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- PDB-8fm9: Nodavirus RNA replication proto-crown, detergent-solubliized C12 ... -

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Basic information

Entry
Database: PDB / ID: 8fm9
TitleNodavirus RNA replication proto-crown, detergent-solubliized C12 multimer
ComponentsRNA-directed RNA polymeraseRNA-dependent RNA polymerase
KeywordsVIRAL PROTEIN / Nodavirus RNA replication and RNA capping complex / Dodecamer ring / Outer mitochondrial membrane protein complex
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
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 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, 1items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 1, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA-directed RNA polymerase
B: RNA-directed RNA polymerase
C: RNA-directed RNA polymerase
D: RNA-directed RNA polymerase
E: RNA-directed RNA polymerase
F: RNA-directed RNA polymerase
G: RNA-directed RNA polymerase
H: RNA-directed RNA polymerase
I: RNA-directed RNA polymerase
J: RNA-directed RNA polymerase
K: RNA-directed RNA polymerase
L: RNA-directed RNA polymerase
M: RNA-directed RNA polymerase
N: RNA-directed RNA polymerase
O: RNA-directed RNA polymerase
P: RNA-directed RNA polymerase
Q: RNA-directed RNA polymerase
R: RNA-directed RNA polymerase
S: RNA-directed RNA polymerase
T: RNA-directed RNA polymerase
U: RNA-directed RNA polymerase
V: RNA-directed RNA polymerase
W: RNA-directed RNA polymerase
X: RNA-directed RNA polymerase


Theoretical massNumber of molelcules
Total (without water)2,735,91824
Polymers2,735,91824
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ...
RNA-directed RNA polymerase / RNA-dependent RNA polymerase / RdRp / RNA replicase / Protein A


Mass: 113996.586 Da / Num. of mol.: 24
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Flock House virus / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q66929, RNA-directed RNA polymerase

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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: Nodavirus RNA replication proto-crown, detergent-solubliized C12 multimer
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Flock House virus
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Image recordingElectron dose: 100 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k)
Image scansWidth: 4096 / Height: 4096

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
1cisTEM2particle selection
2SerialEM3.8image acquisition
4cisTEM2CTF correction
7UCSF ChimeraX1.3model fitting
8Coot0.9.8.1model fitting
9ISOLDE1.3model fitting
11cisTEM2initial Euler assignment
12cisTEM2final Euler assignment
13cisTEM2classification
14cisTEM23D reconstruction
21PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C12 (12 fold cyclic)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 11093 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Details: The final model includes 24 chains representing 12 protein A polypeptides. Chains A-L represent the N-terminal floor segment built into the high-resolution EM density. Chains M-X represent a ...Details: The final model includes 24 chains representing 12 protein A polypeptides. Chains A-L represent the N-terminal floor segment built into the high-resolution EM density. Chains M-X represent a mainchain trace of the polymerase segment rigid body fit into the map and are included to give a more complete picture of the molecule. The polymerase mainchain trace was refined from an Alphafold prediction into a map obtained by symmetry expansion with subtraction. The polymerase map is uploaded as a separate entry. The two segments have separate chain IDs as it is unclear from the density which polymerase connects to which floor segment.
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0011409
ELECTRON MICROSCOPYf_angle_d0.3721766
ELECTRON MICROSCOPYf_dihedral_angle_d3.474329
ELECTRON MICROSCOPYf_chiral_restr0.04621
ELECTRON MICROSCOPYf_plane_restr0.002338

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