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- EMDB-16696: NC-4 nonviral nucleocapsids assembled in vitro -

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Basic information

Entry
Database: EMDB / ID: EMD-16696
TitleNC-4 nonviral nucleocapsids assembled in vitro
Map data
Sample
  • Complex: In vitro-assembled non-viral nucleocapsid NC-4
KeywordsVirus-like particle / RNA carrier / protein engineering / protein design / VIRUS LIKE PARTICLE
Biological speciesAquifex aeolicus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsTetter S / Hilvert D
Funding supportEuropean Union, 1 items
OrganizationGrant numberCountry
H2020 Marie Curie Actions of the European Commission844006European Union
CitationJournal: Nat Commun / Year: 2024
Title: Stimulus-responsive assembly of nonviral nucleocapsids.
Authors: Mao Hori / Angela Steinauer / Stephan Tetter / Jamiro Hälg / Eva-Maria Manz / Donald Hilvert /
Abstract: Controlled assembly of a protein shell around a viral genome is a key step in the life cycle of many viruses. Here we report a strategy for regulating the co-assembly of nonviral proteins and nucleic ...Controlled assembly of a protein shell around a viral genome is a key step in the life cycle of many viruses. Here we report a strategy for regulating the co-assembly of nonviral proteins and nucleic acids into highly ordered nucleocapsids in vitro. By fusing maltose binding protein to the subunits of NC-4, an engineered protein cage that encapsulates its own encoding mRNA, we successfully blocked spontaneous capsid assembly, allowing isolation of the individual monomers in soluble form. To initiate RNA-templated nucleocapsid formation, the steric block can be simply removed by selective proteolysis. Analyses by transmission and cryo-electron microscopy confirmed that the resulting assemblies are structurally identical to their RNA-containing counterparts produced in vivo. Enzymatically triggered cage formation broadens the range of RNA molecules that can be encapsulated by NC-4, provides unique opportunities to study the co-assembly of capsid and cargo, and could be useful for studying other nonviral and viral assemblies.
History
DepositionFeb 14, 2023-
Header (metadata) releaseAug 28, 2024-
Map releaseAug 28, 2024-
UpdateMar 12, 2025-
Current statusMar 12, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16696.png

Downloads & links

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Map

FileDownload / File: emd_16696.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
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AxesZ (Sec.)Y (Row.)X (Col.)
1.24 Å/pix.
x 400 pix.
= 496. Å		1.24 Å/pix.
x 400 pix.
= 496. Å		1.24 Å/pix.
x 400 pix.
= 496. Å

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.24 Å
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Contour LevelBy AUTHOR: 0.0347
Minimum - Maximum-0.118981995 - 0.21366592
Average (Standard dev.)0.00002456655 (±0.0094074)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 496.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16696_msk_1.map
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Half map: #1

Fileemd_16696_half_map_1.map
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Half map: #2

Fileemd_16696_half_map_2.map
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Sample components

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Entire : In vitro-assembled non-viral nucleocapsid NC-4

EntireName: In vitro-assembled non-viral nucleocapsid NC-4
Components
  • Complex: In vitro-assembled non-viral nucleocapsid NC-4

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Supramolecule #1: In vitro-assembled non-viral nucleocapsid NC-4

SupramoleculeName: In vitro-assembled non-viral nucleocapsid NC-4 / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Aquifex aeolicus (bacteria)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 20 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 1.0 µm

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Image processing

Startup modelType of model: INSILICO MODEL
In silico model: Model generated ab initio from the data, with icosahedral symmetry imposed
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 122284
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationNumber classes: 100 / Software - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7a4j


Chain - Source name: PDB / Chain - Initial model type: experimental model
Detailsrigid body fit of in vivo assembled NC-4 (7A4J)
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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