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- PDB-9c2h: SARS-CoV-2 Nucleocapsid Dimerization Domain bound to Fab-NP1E9 an... -

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Basic information

Entry
Database: PDB / ID: 9c2h
TitleSARS-CoV-2 Nucleocapsid Dimerization Domain bound to Fab-NP1E9 and Fab-NP3B4
Components
  • Antibody Fab NP1-E9 Heavy Chain (variable region)
  • Antibody Fab NP1-E9 Light Chain (variable region)
  • Antibody Fab NP3-B4 Heavy Chain (variable region)
  • Antibody Fab NP3-B4 Light Chain (variable region)
  • Nucleoprotein
KeywordsVIRAL PROTEIN / Protein / Fab / immunocomplex
Function / homology
Function and homology information


: / response to host immune response / viral RNA genome packaging / negative regulation of interferon-beta production / poly(U) RNA binding / Maturation of nucleoprotein / intracellular membraneless organelle / positive regulation of NLRP3 inflammasome complex assembly / MHC class I protein binding / CD28 dependent PI3K/Akt signaling ...: / response to host immune response / viral RNA genome packaging / negative regulation of interferon-beta production / poly(U) RNA binding / Maturation of nucleoprotein / intracellular membraneless organelle / positive regulation of NLRP3 inflammasome complex assembly / MHC class I protein binding / CD28 dependent PI3K/Akt signaling / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / protein sequestering activity / VEGFR2 mediated vascular permeability / molecular condensate scaffold activity / NOD1/2 Signaling Pathway / TAK1-dependent IKK and NF-kappa-B activation / DDX58/IFIH1-mediated induction of interferon-alpha/beta / MHC class I protein complex / RNA stem-loop binding / Interleukin-1 signaling / Interferon alpha/beta signaling / viral capsid / PIP3 activates AKT signaling / Transcription of SARS-CoV-2 sgRNAs / host cell endoplasmic reticulum-Golgi intermediate compartment / viral nucleocapsid / host cell Golgi apparatus / Translation of Structural Proteins / Virion Assembly and Release / host extracellular space / Induction of Cell-Cell Fusion / Attachment and Entry / host cell perinuclear region of cytoplasm / ribonucleoprotein complex / SARS-CoV-2 activates/modulates innate and adaptive immune responses / protein homodimerization activity / RNA binding / extracellular region / identical protein binding / cytoplasm
Similarity search - Function
Nucleocapsid protein, betacoronavirus / Nucleocapsid protein, coronavirus / Nucleocapsid protein, C-terminal / Nucleocapsid protein, N-terminal / Nucleocapsid (N) protein, C-terminal domain, coronavirus / Nucleocapsid (N) protein, N-terminal domain, coronavirus / Coronavirus nucleocapsid / Coronavirus nucleocapsid (CoV N) protein N-terminal (NTD) domain profile. / Coronavirus nucleocapsid (CoV N) protein C-terminal (CTD) domain profile.
Similarity search - Domain/homology
Biological speciesSevere acute respiratory syndrome coronavirus 2
Mus sp. (mice)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsLanderas-Bueno, S. / Hariharan, C. / Diaz Avalos, R. / Ollmann Saphire, E.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)NIH R21 AI178427-01 United States
CitationJournal: Nat Commun / Year: 2025
Title: Structural stabilization of the intrinsically disordered SARS-CoV-2 N by binding to RNA sequences engineered from the viral genome fragment.
Authors: Sara Landeras-Bueno / Chitra Hariharan / Ruben Diaz Avalos / Andrew S Norris / Dalton T Snyder / Kathryn M Hastie / Stephanie Harkins / Michelle Zandonatti / Roshan R Rajamanickam / Eduardo ...Authors: Sara Landeras-Bueno / Chitra Hariharan / Ruben Diaz Avalos / Andrew S Norris / Dalton T Snyder / Kathryn M Hastie / Stephanie Harkins / Michelle Zandonatti / Roshan R Rajamanickam / Eduardo Olmedillas / Robyn Miller / Sujan Shresta / Vicki H Wysocki / Erica Ollmann Saphire /
Abstract: The nucleocapsid N is one of four structural proteins of the coronaviruses. Its essential role in genome encapsidation makes it a critical therapeutic target for COVID-19 and related diseases. ...The nucleocapsid N is one of four structural proteins of the coronaviruses. Its essential role in genome encapsidation makes it a critical therapeutic target for COVID-19 and related diseases. However, the inherent disorder of full-length N hampers its structural analysis. Here, we describe a stepwise method using viral-derived RNAs to stabilize SARS-CoV-2 N for EM analysis. We identify pieces of RNA from the SARS-CoV-2 genome that promote the formation of structurally homogeneous N dimers, intermediates of assembly, and filamentous capsid-like structures. Building on these results, we engineer a symmetric RNA to stabilize N protein dimers, the building block of high-order assemblies, for EM studies. We combine domain-specific monoclonal antibodies against N with chemical cross-linking mass spectrometry to validate the spatial arrangement of the N domains within the dimer. Additionally, our cryo-EM analysis reveals novel antigenic sites on the N protein. Our findings provide insights into N protein´s architectural and antigenic principles, which can guide design of pan-coronavirus therapeutics.
History
DepositionMay 31, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 14, 2025Provider: repository / Type: Initial release
Revision 1.0May 14, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 14, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 14, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 14, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 14, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jul 30, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _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
A: Nucleoprotein
B: Antibody Fab NP3-B4 Heavy Chain (variable region)
C: Antibody Fab NP3-B4 Light Chain (variable region)
D: Nucleoprotein
E: Antibody Fab NP1-E9 Heavy Chain (variable region)
F: Antibody Fab NP1-E9 Light Chain (variable region)
G: Antibody Fab NP3-B4 Heavy Chain (variable region)
H: Antibody Fab NP3-B4 Light Chain (variable region)
I: Antibody Fab NP1-E9 Heavy Chain (variable region)
J: Antibody Fab NP1-E9 Light Chain (variable region)


Theoretical massNumber of molelcules
Total (without water)157,86210
Polymers157,86210
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 16422.410 Da / Num. of mol.: 2 / Fragment: UNP Residues 244-364
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0DTC9
#2: Antibody Antibody Fab NP3-B4 Heavy Chain (variable region)


Mass: 26549.525 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus sp. (mice) / Production host: Cricetulus griseus (Chinese hamster)
#3: Antibody Antibody Fab NP3-B4 Light Chain (variable region)


Mass: 11548.821 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus sp. (mice) / Production host: Cricetulus griseus (Chinese hamster)
#4: Antibody Antibody Fab NP1-E9 Heavy Chain (variable region)


Mass: 12557.037 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus sp. (mice) / Production host: Cricetulus griseus (Chinese hamster)
#5: Antibody Antibody Fab NP1-E9 Light Chain (variable region)


Mass: 11853.099 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus sp. (mice) / Production host: Cricetulus griseus (Chinese hamster)
Has protein modificationY

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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: SARS-CoV-2 Nucleocapsid Dimerization Domain bound to Antibody Fabs NP1E9 and NP3B4
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHepesHepes1
2250 mMsodium chlorideNaCl1
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 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 defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC2.15particle selection
4cryoSPARC2.15CTF correction
8PHENIXmodel refinement
10cryoSPARC2.15initial Euler assignment
11cryoSPARC2.15final Euler assignment
12cryoSPARC2.15classification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1520588
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 632077 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0028830
ELECTRON MICROSCOPYf_angle_d0.52411950
ELECTRON MICROSCOPYf_dihedral_angle_d4.9331206
ELECTRON MICROSCOPYf_chiral_restr0.0391270
ELECTRON MICROSCOPYf_plane_restr0.0041528

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