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- EMDB-47165: Cryo-EM structure of DENV2 NS5 in complex with Stem Loop A (SLA) -

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Basic information

Entry
Database: EMDB / ID: EMD-47165
TitleCryo-EM structure of DENV2 NS5 in complex with Stem Loop A (SLA)
Map data
Sample
  • Complex: Full-length DENV2 NS5 bound to viral promoter SLA
    • Complex: DENV2 NS5
      • Protein or peptide: RNA-directed RNA polymerase NS5
    • Complex: SLA70
      • RNA: stem loop A
  • Ligand: ZINC ION
KeywordsRNA-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
Biological speciesdengue virus type 2 / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsObi JO / Fields JK / Snyder AG / Deredge DJ
Funding support United States, 1 items
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, 2024-
Header (metadata) releaseOct 23, 2024-
Map releaseOct 23, 2024-
UpdateJan 8, 2025-
Current statusJan 8, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47165.png

Downloads & links

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Map

FileDownload / File: emd_47165.map.gz / Format: CCP4 / Size: 144.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.72 Å/pix.
x 336 pix.
= 241.248 Å		0.72 Å/pix.
x 336 pix.
= 241.248 Å		0.72 Å/pix.
x 336 pix.
= 241.248 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.718 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.124
Minimum - Maximum-0.35366413 - 0.7910167
Average (Standard dev.)0.00097140454 (±0.034476653)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions336336336
Spacing336336336
CellA=B=C: 241.248 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_47165_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_47165_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : Full-length DENV2 NS5 bound to viral promoter SLA

EntireName: Full-length DENV2 NS5 bound to viral promoter SLA
Components
  • Complex: Full-length DENV2 NS5 bound to viral promoter SLA
    • Complex: DENV2 NS5
      • Protein or peptide: RNA-directed RNA polymerase NS5
    • Complex: SLA70
      • RNA: stem loop A
  • Ligand: ZINC ION

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Supramolecule #1: Full-length DENV2 NS5 bound to viral promoter SLA

SupramoleculeName: Full-length DENV2 NS5 bound to viral promoter SLA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: DENV2 NS5

SupramoleculeName: DENV2 NS5 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: dengue virus type 2

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Supramolecule #3: SLA70

SupramoleculeName: SLA70 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: RNA-directed RNA polymerase NS5

MacromoleculeName: RNA-directed RNA polymerase NS5 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: mRNA (guanine-N7)-methyltransferase
Source (natural)Organism: dengue virus type 2 / Strain: New Guinea C (NGC)
Molecular weightTheoretical: 105.611188 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGENLYFQG GTGNIGETLG EKWKSRLNAL GKSEFQIYKK SGIQEVDRTL AKEGIKRGET DHHAVSRGSA KLRWFVERN MVTPEGKVVD LGCGRGGWSY YCGGLKNVRE VKGLTKGGPG HEEPIPMSTY GWNLVRLQSG VDVFFTPPEK C DTLLCDIG ...String:
MGSSHHHHHH SSGENLYFQG GTGNIGETLG EKWKSRLNAL GKSEFQIYKK SGIQEVDRTL AKEGIKRGET DHHAVSRGSA KLRWFVERN MVTPEGKVVD LGCGRGGWSY YCGGLKNVRE VKGLTKGGPG HEEPIPMSTY GWNLVRLQSG VDVFFTPPEK C DTLLCDIG ESSPNPTVEA GRTLRVLNLV ENWLNNNTQF CIKVLNPYMP SVIEKMEALQ RKYGGALVRN PLSRNSTHEM YW VSNASGN IVSSVNMISR MLINRFTMRH KKATYEPDVD LGSGTRNIGI ESEIPNLDII GKRIEKIKQE HETSWHYDQD HPY KTWAYH GSYETKQTGS ASSMVNGVVR LLTKPWDVVP MVTQMAMTDT TPFGQQRVFK EKVDTRTQEP KEGTKKLMKI TAEW LWKEL GKKKTPRMCT REEFTRKVRS NAALGAIFTD ENKWKSAREA VEDSRFWELV DKERNLHLEG KCETCVYNMM GKREK KLGE FGKAKGSRAI WYMWLGARFL EFEALGFLNE DHWFSRENSL SGVEGEGLHK LGYILRDVSK KEGGAMYADD TAGWDT RIT LEDLKNEEMV TNHMEGEHKK LAEAIFKLTY QNKVVRVQRP TPRGTVMDII SRRDQRGSGQ VGTYGLNTFT NMEAQLI RQ MEGEGVFKSI QHLTVTEEIA VQNWLARVGR ERLSRMAISG DDCVVKPLDD RFASALTALN DMGKVRKDIQ QWEPSRGW N DWTQVPFCSH HFHELIMKDG RVLVVPCRNQ DELIGRARIS QGAGWSLRET ACLGKSYAQM WSLMYFHRRD LRLAANAIC SAVPSHWVPT SRTTWSIHAK HEWMTTEDML TVWNRVWIQE NPWMEDKTPV ESWEEIPYLG KREDQWCGSL IGLTSRATWA KNIQTAINQ VRSLIGNEEY TDYMPSMKRF RREEEEAGVL W

UniProtKB: Genome polyprotein

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Macromolecule #2: stem loop A

MacromoleculeName: stem loop A / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: dengue virus type 2
Molecular weightTheoretical: 22.560412 KDa
SequenceString:
AGUUGUUAGU CUACGUGGAC CGACAAAGAC AGAUUCUUUG AGGGAGCUAA GCUCAACGUA GUUCUAACAG

GENBANK: GENBANK: AF038403.1

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Macromolecule #3: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 8 / Details: 20mM HEPES pH 8.0, 150mM NaCl, 2mM MgCl2, 2mM TCEP
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS TALOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 3257 / Average exposure time: 4.5 sec. / Average electron dose: 46.08 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.1 µm

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 32126
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementOverall B value: 82.2
Output model

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

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