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- EMDB-34401: Cryo-EM structure of the NS5-NS3-SLA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-34401
TitleCryo-EM structure of the NS5-NS3-SLA complex
Map data
Sample
  • Complex: RNA replicase complex bound to the mini-genome RNA
    • Complex: NS5
      • Protein or peptide: Genome polyprotein
    • Complex: NS3
      • Protein or peptide: Genome polyprotein
    • Complex: Mini-genome RNA
      • RNA: RNA
  • Ligand: ZINC ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
Keywordsviral genome replication / VIRAL PROTEIN
Function / homology
Function and homology information


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 / ribonucleoside triphosphate phosphatase activity / double-stranded RNA binding / channel activity / viral capsid / monoatomic ion transmembrane transport / clathrin-dependent endocytosis of virus by host cell ...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 / ribonucleoside triphosphate phosphatase activity / double-stranded RNA binding / channel activity / viral capsid / monoatomic ion transmembrane transport / clathrin-dependent endocytosis of virus by host cell / mRNA (nucleoside-2'-O-)-methyltransferase activity / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / RNA helicase activity / protein dimerization activity / host cell endoplasmic reticulum membrane / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / induction by virus of host autophagy / viral RNA genome replication / serine-type endopeptidase activity / RNA-dependent RNA polymerase activity / virus-mediated perturbation of host defense response / fusion of virus membrane with host endosome membrane / viral envelope / host cell nucleus / virion attachment to host cell / structural molecule activity / virion membrane / proteolysis / extracellular region / ATP binding / membrane / metal ion binding
Similarity search - Function
Flavivirus capsid protein C superfamily / Flavivirus non-structural protein NS2B / Genome polyprotein, Flavivirus / : / mRNA cap 0/1 methyltransferase / mRNA cap 0 and cap 1 methyltransferase (EC 2.1.1.56 and EC 2.1.1.57) domain profile. / Flavivirus non-structural protein NS4A / Flavivirus non-structural protein NS2B / Flavivirus capsid protein C / Flavivirus non-structural protein NS4B ...Flavivirus capsid protein C superfamily / Flavivirus non-structural protein NS2B / Genome polyprotein, Flavivirus / : / mRNA cap 0/1 methyltransferase / mRNA cap 0 and cap 1 methyltransferase (EC 2.1.1.56 and EC 2.1.1.57) domain profile. / Flavivirus non-structural protein NS4A / Flavivirus non-structural protein NS2B / Flavivirus capsid protein C / Flavivirus non-structural protein NS4B / Flavivirus capsid protein C / Flavivirus non-structural protein NS4B / Flavivirus non-structural protein NS4A / Flavivirus NS2B domain profile. / Flavivirus non-structural protein NS2A / Flavivirus non-structural protein NS2A / RNA-directed RNA polymerase, thumb domain, Flavivirus / Flavivirus RNA-directed RNA polymerase, thumb domain / Flavivirus NS3, petidase S7 / Peptidase S7, Flavivirus NS3 serine protease / Flavivirus NS3 protease (NS3pro) domain profile. / RNA-directed RNA polymerase, flavivirus / Flavivirus RNA-directed RNA polymerase, fingers and palm domains / Flavivirus non-structural Protein NS1 / Flavivirus non-structural protein NS1 / Envelope glycoprotein M, flavivirus / Envelope glycoprotein M superfamily, flavivirus / Flavivirus polyprotein propeptide / Flavivirus polyprotein propeptide superfamily / Flavivirus polyprotein propeptide / Flavivirus envelope glycoprotein M / Flavivirus envelope glycoprotein E, stem/anchor domain / Flavivirus envelope glycoprotein E, Stem/Anchor domain / Flavivirus envelope glycoprotein E, Stem/Anchor domain superfamily / Flavivirus glycoprotein E, immunoglobulin-like domain / Flavivirus glycoprotein central and dimerisation domain / Flaviviral glycoprotein E, central domain, subdomain 1 / Flaviviral glycoprotein E, central domain, subdomain 2 / : / Flavivirus NS3 helicase, C-terminal helical domain / Flavivirus glycoprotein, immunoglobulin-like domain / Flavivirus glycoprotein, central and dimerisation domains / 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
Genome polyprotein / Genome polyprotein
Similarity search - Component
Biological speciesDengue virus / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsOsawa T / Ehara H / Sekine S
Funding support Japan, 1 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS) Japan
CitationJournal: Mol Cell / Year: 2023
Title: Structures of dengue virus RNA replicase complexes.
Authors: Takuo Osawa / Mari Aoki / Haruhiko Ehara / Shun-Ichi Sekine /
Abstract: Dengue is a mosquito-borne viral infection caused by dengue virus (DENV), a member of the flaviviruses. The DENV genome is a 5'-capped positive-sense RNA with a unique 5'-stem-loop structure (SLA), ...Dengue is a mosquito-borne viral infection caused by dengue virus (DENV), a member of the flaviviruses. The DENV genome is a 5'-capped positive-sense RNA with a unique 5'-stem-loop structure (SLA), which is essential for RNA replication and 5' capping. The virus-encoded proteins NS5 and NS3 are responsible for viral genome replication, but the structural basis by which they cooperatively conduct the required tasks has remained unclear. Here, we report the cryoelectron microscopy (cryo-EM) structures of SLA-bound NS5 (PC), NS3-bound PC (PC-NS3), and an RNA-elongating NS5-NS3 complex (EC). While SLA bridges the NS5 methyltransferase and RNA-dependent RNA polymerase domains in PC, the NS3 helicase domain displaces it in elongation complex (EC). The SLA- and NS3-binding sites overlap with that of human STAT2. These structures illuminate the key steps in DENV genome replication, namely, SLA-dependent replication initiation, processive RNA elongation, and 5' capping of the nascent genomic RNA, thereby providing foundations to combat flaviviruses.
History
DepositionSep 27, 2022-
Header (metadata) releaseAug 2, 2023-
Map releaseAug 2, 2023-
UpdateAug 23, 2023-
Current statusAug 23, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_34401.png

Downloads & links

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Map

FileDownload / File: emd_34401.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 240 pix.
= 198.84 Å		0.83 Å/pix.
x 240 pix.
= 198.84 Å		0.83 Å/pix.
x 240 pix.
= 198.84 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8285 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.018028572 - 0.03778538
Average (Standard dev.)0.0002481668 (±0.0018372041)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 198.84 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_34401_msk_1.map
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Half map: #2

Fileemd_34401_half_map_1.map
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Half map: #1

Fileemd_34401_half_map_2.map
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Sample components

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Entire : RNA replicase complex bound to the mini-genome RNA

EntireName: RNA replicase complex bound to the mini-genome RNA
Components
  • Complex: RNA replicase complex bound to the mini-genome RNA
    • Complex: NS5
      • Protein or peptide: Genome polyprotein
    • Complex: NS3
      • Protein or peptide: Genome polyprotein
    • Complex: Mini-genome RNA
      • RNA: RNA
  • Ligand: ZINC ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE

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Supramolecule #1: RNA replicase complex bound to the mini-genome RNA

SupramoleculeName: RNA replicase complex bound to the mini-genome RNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Dengue virus

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Supramolecule #2: NS5

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

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Supramolecule #3: NS3

SupramoleculeName: NS3 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Dengue virus

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Supramolecule #4: Mini-genome RNA

SupramoleculeName: Mini-genome RNA / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: Genome polyprotein

MacromoleculeName: Genome polyprotein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Dengue virus
Molecular weightTheoretical: 104.189891 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GPSSGSSGGT GSQGETLGEK WKKRLNQLSR KEFDLYKKSG ITEVDRTEAK EGLKRGEITH HAVSRGSAKL QWFVERNMVI PEGRVIDLG CGRGGWSYYC AGLKKVTEVR GYTKGGPGHE EPVPMSTYGW NIVKLMSGKD VFYLPPEKCD TLLCDIGESS P SPTVEESR ...String:
GPSSGSSGGT GSQGETLGEK WKKRLNQLSR KEFDLYKKSG ITEVDRTEAK EGLKRGEITH HAVSRGSAKL QWFVERNMVI PEGRVIDLG CGRGGWSYYC AGLKKVTEVR GYTKGGPGHE EPVPMSTYGW NIVKLMSGKD VFYLPPEKCD TLLCDIGESS P SPTVEESR TIRVLKMVEP WLKNNQFCIK VLNPYMPTVI EHLERLQRKH GGMLVRNPLS RNSTHEMYWI SNGTGNIVSS VN MVSRLLL NRFTMTYRRP TIEKDVDLGA GTRHVNAEPE TPNMDVIGER IRRIKEEHSS TWHYDDENPY KTWAYHGSYE VKA TGSASS MINGVVKLLT KPWDVVPTVT QMAMTDTTPF GQQRVFKEKV DTRTPKPMPG TRKVMEITAG WLWRTLGRNK RPRL CTREE FTKKVRTNAA MGAVFTEENQ WDSARAAVED EEFWKLVDRE RELHKQGKCG SCVYNMMGKR EKKLGEFGKA KGSRA IWYM WLGARYLEFE ALGFLNEDHW FSRENSYSGV EGEGLHKLGY ILRDISKIPG GAMYADDTAG WDTRITEDDL HNEEKI TQQ MDPEHRQLAN AIFKLTYQNK VVKVQRPTPK GTVMDIISRK DQRGSGQVGT YGLNTFTNME AQLIRQMEGE GVLSKTD LE NPHLLEKKIT QWLETKGVER LKRMAISGDD CVVKPIDDRF ANALLALNDM GKVRKDIPQW QPSKGWHDWQ QVPFCSHH F HELIMKDGRK LVVPCRPQDE LIGRARISQG AGWSLKETAC LGKAYAQMWA LMYFHRRDLR LASNAICSAV PVHWVPTSR TTWSIHAHHQ WMTTEDMLTV WNRVWIEDNP WMEDKTPVTT WEDVPYLGKR EDQWCGSLIG LTSRATWAQN ILTAIQQVRS LIGNEEFLD YMPSMKRFRK EEESEGAIW

UniProtKB: Genome polyprotein

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Macromolecule #2: Genome polyprotein

MacromoleculeName: Genome polyprotein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Dengue virus
Molecular weightTheoretical: 74.753406 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SQDPENLYFQ GSSGSSGADL TVEKAADVTW EEEAEGGGGS GGGGSGVLWD VPSPPETQKA ELEEGVYRIK QQGIFGKTQ VGVGVQKEGV FHTMWHVTRG AVLTYNGKRL EPNWASVKKD LISYGGGWRL SAQWQKGEEV QVIAVEPGKN P KNFQTMPG ...String:
MGSSHHHHHH SQDPENLYFQ GSSGSSGADL TVEKAADVTW EEEAEGGGGS GGGGSGVLWD VPSPPETQKA ELEEGVYRIK QQGIFGKTQ VGVGVQKEGV FHTMWHVTRG AVLTYNGKRL EPNWASVKKD LISYGGGWRL SAQWQKGEEV QVIAVEPGKN P KNFQTMPG IFQTTTGEIG AIALDFKPGT AGSPIINREG KVVGLYGNGV VTKNGGYVSG IAQTNAEPDG PTPELEEEMF KK RNLTIMD LHPGSGKTRK YLPAIVREAI KRRLRTLILA PTRVVAAEME EALKGLPIRY QTTATKSEHT GKEIVDLMCH ATF TMRLLS PVRVPNYNLI IMDEAHFTDP ASIAARGYIS TRVGMGEAAA IFMTATPPGT ADAFPQSNAP IQDEERDIPE RSWN SGNDW ITDFAGKTVW FVPSIKAGND IANCLRKNGK KVIQLSRKTF DTEYQKTKLN DWDFVVTTDI SEMGANFKAD RVIDP RRCL KPVILTDGPE RVILAGPMPV TVASAAQRRG RVGRNPQKEN DQYIFTGQPL NNDEDHAHWT EAKMLLDNIN TPEGII PAL FEPEREKSAA IDGEYRLKGE SRKTFVELMR RGDLPVWLAH KVASEGIKYT DRKWCFDGER NNQILEENMD VEIWTKE GE KKKLRPRWLD ARTYSDPLAL KEFKDFAAGR K

UniProtKB: Genome polyprotein

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Macromolecule #3: RNA

MacromoleculeName: RNA / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 54.128918 KDa
SequenceString:
(GDP)AGUUGUUAG UCUGUGUGGA CCGACAAGGA CAGUUCCAAA UCGGAAGCUU GCUUAACACA GUUCUAACAG UUUGUU UAG AUAGAGAGCA GUAACCUGCU UUCUCUGCAA CAUCAAUCCA GGCACAGAGC GCCGCGAGAU GGAUUGGUGU UGUUGAU CC AACAGGUUCU

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #5: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

ChemComp-GDP:
GUANOSINE-5'-DIPHOSPHATE / GDP, energy-carrying molecule*YM

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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

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 55.46 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.4000000000000001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 31209
Initial angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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