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- EMDB-22982: Nucleocapsid protein from the SARS-CoV-2 coronavirus -

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Basic information

Entry
Database: EMDB / ID: EMD-22982
TitleNucleocapsid protein from the SARS-CoV-2 coronavirusVirus
Map dataNucleocapsid protein from the SARS-CoV-2 CoronavirusVirus
SampleSARS-CoV-2 != Severe acute respiratory syndrome coronavirus 2

SARS-CoV-2

  • Virus: Severe acute respiratory syndrome coronavirus 2
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsCasasanta MA / Kelly DF
CitationJournal: 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
DepositionNov 9, 2020-
Header (metadata) releaseMay 5, 2021-
Map releaseMay 5, 2021-
UpdateMay 5, 2021-
Current statusMay 5, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0035
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0035
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22982.png

Downloads & links

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Map

FileDownload / File: emd_22982.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNucleocapsid protein from the SARS-CoV-2 Coronavirus
Voxel sizeX=Y=Z: 0.93 Å
Density
Contour LevelBy AUTHOR: 0.0035 / Movie #1: 0.0035
Minimum - Maximum-0.0 - 0.0258326
Average (Standard dev.)0.00013124387 (±0.0010522302)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin150150150
Dimensions150150150
Spacing150150150
CellA=B=C: 139.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.930.930.93
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z139.500139.500139.500
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS150150150
NC/NR/NS150150150
D min/max/mean-0.0000.0260.000

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Supplemental data

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Sample components

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Entire : SARS-CoV-2

EntireName: SARS-CoV-2 (virus)
Components
  • Virus: Severe acute respiratory syndrome coronavirus 2

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Supramolecule #1: Severe acute respiratory syndrome coronavirus 2

SupramoleculeName: Severe acute respiratory syndrome coronavirus 2 / type: virus / ID: 1 / Parent: 0 / NCBI-ID: 2697049
Sci species name: Severe acute respiratory syndrome coronavirus 2
Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: Yes
Host (natural)Organism: Homo sapiens (human)
Host systemOrganism: Escherichia coli (E. coli)
Molecular weightExperimental: 50 KDa

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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
Component:
ConcentrationNameFormula
20.0 mMTris
150.0 mMsodium chlorideNaClSodium chloride
10.0 mMmagnesium chlorideMgCl2
10.0 mMcalcium chlorideCaCl2

Details: Solutions were made fresh before diluting protein and adding to the EM grids
GridModel: Homemade / Material: SILICON NITRIDE / Support film - topology: CONTINUOUS / Support film - Film thickness: 20.0 nm
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK III / Details: blot for 7.5 seconds before plunging.
DetailsSample was monodisperse on a silicon nitride microchip coated with 25% Ni-NTA lipids.

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Electron microscopy

MicroscopeTFS TALOS F200C
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 45000
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
TemperatureMin: 75.0 K / Max: 83.0 K
Image recordingFilm or detector model: DIRECT ELECTRON DE-12 (4k x 3k) / Detector mode: INTEGRATING / Number grids imaged: 3 / Average exposure time: 0.25 sec. / Average electron dose: 5.0 e/Å2
Details: Images were collected in movie mode with 0.25 second exposures at 30 frames per second.
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 20000
CTF correctionSoftware - Name: cryoSPARC
Details: CTF correction was performed in Cryosparc before 3D reconstruction.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 1 / Avg.num./class: 20000 / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 20000

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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