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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.