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- PDB-7kvd: Cryo-EM structure of Mal de Rio Cuarto virus P9-1 viroplasm prote... -

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Basic information

Entry
Database: PDB / ID: 7kvd
TitleCryo-EM structure of Mal de Rio Cuarto virus P9-1 viroplasm protein (dodecamer)
Componentsp9-1
KeywordsVIRAL PROTEIN / Fijivirus / MRCV / Wheat / Maize / Reoviridae / viroplasm
Function / homologyReovirus P9-like / Reovirus P9-like family / p9-1
Function and homology information
Biological speciesMal de Rio Cuarto virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.8 Å
AuthorsLlauger, G. / Melero, R. / Monti, D. / Sycz, G. / Huck-Iriart, C. / Cerutti, M.L. / Klinke, S. / Arranz, R. / Carazo, J.M. / Goldbaum, F.A. ...Llauger, G. / Melero, R. / Monti, D. / Sycz, G. / Huck-Iriart, C. / Cerutti, M.L. / Klinke, S. / Arranz, R. / Carazo, J.M. / Goldbaum, F.A. / del Vas, M. / Otero, L.H.
Funding support Argentina, 4items
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, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 15, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 28, 2023Group: Database references / Refinement description
Category: citation / citation_author / pdbx_initial_refinement_model
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2May 29, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / em_3d_fitting_list
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: p9-1
B: p9-1
C: p9-1
D: p9-1
E: p9-1
F: p9-1
G: p9-1
H: p9-1
I: p9-1
J: p9-1
K: p9-1
L: p9-1


Theoretical massNumber of molelcules
Total (without water)539,20012
Polymers539,20012
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, light scattering
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "D"
d_2ens_1chain "B"
d_3ens_1chain "C"
d_4ens_1chain "A"
d_5ens_1chain "E"
d_6ens_1chain "F"
d_7ens_1chain "G"
d_8ens_1chain "H"
d_9ens_1chain "I"
d_10ens_1chain "J"
d_11ens_1chain "K"
d_12ens_1chain "L"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1GLUVALD1 - 266
d_21ens_1GLUVALB1 - 266
d_31ens_1GLUVALC1 - 266
d_41ens_1GLUVALA1 - 266
d_51ens_1GLUVALE1 - 266
d_61ens_1GLUVALF1 - 266
d_71ens_1GLUVALG1 - 266
d_81ens_1GLUVALH1 - 266
d_91ens_1GLUVALI1 - 266
d_101ens_1GLUVALJ1 - 266
d_111ens_1GLUVALK1 - 266
d_121ens_1GLUVALL1 - 266

NCS oper:
IDCodeMatrixVector
1given(0.499997590523, 0.866026781086, -0.00015463765163), (-0.8660267848, 0.499997603985, 6.33850671E-5), (0.0001322116209, 0.0001022279674, 0.999999986035)-25.3669933533, 103.964850591, -0.0174736212172
2given(-0.508153521683, -0.861256904825, 0.00406722167563), (-0.861113252218, 0.507968351048, -0.0212631415559), (0.0162470075953, -0.0143072787482, -0.999765640797)180.56161276, 103.811406661, 95.8141117702
3given(-0.999825763617, 0.00924906460014, -0.0162141053264), (0.00947943477435, 0.999854392808, -0.0141892036269), (0.0160805075751, -0.0143404319053, -0.999767857699)154.805728435, -0.490583983503, 95.8279564943
4given(0.491680043381, -0.870536571561, 0.0204159867624), (-0.870623706084, -0.491897341715, -0.00716712062696), (0.0162818102355, -0.0142507118777, -0.999765882528)103.094288865, 178.266513437, 95.8080143032
5given(0.499987477465, -0.866032633444, -1.374564291E-5), (0.866032633368, 0.499987477623, -1.2700018E-5), (1.78712794E-5, -5.554325E-6, 0.999999999825)102.719106848, -30.010566748, -0.00131413673069
6given(0.999821431776, -0.00927242051567, 0.0164659278207), (-0.0095055327226, -0.999854900461, 0.0141358718018), (0.0163324648745, -0.014289865, -0.999764497444)-0.1167735145, 148.397094679, 95.8041470076
7given(-0.499997575204, -0.86602680279, 4.04899695E-5), (0.866026803729, -0.499997574854, 1.906280932E-5), (3.7359827E-6, 4.45967573E-5, 0.999999998999)180.071192215, 43.9321372196, -0.00307518890824
8given(0.508155651975, 0.861255539392, -0.00409013840595), (0.86111199468, -0.507969914062, 0.0212767249574), (0.0162470299751, -0.0143339552839, -0.99976525832)-25.8543880119, 44.0895072043, 95.8151183668
9given(-0.999999999848, 1.555944E-5, -7.8047884E-6), (-1.555940985E-5, -0.999999999867, -4.8362639E-6), (-7.8048637E-6, -4.8361425E-6, 0.999999999958)154.705956579, 147.903602323, 0.0012045486946
10given(-0.491712213423, 0.870517763497, -0.0204431555123), (0.870605112377, 0.491930102202, 0.00717724527048), (0.0163045230814, -0.0142687765439, -0.999765254719)51.6122903635, -30.3583900977, 95.8063879336
11given(-0.499957578537, 0.866049894311, 1.50688968E-5), (-0.866049894176, -0.499957578739, 1.612140078E-5), (2.14957466E-5, -4.99040001E-6, 0.999999999757)51.9781745758, 177.919186724, -0.00191454521216

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Components

#1: Protein
p9-1


Mass: 44933.328 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mal de Rio Cuarto virus / Plasmid: pRSET / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: D9U542

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Dodecameric complex of Mal de Rio Cuarto virus P9-1 viroplasm protein
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mal de Rio Cuarto virus
Source (recombinant)Organism: Escherichia coli BL21 (bacteria)
Buffer solutionpH: 7.6
Buffer component
IDConc.FormulaBuffer-ID
110 mMTris-HCl1
225 mMNaCl1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: No previous negative glow discharge was applied on the grids in order to avoid protein aggregation
Specimen supportGrid material: COPPER/RHODIUM / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: LEICA EM CPC / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal magnification: 120000 X / Nominal defocus max: 3800 nm / Calibrated defocus min: 800 nm
Image recordingElectron dose: 30 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
EM software
IDNameCategory
1Xmippparticle selection
2Scipionimage acquisition
4GctfCTF correction
7PHENIXmodel fitting
9Scipioninitial Euler assignment
10Scipionfinal Euler assignment
11Scipionclassification
12Scipion3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 202824
SymmetryPoint symmetry: D6 (2x6 fold dihedral)
3D reconstructionResolution: 6.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 22510 / Symmetry type: POINT
Atomic model buildingB value: 175 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 6UCT

6uct


Accession code: 6UCT / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 175.6 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004426748
ELECTRON MICROSCOPYf_angle_d1.005136024
ELECTRON MICROSCOPYf_chiral_restr0.05664080
ELECTRON MICROSCOPYf_plane_restr0.00774560
ELECTRON MICROSCOPYf_dihedral_angle_d19.47713480
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2DELECTRON MICROSCOPYNCS constraints0.000709078327687
ens_1d_3DELECTRON MICROSCOPYNCS constraints0.000706760939058
ens_1d_4DELECTRON MICROSCOPYNCS constraints0.00070873350417
ens_1d_5DELECTRON MICROSCOPYNCS constraints0.000704788029846
ens_1d_6DELECTRON MICROSCOPYNCS constraints0.000734157955246
ens_1d_7DELECTRON MICROSCOPYNCS constraints0.000706328469371
ens_1d_8DELECTRON MICROSCOPYNCS constraints0.000710870516678
ens_1d_9DELECTRON MICROSCOPYNCS constraints0.000706356115303
ens_1d_10DELECTRON MICROSCOPYNCS constraints0.000689877433061
ens_1d_11DELECTRON MICROSCOPYNCS constraints0.000705116422953
ens_1d_12DELECTRON MICROSCOPYNCS constraints0.000707831015661

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