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- EMDB-29072: SARS-CoV-2 Nucleocapsid dimer structure determined from COVID-19 ... -

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Basic information

Entry
Database: EMDB / ID: EMD-29072
TitleSARS-CoV-2 Nucleocapsid dimer structure determined from COVID-19 patients
Map dataSARS-CoV-2 Nucleocapsid dimer structure determined from COVID-19 patients
Sample
  • Complex: Nucleocapsid dimer is comprised of A chain and B chain
    • Protein or peptide: Nucleoprotein
KeywordsSARS-CoV-2 / N protein / COVID-19 / RNA binding protein / VIRAL 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 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.0 Å
AuthorsCasasanta M / Jonaid GM / Kaylor L / Luqiu W / DiCecco L / Solares M / Berry S / Kelly DF
Funding support United States, 3 items
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: 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.
#1: Journal: Microsc Microanal / Year: 2022
Title: Structural insights of the SARS-CoV-2 Nucleocapsid protein: Implications for the inner-workings of rapid antigen tests
Authors: Casasanta M / Jonaid GM / Kaylor L / Luqiu W / DiCecco L / Solares M / Berry S / Kelly DF
History
DepositionDec 12, 2022-
Header (metadata) releaseJan 11, 2023-
Map releaseJan 11, 2023-
UpdateOct 11, 2023-
Current statusOct 11, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29072.png

Downloads & links

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Map

FileDownload / File: emd_29072.map.gz / Format: CCP4 / Size: 2.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSARS-CoV-2 Nucleocapsid dimer structure determined from COVID-19 patients
Voxel sizeX=Y=Z: 1.4 Å
Density
Contour LevelBy AUTHOR: 0.207
Minimum - Maximum-2.5431237 - 29.300049000000001
Average (Standard dev.)-0.000000000002932 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions779184
Spacing847791
CellA: 117.6 Å / B: 107.799995 Å / C: 127.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map 1

Fileemd_29072_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_29072_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 : Nucleocapsid dimer is comprised of A chain and B chain

EntireName: Nucleocapsid dimer is comprised of A chain and B chain
Components
  • Complex: Nucleocapsid dimer is comprised of A chain and B chain
    • Protein or peptide: Nucleoprotein

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Supramolecule #1: Nucleocapsid dimer is comprised of A chain and B chain

SupramoleculeName: Nucleocapsid dimer is comprised of A chain and B chain
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: For each monomer that comprises the dimer structure, residues 1-49 were fit into the map separately from residues 50 - 419.
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: Homo sapiens (human)
Molecular weightTheoretical: 45.689645 KDa
SequenceString: MSDNGPQNQR NAPRITFGGP SDSTGSNQNG ERSGARSKQR RPQGLPNNTA SWFTALTQHG KEDLKFPRGQ GVPINTNSSP DDQIGYYRR ATRRIRGGDG KMKDLSPRWY FYYLGTGPEA GLPYGANKDG IIWVATEGAL NTPKDHIGTR NPANNAAIVL Q LPQGTTLP ...String:
MSDNGPQNQR NAPRITFGGP SDSTGSNQNG ERSGARSKQR RPQGLPNNTA SWFTALTQHG KEDLKFPRGQ GVPINTNSSP DDQIGYYRR ATRRIRGGDG KMKDLSPRWY FYYLGTGPEA GLPYGANKDG IIWVATEGAL NTPKDHIGTR NPANNAAIVL Q LPQGTTLP KGFYAEGSRG GSQASSRSSS RSRNSSRNST PGSSRGTSPA RMAGNGGDAA LALLLLDRLN QLESKMSGKG QQ QQGQTVT KKSAAEASKK PRQKRTATKA YNVTQAFGRR GPEQTQGNFG DQELIRQGTD YKHWPQIAQF APSASAFFGM SRI GMEVTP SGTWLTYTGA IKLDDKDPNF KDQVILLNKH IDAYKTFPPT EPKKDKKKKA DETQALPQRQ KKQQTVTLLP AADL DDFSK QLQQSMSSAD STQA

UniProtKB: Nucleoprotein

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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

Concentration0.1 mg/mL
BufferpH: 7.5
Details: 20 mM Tris (pH 7.5), 150 mM NaCl, 10 mM MgCl2, 10 mM CaCl2
GridModel: Homemade / Material: SILICON NITRIDE
Details: Samples were incubated with Ni-NTA coated microchips for 1 minute prior to plunge freezing into liquid ethane.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK III
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.
DetailsSample was enriched using Ni-NTA coated silicon nitride microchips

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 4 / Number real images: 300 / Average exposure time: 1.0 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 10000
Startup modelType of model: NONE
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 1 / Avg.num./class: 10000 / Software - Name: cryoSPARC
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 10000
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 100
Output model

PDB-8fg2:
SARS-CoV-2 Nucleocapsid dimer structure determined from COVID-19 patients

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