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- PDB-8pzp: Model for influenza A virus helical ribonucleoprotein-like structure -

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Basic information

Entry
Database: PDB / ID: 8pzp
TitleModel for influenza A virus helical ribonucleoprotein-like structure
Components
  • Nucleoprotein
  • RNA (5'P-(UC)6-FAM3')
KeywordsVIRAL PROTEIN / Influenza virus / Nucleocapsid-like / RNA binding protein.
Function / homology
Function and homology information


helical viral capsid / host cell / viral penetration into host nucleus / viral nucleocapsid / ribonucleoprotein complex / symbiont entry into host cell / host cell nucleus / structural molecule activity / RNA binding / identical protein binding
Similarity search - Function
Influenza virus nucleoprotein (NP) / Influenza virus nucleoprotein
Similarity search - Domain/homology
RNA / RNA (> 10) / Nucleoprotein
Similarity search - Component
Biological speciesInfluenza A virus
synthetic construct (others)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 8.7 Å
AuthorsChenavier, F. / Estrozi, L.F. / Zarkadas, E. / Ruigrok, R.W.H. / Schoehn, G. / Ballandras-Colas, A. / Crepin, T.
Funding support France, 1items
OrganizationGrant numberCountry
Grenoble Alliance for Integrated Structural Cell Biology (GRAL)ANR-17-EURE-0003 France
CitationJournal: Sci Adv / Year: 2023
Title: Cryo-EM structure of influenza helical nucleocapsid reveals NP-NP and NP-RNA interactions as a model for the genome encapsidation.
Authors: Florian Chenavier / Leandro F Estrozi / Jean-Marie Teulon / Eleftherios Zarkadas / Lily-Lorette Freslon / Jean-Luc Pellequer / Rob W H Ruigrok / Guy Schoehn / Allison Ballandras-Colas / Thibaut Crépin /
Abstract: Influenza virus genome encapsidation is essential for the formation of a helical viral ribonucleoprotein (vRNP) complex composed of nucleoproteins (NP), the trimeric polymerase, and the viral genome. ...Influenza virus genome encapsidation is essential for the formation of a helical viral ribonucleoprotein (vRNP) complex composed of nucleoproteins (NP), the trimeric polymerase, and the viral genome. Although low-resolution vRNP structures are available, it remains unclear how the viral RNA is encapsidated and how NPs assemble into the helical filament specific of influenza vRNPs. In this study, we established a biological tool, the RNP-like particles assembled from recombinant influenza A virus NP and synthetic RNA, and we present the first subnanometric cryo-electron microscopy structure of the helical NP-RNA complex (8.7 to 5.3 Å). The helical RNP-like structure reveals a parallel double-stranded conformation, allowing the visualization of NP-NP and NP-RNA interactions. The RNA, located at the interface of neighboring NP protomers, interacts with conserved residues previously described as essential for the NP-RNA interaction. The NP undergoes conformational changes to enable RNA binding and helix formation. Together, our findings provide relevant insights for understanding the mechanism for influenza genome encapsidation.
History
DepositionJul 27, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 27, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')


Theoretical massNumber of molelcules
Total (without water)61,1492
Polymers61,1492
Non-polymers00
Water0
1
A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')
x 51
A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')

A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')

A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')

A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')

A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')

A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')

A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')

A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')

A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')

A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')

A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')

A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')

A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')

A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')

A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')

A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')

A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')

A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')

A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')

A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')

A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')

A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')

A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')

A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')

A: Nucleoprotein
B: RNA (5'P-(UC)6-FAM3')


Theoretical massNumber of molelcules
Total (without water)4,647,331152
Polymers4,647,331152
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation73
identity operation1_555x,y,z3
SymmetryHelical symmetry: (Circular symmetry: 2 / N subunits divisor: 1 / Num. of operations: 15 / Rise per n subunits: 24.27 Å / Rotation per n subunits: 57.41 °)

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Components

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


Mass: 57525.965 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (A/WSN/1933(H1N1)) / Gene: NP / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q1K9H2
#2: RNA chain RNA (5'P-(UC)6-FAM3')


Mass: 3623.128 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Influenza A NP-RNA complexCOMPLEXall0MULTIPLE SOURCES
2NucleoproteinCOMPLEX#11RECOMBINANT
3RNA (5'P-(UC)6-FAM3')COMPLEX#21RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Influenza A virus (A/WSN/1933(H1N1))382835
33synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli BL21(DE3) (bacteria)469008
33synthetic construct (others)32630
Buffer solutionpH: 7.5
Details: 20 mM HEPES 150 mM NaCl 5 mM beta-mercaptoethanol 2 mM methyl-PEG8-NHS
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
2150 mMNaClSodium chlorideNaClSodium chloride1
35 mMbeta-mercaptoethanol2-Mercaptoethanol1
42 mMmethyl-PEG8-NHS1
54 uMRNA (5'P-(UC)6-FAM3')1
SpecimenConc.: 0.23 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 20 mM HEPES 150 mM NaCl 5 mM beta-mercaptoethanol Incubation overnight with 0.004 uM RNA (5'P-(UC)6-FAM3') Prior freezing, the sample was incubated 30 min on ice with 2 mM methyl-PEG8-NHS
Specimen supportDetails: 25 mA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 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 FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm / Calibrated defocus min: 800 nm / Calibrated defocus max: 2200 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: 1.9 sec. / Electron dose: 40.8 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 26515
EM imaging opticsEnergyfilter name: GIF Quantum LS

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.2particle selection
2PHENIX1.20.1_4487:model refinement
3EPUimage acquisition
4cryoSPARC3.3.2masking
5cryoSPARC3.3.2CTF correction
6cryoSPARC3.3.2layerline indexing
7UCSF ChimeraX1.6model fitting
10cryoSPARC3.3.2initial Euler assignment
11cryoSPARC3.3.2final Euler assignment
12cryoSPARC3.3.2classification
13cryoSPARC3.3.23D reconstruction
CTF correctionType: NONE
Helical symmertyAngular rotation/subunit: 57.41 ° / Axial rise/subunit: 24.27 Å / Axial symmetry: C2
3D reconstructionResolution: 8.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 225892 / Symmetry type: HELICAL
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 5TJW

5tjw


Pdb chain-ID: A / Accession code: 5TJW / Chain residue range: 21-490 / Details: Fitting of the NPcore by using ChimeraX / Pdb chain residue range: 21-490 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00311786
ELECTRON MICROSCOPYf_angle_d0.82415951
ELECTRON MICROSCOPYf_dihedral_angle_d15.6581863
ELECTRON MICROSCOPYf_chiral_restr0.0471742
ELECTRON MICROSCOPYf_plane_restr0.0052033

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