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- PDB-8pdn: Spiral of assembled human metapneumovirus (HMPV) N-RNA -

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Basic information

Entry
Database: PDB / ID: 8pdn
TitleSpiral of assembled human metapneumovirus (HMPV) N-RNA
Components
  • Nucleoprotein
  • RNA
KeywordsVIRAL PROTEIN / Nucleoprotein / Virus / Nucleocapsid / RNA-binding / HMPV / Pneumoviridae / Mononegavirales
Function / homologyPneumovirus nucleocapsid protein / Pneumovirus nucleocapsid protein / helical viral capsid / viral nucleocapsid / host cell cytoplasm / ribonucleoprotein complex / RNA binding / RNA / Nucleoprotein
Function and homology information
Biological speciesHuman metapneumovirus
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsWhitehead, J.D. / Decool, H. / Leyrat, C. / Carrique, L. / Fix, J. / Eleouet, J.F. / Galloux, M. / Renner, M.
Funding support United Kingdom, 1items
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, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 6, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 20, 2024Group: Database references / Source and taxonomy / Structure summary
Category: em_entity_assembly / em_entity_assembly_naturalsource ...em_entity_assembly / em_entity_assembly_naturalsource / em_entity_assembly_recombinant / entity_name_com / entity_src_gen / struct_ref / struct_ref_seq / struct_ref_seq_dif
Item: _entity_src_gen.gene_src_common_name / _entity_src_gen.gene_src_strain ..._entity_src_gen.gene_src_common_name / _entity_src_gen.gene_src_strain / _entity_src_gen.pdbx_gene_src_gene / _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id / _entity_src_gen.pdbx_gene_src_scientific_name / _struct_ref.db_code / _struct_ref.pdbx_db_accession / _struct_ref.pdbx_seq_one_letter_code / _struct_ref_seq.pdbx_db_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Nucleoprotein
B: Nucleoprotein
C: Nucleoprotein
D: Nucleoprotein
E: Nucleoprotein
F: Nucleoprotein
G: Nucleoprotein
H: Nucleoprotein
I: Nucleoprotein
J: Nucleoprotein
K: Nucleoprotein
L: Nucleoprotein
a: RNA
b: RNA
c: RNA
d: RNA
e: RNA
f: RNA
g: RNA
h: RNA
i: RNA
j: RNA
k: RNA
l: RNA


Theoretical massNumber of molelcules
Total (without water)559,51124
Polymers559,51124
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area56120 Å2
ΔGint-419 kcal/mol
Surface area170310 Å2

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Components

#1: Protein
Nucleoprotein / / Protein N / Nucleocapsid protein


Mass: 44534.570 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human metapneumovirus (strain CAN97-83)
Strain: CAN97-83 / Gene: N / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta2 / References: UniProt: Q6WBA1
#2: RNA chain
RNA /


Mass: 2091.315 Da / Num. of mol.: 12 / Source method: isolated from a natural source
Details: E.coli RNA, co-purified with N. Due to mixed sequence, modelled as poly-C
Source: (natural) Escherichia coli (E. coli) / Strain: Rosetta2

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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

Component
IDNameTypeEntity IDParent-IDSource
1Human metapneumovirus N-RNACOMPLEXall0MULTIPLE SOURCES
2NucleoproteinCOMPLEX#11RECOMBINANT
3RNACOMPLEX#21NATURAL
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
22Human metapneumovirus (strain CAN97-83)694067CAN97-83
33Escherichia coli (E. coli)562
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8 / Details: 25 mM Tris-HCl, pH 8.0, 250 mM NaCl
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2600 nm / Nominal defocus min: 1000 nm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 44.2 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
12cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionDetails: Please see publication for details
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 101868 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Details: Rigid body only
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00733888
ELECTRON MICROSCOPYf_angle_d0.6546104
ELECTRON MICROSCOPYf_dihedral_angle_d17.74112972
ELECTRON MICROSCOPYf_chiral_restr0.0445436
ELECTRON MICROSCOPYf_plane_restr0.0055580

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