Amyloid structure and assembly: insights from scanning transmission electron microscopy.

Amyloid fibrils are filamentous protein aggregates implicated in several common diseases such as Alzheimer's disease and type II diabetes. Similar structures are also the molecular principle of the infectious spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, scrapie in sheep, and of the so-called yeast prions, inherited non-chromosomal elements found in yeast and fungi. Scanning transmission electron microscopy (STEM) is often used to delineate the assembly mechanism and structural properties of amyloid aggregates. In this review we consider specifically contributions and limitations of STEM for the investigation of amyloid assembly pathways, fibril polymorphisms and structural models of amyloid fibrils. This type of microscopy provides the only method to directly measure the mass-per-length (MPL) of individual filaments. Made on both in vitro assembled and ex vivo samples, STEM mass measurements have illuminated the hierarchical relationships between amyloid fibrils and revealed that polymorphic fibrils and various globular oligomers can assemble simultaneously from a single polypeptide. The MPLs also impose strong constraints on possible packing schemes, assisting in molecular model building when combined with high-resolution methods like solid-state nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR).

Structure of hepatitis E virion-sized particle reveals an RNA-dependent viral assembly pathway.

Hepatitis E virus (HEV) induces acute hepatitis in humans with a high fatality rate in pregnant women. There is a need for anti-HEV research to understand the assembly process of HEV native capsid. Here, we produced a large virion-sized and a small T=1 capsid by expressing the HEV capsid protein in insect cells with and without the N-terminal 111 residues, respectively, for comparative structural analysis. The virion-sized capsid demonstrates a T=3 icosahedral lattice and contains RNA fragment in contrast to the RNA-free T=1 capsid. However, both capsids shared common decameric organization. The in vitro assembly further demonstrated that HEV capsid protein had the intrinsic ability to form decameric intermediate. Our data suggest that RNA binding is the extrinsic factor essential for the assembly of HEV native capsids.

The stoichiometry of Gag protein in HIV-1.

The major structural components of HIV-1 are encoded as a single polyprotein, Gag, which is sufficient for virus particle assembly. Initially, Gag forms an approximately spherical shell underlying the membrane of the immature particle. After proteolytic maturation of Gag, the capsid (CA) domain of Gag reforms into a conical shell enclosing the RNA genome. This mature shell contains 1,000-1,500 CA proteins assembled into a hexameric lattice with a spacing of 10 nm. By contrast, little is known about the structure of the immature virus. We used cryo-EM and scanning transmission EM to determine that an average (145 nm diameter) complete immature HIV particle contains approximately 5,000 structural (Gag) proteins, more than twice the number from previous estimates. In the immature virus, Gag forms a hexameric lattice with a spacing of 8.0 nm. Thus, less than half of the CA proteins form the mature core.

Mass mapping of large globin complexes by scanning transmission electron microscopy.

Scanning transmission electron microscopy (STEM) of unstained, freeze-dried biological macromolecules in the dark-field mode provides an image based on the number of electrons elastically scattered by the constituent atoms of the macromolecule. The image of each isolated particle provides information about the projected structure of the latter, and its integrated intensity is directly related to the mass of the selected particle. Particle images can be sorted by shape, providing independent histograms of mass to study assembly/disassembly intermediates. STEM is optimized for low-dose imaging and is suitable for accurate measurement of particle masses over the range from about 30 kDa to 1,000 MDa. This article describes the details of the method developed at the Brookhaven National Laboratory STEM facility and illustrates its application to the mass mapping of large globin complexes.

Cryo-electron microscopy reveals conserved and divergent features of gag packing in immature particles of Rous sarcoma virus and human immunodeficiency virus.

Retrovirus assembly proceeds via multimerisation of the major structural protein, Gag, into a tightly packed, spherical particle that buds from the membrane of the host cell. The lateral packing arrangement of the human immunodeficiency virus type 1 (HIV-1) Gag CA (capsid) domain in the immature virus has been described. Here we have used cryo-electron microscopy (cryo-EM) and image processing to determine the lateral and radial arrangement of Gag in in vivo and in vitro assembled Rous sarcoma virus (RSV) particles and to compare these features with those of HIV-1. We found that the lateral packing arrangement in the vicinity of the inner sub-domain of CA is conserved between these retroviruses. The curvature of the lattice, however, is different. RSV Gag protein adopts a more tightly curved lattice than is seen in HIV-1, and the virions therefore contain fewer copies of Gag. In addition, consideration of the relationship between the radial position of different Gag domains and their lateral spacings in particles of different diameters, suggests that the N-terminal MA (matrix) domain does not form a single, regular lattice in immature retrovirus particles.

Mass determination of native smooth muscle myosin filaments by scanning transmission electron microscopy.

The thick filaments of vertebrate smooth muscle have a fundamentally different arrangement of myosin molecules from the bipolar, helical organization present in striated muscle filaments. This side-polar, non-helical structure is probably critical to the ability of smooth muscles to shorten by large amounts; however, details of myosin organization beyond this general description are unknown. The non-helical arrangement of myosin precludes the use of helical reconstruction methods for structural determination, and a tomographic approach is required. As a first step towards this goal we have determined the number of myosin molecules present at each 14.5 nm repeat in native smooth muscle myosin filaments by scanning transmission electron microscopy. The mass-per-length of myosin filaments was 159 kDa/nm, corresponding to 4.38(+/-0.11) (mean+/-s.e.m.) myosin molecules at each 14.5 nm level. The mass of thin filaments in the preparation (intrinsic control) was 21 kDa/nm, consistent with current models of smooth muscle thin filament structure, and the mass of tobacco mosaic virus (mass standard) was within 5% of the known value. We conclude that native smooth muscle myosin filaments contain four myosin molecules at each 14.5 nm level, two on each side of the side-polar structure.

Structure of the filamentous phage pIV multimer by cryo-electron microscopy.

The homo-multimeric pIV protein constitutes a channel required for the assembly and export of filamentous phage across the outer membrane of Escherichia coli. We present a 22 A-resolution three-dimensional reconstruction of detergent-solubilized pIV by cryo-electron microscopy associated with image analysis. The structure reveals a barrel-like complex, 13.5 nm in diameter and 24 nm in length, with D14 point-group symmetry, consisting of a dimer of unit multimers. Side views of each unit multimer exhibit three cylindrical domains named the N-ring, the M-ring and the C-ring. Gold labeling of pIV engineered to contain a single cysteine residue near the N or C terminus unambiguously identified the N-terminal region as the N-ring, and the C-terminal region was inferred to make up the C-ring. A large pore, ranging in inner diameter from 6.0 nm to 8.8 nm, runs through the middle of the multimer, but a central domain, the pore gate, blocks it. Moreover, the pore diameter at the N-ring is smaller than the phage particle. We therefore propose that the pIV multimer undergoes a large conformational change during phage transport, with reorganization of the central domain to open the pore, and widening at the N-ring in order to accommodate the 6.5 nm diameter phage particle.

Mixtures of wild-type and a pathogenic (E22G) form of Abeta40 in vitro accumulate protofibrils, including amyloid pores.

Although APP mutations associated with inherited forms of Alzheimer's disease (AD) are relatively rare, detailed studies of these mutations may prove critical for gaining important insights into the mechanism(s) and etiology of AD. Here, we present a detailed biophysical characterization of the structural properties of protofibrils formed by the Arctic variant (E22G) of amyloid-beta protein (Abeta40(ARC)) as well as the effect of Abeta40(WT) on the distribution of the protofibrillar species formed by Abeta40(ARC) by characterizing biologically relevant mixtures of both proteins that may mimic the situation in the heterozygous patients. These studies revealed that the Arctic mutation accelerates both Abeta oligomerization and fibrillogenesis in vitro. In addition, Abeta40(ARC) was observed to affect both the morphology and the size distribution of Abeta protofibrils. Electron microscopy examination of the protofibrils formed by Abeta40(ARC) revealed several morphologies, including: (1) relatively compact spherical particles roughly 4-5 nm in diameter; (2) annular pore-like protofibrils; (3) large spherical particles 18-25 nm in diameter; and (4) short filaments with chain-like morphology. Conversion of Abeta40(ARC) protofibrils to fibrils occurred more rapidly than protofibrils formed in mixed solutions of Abeta40(WT)/Abeta40(ARC), suggesting that co-incubation of Abeta40(ARC) with Abeta40(WT) leads to kinetic stabilization of Abeta40(ARC) protofibrils. An increase in the ratio of Abeta(WT)/Abeta(MUT(Arctic)), therefore, may result in the accumulation of potential neurotoxic protofibrils and acceleration of disease progression in familial Alzheimer's disease mutation carriers.

Alpha-synuclein, especially the Parkinson's disease-associated mutants, forms pore-like annular and tubular protofibrils.

Two mutations in the alpha-synuclein gene (A30P and A53T) have been linked to autosomal dominant early-onset Parkinson's disease (PD). Both mutations promote the formation of transient protofibrils (prefibrillar oligomers), suggesting that protofibrils are linked to cytotoxicity. In this work, the effect of these mutations on the structure of alpha-synuclein oligomers was investigated using electron microscopy and digital image processing. The PD-linked mutations (A30P and A53T) were observed to affect both the morphology and the size distribution of alpha-synuclein protofibrils (measured by analytical ultracentrifugation and scanning transmission electron microscopy). The A30P variant was observed to promote the formation of annular, pore-like protofibrils, whereas A53T promotes formation of annular and tubular protofibrillar structures. Wild-type alpha-synuclein also formed annular protofibrils, but only after extended incubation. The formation of pore-like oligomeric structures may explain the membrane permeabilization activity of alpha-synuclein protofibrils. These structures may contribute to the pathogenesis of PD.

Quasi-normal cornified cell envelopes in loricrin knockout mice imply the existence of a loricrin backup system.

The cornified cell envelope, a lipoprotein layer that assembles at the surface of terminally differentiated keratinocytes, is a resilient structure on account of covalent crosslinking of its constituent proteins, principally loricrin, which accounts for up to 60%-80% of total protein. Despite the importance of the cell envelope as a protective barrier, knocking out the loricrin gene in mice results in only mild syndromes. We have investigated the epidermis and forestomach epithelium of these mice by electron microscopy. In both tissues, corneocytes have normal-looking cell envelopes, despite the absence of loricrin, which was confirmed by immunolabeling, and the absence of the distinctive loricrin-containing keratohyalin granules (L-granules). Isolated cell envelopes were normal in thickness (approximately 15 nm) and mass per unit area (approximately 7.3 kDa per nm2); however, metal shadowing revealed an altered substructure on their cytoplasmic surface. Their amino acid compositions indicate altered protein compositions. Analysis of these data implies that the epidermal cell envelopes have elevated levels of the small proline-rich proteins, and cell envelopes of both kinds contain other protein(s) that, like loricrin, are rich in glycine and serine. These observations imply that, in the absence of loricrin, the mechanisms that govern cell envelope assembly function normally but employ different building-blocks.

Gene cloning, purification, and stoichiometry quantification of phi29 anti-receptor gp12 with potential use as special ligand for gene delivery.

Bacterial virus phi29 is the most efficient in vitro DNA packaging system, with which up to 90% of the added DNA can be packaged into purified recombinant procapsid in vitro. The findings that phi29 virions can be assembled with the exclusive use of cloned gene products have bred a thought that phi29 has a potential to be a gene delivery vector since it is a nonpathogenic virus. gp12 of bacterial virus phi29 has been reported to be the anti-receptor that is responsible for binding the virus particle to the host cell. We cloned the gene coding gp12, overexpressed it in Escherichia coli, and purified the gene product to study the properties and functions of gp12 in virus assembly. According to SDS PloyAcrylamide Gel Electrophoresis (SDS-PAGE) analysis and N-terminal sequencing, recombinant gp12 isolated from E. coli had a molecular mass of 80 kDa, and 24 amino acids at N-terminal were cleaved after expression. The purified recombinant gp12 was incorporated into phi29 particles and converted the gp12-lacking assembly intermediates of phi29 into infectious virions in vitro. This purified protein gp12 was able to compete with infectious phi29 virions for binding to the host cell, thus inhibiting the infection by phi29. Scanning Transmission Electron Microscopy (STEM) analysis and sedimentation studies revealed that recombinant gp12 products were assembled into biologically active dimers. Analysis of the dose-response curve showed that 12 dimeric gp12 complexes were assembled onto viral particles and that each virion contained 24 copies of gp12 molecules. The results provide a basis for future research into bacteriophage-host interaction by modifying the anti-receptor protein. The ultimate goal is to re-target the bacteriophage to new host cells for the purpose of gene delivery.

The bipolar filaments formed by herpes simplex virus type 1 SSB/recombination protein (ICP8) suggest a mechanism for DNA annealing.

Herpes simplex virus type 1 encodes a multifunctional protein, ICP8, which serves both as a single-strand binding protein and as a recombinase, catalyzing reactions involved in replication and recombination of the viral genome. In the presence of divalent ions and at low temperature, previous electron microscopic studies showed that ICP8 will form long left-handed helical filaments. Here, electron microscopic image reconstruction reveals that the filaments are bipolar, with an asymmetric unit containing two subunits of ICP8 that constitute a symmetrical dimer. This organization of the filament has been confirmed using scanning transmission electron microscopy. The pitch of the filaments is approximately 250 A, with approximately 6.2 dimers per turn. Docking of a crystal structure of ICP8 into the reconstructed filament shows that the C-terminal domain of ICP8, attached to the body of the subunit by a flexible linker containing approximately 10 residues, is packed into a pocket in the body of a neighboring subunit in the crystal in a similar manner as in the filament. However, the interactions between the large N-terminal domains are quite different in the filament from that observed in the crystal. A previously proposed model for ICP8 binding single-stranded DNA (ssDNA), based upon the crystal structure, leads to a model for a continuous strand of ssDNA near the filament axis. The bipolar nature of the ICP8 filaments means that a second strand of ssDNA would be running through this filament in the opposite orientation, and this provides a potential mechanism for how ICP8 anneals complementary ssDNA into double-stranded DNA, where each strand runs in opposite directions.

Three-dimensional reconstruction of the Shigella T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout.

Type III secretion systems (T3SSs) mediate bacterial protein translocation into eukaryotic cells, a process essential for virulence of many Gram-negative pathogens. They are composed of a cytoplasmic secretion machinery and a base that bridges both bacterial membranes, into which a hollow, external needle is embedded. When isolated, the latter two parts are termed the 'needle complex'. An incomplete understanding of the structure of the needle complex has hampered studies of T3SS function. To estimate the stoichiometry of its components, we measured the mass of its subdomains by scanning transmission electron microscopy (STEM). We determined subunit symmetries by analysis of top and side views within negatively stained samples in low-dose transmission electron microscopy (TEM). Application of 12-fold symmetry allowed generation of a 21-25-A resolution, three-dimensional reconstruction of the needle complex base, revealing many new features and permitting tentative docking of the crystal structure of EscJ, an inner membrane component.

Three-dimensional analysis of budding sites and released virus suggests a revised model for HIV-1 morphogenesis.

Current models of HIV-1 morphogenesis hold that newly synthesized viral Gag polyproteins traffic to and assemble at the cell membrane into spherical protein shells. The resulting late-budding structure is thought to be released by the cellular ESCRT machinery severing the membrane tether connecting it to the producer cell. Using electron tomography and scanning transmission electron microscopy, we find that virions have a morphology and composition distinct from late-budding sites. Gag is arranged as a continuous but incomplete sphere in the released virion. In contrast, late-budding sites lacking functional ESCRT exhibited a nearly closed Gag sphere. The results lead us to propose that budding is initiated by Gag assembly, but is completed in an ESCRT-dependent manner before the Gag sphere is complete. This suggests that ESCRT functions early in HIV-1 release--akin to its role in vesicle formation--and is not restricted to severing the thin membrane tether.

Structural analysis of reconstituted lipoproteins containing the N-terminal domain of apolipoprotein B.

Apolipoproteins play a central role in lipoprotein metabolism, and are directly implicated in cardiovascular diseases, but their structural characterization has been complicated by their structural flexibility and heterogeneity. Here we describe the structural characterization of the N-terminal region of apolipoprotein B (apoB), the major protein component of very low-density lipoprotein and low-density lipoprotein, in the presence of phospholipids. Specifically, we focus on the N-terminal 6.4-17% of apoB (B6.4-17) complexed with the phospholipid dimyristoylphosphatidylcholine in vitro. In addition to circular dichroism spectroscopy and limited proteolysis, our strategy incorporates nanogold-labeling of the protein in the reconstituted lipoprotein complex followed by visualization and molecular weight determination with scanning transmission electron microscopy imaging. Based on the scanning transmission electron microscopy imaging analysis of approximately 1300 individual particles where the B6.4-17 is labeled with nanogold through a six-His tag, most complexes contain either two or three B6.4-17 molecules. Circular dichroism spectroscopy and limited proteolysis of these reconstituted particles indicate that there are no large conformational changes in B6.4-17 upon lipoprotein complex formation. This is in contrast to the large structural changes that occur during apolipoprotein A-I-lipid interactions. The method described here allows a direct measurement of the stoichiometry and molecular weight of individual particles, rather than the average of the entire sample. Thus, it represents a useful strategy to characterize the structure of lipoproteins, which are not structurally uniform, but can still be defined by an ensemble of related patterns.

Mass analysis by scanning transmission electron microscopy and electron diffraction validate predictions of stacked beta-solenoid model of HET-s prion fibrils.

Fungal prions are infectious filamentous polymers of proteins that are soluble in uninfected cells. In its prion form, the HET-s protein of Podospora anserina participates in a fungal self/non-self recognition phenomenon called heterokaryon incompatibility. Like other prion proteins, HET-s has a so-called "prion domain" (its C-terminal region, HET-s-(218-289)) that is responsible for induction and propagation of the prion in vivo and for fibril formation in vitro. Prion fibrils are thought to have amyloid backbones of polymerized prion domains. A relatively detailed model has been proposed for prion domain fibrils of HET-s based on a variety of experimental constraints (Ritter, C., Maddelein, M. L., Siemer, A. B., Luhrs, T., Ernst, M., Meier, B. H., Saupe, S. J., and Riek, R. (2005) Nature 435, 844-848). To test specific predictions of this model, which envisages axial stacking of beta-solenoids with two coils per subunit, we examined fibrils by electron microscopy. Electron diffraction gave a prominent meridional reflection at (0.47 nm)(-1), indicative of cross-beta structure, as predicted. STEM (scanning transmission electron microscopy) mass-per-unit-length measurements yielded 1.02 +/- 0.16 subunits per 0.94 nm, in agreement with the model prediction (1 subunit per 0.94 nm). This is half the packing density of approximately 1 subunit per 0.47 nm previously obtained for fibrils of the yeast prion proteins, Ure2p and Sup35p, whence it follows that the respective amyloid architectures are basically different.

In vitro assembly of a prohead-like structure of the Rhodobacter capsulatus gene transfer agent.

The gene transfer agent (GTA) is a phage-like particle capable of exchanging double-stranded DNA fragments between cells of the photosynthetic bacterium Rhodobacter capsulatus. Here we show that the major capsid protein of GTA, expressed in E. coli, can be assembled into prohead-like structures in the presence of calcium ions in vitro. Transmission electron microscopy (TEM) of uranyl acetate staining material and thin sections of glutaraldehyde-fixed material demonstrates that these associates have spherical structures with diameters in the range of 27-35 nm. The analysis of scanning TEM images revealed particles of mass approximately 4.3 MDa, representing 101+/-11 copies of the monomeric subunit. The establishment of this simple and rapid method to form prohead-like particles permits the GTA system to be used for genome manipulation within the photosynthetic bacterium, for specific targeted drug delivery, and for the construction of biologically based distributed autonomous sensors for environmental monitoring.

Strain-specific morphologies of yeast prion amyloid fibrils.

Mass per length (mpl) measurements on single amyloid fibrils that specifically propagate the [VH], [VK], and [VL] strains of the yeast prion [PSI] reveal unanticipated differences in their structures. Many fibrils have approximately 1.0 prion molecule per 4.7-A cross-beta repeat period, which is consistent with a self-replicating model built by parallel beta-sheet hydrogen-bonding of like prion peptide segments, but other fibrils are definitely heavier. The predominantly straight fibrils of the dominant [VH] strain have a bimodal mpl distribution, corresponding to components with approximately 1.0 and 1.2 prions per repeat. Fibrils of the weaker [VK] strain, which are almost all wavy, have a monodisperse mpl distribution with a mean of 1.15 prions per repeat. The recessive [VL] strain sample has approximately 1.05 prions per repeat in single fibrils and includes approximately 10% double fibrils, which are rare in the duplicate [VH] and [VK] samples. All of these samples were assembled from purified recombinant Sup35 prion protein by seeded growth on nuclei extracted from yeast bearing the three [PSI] strains. Infectious and noninfectious spontaneously assembled fibrils of the recombinant prion protein also display different heterogeneous morphologies. The strain-specific morphological differences we have observed directly confirm the structural prediction of the protein-only prion theory but do not have an obvious molecular explanation.

Hepatitis B small surface antigen particles are octahedral.

The infectious component of hepatitis B (HB) virus (HBV), the Dane particle, has a diameter of approximately 44 nm and consists of a double-layered capsid particle enclosing a circular, incomplete double-stranded DNA genome. The outer capsid layer is formed from the HB surface antigen (HBsAg) and lipid, whereas the inner layer is formed from the HB core Ag assembled into an icosahedral structure. During chronic infection HBsAg is expressed in large excess as noninfectious quasispherical particles and tubules with approximately 22-nm diameter. Here, we report cryo-EM reconstructions of spherical HBsAg particles at approximately 12-A resolution. We show that the particles possess different diameters and have separated them into two predominant populations, both of which have octahedral symmetry. Despite their differing diameters, the two forms of the particle have the same mass and are built through conformational switching of the same building block, a dimer of HBsAg. We propose that this conformational switching, combined with interactions with the underlying core, leads to the formation of HBV Dane particles of different sizes, dictated by the symmetry of the icosahedral core.

Architecture of Ure2p prion filaments: the N-terminal domains form a central core fiber.

The [URE3] prion is an inactive, self-propagating, filamentous form of the Ure2 protein, a regulator of nitrogen catabolism in yeast. The N-terminal "prion" domain of Ure2p determines its in vivo prion properties and in vitro amyloid-forming ability. Here we determined the overall structures of Ure2p filaments and related polymers of the prion domain fused to other globular proteins. Protease digestion of 25-nm diameter Ure2p filaments trimmed them to 4-nm filaments, which mass spectrometry showed to be composed of prion domain fragments, primarily residues approximately 1-70. Fusion protein filaments with diameters of 14-25 nm were also reduced to 4-nm filaments by proteolysis. The prion domain transforms from the most to the least protease-sensitive part upon filament formation in each case, implying that it undergoes a conformational change. Intact filaments imaged by cryo-electron microscopy or after vanadate staining by scanning transmission electron microscopy (STEM) revealed a central 4-nm core with attached globular appendages. STEM mass per unit length measurements of unstained filaments yielded 1 monomer per 0.45 nm in each case. These observations strongly support a unifying model whereby subunits in Ure2p filaments, as well as in fusion protein filaments, are connected by interactions between their prion domains, which form a 4-nm amyloid filament backbone, surrounded by the corresponding C-terminal moieties.