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- PDB-8cbw: CryoEM structure of the Hendra henipavirus nucleocapsid sauronoid... -

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Basic information

Entry
Database: PDB / ID: 8cbw
TitleCryoEM structure of the Hendra henipavirus nucleocapsid sauronoid assembly monomer
Components
  • Nucleocapsid
  • RNA (5'-R(P*UP*UP*UP*UP*UP*U)-3')
KeywordsVIRAL PROTEIN / Nucleoprotein / RNA-binding protein / Sauronoid
Function / homology
Function and homology information


helical viral capsid / viral nucleocapsid / host cell cytoplasm / ribonucleoprotein complex / structural molecule activity / RNA binding
Similarity search - Function
Paramyxovirus nucleocapsid protein / Paramyxovirus nucleocapsid protein
Similarity search - Domain/homology
Biological speciesHendra henipavirus
Escherichia coli BL21 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.485 Å
AuthorsPasschier, T.C. / Maskell, D.P. / Edwards, T.A. / Barr, J.N.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
H2020 Marie Curie Actions of the European Commission721367European Union
CitationJournal: Sci Rep / Year: 2024
Title: The cryoEM structure of the Hendra henipavirus nucleoprotein reveals insights into paramyxoviral nucleocapsid architectures.
Authors: Tim C Passchier / Joshua B R White / Daniel P Maskell / Matthew J Byrne / Neil A Ranson / Thomas A Edwards / John N Barr /
Abstract: We report the first cryoEM structure of the Hendra henipavirus nucleoprotein in complex with RNA, at 3.5 Å resolution, derived from single particle analysis of a double homotetradecameric RNA-bound ...We report the first cryoEM structure of the Hendra henipavirus nucleoprotein in complex with RNA, at 3.5 Å resolution, derived from single particle analysis of a double homotetradecameric RNA-bound N protein ring assembly exhibiting D14 symmetry. The structure of the HeV N protein adopts the common bi-lobed paramyxoviral N protein fold; the N-terminal and C-terminal globular domains are bisected by an RNA binding cleft containing six RNA nucleotides and are flanked by the N-terminal and C-terminal arms, respectively. In common with other paramyxoviral nucleocapsids, the lateral interface between adjacent N and N protomers involves electrostatic and hydrophobic interactions mediated primarily through the N-terminal arm and globular domains with minor contribution from the C-terminal arm. However, the HeV N multimeric assembly uniquely identifies an additional protomer-protomer contact between the N N-terminus and N C-terminal arm linker. The model presented here broadens the understanding of RNA-bound paramyxoviral nucleocapsid architectures and provides a platform for further insight into the molecular biology of HeV, as well as the development of antiviral interventions.
History
DepositionJan 26, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 26, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Nucleocapsid
1: RNA (5'-R(P*UP*UP*UP*UP*UP*U)-3')


Theoretical massNumber of molelcules
Total (without water)60,3332
Polymers60,3332
Non-polymers00
Water00
1
A: Nucleocapsid
1: RNA (5'-R(P*UP*UP*UP*UP*UP*U)-3')
x 28


Theoretical massNumber of molelcules
Total (without water)1,689,33456
Polymers1,689,33456
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation27

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Components

#1: Protein Nucleocapsid


Mass: 58541.320 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hendra henipavirus / Plasmid: pET28a / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta2 / References: UniProt: A0A1L7B858
#2: RNA chain RNA (5'-R(P*UP*UP*UP*UP*UP*U)-3')


Mass: 1792.037 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli BL21(DE3) (bacteria) / Variant: Rosetta 2 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta 2

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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: Sauronoid assembly of Hendra henipavirus nucleoprotein
Type: COMPLEX
Details: Recombinantly expressed Hendra henipavirus nucleoprotein forming sauronoid complexes.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.691779 MDa / Experimental value: NO
Source (natural)Organism: Hendra henipavirus
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: Rosetta 2 / Plasmid: pET28a
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris1
2100 mMSodium ChlorideNaCl1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: PELCO Ultrathin Carbon with Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 281 K

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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: 75000 X / Nominal defocus max: 2800 nm / Nominal defocus min: 700 nm / Calibrated defocus min: 0.7 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 59.7 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3548

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
2EPUimage acquisition
4Gctf1.18CTF correction
7Buccaneermodel fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13Coot0.9.4model refinement
14PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 875262
SymmetryPoint symmetry: D14 (2x14 fold dihedral)
3D reconstructionResolution: 3.485 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 5683 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00289544
ELECTRON MICROSCOPYf_angle_d0.469121688
ELECTRON MICROSCOPYf_dihedral_angle_d4.73813944
ELECTRON MICROSCOPYf_chiral_restr0.03614112
ELECTRON MICROSCOPYf_plane_restr0.00515036

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