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- PDB-9cyt: Cryo-EM structure of MRV outer shell -

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Basic information

Entry
Database: PDB / ID: 9cyt
TitleCryo-EM structure of MRV outer shell
Components
  • Outer capsid protein lambda-2
  • Outer capsid protein mu-1N
  • Outer capsid protein sigma-3
KeywordsVIRAL PROTEIN / Mammalian reovirus / outer shell
Function / homology
Function and homology information


icosahedral viral capsid / host cell surface binding / symbiont-mediated suppression of host PKR/eIFalpha signaling / viral outer capsid / symbiont entry into host cell via permeabilization of host membrane / host cell endoplasmic reticulum / protein serine/threonine kinase inhibitor activity / host cell mitochondrion / viral life cycle / regulation of translation ...icosahedral viral capsid / host cell surface binding / symbiont-mediated suppression of host PKR/eIFalpha signaling / viral outer capsid / symbiont entry into host cell via permeabilization of host membrane / host cell endoplasmic reticulum / protein serine/threonine kinase inhibitor activity / host cell mitochondrion / viral life cycle / regulation of translation / mRNA guanylyltransferase activity / mRNA guanylyltransferase / mRNA (guanine-N7)-methyltransferase / host cell cytoplasm / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / symbiont-mediated suppression of host innate immune response / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / GTP binding / host cell nucleus / host cell plasma membrane / structural molecule activity / RNA binding / zinc ion binding / ATP binding / membrane
Similarity search - Function
Mu1 membrane penetration protein, domain I / Reovirus, outer capsid sigma 3 / Reovirus, outer capsid sigma-3 domain superfamily / Reovirus outer capsid protein, Sigma 3 / Outer capsid protein Mu1/VP4 / Mu1 membrane penetration protein, domain IV / Mu1 membrane penetration protein, domain II / Mu1/VP4 superfamily / Mu1 membrane penetration protein, domain III / Reovirus major virion structural protein Mu-1/Mu-1C (M2) ...Mu1 membrane penetration protein, domain I / Reovirus, outer capsid sigma 3 / Reovirus, outer capsid sigma-3 domain superfamily / Reovirus outer capsid protein, Sigma 3 / Outer capsid protein Mu1/VP4 / Mu1 membrane penetration protein, domain IV / Mu1 membrane penetration protein, domain II / Mu1/VP4 superfamily / Mu1 membrane penetration protein, domain III / Reovirus major virion structural protein Mu-1/Mu-1C (M2) / : / : / : / : / : / : / : / : / Reovirus core-spike protein lambda-2 (L2), 6th domain / Reovirus core-spike protein lambda-2 (L2), 7th domain / Reovirus core-spike protein lambda-2 (L2), ferredoxin-like domain / Reovirus core-spike protein lambda-2 (L2), GTase domain / Reovirus core-spike protein lambda-2 (L2), N-terminal / Reovirus core-spike protein lambda-2 (L2), methyltransferase-1 / Reovirus core-spike protein lambda-2 (L2), methyltransferase-2 / Reovirus core-spike lambda-2 / Reovirus core-spike protein lambda-2 (L2), C-terminal / S-adenosyl-L-methionine-dependent methyltransferase superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Outer capsid protein sigma-3 / Outer capsid protein mu-1 / Outer capsid protein lambda-2
Similarity search - Component
Biological speciesMammalian orthoreovirus 3 Dearing
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsLiu, X.Y. / Xia, X. / Martynowycz, M.W. / Gonen, T. / Zhou, Z.H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI094386 United States
CitationJournal: Nat Commun / Year: 2024
Title: Molecular sociology of virus-induced cellular condensates supporting reovirus assembly and replication.
Authors: Xiaoyu Liu / Xian Xia / Michael W Martynowycz / Tamir Gonen / Z Hong Zhou /
Abstract: Virus-induced cellular condensates, or viral factories, are poorly understood high-density phases where replication of many viruses occurs. Here, by cryogenic electron tomography (cryoET) of focused ...Virus-induced cellular condensates, or viral factories, are poorly understood high-density phases where replication of many viruses occurs. Here, by cryogenic electron tomography (cryoET) of focused ion beam (FIB) milling-produced lamellae of mammalian reovirus (MRV)-infected cells, we visualized the molecular organization and interplay (i.e., "molecular sociology") of host and virus in 3D at two time points post-infection, enabling a detailed description of these condensates and a mechanistic understanding of MRV replication within them. Expanding over time, the condensate fashions host ribosomes at its periphery, and host microtubules, lipid membranes, and viral molecules in its interior, forming a 3D architecture that supports the dynamic processes of viral genome replication and capsid assembly. A total of six MRV assembly intermediates are identified inside the condensate: star core, empty and genome-containing cores, empty and full virions, and outer shell particle. Except for star core, these intermediates are visualized at atomic resolution by cryogenic electron microscopy (cryoEM) of cellular extracts. The temporal sequence and spatial rearrangement among these viral intermediates choreograph the viral life cycle within the condensates. Together, the molecular sociology of MRV-induced cellular condensate highlights the functional advantage of transient enrichment of molecules at the right location and time for viral replication.
History
DepositionAug 2, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 18, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
J: Outer capsid protein lambda-2
A: Outer capsid protein mu-1N
B: Outer capsid protein mu-1N
I: Outer capsid protein sigma-3
D: Outer capsid protein mu-1N
F: Outer capsid protein mu-1N
G: Outer capsid protein sigma-3
E: Outer capsid protein mu-1N
H: Outer capsid protein mu-1N
L: Outer capsid protein sigma-3


Theoretical massNumber of molelcules
Total (without water)725,60910
Polymers725,60910
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Outer capsid protein lambda-2 / Lambda2 / Lambda2(Cap)


Mass: 144098.766 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mammalian orthoreovirus 3 Dearing / Cell line: LLC-MK2
References: UniProt: P11079, mRNA guanylyltransferase, mRNA (guanine-N7)-methyltransferase
#2: Protein
Outer capsid protein mu-1N


Mass: 76334.273 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Mammalian orthoreovirus 3 Dearing / References: UniProt: P11078
#3: Protein Outer capsid protein sigma-3 / Sigma3


Mass: 41168.121 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Mammalian orthoreovirus 3 Dearing / References: UniProt: P03527
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: mammalian reovirus / Type: VIRUS / Entity ID: all / Source: NATURAL
Source (natural)Organism: Mammalian orthoreovirus 3 Dearing
Details of virusEmpty: YES / Enveloped: NO / Isolate: STRAIN / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: LLC-MK2
Buffer solutionpH: 7.4 / Details: Phosphate-buffered saline
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: PELCO Ultrathin Carbon with Lacey Carbon
VitrificationCryogen name: ETHANE / Humidity: 100 %

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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: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 1800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 12101
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
2SerialEM4.1image acquisition
4CTFFIND4.1CTF correction
12RELION3.13D reconstruction
13PHENIX1.19model refinement
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 39141
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 8911 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00434788
ELECTRON MICROSCOPYf_angle_d0.60747413
ELECTRON MICROSCOPYf_dihedral_angle_d4.5324722
ELECTRON MICROSCOPYf_chiral_restr0.0455387
ELECTRON MICROSCOPYf_plane_restr0.0066147

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