Search results for "cryo-electron microscopy"

Real-time Fluorescence Measurement of Enterovirus Uncoating

2019

Viruses need to open, i.e., uncoat, in order to release their genomes for efficient replication and translation. Especially for non-enveloped viruses, such as enteroviruses, the cues leading to uncoating are less well known. The status of the virus has previously been observed mainly by transmission electron microscopy using negative staining, cryo electron microscopy, X-ray crystallography or gradient separation (reviewed in Tuthill et al., 2010, Myllynen et al., 2016, Ruokolainen et al., 2019). However, monitoring of uncoating has been limited by the lack of methods detecting dynamic changes of the virions. Here, we present a real-time fluorescence based protocol, which detects the viral …

Limulus polyphemus Hemocyanin: 10 Å Cryo-EM Structure, Sequence Analysis, Molecular Modelling and Rigid-body Fitting Reveal the Interfaces Between th…

2007

Abstract The blue copper protein hemocyanin from the horseshoe crab Limulus polyphemus is among the largest respiratory proteins found in nature (3.5 MDa) and exhibits a highly cooperative oxygen binding. Its 48 subunits are arranged as eight hexamers (1×6mers) that form the native 8×6mer in a nested hierarchy of 2×6mers and 4×6mers. This quaternary structure is established by eight subunit types (termed I, IIA, II, IIIA, IIIB, IV, V, and VI), of which only type II has been sequenced. Crystal structures of the 1×6mer are available, but for the 8×6mer only a 40 A 3D reconstruction exists. Consequently, the structural parameters of the 8×6mer are not firmly established, and the molecular inte…

Variety of size and form of GRM2 bacterial microcompartment particles

2021

Bacterial microcompartments (BMCs) are bacterial organelles involved in enzymatic processes, such as carbon fixation, choline, ethanolamine and propanediol degradation, and others. Formed of a semi‐permeable protein shell and an enzymatic core, they can enhance enzyme performance and protect the cell from harmful intermediates. With the ability to encapsulate non‐native enzymes, BMCs show high potential for applied use. For this goal, a detailed look into shell form variability is significant to predict shell adaptability. Here we present four novel 3D cryo‐EM maps of recombinant Klebsiella pneumoniae GRM2 BMC shell particles with the resolution in range of 9 to 22 Å and nine novel 2D class…

Hepatitis B core particles as a universal display model: a structure-function basis for development

1999

AbstractBecause it exhibits a remarkable capability to accept mutational intervention and undergo correct folding and self-assembly in all viable prokaryotic and eukaryotic expression systems, hepatitis B core (HBc) protein has been favored over other proposed particulate carriers. Structurally, the unusual α-helical organization of HBc dimeric units allows introduction of foreign peptide sequences into several areas of HBc shells, including their most protruding spikes. Progress toward full resolution of the spatial structure as well as accumulation of chimeric HBc-based structures has brought closer the knowledge-based design of future vaccines, gene therapy tools and other artificial par…

PspA adopts an ESCRT-III-like fold and remodels bacterial membranes

2021

Summary PspA is the main effector of the phage shock protein (Psp) system and preserves the bacterial inner membrane integrity and function. Here, we present the 3.6 A resolution cryoelectron microscopy (cryo-EM) structure of PspA assembled in helical rods. PspA monomers adopt a canonical ESCRT-III fold in an extended open conformation. PspA rods are capable of enclosing lipids and generating positive membrane curvature. Using cryo-EM, we visualized how PspA remodels membrane vesicles into μm-sized structures and how it mediates the formation of internalized vesicular structures. Hotspots of these activities are zones derived from PspA assemblies, serving as lipid transfer platforms and lin…

Localization of GFP-Tagged Proteins at the Electron Microscope

2016

Allosterism of Nautilus pompilius hemocyanin as deduced from 8 Å cryo-EM structures obtained under oxy and deoxy conditions

2008

Hemocyanins are the blue copper-containing respiratory proteins of many molluscs. Nautilus pompilius hemocyanin (NpH) is a cylindrical decamer composed of ten copies of a 350 kDa polypeptide subunit, in turn consisting of seven O2-binding functional units (FUs, termed NpH-a to NpH-g). Ten copies of the subunit segment NpH-a to NpH-f form the cylinder wall (ca. 35 nm in diameter), whereas the ten copies of NpH-g build the internal collar. Recently we published a 9A cryo-EM structure and molecular model of NpH that solved the principal architecture of this protein [1]. Hemocyanins are highly allosteric, and the cooperativity of oxygen binding should be transferred between functional units by …

Structural Studies Reveal that Endosomal Cations Promote Formation of Infectious Coxsackievirus A9 A-Particles, Facilitating RNA and VP4 Release

2022

Coxsackievirus A9 (CVA9), an enterovirus, is a common cause of pediatric aseptic meningitis and neonatal sepsis. During cell entry, enterovirus capsids undergo conformational changes leading to expansion, formation of large pores, externalization of VP1 N termini, and loss of the lipid factor from VP1. Factors such as receptor binding, heat, and acidic pH can trigger capsid expansion in some enteroviruses. Here, we show that fatty acid-free bovine serum albumin or neutral endosomal ionic conditions can independently prime CVA9 for expansion and genome release. Our results showed that CVA9 treatment with albumin or endosomal ions generated a heterogeneous population of virions, which could b…

Formation of two-dimensional crystals of icosahedral RNA viruses.

2007

International audience; The formation of 2D arrays of three small icosahedral RNA viruses with known 3D structures (tomato bushy stunt virus, turnip yellow mosaic virus and bromegrass mosaic virus) has been investigated to determine the role of each component of a negative staining solution containing ammonium molybdate and polyethylene glycol. Virion association was monitored by dynamic light scattering (DLS) and virus array formation was visualised by conventional transmission electron microscopy and cryo-electron microscopy after negative staining. The structural properties of viral arrays prepared in vitro were compared to those of microcrystals found in the leaves of infected plants. A…

An amino-terminal segment of hantavirus nucleocapsid protein presented on hepatitis B virus core particles induces a strong and highly cross-reactive…

2004

AbstractPreviously, we have demonstrated that hepatitis B virus (HBV) core particles tolerate the insertion of the amino-terminal 120 amino acids (aa) of the Puumala hantavirus nucleocapsid (N) protein. Here, we demonstrate that the insertion of 120 amino-terminal aa of N proteins from highly virulent Dobrava and Hantaan hantaviruses allows the formation of chimeric core particles. These particles expose the inserted foreign protein segments, at least in part, on their surface. Analysis by electron cryomicroscopy of chimeric particles harbouring the Puumala virus (PUUV) N segment revealed 90% T = 3 and 10% T = 4 shells. A map computed from T = 3 shells shows additional density splaying out …