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- EMDB-29291: Nodavirus RNA replication protein A polymerase domain, local refi... -

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Basic information

Entry
Database: EMDB / ID: EMD-29291
TitleNodavirus RNA replication protein A polymerase domain, local refinement
Map dataNodavirus RNA replication protein A polymerase domain, local refinement.
Sample
  • Complex: Nodavirus RNA replication protein A polymerase domain, local refinement
    • Protein or peptide: RNA-directed RNA polymerase
KeywordsNodavirus RNA replication protein A polymerase domain / RNA dependent RNA polymerase / VIRAL PROTEIN
Function / homologyNodavirus methyltransferase domain / Nodavirus Vmethyltransferase / host cell mitochondrial outer membrane / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / DNA/RNA polymerase superfamily / membrane / RNA-directed RNA polymerase
Function and homology information
Biological speciesFlock House virus
Methodsingle particle reconstruction / cryo EM / Resolution: 6.3 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 22, 2022-
Header (metadata) releaseFeb 1, 2023-
Map releaseFeb 1, 2023-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29291.png

Downloads & links

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Map

FileDownload / File: emd_29291.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNodavirus RNA replication protein A polymerase domain, local refinement.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.5 Å/pix.
x 192 pix.
= 288. Å		1.5 Å/pix.
x 192 pix.
= 288. Å		1.5 Å/pix.
x 192 pix.
= 288. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.5 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.9
Minimum - Maximum-2.2293763 - 6.23848
Average (Standard dev.)-0.010393302 (±0.20422617)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 288.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Nodavirus RNA replication protein A polymerase domain, local...

Fileemd_29291_half_map_1.map
AnnotationNodavirus RNA replication protein A polymerase domain, local refinement, half map1.
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Nodavirus RNA replication protein A polymerase domain, local...

Fileemd_29291_half_map_2.map
AnnotationNodavirus RNA replication protein A polymerase domain, local refinement, 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 : Nodavirus RNA replication protein A polymerase domain, local refi...

EntireName: Nodavirus RNA replication protein A polymerase domain, local refinement
Components
  • Complex: Nodavirus RNA replication protein A polymerase domain, local refinement
    • Protein or peptide: RNA-directed RNA polymerase

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Supramolecule #1: Nodavirus RNA replication protein A polymerase domain, local refi...

SupramoleculeName: Nodavirus RNA replication protein A polymerase domain, local refinement
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Flock House virus

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Macromolecule #1: RNA-directed RNA polymerase

MacromoleculeName: RNA-directed RNA polymerase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed RNA polymerase
Source (natural)Organism: Flock House virus
Molecular weightTheoretical: 113.996586 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MTLKVILGEH QITRTELLVG IATVSGCGAV VYCISKFWGY GAIAPYPQSG GNRVTRALQR AVIDKTKTPI ETRFYPLDSL RTVTPKRVA DNGHAVSGAV RDAARRLIDE SITAVGGSKF EVNPNPNSST GLRNHFHFAV GDLAQDFRND TPADDAFIVG V DVDYYVTE ...String:
MTLKVILGEH QITRTELLVG IATVSGCGAV VYCISKFWGY GAIAPYPQSG GNRVTRALQR AVIDKTKTPI ETRFYPLDSL RTVTPKRVA DNGHAVSGAV RDAARRLIDE SITAVGGSKF EVNPNPNSST GLRNHFHFAV GDLAQDFRND TPADDAFIVG V DVDYYVTE PDVLLEHMRP VVLHTFNPKK VSGFDADSPF TIKNNLVEYK VSGGAAWVHP VWDWCEAGEF IASRVRTSWK EW FLQLPLR MIGLEKVGYH KIHHCRPWTD CPDRALVYTI PQYVIWRFNW IDTELHVRKL KRIEYQDETK PGWNRLEYVT DKN ELLVSI GREGEHAQIT IEKEKLDMLS GLSATQSVNA RLIGMGHKDP QYTSMIVQYY TGKKVVSPIS PTVYKPTMPR VHWP VTSDA DVPEVSARQY TLPIVSDCMM MPMIKRWETM SESIERRVTF VANDKKPSDR IAKIAETFVK LMNGPFKDLD PLSIE ETIE RLNKPSQQLQ LRAVFEMIGV KPRQLIESFN KNEPGMKSSR IISGFPDILF ILKVSRYTLA YSDIVLHAEH NEHWYY PGR NPTEIADGVC EFVSDCDAEV IETDFSNLDG RVSSWMQRNI AQKAMVQAFR PEYRDEIISF MDTIINCPAK AKRFGFR YE PGVGVKSGSP TTTPHNTQYN GCVEFTALTF EHPDAEPEDL FRLIGPKCGD DGLSRAIIQK SINRAAKCFG LELKVERY N PEIGLCFLSR VFVDPLATTT TIQDPLRTLR KLHLTTRDPT IPLADAACDR VEGYLCTDAL TPLISDYCKM VLRLYGPTA STEQVRNQRR SRNKEKPYWL TCDGSWPQHP QDAHLMKQVL IKRTAIDEDQ VDALIGRFAA MKDVWEKITH DSEESAAACT FDEDGVAPN SVDESLPLLN DAKQTRANPG TSRPHSNGGG SSHGNELPRR TEQRAQGPRQ PARLPKQGKT NGKSDGNITA G ETQRGGIP RGKGPRGGKT NTRRTPPKAG AQPQPSNNRK SRLEEELRRR LTE

UniProtKB: RNA-directed RNA polymerase

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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

BufferpH: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Average electron dose: 100.0 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: 2.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 2.0) / Details: Local refinement / Number images used: 1074227
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM (ver. 2.0)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM (ver. 2.0)
Final 3D classificationSoftware - Name: cisTEM (ver. 2.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT
Output model

PDB-8fmb:
Nodavirus RNA replication protein A polymerase domain, local refinement

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