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- EMDB-45157: SARS-CoV-2 Nucleocapsid Dimerization Domain bound to Fab-NP1E9 an... -

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Basic information

Entry
Database: EMDB / ID: EMD-45157
TitleSARS-CoV-2 Nucleocapsid Dimerization Domain bound to Fab-NP1E9 and Fab-NP3B4
Map data
Sample
  • Complex: SARS-CoV-2 Nucleocapsid Dimerization Domain bound to Antibody Fabs NP1E9 and NP3B4
    • Protein or peptide: Nucleoprotein
    • Protein or peptide: Antibody Fab NP3-B4 Heavy Chain (variable region)
    • Protein or peptide: Antibody Fab NP3-B4 Light Chain (variable region)
    • Protein or peptide: Antibody Fab NP1-E9 Heavy Chain (variable region)
    • Protein or peptide: Antibody Fab NP1-E9 Light Chain (variable region)
KeywordsProtein / Fab / immunocomplex / VIRAL PROTEIN
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 / NOD1/2 Signaling Pathway / TAK1-dependent IKK and NF-kappa-B activation / DDX58/IFIH1-mediated induction of interferon-alpha/beta / molecular condensate scaffold activity / MHC class I protein complex / Interleukin-1 signaling / Interferon alpha/beta signaling / RNA stem-loop binding / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsLanderas-Bueno S / Hariharan C / Diaz Avalos R / Ollmann Saphire E
Funding support United States, 1 items
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, 2024-
Header (metadata) releaseMay 14, 2025-
Map releaseMay 14, 2025-
UpdateJul 30, 2025-
Current statusJul 30, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45157.png

Downloads & links

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Map

FileDownload / File: emd_45157.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.32 Å/pix.
x 320 pix.
= 422.4 Å		1.32 Å/pix.
x 320 pix.
= 422.4 Å		1.32 Å/pix.
x 320 pix.
= 422.4 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.32 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.397
Minimum - Maximum-0.0016681394 - 2.010899
Average (Standard dev.)0.0005506011 (±0.01681343)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 422.40002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_45157_half_map_1.map
Projections & Slices
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Slices (1/2)
Density Histograms

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Histogram (log scale)

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Half map: #2

Fileemd_45157_half_map_2.map
Projections & Slices
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Sample components

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Entire : SARS-CoV-2 Nucleocapsid Dimerization Domain bound to Antibody Fab...

EntireName: SARS-CoV-2 Nucleocapsid Dimerization Domain bound to Antibody Fabs NP1E9 and NP3B4
Components
  • Complex: SARS-CoV-2 Nucleocapsid Dimerization Domain bound to Antibody Fabs NP1E9 and NP3B4
    • Protein or peptide: Nucleoprotein
    • Protein or peptide: Antibody Fab NP3-B4 Heavy Chain (variable region)
    • Protein or peptide: Antibody Fab NP3-B4 Light Chain (variable region)
    • Protein or peptide: Antibody Fab NP1-E9 Heavy Chain (variable region)
    • Protein or peptide: Antibody Fab NP1-E9 Light Chain (variable region)

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Supramolecule #1: SARS-CoV-2 Nucleocapsid Dimerization Domain bound to Antibody Fab...

SupramoleculeName: SARS-CoV-2 Nucleocapsid Dimerization Domain bound to Antibody Fabs NP1E9 and NP3B4
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2

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Macromolecule #1: Nucleoprotein

MacromoleculeName: Nucleoprotein / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 16.42241 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MAHHHHHHVD DDDKMENLYF QGMQTVTKKS AAEASKKPRQ KRTATKAYNV TQAFGRRGPE QTQGNFGDQE LIRQGTDYKH WPQIAQFAP SASAFFGMSR IGMEVTPSGT WLTYTGAIKL DDKDPNFKDQ VILLNKHIDA YKTFP

UniProtKB: Nucleoprotein

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Macromolecule #2: Antibody Fab NP3-B4 Heavy Chain (variable region)

MacromoleculeName: Antibody Fab NP3-B4 Heavy Chain (variable region) / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus sp. (mice)
Molecular weightTheoretical: 26.549525 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: EVQLVESGGG LVQPGGSVKL SCLASGFTFS DYYMSWVRQS PEKGLEWVAE IRLESDNYAT HYAESVKGKF TISRDDSKSR LYLKMNSLR GEDTGIYYCA FDVYYGGAMD YWGQGTTVTV EVQLVESGGG LVQPGGSVKL SCLASGFTFS DYYMSWVRQS P EKGLEWVA ...String:
EVQLVESGGG LVQPGGSVKL SCLASGFTFS DYYMSWVRQS PEKGLEWVAE IRLESDNYAT HYAESVKGKF TISRDDSKSR LYLKMNSLR GEDTGIYYCA FDVYYGGAMD YWGQGTTVTV EVQLVESGGG LVQPGGSVKL SCLASGFTFS DYYMSWVRQS P EKGLEWVA EIRLESDNYA THYAESVKGK FTISRDDSKS RLYLKMNSLR GEDTGIYYCA FDVYYGGAMD YWGQGTTVTV

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Macromolecule #3: Antibody Fab NP3-B4 Light Chain (variable region)

MacromoleculeName: Antibody Fab NP3-B4 Light Chain (variable region) / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus sp. (mice)
Molecular weightTheoretical: 11.548821 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString:
CDIQMTQSPS IMSASPGEKV TMTCSASSSV SYMHWYQQKS STSPKLWIYD TSELASGVPG RFSGSRSGNS YSLTISSMEA EDVATYYCF QGSGYPLTFG GGTKLELK

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Macromolecule #4: Antibody Fab NP1-E9 Heavy Chain (variable region)

MacromoleculeName: Antibody Fab NP1-E9 Heavy Chain (variable region) / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus sp. (mice)
Molecular weightTheoretical: 12.557037 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString:
QVQLQQSGPE LVKPGTLVKI SCKASGYTFT SYDINWVKQR PGQGLEWIGW IYPGDGSTKY NEKFKGKATL TADTSSSTAY MQLNSLTSE NSAVYFCARG LVGAMDYWGQ GTSVTV

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Macromolecule #5: Antibody Fab NP1-E9 Light Chain (variable region)

MacromoleculeName: Antibody Fab NP1-E9 Light Chain (variable region) / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus sp. (mice)
Molecular weightTheoretical: 11.853099 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString:
DIVMTQSQKF MSTSVGDRVS VTCKASQNVL NNVAWYQQKP GQSPKALIYS ASYRYSGVPD RFTGSGSGTD FTLTISNVQS EDLAEYFCQ QYNSYPLTFG DGTKLELK

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.15 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMHepesHepes
250.0 mMNaClsodium chloride
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1520588
CTF correctionSoftware - Name: cryoSPARC (ver. 2.15) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 632077
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.15)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.15)

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