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- PDB-8fd5: Nucleocapsid monomer structure from SARS-CoV-2 -

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Basic information

Entry
Database: PDB / ID: 8fd5
TitleNucleocapsid monomer structure from SARS-CoV-2
ComponentsNucleoprotein
KeywordsVIRAL PROTEIN / SARS-CoV-2 / N protein / COVID-19 / RNA binding protein
Function / homology
Function and homology information


cytoplasmic capsid assembly / viral RNA genome packaging / response to host immune response / negative regulation of interferon-beta production / RNA stem-loop binding / Maturation of nucleoprotein / positive regulation of NLRP3 inflammasome complex assembly / intracellular non-membrane-bounded organelle / CD28 dependent PI3K/Akt signaling / MHC class I protein binding ...cytoplasmic capsid assembly / viral RNA genome packaging / response to host immune response / negative regulation of interferon-beta production / RNA stem-loop binding / Maturation of nucleoprotein / positive regulation of NLRP3 inflammasome complex assembly / intracellular non-membrane-bounded organelle / CD28 dependent PI3K/Akt signaling / MHC class I protein binding / SARS-CoV-2 targets host intracellular signalling and regulatory pathways / molecular condensate scaffold activity / protein sequestering activity / VEGFR2 mediated vascular permeability / TAK1-dependent IKK and NF-kappa-B activation / DDX58/IFIH1-mediated induction of interferon-alpha/beta / NOD1/2 Signaling Pathway / MHC class I protein complex / Interleukin-1 signaling / viral capsid / Interferon alpha/beta signaling / PIP3 activates AKT signaling / Transcription of SARS-CoV-2 sgRNAs / host cell endoplasmic reticulum-Golgi intermediate compartment / host cell Golgi apparatus / viral nucleocapsid / 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 (N) protein, C-terminal domain, coronavirus / Nucleocapsid (N) protein, N-terminal domain, coronavirus / Coronavirus nucleocapsid (CoV N) protein N-terminal (NTD) domain profile. / Coronavirus nucleocapsid (CoV N) protein C-terminal (CTD) domain profile. / Nucleocapsid protein, coronavirus / Nucleocapsid protein, N-terminal / Coronavirus nucleocapsid / Nucleocapsid protein, C-terminal
Similarity search - Domain/homology
Biological speciesSevere acute respiratory syndrome coronavirus 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.57 Å
AuthorsCasasanta, M. / Jonaid, G.M. / Kaylor, L. / Luqiu, W. / DiCecco, L. / Solares, M. / Berry, S. / Kelly, D.F.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA193578 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA227261 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA219700 United States
Citation
Journal: Microsc Microanal / Year: 2023
Title: Structural Insights of the SARS-CoV-2 Nucleocapsid Protein: Implications for the Inner-workings of Rapid Antigen Tests.
Authors: Michael A Casasanta / G M Jonaid / Liam Kaylor / William Y Luqiu / Liza-Anastasia DiCecco / Maria J Solares / Samantha Berry / William J Dearnaley / Deborah F Kelly /
Abstract: The nucleocapsid (N) protein is an abundant component of SARS-CoV-2 and a key analyte for lateral-flow rapid antigen tests. Here, we present new structural insights for the SARS-CoV-2 N protein using ...The nucleocapsid (N) protein is an abundant component of SARS-CoV-2 and a key analyte for lateral-flow rapid antigen tests. Here, we present new structural insights for the SARS-CoV-2 N protein using cryo-electron microscopy (EM) and molecular modeling tools. Epitope mapping based on structural data supported host-immune interactions in the C-terminal portion of the protein, while other regions revealed protein-protein interaction sites. Complementary modeling results suggested that N protein structures from known variants of concern (VOC) are nearly 100% conserved at specific antibody-binding sites. Collectively, these results suggest that rapid tests that target the nucleocapsid C-terminal domain should have similar accuracy across all VOCs. In addition, our combined structural modeling workflow may guide the design of immune therapies to counter viral processes as we plan for future variants and pandemics.
#1: Journal: Nanoscale / Year: 2021
Title: Microchip-based structure determination of low-molecular weight proteins using cryo-electron microscopy.
Authors: Michael A Casasanta / G M Jonaid / Liam Kaylor / William Y Luqiu / Maria J Solares / Mariah L Schroen / William J Dearnaley / Jarad Wilson / Madeline J Dukes / Deborah F Kelly /
Abstract: Interest in cryo-Electron Microscopy (EM) imaging has skyrocketed in recent years due to its pristine views of macromolecules and materials. As advances in instrumentation and computing algorithms ...Interest in cryo-Electron Microscopy (EM) imaging has skyrocketed in recent years due to its pristine views of macromolecules and materials. As advances in instrumentation and computing algorithms spurred this progress, there is renewed focus to address specimen-related challenges. Here we contribute a microchip-based toolkit to perform complementary structural and biochemical analysis on low-molecular weight proteins. As a model system, we used the SARS-CoV-2 nucleocapsid (N) protein (48 kDa) due to its stability and important role in therapeutic development. Cryo-EM structures of the N protein monomer revealed a flexible N-terminal "top hat" motif and a helical-rich C-terminal domain. To complement our structural findings, we engineered microchip-based immunoprecipitation assays that led to the discovery of the first antibody binding site on the N protein. The data also facilitated molecular modeling of a variety of pandemic and common cold-related coronavirus proteins. Such insights may guide future pandemic-preparedness protocols through immuno-engineering strategies to mitigate viral outbreaks.
History
DepositionDec 2, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 11, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 11, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Nucleoprotein


Theoretical massNumber of molelcules
Total (without water)45,6901
Polymers45,6901
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 45689.645 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Production host: Escherichia coli (E. coli) / References: UniProt: P0DTC9

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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: A chain contains the full-length N protein / Type: COMPLEX
Details: Residues 1-49 were fit into the map separately from residues 50 - 419. The 2 fragments were fit as individual rigid bodies, then refined and rebuilt. There is a missing peptide linkage ...Details: Residues 1-49 were fit into the map separately from residues 50 - 419. The 2 fragments were fit as individual rigid bodies, then refined and rebuilt. There is a missing peptide linkage between residues 49 and 50.
Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Details: 20 mM Tris (pH 7.5), 150 mM NaCl, 10 mM MgCl2, 10 mM CaCl2
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was enriched using Ni-NTA coated silicon nitride microchips
Specimen supportDetails: Protein samples were added to the Ni-NTA-coated microchips and incubated for 1 minute at room temperature prior to vitrification.
Grid material: SILICON NITRIDE / Grid type: Homemade
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K
Details: A Mark III Vitrobot was used to plunge samples into liquid ethane, operating at room temperature and 100% humidity with 3 - 4 seconds blot time

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: TFS TALOS F200C
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 45000 X / Nominal defocus max: 5000 nm / Nominal defocus min: 1000 nm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Image recordingAverage exposure time: 1 sec. / Electron dose: 60 e/Å2 / Detector mode: INTEGRATING / Film or detector model: DIRECT ELECTRON DE-12 (4k x 3k) / Num. of grids imaged: 10 / Num. of real images: 200
Image scansSampling size: 6 µm / Movie frames/image: 30

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameCategory
1RELIONparticle selection
2SerialEMimage acquisition
4RELIONCTF correction
7PHENIXmodel fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
12RELION3D reconstruction
13ISOLDEmodel refinement
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 20000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.57 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 20000 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 100 / Protocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0063276
ELECTRON MICROSCOPYf_angle_d1.1074416
ELECTRON MICROSCOPYf_dihedral_angle_d46.831456
ELECTRON MICROSCOPYf_chiral_restr0.057457
ELECTRON MICROSCOPYf_plane_restr0.008599

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