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- PDB-6zts: Assembly intermediates of orthoreovirus captured in the cell -

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Basic information

Entry
Database: PDB / ID: 6zts
TitleAssembly intermediates of orthoreovirus captured in the cell
ComponentsLambda-1
KeywordsVIRUS LIKE PARTICLE / Assemble Intermediates / Orthoreovirus / cryo-electron tomography / cellular lamellae
Function / homologyZinc finger C2H2-type / Lambda-1
Function and homology information
Biological speciesMammalian orthoreovirus
MethodELECTRON MICROSCOPY / electron tomography / cryo EM
AuthorsSutton, G.C. / Stuart, D.I.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/N00065X/1 United Kingdom
Wellcome Trust206422/Z/17/Z United Kingdom
CitationJournal: Nat Commun / Year: 2020
Title: Assembly intermediates of orthoreovirus captured in the cell.
Authors: Geoff Sutton / Dapeng Sun / Xiaofeng Fu / Abhay Kotecha / Corey W Hecksel / Daniel K Clare / Peijun Zhang / David I Stuart / Mark Boyce /
Abstract: Traditionally, molecular assembly pathways for viruses are inferred from high resolution structures of purified stable intermediates, low resolution images of cell sections and genetic approaches. ...Traditionally, molecular assembly pathways for viruses are inferred from high resolution structures of purified stable intermediates, low resolution images of cell sections and genetic approaches. Here, we directly visualise an unsuspected 'single shelled' intermediate for a mammalian orthoreovirus in cryo-preserved infected cells, by cryo-electron tomography of cellular lamellae. Particle classification and averaging yields structures to 5.6 Å resolution, sufficient to identify secondary structural elements and produce an atomic model of the intermediate, comprising 120 copies each of protein λ1 and σ2. This λ1 shell is 'collapsed' compared to the mature virions, with molecules pushed inwards at the icosahedral fivefolds by ~100 Å, reminiscent of the first assembly intermediate of certain prokaryotic dsRNA viruses. This supports the supposition that these viruses share a common ancestor, and suggests mechanisms for the assembly of viruses of the Reoviridae. Such methodology holds promise for dissecting the replication cycle of many viruses.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJul 20, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 16, 2020Provider: repository / Type: Initial release

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Assembly

Deposited unit
A: Lambda-1
B: Lambda-1


Theoretical massNumber of molelcules
Total (without water)219,2062
Polymers219,2062
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1740 Å2
ΔGint-5 kcal/mol
Surface area95380 Å2
MethodPISA

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Components

#1: Protein Lambda-1 /


Mass: 109603.016 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Mammalian orthoreovirus / References: UniProt: A0A023VYS9

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: CELL / 3D reconstruction method: electron tomography

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Sample preparation

ComponentName: Assembly intermediates of orthoreovirus / Type: VIRUS / Entity ID: #1 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Mammalian orthoreovirus
Details of virusEmpty: YES / Enveloped: NO / Isolate: OTHER / Type: VIRION
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: NITROGEN

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DARK FIELD
Image recordingElectron dose: 2 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionNum. of particles: 41

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