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- EMDB-51186: CryoEM structure of influenza A RNP-like particle double-stranded... -

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Basic information

Entry
Database: EMDB / ID: EMD-51186
TitleCryoEM structure of influenza A RNP-like particle double-stranded assembled with a 14-mer RNA.
Map dataGlobal map of parallel double strand helical filament, locally refined on one strand.
Sample
  • Complex: complex NP-RNA
    • Complex: RNA
      • RNA: RNA (5'-P(UC)7-FAM3')
    • Complex: Nucleoprotein
      • Protein or peptide: Nucleoprotein
KeywordsNucleoprotein / RNA / complex / RNP-like / influenza virus / VIRAL PROTEIN
Function / homology
Function and homology information


helical viral capsid / viral penetration into host nucleus / host cell / 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
Biological speciessynthetic construct (others) / Influenza A virus
Methodhelical reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsChenavier F / Ruigrok RWH / Schoehn G / Ballandras-Colas A / Crepin T
Funding support France, 3 items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-23-CE11-01 France
Grenoble Alliance for Integrated Structural Cell Biology (GRAL)ANR-17-EURE-0003 France
Grenoble Instruct-ERIC Center (ISBG)ANR-10-INSB-0005-02 France
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Influenza a virus antiparallel helical nucleocapsid-like pseudo-atomic structure.
Authors: Florian Chenavier / Eleftherios Zarkadas / Lily-Lorette Freslon / Alice J Stelfox / Guy Schoehn / Rob W H Ruigrok / Allison Ballandras-Colas / Thibaut Crépin /
Abstract: Influenza A viruses are responsible for human seasonal epidemics and severe animal pandemics with a risk of zoonotic transmission to humans. The viral segmented RNA genome is encapsidated by ...Influenza A viruses are responsible for human seasonal epidemics and severe animal pandemics with a risk of zoonotic transmission to humans. The viral segmented RNA genome is encapsidated by nucleoproteins (NP) and attached to the heterotrimeric polymerase, forming the viral ribonucleoproteins (vRNPs). Flexible helical vRNPs are central for viral transcription and replication. In this study, we present an advanced biological tool, the antiparallel helical RNP-like complex, assembled from recombinant N-terminally truncated NP and short synthetic RNA. The 3.0 Å cryo-electron microscopy structure details for the first time the whole RNA pathway across NP as well as NP-NP interactions that drive the antiparallel helical assembly accommodating major and minor grooves. Our findings show that the surface of the protein can harbour several conformations of the RNA, confirming that the number of nucleobases that binds to NP is not fixed, but ranges probably between 20 and 24. Taking all together, our data provide details to further understand the genome encapsidation and explain the inherent flexibility of influenza A virus vRNPs.
History
DepositionJul 29, 2024-
Header (metadata) releaseJan 15, 2025-
Map releaseJan 15, 2025-
UpdateFeb 12, 2025-
Current statusFeb 12, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_51186.png

Downloads & links

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Map

FileDownload / File: emd_51186.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGlobal map of parallel double strand helical filament, locally refined on one strand.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.15 Å/pix.
x 400 pix.
= 458. Å		1.15 Å/pix.
x 400 pix.
= 458. Å		1.15 Å/pix.
x 400 pix.
= 458. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.145 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.326
Minimum - Maximum-0.62136686 - 1.4438078
Average (Standard dev.)0.000015435597 (±0.03378026)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 458.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_51186_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: EMready sharpened map of parallel double strand helical...

Fileemd_51186_additional_1.map
AnnotationEMready sharpened map of parallel double strand helical filament, locally refined on one strand.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: B-factor sharpened map of parallel double strand helical...

Fileemd_51186_additional_2.map
AnnotationB-factor sharpened map of parallel double strand helical filament, locally refined on one strand.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 1

Fileemd_51186_half_map_1.map
AnnotationHalf-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 2

Fileemd_51186_half_map_2.map
AnnotationHalf-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : complex NP-RNA

EntireName: complex NP-RNA
Components
  • Complex: complex NP-RNA
    • Complex: RNA
      • RNA: RNA (5'-P(UC)7-FAM3')
    • Complex: Nucleoprotein
      • Protein or peptide: Nucleoprotein

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Supramolecule #1: complex NP-RNA

SupramoleculeName: complex NP-RNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: RNA

SupramoleculeName: RNA / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: synthetic construct (others)

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Supramolecule #3: Nucleoprotein

SupramoleculeName: Nucleoprotein / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Influenza A virus

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Macromolecule #1: RNA (5'-P(UC)7-FAM3')

MacromoleculeName: RNA (5'-P(UC)7-FAM3') / type: rna / ID: 1 / Number of copies: 2
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.234475 KDa
SequenceString:
UCUCUCUCUC UCUC

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Macromolecule #2: Nucleoprotein

MacromoleculeName: Nucleoprotein / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Influenza A virus
Molecular weightTheoretical: 56.150395 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: HMTDGERQNA TEIRASVGKM IDGIGRFYIQ MCTELKLSDY EGRLIQNSLT IERMVLSAFD ERRNKYLEEH PSAGKDPKKT GGPIYRRVD GKWRRELILY DKEEIRRIWR QANNGDDATA GLTHMMIWHS NLNDATYQRT RALVRTGMDP RMCSLMQGST L PRRSGAAG ...String:
HMTDGERQNA TEIRASVGKM IDGIGRFYIQ MCTELKLSDY EGRLIQNSLT IERMVLSAFD ERRNKYLEEH PSAGKDPKKT GGPIYRRVD GKWRRELILY DKEEIRRIWR QANNGDDATA GLTHMMIWHS NLNDATYQRT RALVRTGMDP RMCSLMQGST L PRRSGAAG AAVKGVGTMV MELIRMIKRG INDRNFWRGE NGRRTRIAYE RMCNILKGKF QTAAQRTMVD QVRESRNPGN AE FEDLIFL ARSALILRGS VAHKSCLPAC VYGSAVASGY DFEREGYSLV GIDPFRLLQN SQVYSLIRPN ENPAHKSQLV WMA CHSAAF EDLRVSSFIR GTKVVPRGKL STRGVQIASN ENMETMESST LELRSRYWAI RTRSGGNTNQ QRASSGQISI QPTF SVQRN LPFDRPTIMA AFTGNTEGRT SDMRTEIIRL MESARPEDVS FQGRGVFELS DEKATSPIVP SFDMSNEGSY FFGDN AEEY DNLEHHHHHH

UniProtKB: Nucleoprotein

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.23 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMHEPES
150.0 mMNaClNaCl
5.0 mMbeta-mercaptoethanol
2.0 mMmethyl-PEG8-NHS
4.0 uMRNA (5'P-(UC)6-FAM3')

Details: 20 mM HEPES 150 mM NaCl 5 mM beta-mercaptoethanol 2 mM methyl-PEG8-NHS
VitrificationCryogen name: ETHANE / Chamber humidity: 98 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: 5 seconds blot at force 0.
Details20 mM HEPES 150 mM NaCl 5 mM betamercaptoethanol Incubation overnight with 0.004 uM RNA. Prior freezing, the sample was incubated with 2 mM methyl PEG8 N hydroxysuccinimide ester reagent.

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / 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 / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 36000
Sample stageCooling holder cryogen: NITROGEN

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 24.55 Å
Applied symmetry - Helical parameters - Δ&Phi: 57.27 °
Applied symmetry - Helical parameters - Axial symmetry: C2 (2 fold cyclic)
Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 242730
Startup modelType of model: NONE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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