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- EMDB-29217: Nodavirus RNA replication proto-crown from baculovirus-expressed ... -

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Basic information

Entry
Database: EMDB / ID: EMD-29217
TitleNodavirus RNA replication proto-crown from baculovirus-expressed viral protein A minus RNA1 template
Map dataNodavirus RNA replication proto-crown from baculovirus-expressed viral protein A minus RNA1 template
Sample
  • Complex: Flock House virus
    • Protein or peptide: Flock House nodavirus protein A
Biological speciesFlock House virus
Methodsubtomogram averaging / cryo EM / Resolution: 12.6 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

Downloads & links

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Map

FileDownload / File: emd_29217.map.gz / Format: CCP4 / Size: 70.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNodavirus RNA replication proto-crown from baculovirus-expressed viral protein A minus RNA1 template
Voxel sizeX=Y=Z: 1.645 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-1.1233082 - 0.9286308
Average (Standard dev.)0.0006483907 (±0.08817936)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions264264264
Spacing264264264
CellA=B=C: 434.28 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Nodavirus RNA replication proto-crown from baculovirus-expressed viral protein...

Fileemd_29217_half_map_1.map
AnnotationNodavirus RNA replication proto-crown from baculovirus-expressed viral protein A minus RNA1 template, unmasked and unfiltered half map1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Nodavirus RNA replication proto-crown from baculovirus-expressed viral protein...

Fileemd_29217_half_map_2.map
AnnotationNodavirus RNA replication proto-crown from baculovirus-expressed viral protein A minus RNA1 template, unmasked and unfiltered half map2.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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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
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
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 3.5 µm
SoftwareName: SerialEM (ver. 3.8)
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 4.86 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 34 / Number images used: 5628 / Software - Name: emClarity (ver. 1.5.3)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C12 (12 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 12.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.0.0) / Number subtomograms used: 1685
FSC plot (resolution estimation)

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