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- EMDB-18969: Structure of the SFTSV L protein stalled in a transcription-speci... -

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Entry
Database: EMDB / ID: EMD-18969
TitleStructure of the SFTSV L protein stalled in a transcription-specific early elongation state with bound capped RNA [TRANSCRIPTION-EARLY-ELONGATION]
Map data
Sample
  • Complex: SFTSV L protein in complex with 5' and 3' RNA, as well as RNA acting as a primer.
    • Protein or peptide: RNA-directed RNA polymerase L
    • RNA: RNA (5'-R(*AP*CP*AP*CP*AP*GP*AP*GP*AP*CP*GP*CP*CP*CP*AP*G)-3')
    • RNA: RNA (5'-R(P*CP*UP*GP*GP*GP*CP*GP*GP*UP*AP*AP*AP*UP*GP*U)-3')
    • RNA: RNA (5'-R(P*AP*CP*A)-3')
  • RNA: RNA (5'-R(*(M7G)*AP*AP*A)-3')
  • Ligand: 5'-O-[(S)-hydroxy{[(S)-hydroxy(phosphonooxy)phosphoryl]amino}phosphoryl]uridine
  • Ligand: MAGNESIUM ION
KeywordsSFTSV / RNA-DEPENDENT RNA POLYMERASE / VIRAL RNA / VIRAL PROTEIN
Function / homology
Function and homology information


host cell endoplasmic reticulum / endoplasmic reticulum-Golgi intermediate compartment / 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: 3.4 Å
AuthorsWilliams HM / Thorkelsson SR / Vogel D / Busch C / Milewski M / Cusack S / Grunewald K / Quemin ERJ / Rosenthal M
Funding support Germany, 3 items
OrganizationGrant numberCountry
Leibniz AssociationK72/2017 Germany
German Research Foundation (DFG)NST 152/772-1, 774-1, 775-1 and 776-1 Germany
German Federal Ministry for Education and Research01KI2019 Germany
Citation
Journal: Nucleic Acids Res / Year: 2024
Title: Structural snapshots of phenuivirus cap-snatching and transcription.
Authors: Harry M Williams / Sigurdur R Thorkelsson / Dominik Vogel / Carola Busch / Morlin Milewski / Stephen Cusack / Kay Grünewald / Emmanuelle R J Quemin / Maria Rosenthal /
Abstract: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a human pathogen that is now endemic to several East Asian countries. The viral large (L) protein catalyzes viral transcription by ...Severe fever with thrombocytopenia syndrome virus (SFTSV) is a human pathogen that is now endemic to several East Asian countries. The viral large (L) protein catalyzes viral transcription by stealing host mRNA caps via a process known as cap-snatching. Here, we establish an in vitro cap-snatching assay and present three high-quality electron cryo-microscopy (cryo-EM) structures of the SFTSV L protein in biologically relevant, transcription-specific states. In a priming-state structure, we show capped RNA bound to the L protein cap-binding domain (CBD). The L protein conformation in this priming structure is significantly different from published replication-state structures, in particular the N- and C-terminal domains. The capped-RNA is positioned in a way that it can feed directly into the RNA-dependent RNA polymerase (RdRp) ready for elongation. We also captured the L protein in an early-elongation state following primer-incorporation demonstrating that this priming conformation is retained at least in the very early stages of primer extension. This structural data is complemented by in vitro biochemical and cell-based assays. Together, these insights further our mechanistic understanding of how SFTSV and other bunyaviruses incorporate stolen host mRNA fragments into their viral transcripts thereby allowing the virus to hijack host cell translation machinery.
#1: Journal: BiorXiv / Year: 2023
Title: Structural snapshots of phenuivirus cap-snatching and transcription
Authors: Williams HM / Thorkelsson SR / Vogel D / Busch C / Milewski M / Cusack S / Grunewald K / Quemin ERJ / Rosenthal M
History
DepositionNov 23, 2023-
Header (metadata) releaseApr 24, 2024-
Map releaseApr 24, 2024-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18969.png

Downloads & links

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Map

FileDownload / File: emd_18969.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.67 Å
Density
Contour LevelBy AUTHOR: 0.0013
Minimum - Maximum-0.005434659 - 0.010890229
Average (Standard dev.)0.000025117843 (±0.00043078713)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 214.40001 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_18969_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_18969_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : SFTSV L protein in complex with 5' and 3' RNA, as well as RNA act...

EntireName: SFTSV L protein in complex with 5' and 3' RNA, as well as RNA acting as a primer.
Components
  • Complex: SFTSV L protein in complex with 5' and 3' RNA, as well as RNA acting as a primer.
    • Protein or peptide: RNA-directed RNA polymerase L
    • RNA: RNA (5'-R(*AP*CP*AP*CP*AP*GP*AP*GP*AP*CP*GP*CP*CP*CP*AP*G)-3')
    • RNA: RNA (5'-R(P*CP*UP*GP*GP*GP*CP*GP*GP*UP*AP*AP*AP*UP*GP*U)-3')
    • RNA: RNA (5'-R(P*AP*CP*A)-3')
  • RNA: RNA (5'-R(*(M7G)*AP*AP*A)-3')
  • Ligand: 5'-O-[(S)-hydroxy{[(S)-hydroxy(phosphonooxy)phosphoryl]amino}phosphoryl]uridine
  • Ligand: MAGNESIUM ION

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Supramolecule #1: SFTSV L protein in complex with 5' and 3' RNA, as well as RNA act...

SupramoleculeName: SFTSV L protein in complex with 5' and 3' RNA, as well as RNA acting as a primer.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Details: SFTSV L protein expressed recombinantly in complex with several RNA species.
Source (natural)Organism: SFTS virus AH12
Molecular weightTheoretical: 240 KDa

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Macromolecule #1: RNA-directed RNA polymerase L

MacromoleculeName: RNA-directed RNA polymerase L / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
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

UniProtKB: RNA-directed RNA polymerase L

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Macromolecule #2: RNA (5'-R(*AP*CP*AP*CP*AP*GP*AP*GP*AP*CP*GP*CP*CP*CP*AP*G)-3')

MacromoleculeName: RNA (5'-R(*AP*CP*AP*CP*AP*GP*AP*GP*AP*CP*GP*CP*CP*CP*AP*G)-3')
type: rna / ID: 2
Details: RNA corresponds to 5i end of SFTSV L genome segment.
Number of copies: 1
Source (natural)Organism: SFTS virus AH12
Molecular weightTheoretical: 6.451975 KDa
SequenceString:
ACACAGAGAC GCCCAGAUGA

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Macromolecule #3: RNA (5'-R(P*CP*UP*GP*GP*GP*CP*GP*GP*UP*AP*AP*AP*UP*GP*U)-3')

MacromoleculeName: RNA (5'-R(P*CP*UP*GP*GP*GP*CP*GP*GP*UP*AP*AP*AP*UP*GP*U)-3')
type: rna / ID: 3
Details: RNA corresponds to 3' end of SFTSV L genome segment.
Number of copies: 1
Source (natural)Organism: SFTS virus AH12
Molecular weightTheoretical: 6.480872 KDa
SequenceString:
GAUCUGGGCG GUAAAUGUGU

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Macromolecule #4: RNA (5'-R(P*AP*CP*A)-3')

MacromoleculeName: RNA (5'-R(P*AP*CP*A)-3') / type: rna / ID: 4 / Details: Growing strand RNA. / Number of copies: 1
Source (natural)Organism: SFTS virus AH12
Molecular weightTheoretical: 918.636 Da
SequenceString:
ACA

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Macromolecule #5: RNA (5'-R(*(M7G)*AP*AP*A)-3')

MacromoleculeName: RNA (5'-R(*(M7G)*AP*AP*A)-3') / type: rna / ID: 5
Details: The ADM ligand is actually the m7GTP cap and the first A of the sequence. Hence, it corresponds to 2 nts *NOT* 1 nt. I have deleted the first 'real' A from the sequence to reflect this.
Number of copies: 1
Source (natural)Organism: SFTS virus AH12
Molecular weightTheoretical: 5.863617 KDa
SequenceString:
(M7G)AAAAACGCA ACCAACAC

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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: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 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

Concentration0.8 mg/mL
BufferpH: 7
GridModel: Quantifoil R2/1 / Material: GOLD / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 27095 / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2998852
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8as7

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 264083
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-8r6y:
Structure of the SFTSV L protein stalled in a transcription-specific early elongation state with bound capped RNA [TRANSCRIPTION-EARLY-ELONGATION]

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