scholarly journals Structural identification of individual helical amyloid filaments by integration of cryo-electron microscopy-derived maps in comparative morphometric atomic force microscopy image analysis

2021 ◽  
Author(s):  
Liisa Lutter ◽  
Youssra Al-Hilaly ◽  
Christopher J. Serpell ◽  
Mick F. Tuite ◽  
Claude M. Wischik ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Atomic Force Microscopy ◽  
Image Analysis ◽  
Structural Analysis ◽  
Amyloid Fibrils ◽  
Structural Identification ◽  
Force Microscopy ◽  
Atomic Force ◽  
Cryo Electron Microscopy

The presence of amyloid fibrils is a hallmark of more than 50 human disorders, including neurodegenerative diseases and systemic amyloidoses. A key unresolved challenge in understanding the involvement of amyloid in disease is to explain the relationship between individual structural polymorphs of amyloid fibrils, in potentially mixed populations, and the specific pathologies with which they are associated. Although cryo-electron microscopy (cryo-EM) and solid-state nuclear magnetic resonance (ssNMR) spectroscopy methods have been successfully employed in recent years to determine the structures of amyloid fibrils with high resolution detail, they rely on ensemble averaging of fibril structures in the entire sample or significant subpopulations. Here, we report a method for structural identification of individual fibril structures imaged by atomic force microscopy (AFM) by integration of high-resolution maps of amyloid fibrils determined by cryo-EM in comparative AFM image analysis. This approach was demonstrated using the hitherto structurally unresolved amyloid fibrils formed in vitro from a fragment of tau (297-391), termed 'dGAE'. Our approach established unequivocally that dGAE amyloid fibrils bear no structural relationship to heparin-induced tau fibrils formed in vitro. Furthermore, our comparative analysis resulted in the prediction that dGAE fibrils are closely related structurally to the paired helical filaments (PHFs) isolated from Alzheimer's disease (AD) brain tissue characterised by cryo-EM. These results show the utility of individual particle structural analysis using AFM, provide a workflow of how cryo-EM data can be incorporated into AFM image analysis and facilitate an integrated structural analysis of amyloid polymorphism.

scholarly journals Cryo-electron Microscopy Structure, Assembly, and Mechanics Show Morphogenesis and Evolution of Human Picobirnavirus

2020 ◽  
Vol 94 (24) ◽  
Author(s):  
Álvaro Ortega-Esteban ◽  
Carlos P. Mata ◽  
María J. Rodríguez-Espinosa ◽  
Daniel Luque ◽  
Nerea Irigoyen ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Atomic Force Microscopy ◽  
Nucleic Acids ◽  
Capsid Protein ◽  
Cycle Phase ◽  
Human Virus ◽  
Double Stranded Rna ◽  
Force Microscopy ◽  
Atomic Force ◽  
Cryo Electron Microscopy

ABSTRACT Despite their diversity, most double-stranded-RNA (dsRNA) viruses share a specialized T=1 capsid built from dimers of a single protein that provides a platform for genome transcription and replication. This ubiquitous capsid remains structurally undisturbed throughout the viral cycle, isolating the genome to avoid triggering host defense mechanisms. Human picobirnavirus (hPBV) is a dsRNA virus frequently associated with gastroenteritis, although its pathogenicity is yet undefined. Here, we report the cryo-electron microscopy (cryo-EM) structure of hPBV at 2.6-Å resolution. The capsid protein (CP) is arranged in a single-shelled, ∼380-Å-diameter T=1 capsid with a rough outer surface similar to that of dsRNA mycoviruses. The hPBV capsid is built of 60 quasisymmetric CP dimers (A and B) stabilized by domain swapping, and only the CP-A N-terminal basic region interacts with the packaged nucleic acids. hPBV CP has an α-helical domain with a fold similar to that of fungal partitivirus CP, with many domain insertions in its C-terminal half. In contrast to dsRNA mycoviruses, hPBV has an extracellular life cycle phase like complex reoviruses, which indicates that its own CP probably participates in cell entry. Using an in vitro reversible assembly/disassembly system of hPBV, we isolated tetramers as possible assembly intermediates. We used atomic force microscopy to characterize the biophysical properties of hPBV capsids with different cargos (host nucleic acids or proteins) and found that the CP N-terminal segment not only is involved in nucleic acid interaction/packaging but also modulates the mechanical behavior of the capsid in conjunction with the cargo. IMPORTANCE Despite intensive study, human virus sampling is still sparse, especially for viruses that cause mild or asymptomatic disease. Human picobirnavirus (hPBV) is a double-stranded-RNA virus, broadly dispersed in the human population, but its pathogenicity is uncertain. Here, we report the hPBV structure derived from cryo-electron microscopy (cryo-EM) and reconstruction methods using three capsid protein variants (of different lengths and N-terminal amino acid compositions) that assemble as virus-like particles with distinct properties. The hPBV near-atomic structure reveals a quasisymmetric dimer as the structural subunit and tetramers as possible assembly intermediates that coassemble with nucleic acids. Our structural studies and atomic force microscopy analyses indicate that hPBV capsids are potentially excellent nanocages for gene therapy and targeted drug delivery in humans.

Improved unroofing protocols for cryo-electron microscopy, atomic force microscopy and freeze-etching electron microscopy and the associated mechanisms

Microscopy ◽  
2020 ◽  
Vol 69 (6) ◽  
pp. 350-359
Author(s):  
Nobuhiro Morone ◽  
Eiji Usukura ◽  
Akihiro Narita ◽  
Jiro Usukura
Keyword(s):  
Electron Microscopy ◽  
Atomic Force Microscopy ◽  
Cell Membrane ◽  
Apical Cell ◽  
Force Microscopy ◽  
Atomic Force ◽  
Cryo Electron Microscopy ◽  
Cytoplasmic Surface ◽  
A Cell ◽  
Freeze Etching

Abstract Unroofing, which is the mechanical shearing of a cell to expose the cytoplasmic surface of the cell membrane, is a unique preparation method that allows membrane cytoskeletons to be observed by cryo-electron microscopy, atomic force microscopy, freeze-etching electron microscopy and other methods. Ultrasound and adhesion have been known to mechanically unroof cells. In this study, unroofing using these two means was denoted sonication unroofing and adhesion unroofing, respectively. We clarified the mechanisms by which cell membranes are removed in these unroofing procedures and established efficient protocols for each based on the mechanisms. In sonication unroofing, fine bubbles generated by sonication adhered electrostatically to apical cell surfaces and then removed the apical (dorsal) cell membrane with the assistance of buoyancy and water flow. The cytoplasmic surface of the ventral cell membrane remaining on the grids became observable by this method. In adhesion unroofing, grids charged positively by coating with Alcian blue were pressed onto the cells, thereby tightly adsorbing the dorsal cell membrane. Subsequently, a part of the cell membrane strongly adhered to the grids was peeled from the cells and transferred onto the grids when the grids were lifted. This method thus allowed the visualization of the cytoplasmic surface of the dorsal cell membrane. This paper describes robust, improved protocols for the two unroofing methods in detail. In addition, micro-unroofing (perforation) likely due to nanobubbles is introduced as a new method to make cells transparent to electron beams.

Chemical and Structural Analysis of Nitridated Sapphire

1997 ◽  
Vol 482 ◽  
Author(s):  
Y. Cho ◽  
S. Rouvimov ◽  
Y. Kim ◽  
Z. Liliental-Weber ◽  
E. R. Weber
Keyword(s):  
Electron Microscopy ◽  
Atomic Force Microscopy ◽  
Structural Analysis ◽  
Photoelectron Spectroscopy ◽  
High Resolution Electron Microscopy ◽  
Cross Sectional ◽  
Force Microscopy ◽  
Sapphire Substrates ◽  
Atomic Force ◽  
Resolution Electron

AbstractThe incorporation of nitrogen into sapphire substrates during nitridation was studied by xray photoelectron spectroscopy (XPS). An increase in the intensity of nitrogen 1s peak in XPS was observed upon longer nitridation. The surface morphology of the substrates was characterized by atomic force microscopy (AFM). High resolution electron microscopy (HREM) was employed for structural analysis. The cross sectional TEM showed a thin layer of AlN buried between amorphous AlNxO1−x and sapphire. This is the first direct observation of AlN on sapphire. The TEM images show a deeper penetration depth of nitrogen into a longer nitridated sapphire.

Template Assisted Ni Nanowires Fabrication

2019 ◽  
Vol 946 ◽  
pp. 235-241 ◽  
Author(s):  
D.I. Tishkevich ◽  
A.I. Vorobjova ◽  
D.A. Vinnik
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Atomic Force Microscopy ◽  
Structural Analysis ◽  
Specific Magnetization ◽  
X Ray ◽  
Force Microscopy ◽  
Alumina Membranes ◽  
Atomic Force ◽  
Scanning Electron

Through-pores alumina membranes of 50 μm thickness and 70 × 70 mm size have been fabricated to deposit Ni nanowires by electrochemical processing. Due to highly ordered microstructure of the membranes, the pores were filled by nanowires almost to 100%. The membrane nanowires composite morphology; structure and chemical features have been studied by scanning electron microscopy, atomic-force microscopy and X-ray structural analysis. To measure the specific magnetization σ as a function of temperature in the range of 77–1400 K, the pondero-motive method was used.

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