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- PDB-9gas: Focused reconstruction on strand 2 of the influenza A RNP-like pa... -

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Basic information

Entry
Database: PDB / ID: 9gas
TitleFocused reconstruction on strand 2 of the influenza A RNP-like particle double-stranded assembled with a 18-mer RNA.
Components
  • Nucleoprotein
  • RNA (5'-P(UC)9-FAM3')
KeywordsVIRAL PROTEIN / Nucleoprotein / RNA / complex / RNP-like / influenza virus
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
: / RNA / RNA (> 10) / Nucleoprotein
Similarity search - Component
Biological speciesInfluenza A virus
synthetic construct (others)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.08 Å
AuthorsChenavier, F. / Ruigrok, R.W.H. / Schoehn, G. / Ballandras-Colas, A. / Crepin, T.
Funding support France, 3items
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, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 15, 2025Provider: repository / Type: Initial release
Revision 1.1Feb 12, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
D: RNA (5'-P(UC)9-FAM3')
C: RNA (5'-P(UC)9-FAM3')
B: Nucleoprotein
A: Nucleoprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)123,7095
Polymers123,2154
Non-polymers4941
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: RNA chain RNA (5'-P(UC)9-FAM3')


Mass: 5457.170 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: Protein Nucleoprotein / Nucleocapsid protein / Protein N


Mass: 56150.395 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus / Gene: NP / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q1K9H2
#3: Chemical ChemComp-A1IJK / 2-[3,6-bis(oxidanylidene)-4,5-dihydroxanthen-9-yl]-4-[3-[(2R)-2-oxidanylpropoxy]propylcarbamoyl]benzoic acid


Mass: 493.505 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H27NO8
Has ligand of interestY
Has protein modificationN

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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
1complex NP-RNACOMPLEX#1-#20MULTIPLE SOURCES
2RNACOMPLEX#11RECOMBINANT
3NucleoproteinCOMPLEX#21RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22synthetic construct (others)32630
33Influenza A virus11320
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22synthetic construct (others)32630
33Escherichia coli BL21(DE3) (bacteria)469008
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 mMNaClNaCl1
35 mMbeta-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 betamercaptoethanol Incubation overnight with 0.004 uM RNA. Prior freezing, the sample was incubated with 2 mM methyl PEG8 N hydroxysuccinimide ester reagent.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 98 % / Chamber temperature: 298 K / Details: 5 seconds blot at force 0

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 36000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 53 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
2PHENIX1.20.1_4487:model refinement
3SerialEMimage acquisition
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 56.13 ° / Axial rise/subunit: 27.3 Å / Axial symmetry: C1
3D reconstructionResolution: 3.08 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 276761 / Symmetry type: HELICAL

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