|Entry||Database: PDB / ID: 6oj3|
|Title||In situ structure of rotavirus VP1 RNA-dependent RNA polymerase (TLP)|
|Keywords||VIRAL PROTEIN/TRANSFERASE / Rotavirus / RNA-dependent RNA polymerase / VP1 / VP2 / VIRAL PROTEIN-TRANSFERASE complex|
|Function / homology|
Function and homology information
T=2 icosahedral viral capsid / viral inner capsid / viral genome replication / viral nucleocapsid / RNA-directed RNA polymerase / RNA-directed 5'-3' RNA polymerase activity / DNA-templated transcription / nucleotide binding / RNA binding
Similarity search - Function
Rotavirus VP2 protein / Rotavirus VP2 / Viral RNA-directed RNA polymerase, 4-helical domain / Rotavirus VP1 C-terminal domain / Rotavirus VP1 RNA-directed RNA polymerase, C-terminal / RNA-directed RNA polymerase, luteovirus / Viral RNA-directed RNA-polymerase / RNA-directed RNA polymerase, reovirus / RdRp of Reoviridae dsRNA viruses catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
RNA-directed RNA polymerase / Inner capsid protein VP2
Similarity search - Component
|Biological species||Rotavirus A|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å|
|Authors||Jenni, S. / Salgado, E.N. / Herrmann, T. / Li, Z. / Grant, T. / Grigorieff, N. / Trapani, S. / Estrozi, L.F. / Harrison, S.C.|
|Funding support|| United States, 2items |
|Citation||Journal: J Mol Biol / Year: 2019|
Title: In situ Structure of Rotavirus VP1 RNA-Dependent RNA Polymerase.
Authors: Simon Jenni / Eric N Salgado / Tobias Herrmann / Zongli Li / Timothy Grant / Nikolaus Grigorieff / Stefano Trapani / Leandro F Estrozi / Stephen C Harrison /
Abstract: Rotaviruses, like other non-enveloped, double-strand RNA viruses, package an RNA-dependent RNA polymerase (RdRp) with each duplex of their segmented genomes. Rotavirus cell entry results in loss of ...Rotaviruses, like other non-enveloped, double-strand RNA viruses, package an RNA-dependent RNA polymerase (RdRp) with each duplex of their segmented genomes. Rotavirus cell entry results in loss of an outer protein layer and delivery into the cytosol of an intact, inner capsid particle (the "double-layer particle," or DLP). The RdRp, designated VP1, is active inside the DLP; each VP1 achieves many rounds of mRNA transcription from its associated genome segment. Previous work has shown that one VP1 molecule lies close to each 5-fold axis of the icosahedrally symmetric DLP, just beneath the inner surface of its protein shell, embedded in tightly packed RNA. We have determined a high-resolution structure for the rotavirus VP1 RdRp in situ, by local reconstruction of density around individual 5-fold positions. We have analyzed intact virions ("triple-layer particles"), non-transcribing DLPs and transcribing DLPs. Outer layer dissociation enables the DLP to synthesize RNA, in vitro as well as in vivo, but appears not to induce any detectable structural change in the RdRp. Addition of NTPs, Mg, and S-adenosylmethionine, which allows active transcription, results in conformational rearrangements, in both VP1 and the DLP capsid shell protein, that allow a transcript to exit the polymerase and the particle. The position of VP1 (among the five symmetrically related alternatives) at one vertex does not correlate with its position at other vertices. This stochastic distribution of site occupancies limits long-range order in the 11-segment, double-strand RNA genome.
|Structure viewer||Molecule: |
Downloads & links
A: Inner capsid protein VP2
B: Inner capsid protein VP2
C: Inner capsid protein VP2
D: Inner capsid protein VP2
E: Inner capsid protein VP2
F: Inner capsid protein VP2
G: Inner capsid protein VP2
H: Inner capsid protein VP2
I: Inner capsid protein VP2
J: Inner capsid protein VP2
P: RNA-directed RNA polymerase
Mass: 103425.992 Da / Num. of mol.: 10 / Source method: isolated from a natural source
Source: (natural) Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B)
Cell line: MA104 / Strain: RVA/Monkey/United States/RRV/1975/G3P5B / References: UniProt: B3F2X3
Mass: 125276.305 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B)
Cell line: MA104 / Strain: RVA/Monkey/United States/RRV/1975/G3P5B / References: UniProt: B3F2X2, RNA-directed RNA polymerase
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction|
|Component||Name: Rhesus rotavirus / Type: VIRUS / Details: TLP / Entity ID: all / Source: NATURAL|
|Source (natural)||Organism: Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B)|
Strain: RVA/Monkey/United States/RRV/1975/G3P5B
|Details of virus||Empty: NO / Enveloped: NO / Isolate: SEROTYPE / Type: VIRION|
|Buffer solution||pH: 7.5|
|Specimen||Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Details: unspecified|
|Vitrification||Cryogen name: ETHANE|
-Electron microscopy imaging
Model: Tecnai Polara / Image courtesy: FEI Company
|Microscopy||Model: FEI POLARA 300|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy|
|Image recording||Electron dose: 64 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)|
|EM software||Name: cisTEM / Version: 1.01 / Category: 3D reconstruction / Details: FrealignX|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|Symmetry||Point symmetry: C1 (asymmetric)|
|3D reconstruction||Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30606 / Symmetry type: POINT|
|Atomic model building||Protocol: AB INITIO MODEL|
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