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- EMDB-42509: Intracellular cryo-tomography structure of EBOV nucleocapsid at 8... -

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Basic information

Entry
Database: EMDB / ID: EMD-42509
TitleIntracellular cryo-tomography structure of EBOV nucleocapsid at 8.9 Angstrom
Map datamap sharp
Sample
  • Virus: Complex
    • Complex: NP core with RNA
      • Protein or peptide: Nucleoprotein
      • RNA: RNA (5'-R(*AP*AP*AP*AP*AP*A)-3')
    • Complex: Outer NP with VP35 NP-binding peptide
      • Protein or peptide: Polymerase cofactor VP35
    • Complex: VP24 second layer of nucleocapsid
      • Protein or peptide: Membrane-associated protein VP24
    • Complex: VP35 CTD
KeywordsVIRAL PROTEIN / nucleoprotein / nucleocapsid / Ebola virus / EBOV / filovirus / subtomogram averaging / cryo-ET / FIB / intracellular / in situ
Function / homology
Function and homology information


suppression by virus of host intracellular interferon activity / suppression by virus of host cytokine production / host cell endomembrane system / symbiont-mediated suppression of host defenses / symbiont-mediated suppression of host RNAi-mediated antiviral immune response / negative regulation of miRNA-mediated gene silencing / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF7 activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IKBKE activity / suppression by virus of host type I interferon production / symbiont-mediated suppression of host antigen processing and presentation of peptide antigen via MHC class II ...suppression by virus of host intracellular interferon activity / suppression by virus of host cytokine production / host cell endomembrane system / symbiont-mediated suppression of host defenses / symbiont-mediated suppression of host RNAi-mediated antiviral immune response / negative regulation of miRNA-mediated gene silencing / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF7 activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IKBKE activity / suppression by virus of host type I interferon production / symbiont-mediated suppression of host antigen processing and presentation of peptide antigen via MHC class II / positive regulation of protein sumoylation / viral RNA genome packaging / molecular sequestering activity / helical viral capsid / viral transcription / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT1 activity / viral genome replication / viral budding from plasma membrane / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / symbiont-mediated suppression of host toll-like receptor signaling pathway / viral nucleocapsid / host cell cytoplasm / symbiont-mediated suppression of host innate immune response / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / ribonucleoprotein complex / negative regulation of gene expression / host cell plasma membrane / virion membrane / structural molecule activity / RNA binding / membrane
Similarity search - Function
Filovirus membrane-associated VP24 / Filovirus membrane-associated protein VP24 / Ebola nucleoprotein / Ebola nucleoprotein / Filoviruses VP35 interferon inhibitory domain, beta-sheet subdomain / Filoviridae VP35 protein / Filoviruses VP35 interferon inhibitory domain / Filoviruses VP35 interferon inhibitory domain, helical subdomain / Filoviridae VP35 / Filoviruses VP35 interferon inhibitory domain profile.
Similarity search - Domain/homology
Nucleoprotein / Polymerase cofactor VP35 / Membrane-associated protein VP24
Similarity search - Component
Biological speciesHomo sapiens (human) / Ebola virus - Mayinga, Zaire, 1976 / Zaire (virus)
Methodsubtomogram averaging / cryo EM / Resolution: 8.9 Å
AuthorsWatanabe R / Zyla D / Saphire EO
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Cell / Year: 2024
Title: Intracellular Ebola virus nucleocapsid assembly revealed by in situ cryo-electron tomography.
Authors: Reika Watanabe / Dawid Zyla / Diptiben Parekh / Connor Hong / Ying Jones / Sharon L Schendel / William Wan / Guillaume Castillon / Erica Ollmann Saphire /
Abstract: Filoviruses, including the Ebola and Marburg viruses, cause hemorrhagic fevers with up to 90% lethality. The viral nucleocapsid is assembled by polymerization of the nucleoprotein (NP) along the ...Filoviruses, including the Ebola and Marburg viruses, cause hemorrhagic fevers with up to 90% lethality. The viral nucleocapsid is assembled by polymerization of the nucleoprotein (NP) along the viral genome, together with the viral proteins VP24 and VP35. We employed cryo-electron tomography of cells transfected with viral proteins and infected with model Ebola virus to illuminate assembly intermediates, as well as a 9 Å map of the complete intracellular assembly. This structure reveals a previously unresolved third and outer layer of NP complexed with VP35. The intrinsically disordered region, together with the C-terminal domain of this outer layer of NP, provides the constant width between intracellular nucleocapsid bundles and likely functions as a flexible tether to the viral matrix protein in the virion. A comparison of intracellular nucleocapsids with prior in-virion nucleocapsid structures reveals that the nucleocapsid further condenses vertically in the virion. The interfaces responsible for nucleocapsid assembly are highly conserved and offer targets for broadly effective antivirals.
History
DepositionOct 27, 2023-
Header (metadata) releaseOct 2, 2024-
Map releaseOct 2, 2024-
UpdateOct 2, 2024-
Current statusOct 2, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42509.png

Downloads & links

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Map

FileDownload / File: emd_42509.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmap sharp
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
2.15 Å/pix.
x 224 pix.
= 481.376 Å		2.15 Å/pix.
x 224 pix.
= 481.376 Å		2.15 Å/pix.
x 224 pix.
= 481.376 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 2.149 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0037
Minimum - Maximum-0.013077345 - 0.025204882
Average (Standard dev.)0.000066310444 (±0.001457545)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 481.37598 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half 2

Fileemd_42509_half_map_1.map
Annotationhalf 2
Projections & Slices
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Half map: half 1

Fileemd_42509_half_map_2.map
Annotationhalf 1
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Complex

EntireName: Complex
Components
  • Virus: Complex
    • Complex: NP core with RNA
      • Protein or peptide: Nucleoprotein
      • RNA: RNA (5'-R(*AP*AP*AP*AP*AP*A)-3')
    • Complex: Outer NP with VP35 NP-binding peptide
      • Protein or peptide: Polymerase cofactor VP35
    • Complex: VP24 second layer of nucleocapsid
      • Protein or peptide: Membrane-associated protein VP24
    • Complex: VP35 CTD

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Supramolecule #1: Complex

SupramoleculeName: Complex / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Details: 3 virus proteins were expressed from a plasmid in HEK 293T cells
Virus type: VIRUS-LIKE PARTICLE / Virus isolate: OTHER / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Homo sapiens (human)

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Supramolecule #2: NP core with RNA

SupramoleculeName: NP core with RNA / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Outer NP with VP35 NP-binding peptide

SupramoleculeName: Outer NP with VP35 NP-binding peptide / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1, #4
Source (natural)Organism: Ebola virus - Mayinga, Zaire, 1976

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Supramolecule #4: VP24 second layer of nucleocapsid

SupramoleculeName: VP24 second layer of nucleocapsid / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Ebola virus - Mayinga, Zaire, 1976

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Supramolecule #5: VP35 CTD

SupramoleculeName: VP35 CTD / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: Ebola virus - Mayinga, Zaire, 1976

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Macromolecule #1: Nucleoprotein

MacromoleculeName: Nucleoprotein / type: protein_or_peptide / ID: 1 / Details: The N-terminal part of nucleoprotein / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Ebola virus - Mayinga, Zaire, 1976
Molecular weightTheoretical: 83.3875 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDSRPQKIWM APSLTESDMD YHKILTAGLS VQQGIVRQRV IPVYQVNNLE EICQLIIQAF EAGVDFQESA DSFLLMLCLH HAYQGDYKL FLESGAVKYL EGHGFRFEVK KRDGVKRLEE LLPAVSSGKN IKRTLAAMPE EETTEANAGQ FLSFASLFLP K LVVGEKAC ...String:
MDSRPQKIWM APSLTESDMD YHKILTAGLS VQQGIVRQRV IPVYQVNNLE EICQLIIQAF EAGVDFQESA DSFLLMLCLH HAYQGDYKL FLESGAVKYL EGHGFRFEVK KRDGVKRLEE LLPAVSSGKN IKRTLAAMPE EETTEANAGQ FLSFASLFLP K LVVGEKAC LEKVQRQIQV HAEQGLIQYP TAWQSVGHMM VIFRLMRTNF LIKFLLIHQG MHMVAGHDAN DAVISNSVAQ AR FSGLLIV KTVLDHILQK TERGVRLHPL ARTAKVKNEV NSFKAALSSL AKHGEYAPFA RLLNLSGVNN LEHGLFPQLS AIA LGVATA HGSTLAGVNV GEQYQQLREA ATEAEKQLQQ YAESRELDHL GLDDQEKKIL MNFHQKKNEI SFQQTNAMVT LRKE RLAKL TEAITAASLP KTSGHYDDDD DIPFPGPIND DDNPGHQDDD PTDSQDTTIP DVVVDPDDGS YGEYQSYSEN GMNAP DDLV LFDLDEDDED TKPVPNRSTK GGQQKNSQKG QHIEGRQTQS RPIQNVPGPH RTIHHASAPL TDNDRRNEPS GSTSPR MLT PINEEADPLD DADDETSSLP PLESDDEEQD RDGTSNRTPT VAPPAPVYRD HSEKKELPQD EQQDQDHTQE ARNQDSD NT QSEHSFEEMY RHILRSQGPF DAVLYYHMMK DEPVVFSTSD GKEYTYPDSL EEEYPPWLTE KEAMNEENRF VTLDGQQF Y WPVMNHKNKF MAILQHHQ

UniProtKB: Nucleoprotein

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Macromolecule #3: Membrane-associated protein VP24

MacromoleculeName: Membrane-associated protein VP24 / type: protein_or_peptide / ID: 3 / Details: VP24 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Ebola virus - Mayinga, Zaire, 1976
Molecular weightTheoretical: 28.250811 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAKATGRYNL ISPKKDLEKG VVLSDLCNFL VSQTIQGWKV YWAGIEFDVT HKGMALLHRL KTNDFAPAWS MTRNLFPHLF QNPNSTIES PLWALRVILA AGIQDQLIDQ SLIEPLAGAL GLISDWLLTT NTNHFNMRTQ RVKEQLSLKM LSLIRSNILK F INKLDALH ...String:
MAKATGRYNL ISPKKDLEKG VVLSDLCNFL VSQTIQGWKV YWAGIEFDVT HKGMALLHRL KTNDFAPAWS MTRNLFPHLF QNPNSTIES PLWALRVILA AGIQDQLIDQ SLIEPLAGAL GLISDWLLTT NTNHFNMRTQ RVKEQLSLKM LSLIRSNILK F INKLDALH VVNYNGLLSS IEIGTQNHTI IITRTNMGFL VELQEPDKSA MNRMKPGPAK FSLLHESTLK AFTQGSSTRM QS LILEFNS SLAI

UniProtKB: Membrane-associated protein VP24

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Macromolecule #4: Polymerase cofactor VP35

MacromoleculeName: Polymerase cofactor VP35 / type: protein_or_peptide / ID: 4 / Details: C-terminal domain of VP35 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Ebola virus - Mayinga, Zaire, 1976
Molecular weightTheoretical: 37.403277 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MTTRTKGRGH TAATTQNDRM PGPELSGWIS EQLMTGRIPV SDIFCDIENN PGLCYASQMQ QTKPNPKTRN SQTQTDPICN HSFEEVVQT LASLATVVQQ QTIASESLEQ RITSLENGLK PVYDMAKTIS SLNRVCAEMV AKYDLLVMTT GRATATAAAT E AYWAEHGQ ...String:
MTTRTKGRGH TAATTQNDRM PGPELSGWIS EQLMTGRIPV SDIFCDIENN PGLCYASQMQ QTKPNPKTRN SQTQTDPICN HSFEEVVQT LASLATVVQQ QTIASESLEQ RITSLENGLK PVYDMAKTIS SLNRVCAEMV AKYDLLVMTT GRATATAAAT E AYWAEHGQ PPPGPSLYEE SAIRGKIESR DETVPQSVRE AFNNLNSTTS LTEENFGKPD ISAKDLRNIM YDHLPGFGTA FH QLVQVIC KLGKDSNSLD IIHAEFQASL AEGDSPQCAL IQITKRVPIF QDAAPPVIHI RSRGDIPRAC QKSLRPVPPS PKI DRGWVC VFQLQDGKTL GLKI

UniProtKB: Polymerase cofactor VP35

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Macromolecule #2: RNA (5'-R(*AP*AP*AP*AP*AP*A)-3')

MacromoleculeName: RNA (5'-R(*AP*AP*AP*AP*AP*A)-3') / type: rna / ID: 2 / Details: Sample RNA sequence / Number of copies: 2
Source (natural)Organism: Zaire (virus)
Molecular weightTheoretical: 1.930277 KDa
SequenceString:
AAAAAA

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.2
GridModel: Quantifoil / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE
DetailsFIB-milled-plunge-frozen cell expressing EBOV NP(601-739 truncated), VP24 and VP35

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 3.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 2.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number subtomograms used: 28000
ExtractionNumber tomograms: 25 / Number images used: 102000
Final 3D classificationNumber classes: 3 / Avg.num./class: 33000 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT
Output model

PDB-8usn:
Intracellular cryo-tomography structure of EBOV nucleocapsid at 8.9 Angstrom

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