How much can inelastically scattered electrons contribute to electron cryotomography of biological specimens?

AbstractThe lipid-enveloped influenza C virus contains a single surface glycoprotein, the haemagglutinin-esterase-fusion (HEF) protein, that mediates receptor binding, receptor destruction, and membrane fusion at the low pH of the endosome. Here we apply electron cryotomography and subtomogram averaging to describe the structural basis for hexagonal lattice formation by HEF on the viral surface. The conformation of the glycoprotein in situ is distinct from the structure of the isolated trimeric ectodomain, showing that a splaying of the membrane distal domains is required to mediate contacts that form the lattice. The splaying of these domains is also coupled to changes in the structure of the stem region which is involved in membrane fusion, thereby linking HEF’s membrane fusion conformation with its assembly on the virus surface. The glycoprotein lattice can form independent of other virion components but we show a major role for the matrix layer in particle formation.

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Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes

Nature Communications ◽

10.1038/s41467-021-21695-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Shirley Tremel ◽

Yohei Ohashi ◽

Dustin R. Morado ◽

Jessie Bertram ◽

Olga Perisic ◽

...

Keyword(s):

Complex I ◽

Kinase Domain ◽

Beclin 1 ◽

Structural Basis ◽

Rab Gtpases ◽

Lipid Kinase ◽

Complex Ii ◽

Cellular Processes ◽

Specific Complex ◽

Electron Cryotomography

AbstractThe lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a–GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations.

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Visualizing the Molecular Machines of Bacterial Motility In-situ with Electron Cryotomography

Microscopy and Microanalysis ◽

10.1017/s1431927606064439 ◽

2006 ◽

Vol 12 (S02) ◽

pp. 398-399

Author(s):

GJ Jensen ◽

GE Murphy ◽

GP Henderson ◽

Z Li ◽

A Komeili ◽

...

Keyword(s):

Molecular Machines ◽

Bacterial Motility ◽

Electron Cryotomography

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2005

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Electron Cryotomography of Tula Hantavirus Suggests a Unique Assembly Paradigm for Enveloped Viruses

Journal of Virology ◽

10.1128/jvi.00057-10 ◽

2010 ◽

Vol 84 (10) ◽

pp. 4889-4897 ◽

Cited By ~ 95

Author(s):

Juha T. Huiskonen ◽

Jussi Hepojoki ◽

Pasi Laurinmäki ◽

Antti Vaheri ◽

Hilkka Lankinen ◽

...

Keyword(s):

Three Dimensional ◽

Hemorrhagic Fever ◽

Membrane Curvature ◽

Emerging Viruses ◽

Enveloped Viruses ◽

Viral Membrane ◽

Density Maps ◽

Virion Structure ◽

Electron Cryotomography ◽

Assembly Principles

ABSTRACT Hantaviruses (family Bunyaviridae) are rodent-borne emerging viruses that cause a serious, worldwide threat to human health. Hantavirus diseases include hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome. Virions are enveloped and contain a tripartite single-stranded negative-sense RNA genome. Two types of glycoproteins, GN and GC, are embedded in the viral membrane and form protrusions, or “spikes.” The membrane encloses a ribonucleoprotein core, which consists of the RNA segments, the nucleocapsid protein, and the RNA-dependent RNA polymerase. Detailed information on hantavirus virion structure and glycoprotein spike composition is scarce. Here, we have studied the structures of Tula hantavirus virions using electron cryomicroscopy and tomography. Three-dimensional density maps show how the hantavirus surface glycoproteins, membrane, and ribonucleoprotein are organized. The structure of the GN-GC spike complex was solved to 3.6-nm resolution by averaging tomographic subvolumes. Each spike complex is a square-shaped assembly with 4-fold symmetry. Spike complexes formed ordered patches on the viral membrane by means of specific lateral interactions. These interactions may be sufficient for creating membrane curvature during virus budding. In conclusion, the structure and assembly principles of Tula hantavirus exemplify a unique assembly paradigm for enveloped viruses.

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