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- PDB-8kdb: Cryo-EM structure of the human parainfluenza virus hPIV3 L-P poly... -

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Basic information

Entry
Database: PDB / ID: 8kdb
TitleCryo-EM structure of the human parainfluenza virus hPIV3 L-P polymerase in dimeric form
Components
  • Phosphoprotein
  • RNA-directed RNA polymerase L
KeywordsVIRAL PROTEIN / dimeric polymerase / parainfluenza virus / L-P polymerase / cryo-EM structure / non-segmented negative-strand RNA virus / L-L dimerization / RNA replication / RdRp active site
Function / homology
Function and homology information


GDP polyribonucleotidyltransferase / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / viral genome replication / Transferases; Transferring one-carbon groups; Methyltransferases / virion component / host cell cytoplasm / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / RNA-directed RNA polymerase / RNA-directed RNA polymerase activity / GTPase activity ...GDP polyribonucleotidyltransferase / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / viral genome replication / Transferases; Transferring one-carbon groups; Methyltransferases / virion component / host cell cytoplasm / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / RNA-directed RNA polymerase / RNA-directed RNA polymerase activity / GTPase activity / DNA-templated transcription / RNA binding / ATP binding
Similarity search - Function
Paramyxovirinae P phosphoprotein C-terminal domain / Paramyxovirinae P phosphoprotein C-terminal region / RNA polymerase, phosphoprotein P, C-terminal XD, paramyxovirinae / RNA-directed RNA polymerase, paramyxovirus / Mononegavirus L protein 2-O-ribose methyltransferase / RNA-directed RNA polymerase L, C-terminal / Mononegavirus L protein 2'-O-ribose methyltransferase domain profile. / Mononegavirales RNA-directed RNA polymerase catalytic domain / Mononegavirales mRNA-capping domain V / Mononegavirales RNA dependent RNA polymerase ...Paramyxovirinae P phosphoprotein C-terminal domain / Paramyxovirinae P phosphoprotein C-terminal region / RNA polymerase, phosphoprotein P, C-terminal XD, paramyxovirinae / RNA-directed RNA polymerase, paramyxovirus / Mononegavirus L protein 2-O-ribose methyltransferase / RNA-directed RNA polymerase L, C-terminal / Mononegavirus L protein 2'-O-ribose methyltransferase domain profile. / Mononegavirales RNA-directed RNA polymerase catalytic domain / Mononegavirales mRNA-capping domain V / Mononegavirales RNA dependent RNA polymerase / Mononegavirales mRNA-capping region V / RdRp of negative ssRNA viruses with non-segmented genomes catalytic domain profile.
Similarity search - Domain/homology
Phosphoprotein / RNA-directed RNA polymerase L
Similarity search - Component
Biological speciesHuman respirovirus 3
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsXie, J. / Wang, L. / Zhai, G. / Wu, D. / Lin, Z. / Wang, M. / Yan, X. / Gao, L. / Huang, X. / Fearns, R. / Chen, S.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis for dimerization of a paramyxovirus polymerase complex.
Authors: Jin Xie / Mohamed Ouizougun-Oubari / Li Wang / Guanglei Zhai / Daitze Wu / Zhaohu Lin / Manfu Wang / Barbara Ludeke / Xiaodong Yan / Tobias Nilsson / Lu Gao / Xinyi Huang / Rachel Fearns / Shuai Chen /
Abstract: The transcription and replication processes of non-segmented, negative-strand RNA viruses (nsNSVs) are catalyzed by a multi-functional polymerase complex composed of the large protein (L) and a ...The transcription and replication processes of non-segmented, negative-strand RNA viruses (nsNSVs) are catalyzed by a multi-functional polymerase complex composed of the large protein (L) and a cofactor protein, such as phosphoprotein (P). Previous studies have shown that the nsNSV polymerase can adopt a dimeric form, however, the structure of the dimer and its function are poorly understood. Here we determine a 2.7 Å cryo-EM structure of human parainfluenza virus type 3 (hPIV3) L-P complex with the connector domain (CD') of a second L built, while reconstruction of the rest of the second L-P obtains a low-resolution map of the ring-like L core region. This study reveals detailed atomic features of nsNSV polymerase active site and distinct conformation of hPIV3 L with a unique β-strand latch. Furthermore, we report the structural basis of L-L dimerization, with CD' located at the putative template entry of the adjoining L. Disruption of the L-L interface causes a defect in RNA replication that can be overcome by complementation, demonstrating that L dimerization is necessary for hPIV3 genome replication. These findings provide further insight into how nsNSV polymerases perform their functions, and suggest a new avenue for rational drug design.
History
DepositionAug 9, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 24, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: RNA-directed RNA polymerase L
B: Phosphoprotein
C: Phosphoprotein
D: Phosphoprotein
E: Phosphoprotein
F: Phosphoprotein
G: RNA-directed RNA polymerase L
hetero molecules


Theoretical massNumber of molelcules
Total (without water)862,71910
Polymers862,5647
Non-polymers1553
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein RNA-directed RNA polymerase L


Mass: 260103.703 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: NP_067153.2 / Source: (gene. exp.) Human respirovirus 3 / Cell line (production host): Sf21 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O89238
#2: Protein
Phosphoprotein


Mass: 68471.312 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Details: NP_067149.1 / Source: (gene. exp.) Human respirovirus 3 / Cell line (production host): Sf21 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O89234
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: Human parainfluenza virus hPIV3 L-P polymerase in dimeric form
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Human respirovirus 3
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: Sf21
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

Image processing
IDImage recording-ID
11
21
CTF correction
IDEM image processing-IDType
11PHASE FLIPPING AND AMPLITUDE CORRECTION
22PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstruction
IDResolution (Å)Resolution methodNum. of particlesImage processing-IDEntry-IDSymmetry type
12.7FSC 0.143 CUT-OFF10295618KDBPOINT
22.7FSC 0.143 CUT-OFF10295618KDBPOINT
32.7FSC 0.143 CUT-OFF10295628KDBPOINT
42.7FSC 0.143 CUT-OFF10295628KDBPOINT
RefinementHighest resolution: 2.7 Å

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