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- EMDB-28996: Nodavirus RNA replication crown from BSRT7/5 cells expressing vir... -

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Basic information

Entry
Database: EMDB / ID: EMD-28996
TitleNodavirus RNA replication crown from BSRT7/5 cells expressing viral protein A and fsRNA1 template in presence of B2
Map dataNodavirus RNA replication crown from BSRT7/5 cells expressing viral protein A and fsRNA1 template in presence of B2
Sample
  • Complex: Flock house nodavirus protein A
    • Protein or peptide: Flock House nodavirus protein A
KeywordsNodavirus RNA replication and RNA capping complex / Dodecamer ring / Outer mitochondrial membrane protein complex / VIRAL PROTEIN
Biological speciesFlock House virus
Methodsubtomogram averaging / cryo EM / Resolution: 26.7 Å
Authorsden Boon J / Zhan H / Unchwaniwala N / Horswill M / Slavik K / Pennington J / Navine A / Ahlquist P
Funding support United States, 2 items
OrganizationGrant numberCountry
Other private United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Viruses / Year: 2022
Title: Multifunctional Protein A Is the Only Viral Protein Required for Nodavirus RNA Replication Crown Formation.
Authors: Johan A den Boon / Hong Zhan / Nuruddin Unchwaniwala / Mark Horswill / Kailey Slavik / Janice Pennington / Amanda Navine / Paul Ahlquist /
Abstract: Positive-strand RNA virus RNA genome replication occurs in membrane-associated RNA replication complexes (RCs). Nodavirus RCs are outer mitochondrial membrane invaginations whose necked openings to ...Positive-strand RNA virus RNA genome replication occurs in membrane-associated RNA replication complexes (RCs). Nodavirus RCs are outer mitochondrial membrane invaginations whose necked openings to the cytosol are "crowned" by a 12-fold symmetrical proteinaceous ring that functions as the main engine of RNA replication. Similar protein crowns recently visualized at the openings of alphavirus and coronavirus RCs highlight their broad conservation and functional importance. Using cryo-EM tomography, we earlier showed that the major nodavirus crown constituent is viral protein A, whose polymerase, RNA capping, membrane interaction and multimerization domains drive RC formation and function. Other viral proteins are strong candidates for unassigned EM density in the crown. RNA-binding RNAi inhibitor protein B2 co-immunoprecipitates with protein A and could form crown subdomains that protect nascent viral RNA and dsRNA templates. Capsid protein may interact with the crown since nodavirus virion assembly has spatial and other links to RNA replication. Using cryoelectron tomography and complementary approaches, we show that, even when formed in mammalian cells, nodavirus RC crowns generated without B2 and capsid proteins are functional and structurally indistinguishable from mature crowns in infected cells expressing all viral proteins. Thus, the only nodaviral factors essential to form functional RCs and crowns are RNA replication protein A and an RNA template. We also resolve apparent conflicts in prior results on B2 localization in infected cells, revealing at least two distinguishable pools of B2. The results have significant implications for crown structure, assembly, function and control as an antiviral target.
History
DepositionDec 1, 2022-
Header (metadata) releaseDec 14, 2022-
Map releaseDec 14, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28996.png

Downloads & links

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Map

FileDownload / File: emd_28996.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNodavirus RNA replication crown from BSRT7/5 cells expressing viral protein A and fsRNA1 template in presence of B2
Voxel sizeX=Y=Z: 5.4 Å
Density
Contour LevelBy AUTHOR: 22.0
Minimum - Maximum-96.935569999999998 - 101.755200000000002
Average (Standard dev.)-6.9738407 (±16.338602000000002)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 432.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Nodavirus RNA replication crown from BSRT7/5 cells expressing...

Fileemd_28996_half_map_1.map
AnnotationNodavirus RNA replication crown from BSRT7/5 cells expressing viral protein A and fsRNA1 template in presence of B2, half map1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Nodavirus RNA replication crown from BSRT7/5 cells expressing...

Fileemd_28996_half_map_2.map
AnnotationNodavirus RNA replication crown from BSRT7/5 cells expressing viral protein A and fsRNA1 template in presence of B2, half map2.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Flock house nodavirus protein A

EntireName: Flock house nodavirus protein A
Components
  • Complex: Flock house nodavirus protein A
    • Protein or peptide: Flock House nodavirus protein A

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Supramolecule #1: Flock house nodavirus protein A

SupramoleculeName: Flock house nodavirus protein A / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Flock House virus / Tissue: BSRT7/5 cells / Organelle: mitochondria / Location in cell: outer mitochondrial membrane

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Macromolecule #1: Flock House nodavirus protein A

MacromoleculeName: Flock House nodavirus protein A / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
SequenceString: MTLKVILGEH QITRTELLVG IATVSGCGAV VYCISKFWGY GAIAPYPQSG GNRVTRALQR AVIDKTKTPI ETRFYPLDSL RTVTPKRVAD NGHAVSGAVR DAARRLIDES ITAVGGSKFE VNPNPNSSTG LRNHFHFAVG DLAQDFRNDT PADDAFIVGV DVDYYVTEPD ...String:
MTLKVILGEH QITRTELLVG IATVSGCGAV VYCISKFWGY GAIAPYPQSG GNRVTRALQR AVIDKTKTPI ETRFYPLDSL RTVTPKRVAD NGHAVSGAVR DAARRLIDES ITAVGGSKFE VNPNPNSSTG LRNHFHFAVG DLAQDFRNDT PADDAFIVGV DVDYYVTEPD VLLEHMRPVV LHTFNPKKVS GFDADSPFTI KNNLVEYKVS GGAAWVHPVW DWCEAGEFIA SRVRTSWKEW FLQLPLRMIG LEKVGYHKIH HCRPWTDCPD RALVYTIPQY VIWRFNWIDT ELHVRKLKRI EYQDETKPGW NRLEYVTDKN ELLVSIGREG EHAQITIEKE KLDMLSGLSA TQSVNARLIG MGHKDPQYTS MIVQYYTGKK VVSPISPTVY KPTMPRVHWP VTSDADVPEV SARQYTLPIV SDCMMMPMIK RWETMSESIE RRVTFVANDK KPSDRIAKIA ETFVKLMNGP FKDLDPLSIE ETIERLNKPS QQLQLRAVFE MIGVKPRQLI ESFNKNEPGM KSSRIISGFP DILFILKVSR YTLAYSDIVL HAEHNEHWYY PGRNPTEIAD GVCEFVSDCD AEVIETDFSN LDGRVSSWMQ RNIAQKAMVQ AFRPEYRDEI ISFMDTIINC PAKAKRFGFR YEPGVGVKSG SPTTTPHNTQ YNGCVEFTAL TFEHPDAEPE DLFRLIGPKC GDDGLSRAII QKSINRAAKC FGLELKVERY NPEIGLCFLS RVFVDPLATT TTIQDPLRTL RKLHLTTRDP TIPLADAACD RVEGYLCTDA LTPLISDYCK MVLRLYGPTA STEQVRNQRR SRNKEKPYWL TCDGSWPQHP QDAHLMKQVL IKRTAIDEDQ VDALIGRFAA MKDVWEKITH DSEESAAACT FDEDGVAPNS VDESLPLLND AKQTRANPGT SRPHSNGGGS SHGNELPRRT EQRAQGPRQP ARLPKQGKTN GKSDGNITAG ETQRGGIPRG KGPRGGKTNT RRTPPKAGAQ PQPSNNRK

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
GridModel: Quantifoil / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 4.86 e/Å2 / Details: Grid ID: N56
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 19500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 3.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C12 (12 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 26.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: PEET (ver. 1.15) / Number subtomograms used: 223
ExtractionNumber tomograms: 1 / Number images used: 223 / Method: volumes picked manually / Software - Name: IMOD (ver. 4.11)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: PEET (ver. 1.15)
FSC plot (resolution estimation)

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