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- EMDB-16670: Structure of the SNV L protein bound to 5' RNA -

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Basic information

Entry
Database: EMDB / ID: EMD-16670
TitleStructure of the SNV L protein bound to 5' RNA
Map data
Sample
  • Complex: Structure of Sin nombre virus L protein bound to 5' RNA
    • Protein or peptide: RNA-directed RNA polymerase L
    • RNA: RNA (5'-R(P*AP*GP*UP*AP*GP*UP*AP*GP*AP*CP*U)-3')
KeywordsNegative-strand viruses / Bunyaviruses / Hantaviruses / RNA-dependent RNA polymerase / viral genome replication and transcription / VIRUS PROTEIN / VIRAL PROTEIN
Function / homology
Function and homology information


cap snatching / endonuclease activity / host cell perinuclear region of cytoplasm / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / DNA-templated transcription
Similarity search - Function
RNA-directed RNA polymerase, hantavirus / RNA-directed RNA polymerase, hantavirus, N-terminal / : / RNA dependent RNA polymerase / Cap-snatching endonuclease / : / 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 speciesSin Nombre orthohantavirus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsMeier K / Thorkelsson SR / Durieux Trouilleton Q / Vogel D / Yu D / Kosinski J / Cusack S / Malet H / Grunewald K / Quemin ERJ / Rosenthal M
Funding support Germany, France, 5 items
OrganizationGrant numberCountry
Leibniz AssociationK72/2017) Germany
German Research Foundation (DFG)5979/2-1, INST 152/772-1, 774-1, 775-1, 776-1 Germany
Alexander von Humboldt Foundation Germany
German Federal Ministry for Education and Research01KI2019 Germany
French Infrastructure for Integrated Structural Biology (FRISBI)ANR-10-INBS-05-02 France
CitationJournal: PLoS Pathog / Year: 2023
Title: Structural and functional characterization of the Sin Nombre virus L protein.
Authors: Kristina Meier / Sigurdur R Thorkelsson / Quentin Durieux Trouilleton / Dominik Vogel / Dingquan Yu / Jan Kosinski / Stephen Cusack / Hélène Malet / Kay Grünewald / Emmanuelle R J Quemin / Maria Rosenthal /
Abstract: The Bunyavirales order is a large and diverse group of segmented negative-strand RNA viruses. Several virus families within this order contain important human pathogens, including Sin Nombre virus ...The Bunyavirales order is a large and diverse group of segmented negative-strand RNA viruses. Several virus families within this order contain important human pathogens, including Sin Nombre virus (SNV) of the Hantaviridae. Despite the high epidemic potential of bunyaviruses, specific medical countermeasures such as vaccines or antivirals are missing. The multifunctional ~250 kDa L protein of hantaviruses, amongst other functional domains, harbors the RNA-dependent RNA polymerase (RdRp) and an endonuclease and catalyzes transcription as well as replication of the viral RNA genome, making it a promising therapeutic target. The development of inhibitors targeting these key processes requires a profound understanding of the catalytic mechanisms. Here, we established expression and purification protocols of the full-length SNV L protein bearing the endonuclease mutation K124A. We applied different biochemical in vitro assays to provide an extensive characterization of the different enzymatic functions as well as the capacity of the hantavirus L protein to interact with the viral RNA. By using single-particle cryo-EM, we obtained a 3D model including the L protein core region containing the RdRp, in complex with the 5' promoter RNA. This first high-resolution model of a New World hantavirus L protein shows striking similarity to related bunyavirus L proteins. The interaction of the L protein with the 5' RNA observed in the structural model confirms our hypothesis of protein-RNA binding based on our biochemical data. Taken together, this study provides an excellent basis for future structural and functional studies on the hantavirus L protein and for the development of antiviral compounds.
History
DepositionFeb 8, 2023-
Header (metadata) releaseJul 19, 2023-
Map releaseJul 19, 2023-
UpdateAug 23, 2023-
Current statusAug 23, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16670.png

Downloads & links

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Map

FileDownload / File: emd_16670.map.gz / Format: CCP4 / Size: 178 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 360 pix.
= 306. Å		0.85 Å/pix.
x 360 pix.
= 306. Å		0.85 Å/pix.
x 360 pix.
= 306. Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015
Minimum - Maximum-0.054668244 - 0.11075268
Average (Standard dev.)0.00008499002 (±0.001901221)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 306.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_16670_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_16670_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Structure of Sin nombre virus L protein bound to 5' RNA

EntireName: Structure of Sin nombre virus L protein bound to 5' RNA
Components
  • Complex: Structure of Sin nombre virus L protein bound to 5' RNA
    • Protein or peptide: RNA-directed RNA polymerase L
    • RNA: RNA (5'-R(P*AP*GP*UP*AP*GP*UP*AP*GP*AP*CP*U)-3')

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Supramolecule #1: Structure of Sin nombre virus L protein bound to 5' RNA

SupramoleculeName: Structure of Sin nombre virus L protein bound to 5' RNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Sin Nombre orthohantavirus
Molecular weightTheoretical: 254 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: Sin Nombre orthohantavirus
Molecular weightTheoretical: 246.846797 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MEKYREIHQR VKEIPPGGAS ALECLDLLDR LYAVRHDVVD QMIKHDWSDN KDMERPIGQV LLMAGVPNDI IQGMEKKVIP TSPSGQILK SFFRMTPDNY KITGALIEFI EVTVTADVAK GIREAKLKYE SGLQFIESLL SQEHKKGNIN QVYKITFDVV A VKTDGSNI ...String:
MEKYREIHQR VKEIPPGGAS ALECLDLLDR LYAVRHDVVD QMIKHDWSDN KDMERPIGQV LLMAGVPNDI IQGMEKKVIP TSPSGQILK SFFRMTPDNY KITGALIEFI EVTVTADVAK GIREAKLKYE SGLQFIESLL SQEHKKGNIN QVYKITFDVV A VKTDGSNI STQWPSRRND GVVQHMRLVQ ADINYVREHL IKPDERASLE AMFNLKFHVG GPKLRYFNIP DYKPQSLCQP EI TNLIQYC KHWLTEDHDF VFKEVTGNNV MSSFENNEDV YMSRYKESRK PRNFLLIQGS IQGPYLPSTI SSDQCDTRIG CLE VLKVHP ETPVQAIAVD MAYKYMELNR DEIINYYNPR VHFQATQSVK EPGTFKLGLS QLNPMSKSIL DQVGKHKSEK GLFG EPLES INISSQIQQN ECSRIIESIL SNLEINVGEV TMNLANPRKT TGVDELLGKF YENELSKYLI SILRKTAAWH IGHLI RDIT ESLIAHAGLK RSKYWSIHAY DHGGVILFIL PSKSLEVVGS YIRYFTVFKD GIGLIDEENL DSKVDIDGVQ WCFSKV MSI DLNRLLALNI AFEKALLATA TWFQYYTEDQ GHFPLQHALR SVFSFHFLLC VSQKMKICAI FDNLRYLIPA VTSLYSG YE LLIEKFFERP FKSALEVYLY NIIKALLISL AQNNKVRFYS KVRLLGLTVD HSTVGASGVY PSLMSRVVYK HYRSLISE A TTCFFLFEKG LHGNLNEEAK IHLETVEWAR KFEAKERKYG DILMREGYTI DAIRVGDVQV EQQLFCQEVV ELSAEELNK YLQAKSQVLS SNIMNKHWDK PYFSQTRNIS LKGMSGALQE DGHLAASVTL IEAIRFLNRS QTNPNVIDMY EQTKQHKAQA RIVRKYQRT EADRGFFITT LPTRVRLEII EDYYDAIARV VPEEYISYGG DKKILNIQTA LEKALRWASG SSEITTSTGN V IKFKRRLM YVSADATKWS PGDNSAKFKR FTQALYDGLS DEKLKCCVVD ALRHVYETEF FMSRKLHRYI DSMDEHSEAV QD FLDFFKG GVSATVKGNW LQGNLNKCSS LFGAAVSLLF RRIWAELFPE LECFFEFAHH SDDALFIYGY LEPEDDGTDW FLY VSQQIQ AGNYHWHAVN QEMWKSMFNL HEHLLLMGSI KVSPKKTTVS PTNAEFLSTF FEGCAVSIPF IKILLGSLSD LPGL GFFDD LAAAQSRCVK AMDLGASPQL AQLAVVICTS KVERLYGTAD GMVNSPVAFL KVTKAHVPIP LGGDGSMSIM ELATA GIGM ADKNILKQAF YSYKHTRRDG DRYVLGLFKF LMSLSEDVFQ HDRLGEFSFV GKVQWKVFTP KNEFEFYDQF SQSYLK SWT NQHPVYDYII PRGRDNLLVY LVRKLNDPSI VTAMTMQSPL QLRFRMQAKQ HMKVCKLDGE WVTFREVLAA ADSFATK YN PTEKDLDLFN TLVSCTFSKE YAWKDFLNEV RCEVVPTKHV HRSKIARTFT VREKDQAIQN PITAVIGYKY ASTVDEIS D VLDSSFFPDS LSADLQVMKE GVYRELGLDI GLPEVLKRIA PLLYKAGRSR VVIVEGNVEG TAESICSYWL RSMSLVKTI KVRPKKEVLR AVSLYSTKEN IGLQDDVAAT RLCIEVWRWC KANDQNVNDW LNALYFEKQT LMDWVERFRR KGVVPVDPEI QCIALLLYD VLGYKSVLQM QANRRAYSGK QYDAYCVQTY NEETKLYEGD LRVTFNFGLD CARLEIFWDK KEYILETSIT Q RHVLKLMM EEVTQELLRC GMRFKTEQVS HTKSLVLFKT ESGFEWGKPN VPCIVFKHCA LRTGLRTKQA INKEFMINVQ AD GFRAIAQ MDVESPRFLL AHAYHTLRDV RYQAVQAVGN VWFQTNQHKL FINPIISSGL LENFMKGLPA AIPPAAYSLI MNK AKISVD LFMFNELLAL VNPRNVLNLD GIEETSEGYS TVTSISSRQW SEEVSLMADD NIDDEEEFTI ALDDIDFEQI NLDE DIQHF LQDESAYTGD LTIQTEEVEV KRIRGVTRVL EPVKLIKSWV SKGLAIDKVY NPIGIVLMAR YMSKNYDFSK IPLAL LNPY DLTEFESVVK GWGETVNDRF LEVDNDAQRL IREKNILPED ILPDSLFSFR HVDVLLKRLF PRDPVSSFY

UniProtKB: RNA-directed RNA polymerase L

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

MacromoleculeName: RNA (5'-R(P*AP*GP*UP*AP*GP*UP*AP*GP*AP*CP*U)-3') / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Sin Nombre orthohantavirus
Molecular weightTheoretical: 5.796515 KDa
SequenceString:
UAGUAGUAGA CUCCGAGA

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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

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 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: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 514913
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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