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- EMDB-29218: Nodavirus RNA replication crown from flock house virus-infected cells -

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Basic information

Entry
Database: EMDB / ID: EMD-29218
TitleNodavirus RNA replication crown from flock house virus-infected cells
Map dataNodavirus RNA replication crown
Sample
  • Complex: Flock House virus
    • Protein or peptide: Flock House nodavirus protein A
Biological speciesFlock House virus
Methodsubtomogram averaging / cryo EM / Resolution: 10.3 Å
AuthorsZhan H / Unchwaniwala N / Rebolledo Viveros A / Pennington J / Horswill M / Broadberry R / Myers J / den Boon J / Grant T / Ahlquist P
Funding support United States, 1 items
OrganizationGrant numberCountry
Other private United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Nodavirus RNA replication crown architecture reveals proto-crown precursor and viral protein A conformational switching.
Authors: Hong Zhan / Nuruddin Unchwaniwala / Andrea Rebolledo-Viveros / Janice Pennington / Mark Horswill / Roma Broadberry / Jonathan Myers / Johan A den Boon / Timothy Grant / Paul Ahlquist /
Abstract: Positive-strand RNA viruses replicate their genomes in virus-induced membrane vesicles, and the resulting RNA replication complexes are a major target for virus control. Nodavirus studies first ...Positive-strand RNA viruses replicate their genomes in virus-induced membrane vesicles, and the resulting RNA replication complexes are a major target for virus control. Nodavirus studies first revealed viral RNA replication proteins forming a 12-fold symmetric "crown" at the vesicle opening to the cytosol, an arrangement recently confirmed to extend to distantly related alphaviruses. Using cryoelectron microscopy (cryo-EM), we show that mature nodavirus crowns comprise two stacked 12-mer rings of multidomain viral RNA replication protein A. Each ring contains an ~19 nm circle of C-proximal polymerase domains, differentiated by strikingly diverged positions of N-proximal RNA capping/membrane binding domains. The lower ring is a "proto-crown" precursor that assembles prior to RNA template recruitment, RNA synthesis, and replication vesicle formation. In this proto-crown, the N-proximal segments interact to form a toroidal central floor, whose 3.1 Å resolution structure reveals many mechanistic details of the RNA capping/membrane binding domains. In the upper ring, cryo-EM fitting indicates that the N-proximal domains extend radially outside the polymerases, forming separated, membrane-binding "legs." The polymerase and N-proximal domains are connected by a long linker accommodating the conformational switch between the two rings and possibly also polymerase movements associated with RNA synthesis and nonsymmetric electron density in the lower center of mature crowns. The results reveal remarkable viral protein multifunctionality, conformational flexibility, and evolutionary plasticity and insights into (+)RNA virus replication and control.
History
DepositionDec 19, 2022-
Header (metadata) releaseFeb 1, 2023-
Map releaseFeb 1, 2023-
UpdateFeb 1, 2023-
Current statusFeb 1, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29218.png

Downloads & links

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Map

FileDownload / File: emd_29218.map.gz / Format: CCP4 / Size: 139.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNodavirus RNA replication crown
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.16 Å/pix.
x 332 pix.
= 715.792 Å		2.16 Å/pix.
x 332 pix.
= 715.792 Å		2.16 Å/pix.
x 332 pix.
= 715.792 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.156 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.175
Minimum - Maximum-0.4038698 - 0.6038459
Average (Standard dev.)-0.0014832467 (±0.044144254)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions332332332
Spacing332332332
CellA=B=C: 715.792 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Nodavirus RNA replication crown unmasked half map1

Fileemd_29218_half_map_1.map
AnnotationNodavirus RNA replication crown unmasked 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 unmasked half map2

Fileemd_29218_half_map_2.map
AnnotationNodavirus RNA replication crown unmasked 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 virus

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

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Supramolecule #1: Flock House virus

SupramoleculeName: Flock House virus / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Details: Nodavirus RNA replication crown from flock house virus-infected Drosophila S2 cells
Source (natural)Organism: Flock House virus

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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
Source (natural)Organism: Flock House virus
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 VDESLPMLND 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 R2/2 / 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
SoftwareName: SerialEM (ver. 3.8)
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 4.86 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 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: 10.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.0.0) / Number subtomograms used: 4640
ExtractionNumber tomograms: 59 / Number images used: 4640 / Software - Name: emClarity (ver. 1.5.3)
FSC plot (resolution estimation)

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