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- EMDB-15614: Structure of the SFTSV L protein stalled at late elongation [LATE... -

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Entry
Database: EMDB / ID: EMD-15614
TitleStructure of the SFTSV L protein stalled at late elongation [LATE-ELONGATION]
Map data
Sample
  • Complex: Structure of the SFTSV L protein stalled at late elongation [LATE-ELONGATION]
    • Complex: RNA-dependent RNA-polymerase L protein
      • Protein or peptide: RNA-directed RNA polymerase
    • Complex: RNA
      • RNA: RNA (5'-R(P*AP*AP*AP*AP*AP*AP*GP*AP*UP*CP*UP*GP*GP*GP*CP*GP*GP*UP*CP*UP*UP*UP*GP*UP*GP*U)-3')
      • RNA: RNA (5'-R(P*AP*CP*AP*CP*AP*AP*AP*GP*AP*CP*CP*GP*CP*CP*CP*AP*GP*AP*CP*CP*UP*UP*UP*UP*UP*U)-3')
      • RNA: RNA (5'-R(P*AP*CP*AP*CP*AP*GP*AP*GP*AP*CP*GP*CP*CP*CP*AP*GP*AP*UP*GP*A)-3')
  • Ligand: MAGNESIUM ION
  • Ligand: 5'-O-[(S)-hydroxy{[(S)-hydroxy(phosphonooxy)phosphoryl]amino}phosphoryl]uridine
  • Ligand: water
Function / homology
Function and homology information


host cell endoplasmic reticulum / host cell endoplasmic reticulum-Golgi intermediate compartment / host cell Golgi apparatus / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / DNA-templated transcription / metal ion binding
Similarity search - Function
RNA-directed RNA polymerase L, PA-C-like domain / RNA-directed RNA polymerase L, PA-C-like domain / RNA-directed RNA polymerase L, N-terminal / L protein N-terminus / RNA-dependent RNA polymerase, bunyaviral / Bunyavirus RNA dependent RNA polymerase / RNA-directed RNA polymerase, negative-strand RNA virus / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile.
Similarity search - Domain/homology
RNA-directed RNA polymerase L
Similarity search - Component
Biological speciesSFTS virus AH12
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsWilliams HM / Thorkelsson SR / Vogel D / Milewski M / Busch C / Cusack S / Grunewald K / Quemin ERJ / Rosenthal M
Funding support Germany, 3 items
OrganizationGrant numberCountry
Leibniz AssociationK72/2017 Germany
German Research Foundation (DFG)INST 152/772-1, 774-1, 775-1 and 776-1 Germany
German Federal Ministry for Education and Research01KI2019 Germany
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Structural insights into viral genome replication by the severe fever with thrombocytopenia syndrome virus L protein.
Authors: Harry M Williams / Sigurdur R Thorkelsson / Dominik Vogel / Morlin Milewski / Carola Busch / Stephen Cusack / Kay Grünewald / Emmanuelle R J Quemin / Maria Rosenthal /
Abstract: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a phenuivirus that has rapidly become endemic in several East Asian countries. The large (L) protein of SFTSV, which includes the RNA- ...Severe fever with thrombocytopenia syndrome virus (SFTSV) is a phenuivirus that has rapidly become endemic in several East Asian countries. The large (L) protein of SFTSV, which includes the RNA-dependent RNA polymerase (RdRp), is responsible for catalysing viral genome replication and transcription. Here, we present 5 cryo-electron microscopy (cryo-EM) structures of the L protein in several states of the genome replication process, from pre-initiation to late-stage elongation, at a resolution of up to 2.6 Å. We identify how the L protein binds the 5' viral RNA in a hook-like conformation and show how the distal 5' and 3' RNA ends form a duplex positioning the 3' RNA terminus in the RdRp active site ready for initiation. We also observe the L protein stalled in the early and late stages of elongation with the RdRp core accommodating a 10-bp product-template duplex. This duplex ultimately splits with the template binding to a designated 3' secondary binding site. The structural data and observations are complemented by in vitro biochemical and cell-based mini-replicon assays. Altogether, our data provide novel key insights into the mechanism of viral genome replication by the SFTSV L protein and will aid drug development against segmented negative-strand RNA viruses.
History
DepositionAug 19, 2022-
Header (metadata) releaseJan 18, 2023-
Map releaseJan 18, 2023-
UpdateMar 1, 2023-
Current statusMar 1, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15614.png

Downloads & links

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Map

FileDownload / File: emd_15614.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 320 pix.
= 272. Å		0.85 Å/pix.
x 320 pix.
= 272. Å		0.85 Å/pix.
x 320 pix.
= 272. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.019
Minimum - Maximum-0.08445003 - 0.18688996
Average (Standard dev.)4.2831503e-05 (±0.006711456)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 272.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_15614_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_15614_half_map_2.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Structure of the SFTSV L protein stalled at late elongation [LATE...

EntireName: Structure of the SFTSV L protein stalled at late elongation [LATE-ELONGATION]
Components
  • Complex: Structure of the SFTSV L protein stalled at late elongation [LATE-ELONGATION]
    • Complex: RNA-dependent RNA-polymerase L protein
      • Protein or peptide: RNA-directed RNA polymerase
    • Complex: RNA
      • RNA: RNA (5'-R(P*AP*AP*AP*AP*AP*AP*GP*AP*UP*CP*UP*GP*GP*GP*CP*GP*GP*UP*CP*UP*UP*UP*GP*UP*GP*U)-3')
      • RNA: RNA (5'-R(P*AP*CP*AP*CP*AP*AP*AP*GP*AP*CP*CP*GP*CP*CP*CP*AP*GP*AP*CP*CP*UP*UP*UP*UP*UP*U)-3')
      • RNA: RNA (5'-R(P*AP*CP*AP*CP*AP*GP*AP*GP*AP*CP*GP*CP*CP*CP*AP*GP*AP*UP*GP*A)-3')
  • Ligand: MAGNESIUM ION
  • Ligand: 5'-O-[(S)-hydroxy{[(S)-hydroxy(phosphonooxy)phosphoryl]amino}phosphoryl]uridine
  • Ligand: water

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Supramolecule #1: Structure of the SFTSV L protein stalled at late elongation [LATE...

SupramoleculeName: Structure of the SFTSV L protein stalled at late elongation [LATE-ELONGATION]
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#4
Molecular weightTheoretical: 238 KDa

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Supramolecule #2: RNA-dependent RNA-polymerase L protein

SupramoleculeName: RNA-dependent RNA-polymerase L protein / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: SFTS virus AH12

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Supramolecule #3: RNA

SupramoleculeName: RNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#4
Source (natural)Organism: SFTS virus AH12

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Macromolecule #1: RNA-directed RNA polymerase

MacromoleculeName: RNA-directed RNA polymerase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed RNA polymerase
Source (natural)Organism: SFTS virus AH12
Molecular weightTheoretical: 235.6985 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MNLEVLCGRI NVENGLSLGE PGLYDQIYDR PGLPDLDVTV DATGVTVDIG AVPDSASQLG SSINAGLITI QLSEAYKINH DFTFSGLSK TTDRRLSEVF PITHDGSDGM TPAVIHTRLD GTIVVVEFST TRSHNIGGLE AAYRTKIEKY RDPISRRVDI M ENPRVFFG ...String:
MNLEVLCGRI NVENGLSLGE PGLYDQIYDR PGLPDLDVTV DATGVTVDIG AVPDSASQLG SSINAGLITI QLSEAYKINH DFTFSGLSK TTDRRLSEVF PITHDGSDGM TPAVIHTRLD GTIVVVEFST TRSHNIGGLE AAYRTKIEKY RDPISRRVDI M ENPRVFFG VIVVSSGGVL SNMPLTQDEA EELMYRFCIA NEIYTKARSM DADIELQKSE EELEAISRAL SFFSLFEPNI ER VEGTFPN SEIKMLEQFL STPADVDFIT KTLKAKEVEA YADLCDSHYL KPEKTIQERL EINRCEAIDK TQDLLAGLHA RSN KQTSLN RGTVKLPPWL PKPSSESIDI KTDSGFGSLM DHGAYGELWA KCLLDVSLGN VEGVVSDPAK ELDIAISDDP EKDT PKEAK ITYRRFKPAL SSSARQEFSL QGVEGKKWKR MAANQKKEKE SHETLSPFLD VEDIGDFLTF NNLLTDSRYG DESIQ RAVS ILLEKASAMQ DTELTHALND SFKRNLSSNV VQWSLWVSCL AQELASALKQ HCRAGEFIIK KLKFWPIYVI IKPTKS SSH IFYSLGIRKA DVTRRLTGRV FSDTIDAGEW ELTEFKSLKT CKLTNLVNLP CTMLNSIAFW REKLGVAPWL VRKPCSE LR EQVGLTFLIS LEDKSKTEEI ITLTRYTQME GFVSPPMLPK PQKMLGKLDG PLRTKLQVYL LRKHLDCMVR IASQPFSL I PREGRVEWGG TFHAISGRST NLENMVNSWY IGYYKNKEES TELNALGEMY KKIVEMEEDK PSSPEFLGWG DTDSPKKHE FSRSFLRAAC SSLEREIAQR HGRQWKQNLE ERVLREIGTK NILDLASMKA TSNFSKDWEL YSEVQTKEYH RSKLLEKMAT LIEKGVMWY IDAVGQAWKA VLDDGCMRIC LFKKNQHGGL REIYVMDANA RLVQFGVETM ARCVCELSPH ETVANPRLKN S IIENHGLK SARSLGPGSI NINSSNDAKK WNQGHYTTKL ALVLCWFMPA KFHRFIWAAI SMFRRKKMMV DLRFLAHLSS KS ESRSSDP FREAMTDAFH GNRDVSWMDK GRTYIKTETG MMQGILHFTS SLLHSCVQSF YKSYFVSKLK EGYMGESISG VVD VIEGSD DSAIMISIRP KSDMDEVRSR FFVANLLHSV KFLNPLFGIY SSEKSTVNTV YCVEYNSEFH FHRHLVRPTL RWIA ASHQI SETEALASRQ EDYSNLLTQC LEGGASFSLT YLIQCAQLLH HYMLLGLCLH PLFGTFMGML ISDPDPALGF FLMDN PAFA GGAGFRFNLW RACKTTDLGR KYAYYFNEIQ GKTKGDEDYR ALDATSGGTL SHSVMVYWGD RKKYQALLNR MGLPED WVE QIDENPGVLY RRAANKKELL LKLAEKVHSP GVTSSLSKGH VVPRVVAAGV YLLSRHCFRF SSSIHGRGST QKASLIK LL MMSSISAMKH GGSLNPNQER MLFPQAQEYD RVCTLLEEVE HLTGKFVVRE RNIVRSRIDL FQEPVDLRCK AEDLVSEV W FGLKRTKLGP RLLKEEWDKL RASFAWLSTD PSETLRDGPF LSHVQFRNFI AHVDAKSRSV RLLGAPVKKS GGVTTISQV VRMNFFPGFS LEAEKSLDNQ ERLESISILK HVLFMVLNGP YTEEYKLEMI IEAFSTLVIP QPSEVIRKSR TMTLCLLSNY LSSRGGSIL DQIERAQSGT LGGFSKPQKT FVRPGGGVGY KGKGVWTGVM EDTHVQILID GDGTSNWLEE IRLSSDARLY D VIESIRRL CDDLGINNRV ASAYRGHCMV RLSGFKIKPA SRTDGCPVRI MERGFRIREL QNPDEVKMRV RGDILNLSVT IQ EGRVMNI LSYRPRDTDI SESAAAYLWS NRDLFSFGKK EPSCSWICLK TLDNWAWSHA SVLLANDRKT QGIDNRAMGN IFR DCLEGS LRKQGLMRSK LTEMVEKNVV PLTTQELVDI LEEDIDFSDV IAVELSEGSL DIESIFDGAP ILWSAEVEEF GEGV VAVSY SSKYYHLTLM DQAAITMCAI MGKEGCRGLL TEKRCMAAIR EQVRPFLIFL QIPEDSISWV SDQFCDSRGL DEEST IMWG

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Macromolecule #2: RNA (5'-R(P*AP*AP*AP*AP*AP*AP*GP*AP*UP*CP*UP*GP*GP*GP*CP*GP*GP*UP...

MacromoleculeName: RNA (5'-R(P*AP*AP*AP*AP*AP*AP*GP*AP*UP*CP*UP*GP*GP*GP*CP*GP*GP*UP*CP*UP*UP*UP*GP*UP*GP*U)-3')
type: rna / ID: 2
Details: Additional 6A's added to template RNA (nt 21 - 26). Start of 3' cRNA bound to secondary binding site, remainder defined in the L protein core - nts in between are not clearly defined in the ...Details: Additional 6A's added to template RNA (nt 21 - 26). Start of 3' cRNA bound to secondary binding site, remainder defined in the L protein core - nts in between are not clearly defined in the EM map (although clearly there in the blurred map) and so not fit in the PDB.
Number of copies: 1
Source (natural)Organism: SFTS virus AH12
Molecular weightTheoretical: 8.386002 KDa
SequenceString:
AAAAAAGAUC UGGGCGGUCU UUGUGU

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Macromolecule #3: RNA (5'-R(P*AP*CP*AP*CP*AP*AP*AP*GP*AP*CP*CP*GP*CP*CP*CP*AP*GP*AP...

MacromoleculeName: RNA (5'-R(P*AP*CP*AP*CP*AP*AP*AP*GP*AP*CP*CP*GP*CP*CP*CP*AP*GP*AP*CP*CP*UP*UP*UP*UP*UP*U)-3')
type: rna / ID: 3
Details: Additional 6A's added to template RNA (nt 21 - 26) so poly-U stretch here is artificial. At position 19 on the 3' cRNA we have a A. However, our investigations show that a C is mis- ...Details: Additional 6A's added to template RNA (nt 21 - 26) so poly-U stretch here is artificial. At position 19 on the 3' cRNA we have a A. However, our investigations show that a C is mis-incorporated at this point in the growing strand. If I place the correct growing strand sequence in the box, it is flagged as incorrect because in our structure we have C instead of the expected A. To get around this, I have changed the sample sequence to reflect what we see in our experimental setup.
Number of copies: 1
Source (natural)Organism: SFTS virus AH12
Molecular weightTheoretical: 8.207938 KDa
SequenceString:
ACACAAAGAC CGCCCAGACC UUUUUU

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Macromolecule #4: RNA (5'-R(P*AP*CP*AP*CP*AP*GP*AP*GP*AP*CP*GP*CP*CP*CP*AP*GP*AP*UP...

MacromoleculeName: RNA (5'-R(P*AP*CP*AP*CP*AP*GP*AP*GP*AP*CP*GP*CP*CP*CP*AP*GP*AP*UP*GP*A)-3')
type: rna / ID: 4 / Details: Excess 5' cRNA bound to endonuclease domain. / Number of copies: 2
Source (natural)Organism: SFTS virus AH12
Molecular weightTheoretical: 6.451975 KDa
SequenceString:
ACACAGAGAC GCCCAGAUGA

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: 5'-O-[(S)-hydroxy{[(S)-hydroxy(phosphonooxy)phosphoryl]amino}phos...

MacromoleculeName: 5'-O-[(S)-hydroxy{[(S)-hydroxy(phosphonooxy)phosphoryl]amino}phosphoryl]uridine
type: ligand / ID: 6 / Number of copies: 1 / Formula: 2KH
Molecular weightTheoretical: 483.156 Da
Chemical component information

ChemComp-2KH:
5'-O-[(S)-hydroxy{[(S)-hydroxy(phosphonooxy)phosphoryl]amino}phosphoryl]uridine

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 32 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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: 7
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 53.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7alp

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 60000
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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