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- EMDB-17613: 10-mer ring of human metapneumovirus (HMPV) N-RNA -

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Basic information

Entry
Database: EMDB / ID: EMD-17613
Title10-mer ring of human metapneumovirus (HMPV) N-RNA
Map data
Sample
  • Complex: Human metapneumovirus N-RNA
    • Complex: Nucleoprotein
      • Protein or peptide: Nucleoprotein
    • Complex: RNA
      • RNA: RNA
KeywordsNucleoprotein / Virus / Nucleocapsid / RNA-binding / HMPV / Pneumoviridae / Mononegavirales / VIRAL PROTEIN
Function / homologyPneumovirus nucleocapsid protein / Pneumovirus nucleocapsid protein / helical viral capsid / viral nucleocapsid / host cell cytoplasm / ribonucleoprotein complex / RNA binding / Nucleoprotein
Function and homology information
Biological speciesHuman metapneumovirus (strain CAN97-83) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsWhitehead JD / Decool H / Leyrat C / Carrique L / Fix J / Eleouet JF / Galloux M / Renner M
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
CitationJournal: Nat Commun / Year: 2023
Title: Structure of the N-RNA/P interface indicates mode of L/P recruitment to the nucleocapsid of human metapneumovirus.
Authors: Jack D Whitehead / Hortense Decool / Cédric Leyrat / Loic Carrique / Jenna Fix / Jean-François Eléouët / Marie Galloux / Max Renner /
Abstract: Human metapneumovirus (HMPV) is a major cause of respiratory illness in young children. The HMPV polymerase (L) binds an obligate cofactor, the phosphoprotein (P). During replication and ...Human metapneumovirus (HMPV) is a major cause of respiratory illness in young children. The HMPV polymerase (L) binds an obligate cofactor, the phosphoprotein (P). During replication and transcription, the L/P complex traverses the viral RNA genome, which is encapsidated within nucleoproteins (N). An essential interaction between N and a C-terminal region of P tethers the L/P polymerase to the template. This N-P interaction is also involved in the formation of cytoplasmic viral factories in infected cells, called inclusion bodies. To define how the polymerase component P recognizes N-encapsidated RNA (N-RNA) we employed cryogenic electron microscopy (cryo-EM) and molecular dynamics simulations, coupled to activity assays and imaging of inclusion bodies in cells. We report a 2.9 Å resolution structure of a triple-complex between multimeric N, bound to both RNA and the C-terminal region of P. Furthermore, we also present cryo-EM structures of assembled N in different oligomeric states, highlighting the plasticity of N. Combined with our functional assays, these structural data delineate in molecular detail how P attaches to N-RNA whilst retaining substantial conformational dynamics. Moreover, the N-RNA-P triple complex structure provides a molecular blueprint for the design of therapeutics to potentially disrupt the attachment of L/P to its template.
History
DepositionJun 12, 2023-
Header (metadata) releaseDec 6, 2023-
Map releaseDec 6, 2023-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17613.png

Downloads & links

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Map

FileDownload / File: emd_17613.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.05 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-1.4599011 - 2.641763
Average (Standard dev.)-0.00078658125 (±0.0419382)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 419.99997 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_17613_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_17613_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human metapneumovirus N-RNA

EntireName: Human metapneumovirus N-RNA
Components
  • Complex: Human metapneumovirus N-RNA
    • Complex: Nucleoprotein
      • Protein or peptide: Nucleoprotein
    • Complex: RNA
      • RNA: RNA

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Supramolecule #1: Human metapneumovirus N-RNA

SupramoleculeName: Human metapneumovirus N-RNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 460 KDa

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Supramolecule #2: Nucleoprotein

SupramoleculeName: Nucleoprotein / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Human metapneumovirus (strain CAN97-83) / Strain: CAN97-83

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Supramolecule #3: RNA

SupramoleculeName: RNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Nucleoprotein

MacromoleculeName: Nucleoprotein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Human metapneumovirus (strain CAN97-83) / Strain: CAN97-83
Molecular weightTheoretical: 43.57652 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSLQGIHLSD LSYKHAILKE SQYTIKRDVG TTTAVTPSSL QQEITLLCGE ILYAKHADYK YAAEIGIQYI STALGSERVQ QILRNSGSE VQVVLTRTYS LGKIKNNKGE DLQMLDIHGV EKSWVEEIDK EARKTMATLL KESSGNIPQN QRPSAPDTPI I LLCVGALI ...String:
MSLQGIHLSD LSYKHAILKE SQYTIKRDVG TTTAVTPSSL QQEITLLCGE ILYAKHADYK YAAEIGIQYI STALGSERVQ QILRNSGSE VQVVLTRTYS LGKIKNNKGE DLQMLDIHGV EKSWVEEIDK EARKTMATLL KESSGNIPQN QRPSAPDTPI I LLCVGALI FTKLASTIEV GLETTVRRAN RVLSDALKRY PRMDIPKIAR SFYDLFEQKV YHRSLFIEYG KALGSSSTGS KA ESLFVNI FMQAYGAGQT MLRWGVIARS SNNIMLGHVS VQAELKQVTE VYDLVREMGP ESGLLHLRQS PKAGLLSLAN CPN FASVVL GNASGLGIIG MYRGRVPNTE LFSAAESYAK SLKESNKINF SSLGLTDEEK EAAEHFLNVS DDSQNDYE

UniProtKB: Nucleoprotein

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Macromolecule #2: RNA

MacromoleculeName: RNA / type: rna / ID: 2
Details: E.coli RNA, co-purified with N. Due to mixed sequence, modelled as poly-C
Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli) / Strain: Rosetta2
Molecular weightTheoretical: 2.091315 KDa
SequenceString:
CCCCCCC

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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

Concentration1 mg/mL
BufferpH: 8 / Details: 25 mM Tris-HCl, pH 8.0, 250 mM NaCl
GridModel: Quantifoil R2/1 / Material: COPPER
VitrificationCryogen name: ETHANE-PROPANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 44.2 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionDetails: Please see publication for details
Startup modelType of model: OTHER / Details: CryoSPARC ab initio
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC / Details: CryoSPARC
Final angle assignmentType: OTHER / Software - Name: cryoSPARC / Details: CryoSPARC
Final reconstructionApplied symmetry - Point group: C10 (10 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 509664
FSC plot (resolution estimation)

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