately 12 nm × 9 nm × 4 nm. The major difference between this model and previous models is the absence of a central nodule. There are, however, two small additional protein-dense regions at 5.0 nm from the centre of the rod connecting the two ends. A rough estimate of the relative heights of the different parts of the molecule was obtained using the two different projections of the molecule obtained from the two different crystalline forms. The molecule appears to be slightly bent at the centre, as predicted by Doolittle (1977).     

Subunit structure of junctional feet in triads of skeletal muscle: a freeze-drying, rotary-shadowing study

Isolated heavy sarcoplasmic reticulum vesicles retain junctional specializations (feet) on their outer surface. We have obtained en face three-dimensional views of the feet by shadowing and replicating the surfaces of freeze-dried isolated vesicles. Feet are clearly visible as large structures located on raised platforms. New details of foot structure include a four subunit structure and the fact that adjacent feet do not abut directly corner to corner but are offset by half a subunit. Feet aligned within rows were observed to be rotated at a slight angle off the long axis of the row creating a center-to-center spacing (32.5 nm) slightly less than the average diagonal of the feet (35.3 nm). Comparison with previous information from thin sections and freeze-fracture showed that this approach to the study of membranes faithfully preserves structure and allows better visualization of surface details than either thin-sectioning or negative-staining.