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- EMDB-18044: Focused reconstruction of influenza A RNP-like particle -

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Basic information

Entry
Database: EMDB / ID: EMD-18044
TitleFocused reconstruction of influenza A RNP-like particle
Map dataFocused reconstruction map post-processed.
Sample
  • Complex: Influenza A NP-RNA complex
    • Complex: Nucleoprotein
      • Protein or peptide: Nucleoprotein
    • Complex: RNA (5'P-(UC)6-FAM3')
      • RNA: RNA (5'P-(UC)6-FAM3')
KeywordsInfluenza virus / Nucleocapsid-like / RNA binding protein. / VIRAL PROTEIN
Function / homology
Function and homology information


helical viral capsid / viral penetration into host nucleus / viral nucleocapsid / symbiont entry into host cell / ribonucleoprotein complex / 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 speciesInfluenza A virus / synthetic construct (others)
Methodhelical reconstruction / cryo EM / Resolution: 5.3 Å
AuthorsChenavier F / Estrozi LF / Zarkadas E / Ruigrok RWH / Schoehn G / Ballandras-Colas A / Crepin T
Funding support France, 1 items
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, 2023-
Header (metadata) releaseDec 27, 2023-
Map releaseDec 27, 2023-
UpdateDec 27, 2023-
Current statusDec 27, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18044.png

Downloads & links

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Map

FileDownload / File: emd_18044.map.gz / Format: CCP4 / Size: 4.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFocused reconstruction map post-processed.
Voxel sizeX=Y=Z: 1.68 Å
Density
Contour LevelBy AUTHOR: 0.112
Minimum - Maximum-0.047526892 - 1.7618601
Average (Standard dev.)0.0051084147 (±0.048385072)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions106106106
Spacing106106106
CellA=B=C: 178.08 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18044_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Focused reconstruction map non post-processed.

Fileemd_18044_additional_1.map
AnnotationFocused reconstruction map non post-processed.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 1.

Fileemd_18044_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_18044_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 : Influenza A NP-RNA complex

EntireName: Influenza A NP-RNA complex
Components
  • Complex: Influenza A NP-RNA complex
    • Complex: Nucleoprotein
      • Protein or peptide: Nucleoprotein
    • Complex: RNA (5'P-(UC)6-FAM3')
      • RNA: RNA (5'P-(UC)6-FAM3')

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Supramolecule #1: Influenza A NP-RNA complex

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

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Supramolecule #2: Nucleoprotein

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

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Supramolecule #3: RNA (5'P-(UC)6-FAM3')

SupramoleculeName: RNA (5'P-(UC)6-FAM3') / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: Nucleoprotein

MacromoleculeName: Nucleoprotein / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Influenza A virus
Molecular weightTheoretical: 57.525965 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MATKGTKRSY EQMETDGERQ NATEIRASVG KMIDGIGRFY IQMCTELKLS DYEGRLIQNS LTIERMVLSA FDERRNKYLE EHPSAGKDP KKTGGPIYRR VDGKWRRELI LYDKEEIRRI WRQANNGDDA TAGLTHMMIW HSNLNDATYQ RTRALVRTGM D PRMCSLMQ ...String:
MATKGTKRSY EQMETDGERQ NATEIRASVG KMIDGIGRFY IQMCTELKLS DYEGRLIQNS LTIERMVLSA FDERRNKYLE EHPSAGKDP KKTGGPIYRR VDGKWRRELI LYDKEEIRRI WRQANNGDDA TAGLTHMMIW HSNLNDATYQ RTRALVRTGM D PRMCSLMQ GSTLPRRSGA AGAAVKGVGT MVMELIRMIK RGINDRNFWR GENGRRTRIA YERMCNILKG KFQTAAQRTM VD QVRESRN PGNAEFEDLI FLARSALILR GSVAHKSCLP ACVYGSAVAS GYDFEREGYS LVGIDPFRLL QNSQVYSLIR PNE NPAHKS QLVWMACHSA AFEDLRVSSF IRGTKVVPRG KLSTRGVQIA SNENMETMES STLELRSRYW AIRTRSGGNT NQQR ASSGQ ISIQPTFSVQ RNLPFDRPTI MAAFTGNTEG RTSDMRTEII RLMESARPED VSFQGRGVFE LSDEKATSPI VPSFD MSNE GSYFFGDNAE EYDNLEHHHH HH

UniProtKB: Nucleoprotein

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Macromolecule #2: RNA (5'P-(UC)6-FAM3')

MacromoleculeName: RNA (5'P-(UC)6-FAM3') / type: rna / ID: 2 / Number of copies: 2
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.623128 KDa
SequenceString:
UCUCUCUCUC UC

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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 mMNaClSodium chlorideNaClSodium chloride
5.0 mMbeta-mercaptoethanol2-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
GridModel: Quantifoil R0.6/1 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Details: 25 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
Details20 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

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 2.2 µm / Calibrated defocus min: 0.8 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Quantum LS
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 26515 / Average exposure time: 1.9 sec. / Average electron dose: 40.8 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC (ver. 3.3.2)
Software - details: Local refine of signal subtracted particles
Final reconstructionApplied symmetry - Helical parameters - Δz: 24.27 Å
Applied symmetry - Helical parameters - Δ&Phi: 57.41 °
Applied symmetry - Helical parameters - Axial symmetry: C2 (2 fold cyclic)
Resolution.type: BY AUTHOR / Resolution: 5.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 59232
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5tjw


Chain - Chain ID: A / Chain - Residue range: 21-490 / Chain - Source name: PDB / Chain - Initial model type: experimental model / Details: Fitting of the NPcore by using ChimeraX
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8pzq:
Model for focused reconstruction of influenza A RNP-like particle

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