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- EMDB-23047: Cryo-EM structure of Mal de Rio Cuarto virus P9-1 viroplasm prote... -

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Entry
Database: EMDB / ID: EMD-23047
TitleCryo-EM structure of Mal de Rio Cuarto virus P9-1 viroplasm protein (dodecamer)
Map dataCryo-EM structure of Mal de Rio Cuarto virus P9-1 viroplasm protein (dodecamer)
Sample
  • Complex: Dodecameric complex of Mal de Rio Cuarto virus P9-1 viroplasm protein
    • Protein or peptide: p9-1
KeywordsFijivirus / MRCV / Wheat / Maize / Reoviridae / viroplasm / VIRAL PROTEIN
Function / homologyReovirus P9-like / Reovirus P9-like family / p9-1
Function and homology information
Biological speciesMal de Rio Cuarto virus
Methodsingle particle reconstruction / cryo EM / Resolution: 6.8 Å
AuthorsLlauger G / Melero R
Funding support Argentina, 4 items
OrganizationGrant numberCountry
National Research Council (NRC, Argentina)PICT 2015-0621 Argentina
National Research Council (NRC, Argentina)PICT 2016-1425 Argentina
National Research Council (NRC, Argentina)PICT-2014-3754 Argentina
National Research Council (NRC, Argentina)PICT-2017-2537 Argentina
CitationJournal: mBio / Year: 2023
Title: A Fijivirus Major Viroplasm Protein Shows RNA-Stimulated ATPase Activity by Adopting Pentameric and Hexameric Assemblies of Dimers.
Authors: Gabriela Llauger / Roberto Melero / Demián Monti / Gabriela Sycz / Cristián Huck-Iriart / María L Cerutti / Sebastián Klinke / Evelyn Mikkelsen / Ariel Tijman / Rocío Arranz / Victoria ...Authors: Gabriela Llauger / Roberto Melero / Demián Monti / Gabriela Sycz / Cristián Huck-Iriart / María L Cerutti / Sebastián Klinke / Evelyn Mikkelsen / Ariel Tijman / Rocío Arranz / Victoria Alfonso / Sofía M Arellano / Fernando A Goldbaum / Yann G J Sterckx / José-María Carazo / Sergio B Kaufman / Pablo D Dans / Mariana Del Vas / Lisandro H Otero /
Abstract: Fijiviruses replicate and package their genomes within viroplasms in a process involving RNA-RNA and RNA-protein interactions. Here, we demonstrate that the 24 C-terminal residues (C-arm) of the P9-1 ...Fijiviruses replicate and package their genomes within viroplasms in a process involving RNA-RNA and RNA-protein interactions. Here, we demonstrate that the 24 C-terminal residues (C-arm) of the P9-1 major viroplasm protein of the mal de Río Cuarto virus (MRCV) are required for its multimerization and the formation of viroplasm-like structures. Using an integrative structural approach, the C-arm was found to be dispensable for P9-1 dimer assembly but essential for the formation of pentamers and hexamers of dimers (decamers and dodecamers), which favored RNA binding. Although both P9-1 and P9-1ΔC-arm catalyzed ATP with similar activities, an RNA-stimulated ATPase activity was only detected in the full-length protein, indicating a C-arm-mediated interaction between the ATP catalytic site and the allosteric RNA binding sites in the (do)decameric assemblies. A stronger preference to bind phosphate moieties in the decamer was predicted, suggesting that the allosteric modulation of ATPase activity by RNA is favored in this structural conformation. Our work reveals the structural versatility of a fijivirus major viroplasm protein and provides clues to its mechanism of action. The mal de Río Cuarto virus (MRCV) causes an important maize disease in Argentina. MRCV replicates in several species of plants and planthopper vectors. The viral factories, also called viroplasms, have been studied in detail in animal reovirids. This work reveals that a major viroplasm protein of MRCV forms previously unidentified structural arrangements and provides evidence that it may simultaneously adopt two distinct quaternary assemblies. Furthermore, our work uncovers an allosteric communication between the ATP and RNA binding sites that is favored in the multimeric arrangements. Our results contribute to the understanding of plant reovirids viroplasm structure and function and pave the way for the design of antiviral strategies for disease control.
History
DepositionNov 27, 2020-
Header (metadata) releaseJun 15, 2022-
Map releaseJun 15, 2022-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_23047.png

Downloads & links

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Map

FileDownload / File: emd_23047.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of Mal de Rio Cuarto virus P9-1 viroplasm protein (dodecamer)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.85 Å/pix.
x 240 pix.
= 204. Å		0.85 Å/pix.
x 240 pix.
= 204. Å		0.85 Å/pix.
x 240 pix.
= 204. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.4
Minimum - Maximum-8.463305 - 12.778477000000001
Average (Standard dev.)0.000000000000179 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 204.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Dodecameric complex of Mal de Rio Cuarto virus P9-1 viroplasm protein

EntireName: Dodecameric complex of Mal de Rio Cuarto virus P9-1 viroplasm protein
Components
  • Complex: Dodecameric complex of Mal de Rio Cuarto virus P9-1 viroplasm protein
    • Protein or peptide: p9-1

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Supramolecule #1: Dodecameric complex of Mal de Rio Cuarto virus P9-1 viroplasm protein

SupramoleculeName: Dodecameric complex of Mal de Rio Cuarto virus P9-1 viroplasm protein
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mal de Rio Cuarto virus

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Macromolecule #1: p9-1

MacromoleculeName: p9-1 / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Mal de Rio Cuarto virus
Molecular weightTheoretical: 44.933328 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MRGSHHHHHH GMASMTGGQQ MGRDLYDDDD KDRWGSELEI CSWYHGGRGN SIADLERRTF GSYKIEEITI KNDQQQKTTN QQQISNNEQ RISTKKIPIL DDGIFDLINY LLNGTHFDKT HYCGFDYSHL PTLERDFNTA SNYVSENYSI IVEEIDLNKY E RSESISLK ...String:
MRGSHHHHHH GMASMTGGQQ MGRDLYDDDD KDRWGSELEI CSWYHGGRGN SIADLERRTF GSYKIEEITI KNDQQQKTTN QQQISNNEQ RISTKKIPIL DDGIFDLINY LLNGTHFDKT HYCGFDYSHL PTLERDFNTA SNYVSENYSI IVEEIDLNKY E RSESISLK SPDFTVVLEY FKKHVEGQTE QEENKTESTS SELPAKIVRE LPLLPIMCRE SEDSISEDIL EGEGAVIQVL KM FMKGFLV HLGENPNSYD RQLTIEKYRP LLISIIGYEF TVGTRATHTK INHIYYQLAT FDNYPFDLLR FQLQSLIDTP NVI KERIEK DGLFKVITTT NARGQYQSVL LRGINGSESY LNLKRYRKFK VRVVGNVDNV IKNDFSSLKL DV

UniProtKB: p9-1

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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: 7.6
Component:
ConcentrationFormula
10.0 mMTris-HClTris
25.0 mMNaClSodium chloride
GridModel: Quantifoil R2/2 / Material: COPPER/RHODIUM / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: LEICA EM CPC
DetailsNo previous negative glow discharge was applied on the grids in order to avoid protein aggregation

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus min: 0.8 µm / Illumination mode: OTHER / Imaging mode: OTHER / Nominal defocus max: 3.8000000000000003 µm / Nominal magnification: 120000
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Average electron dose: 30.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 202824
Startup modelType of model: NONE
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: Scipion
Final 3D classificationSoftware - Name: Scipion
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: Scipion
Final reconstructionApplied symmetry - Point group: D6 (2x6 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 6.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Scipion / Number images used: 22510

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Atomic model buiding 1

Initial modelPDB ID:

6uct


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 175 / Target criteria: Correlation coefficient
Output model

PDB-7kvd:
Cryo-EM structure of Mal de Rio Cuarto virus P9-1 viroplasm protein (dodecamer)

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