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- PDB-9dtt: Cryo-EM structure of DENV2 NS5 in complex with Stem Loop A (SLA) -

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Basic information

Entry
Database: PDB / ID: 9dtt
TitleCryo-EM structure of DENV2 NS5 in complex with Stem Loop A (SLA)
Components
  • RNA-directed RNA polymerase NS5
  • stem loop A
KeywordsVIRAL PROTEIN/RNA / RNA-dependent RNA polymerase / flavivirus / dengue / NS5 / VIRAL PROTEIN-RNA complex
Function / homology
Function and homology information


flavivirin / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of host TYK2 activity / host cell mitochondrion / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT2 activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / viral capsid / double-stranded RNA binding / nucleoside-triphosphate phosphatase / channel activity / monoatomic ion transmembrane transport ...flavivirin / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of host TYK2 activity / host cell mitochondrion / symbiont-mediated suppression of host JAK-STAT cascade via inhibition of STAT2 activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / viral capsid / double-stranded RNA binding / nucleoside-triphosphate phosphatase / channel activity / monoatomic ion transmembrane transport / mRNA (guanine-N7)-methyltransferase / methyltransferase cap1 / clathrin-dependent endocytosis of virus by host cell / methyltransferase cap1 activity / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / RNA helicase activity / protein dimerization activity / host cell perinuclear region of cytoplasm / host cell endoplasmic reticulum membrane / RNA helicase / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / symbiont-mediated activation of host autophagy / RNA-directed RNA polymerase / serine-type endopeptidase activity / viral RNA genome replication / RNA-directed RNA polymerase activity / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / host cell nucleus / virion membrane / structural molecule activity / ATP hydrolysis activity / proteolysis / extracellular region / ATP binding / metal ion binding / membrane
Similarity search - Function
Flavivirus non-structural protein NS2B / Flavivirus capsid protein C superfamily / Genome polyprotein, Flavivirus / : / Flavivirus non-structural protein NS4A / Flavivirus non-structural protein NS2B / Flavivirus non-structural protein NS4B / mRNA cap 0/1 methyltransferase / Flavivirus non-structural protein NS4B / Flavivirus non-structural protein NS4A ...Flavivirus non-structural protein NS2B / Flavivirus capsid protein C superfamily / Genome polyprotein, Flavivirus / : / Flavivirus non-structural protein NS4A / Flavivirus non-structural protein NS2B / Flavivirus non-structural protein NS4B / mRNA cap 0/1 methyltransferase / Flavivirus non-structural protein NS4B / Flavivirus non-structural protein NS4A / Flavivirus NS2B domain profile. / mRNA cap 0 and cap 1 methyltransferase (EC 2.1.1.56 and EC 2.1.1.57) domain profile. / Flavivirus non-structural protein NS2A / Flavivirus non-structural protein NS2A / Flavivirus NS3, petidase S7 / Peptidase S7, Flavivirus NS3 serine protease / Flavivirus NS3 protease (NS3pro) domain profile. / RNA-directed RNA polymerase, thumb domain, Flavivirus / Flavivirus RNA-directed RNA polymerase, thumb domain / RNA-directed RNA polymerase, flavivirus / Flavivirus RNA-directed RNA polymerase, fingers and palm domains / Flavivirus capsid protein C / Flavivirus capsid protein C / Flavivirus non-structural Protein NS1 / Flavivirus non-structural protein NS1 / Envelope glycoprotein M, flavivirus / Envelope glycoprotein M superfamily, flavivirus / Flavivirus envelope glycoprotein M / Flavivirus polyprotein propeptide / Flavivirus polyprotein propeptide superfamily / Flavivirus polyprotein propeptide / Flavivirus envelope glycoprotein E, stem/anchor domain / : / Flavivirus NS3 helicase, C-terminal helical domain / Flavivirus envelope glycoprotein E, Stem/Anchor domain / Flavivirus envelope glycoprotein E, Stem/Anchor domain superfamily / Flavivirus glycoprotein E, immunoglobulin-like domain / Flavivirus glycoprotein, immunoglobulin-like domain / Flavivirus glycoprotein central and dimerisation domain / Flavivirus glycoprotein, central and dimerisation domains / Flaviviral glycoprotein E, central domain, subdomain 1 / Flaviviral glycoprotein E, central domain, subdomain 2 / Ribosomal RNA methyltransferase, FtsJ domain / FtsJ-like methyltransferase / Flavivirus/Alphavirus glycoprotein, immunoglobulin-like domain superfamily / Flavivirus glycoprotein, central and dimerisation domain superfamily / Flaviviral glycoprotein E, dimerisation domain / DEAD box, Flavivirus / Flavivirus DEAD domain / helicase superfamily c-terminal domain / Immunoglobulin E-set / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / RNA-directed RNA polymerase, catalytic domain / RdRp of positive ssRNA viruses catalytic domain profile. / S-adenosyl-L-methionine-dependent methyltransferase superfamily / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
: / RNA / RNA (> 10) / Genome polyprotein
Similarity search - Component
Biological speciesdengue virus type 2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsObi, J.O. / Fields, J.K. / Snyder, A.G. / Deredge, D.J.
Funding support United States, 1items
OrganizationGrant numberCountry
Not funded United States
CitationJournal: bioRxiv / Year: 2024
Title: Structural Dynamics of the Dengue Virus Non-structural 5 (NS5) Interactions with Promoter Stem Loop A (SLA).
Authors: Juliet O Obi / Kyle C Kihn / Linfah McQueen / James K Fields / Greg A Snyder / Daniel J Deredge /
Abstract: The dengue virus (DENV) NS5 protein plays a central role in dengue viral RNA synthesis which makes it an attractive target for antiviral drug development. DENV NS5 is known to interact with the stem- ...The dengue virus (DENV) NS5 protein plays a central role in dengue viral RNA synthesis which makes it an attractive target for antiviral drug development. DENV NS5 is known to interact with the stem-loop A (SLA) promoter at the 5'-untranslated region (5'-UTR) of the viral genome as a molecular recognition signature for the initiation of negative strand synthesis at the 3' end of the viral genome. However, the conformational dynamics involved in these interactions are yet to be fully elucidated. Our study explores the structural dynamics of NS5 from DENV serotype 2 (DENV2 NS5) in complex with SLA, employing surface plasmon resonance (SPR), hydrogen - deuterium exchange coupled to mass spectrometry (HDX-MS), computational modeling, and cryoEM single particle analysis to delineate the molecular details of their interaction. Our findings indicate that DENV2 NS5 binds SLA in a closed conformation with significant interdomain cooperation between the methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, a feature integral to the interaction. Our HDX-MS studies reveal SLA-induced conformational changes in both domains of DENV2 NS5, reflecting a potential mechanism for dengue NS5's multifunctional role in viral replication. Lastly, our cryoEM structure provides the first visualization of the DENV2 NS5-SLA complex, confirming a conserved SLA binding mode across DENV serotypes. These insights obtained from our study enhance our understanding of dengue NS5's complex conformational landscape, supporting the potential development of antiviral strategies targeting dengue NS5's conformational states.
History
DepositionOct 1, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 23, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 8, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA-directed RNA polymerase NS5
B: stem loop A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)128,3024
Polymers128,1722
Non-polymers1312
Water00
1


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

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Components

#1: Protein RNA-directed RNA polymerase NS5 / Non-structural protein 5


Mass: 105611.188 Da / Num. of mol.: 1 / Fragment: UNP residues 2492-3391
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) dengue virus type 2 / Strain: New Guinea C (NGC) / Production host: Escherichia coli (E. coli)
References: UniProt: P14340, mRNA (guanine-N7)-methyltransferase, methyltransferase cap1, RNA-directed RNA polymerase
#2: RNA chain stem loop A / SLA


Mass: 22560.412 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) dengue virus type 2 / References: GenBank: AF038403.1
#3: 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
Has protein modificationN

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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

Component
IDNameTypeEntity IDParent-IDSource
1Full-length DENV2 NS5 bound to viral promoter SLACOMPLEX#1-#20MULTIPLE SOURCES
2DENV2 NS5COMPLEX#11RECOMBINANT
3SLA70COMPLEX#21NATURAL
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
31NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12dengue virus type 211060
23synthetic construct (others)32630
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8 / Details: 20mM HEPES pH 8.0, 150mM NaCl, 2mM MgCl2, 2mM TCEP
SpecimenConc.: 0.5 mg/ml / Embedding 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-PROPANE

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Electron microscopy imaging

MicroscopyModel: TFS TALOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1100 nm / Cs: 2.7 mm
Image recordingAverage exposure time: 4.5 sec. / Electron dose: 46.08 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 3257

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Processing

EM softwareName: PHENIX / Version: 1.21.1_5286: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 32126 / Symmetry type: POINT
Atomic model buildingB value: 82.2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0028588
ELECTRON MICROSCOPYf_angle_d0.46711915
ELECTRON MICROSCOPYf_dihedral_angle_d17.3211876
ELECTRON MICROSCOPYf_chiral_restr0.0361335
ELECTRON MICROSCOPYf_plane_restr0.0031270

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