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- EMDB-11093: Cryo-EM map of La Crosse virus polymerase at pre-initiation stage -

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Basic information

Entry
Database: EMDB / ID: EMD-11093
TitleCryo-EM map of La Crosse virus polymerase at pre-initiation stage
Map dataLa Crosse full length polymerase at pre-initiation stage
Sample
  • Complex: La Crosse virus polymerase at pre-initiation stage
    • Complex: La Crosse virus polymerase at pre-initiation stage
      • RNA: 5'vRNA 1-10
      • RNA: 5'vRNA 9-16
      • RNA: 3'vRNA 1-16
      • Protein or peptide: RNA-directed RNA polymerase L
    • Complex: La Crosse virus polymerase at pre-initiation stage
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
Function / homology
Function and homology information


host cell endoplasmic reticulum / virion component / host cell endoplasmic reticulum-Golgi intermediate compartment / host cell Golgi apparatus / Hydrolases; Acting on ester bonds / hydrolase activity / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / nucleotide binding ...host cell endoplasmic reticulum / virion component / host cell endoplasmic reticulum-Golgi intermediate compartment / host cell Golgi apparatus / Hydrolases; Acting on ester bonds / hydrolase activity / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / nucleotide binding / DNA-templated transcription / RNA binding / metal ion binding
Similarity search - Function
RNA-directed RNA polymerase, orthobunyavirus / : / Virus, RNA-directed RNA polymerase L, thumb ring 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 speciesBunyavirus La Crosse / La Crosse orthobunyavirus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsArragain B / Effantin G / Gerlach P / Reguera J / Schoehn G / Cusack S / Malet H
Funding support France, 1 items
OrganizationGrant numberCountry
French National Research AgencyANR-19-CE11-0024-02 France
CitationJournal: Nat Commun / Year: 2020
Title: Pre-initiation and elongation structures of full-length La Crosse virus polymerase reveal functionally important conformational changes.
Authors: Benoît Arragain / Grégory Effantin / Piotr Gerlach / Juan Reguera / Guy Schoehn / Stephen Cusack / Hélène Malet /
Abstract: Bunyavirales is an order of segmented negative-strand RNA viruses comprising several life-threatening pathogens against which no effective treatment is currently available. Replication and ...Bunyavirales is an order of segmented negative-strand RNA viruses comprising several life-threatening pathogens against which no effective treatment is currently available. Replication and transcription of the RNA genome constitute essential processes performed by the virally encoded multi-domain RNA-dependent RNA polymerase. Here, we describe the complete high-resolution cryo-EM structure of La Crosse virus polymerase. It reveals the presence of key protruding C-terminal domains, notably the cap-binding domain, which undergoes large movements related to its role in transcription initiation, and a zinc-binding domain that displays a fold not previously observed. We capture the polymerase structure at pre-initiation and elongation states, uncovering the coordinated movement of the priming loop, mid-thumb ring linker and lid domain required for the establishment of a ten-base-pair template-product RNA duplex before strand separation into respective exit tunnels. These structural details and the observed dynamics of key functional elements will be instrumental for structure-based development of polymerase inhibitors.
History
DepositionMay 28, 2020-
Header (metadata) releaseJul 29, 2020-
Map releaseJul 29, 2020-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6z6g
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11093.png

Downloads & links

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Map

FileDownload / File: emd_11093.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLa Crosse full length polymerase at pre-initiation stage
Voxel sizeX=Y=Z: 0.826 Å
Density
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.013
Minimum - Maximum-0.038388956 - 0.08880791
Average (Standard dev.)0.00023802175 (±0.0025055835)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 247.79999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8260.8260.826
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z247.800247.800247.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0380.0890.000

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Supplemental data

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Sample components

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Entire : La Crosse virus polymerase at pre-initiation stage

EntireName: La Crosse virus polymerase at pre-initiation stage
Components
  • Complex: La Crosse virus polymerase at pre-initiation stage
    • Complex: La Crosse virus polymerase at pre-initiation stage
      • RNA: 5'vRNA 1-10
      • RNA: 5'vRNA 9-16
      • RNA: 3'vRNA 1-16
      • Protein or peptide: RNA-directed RNA polymerase L
    • Complex: La Crosse virus polymerase at pre-initiation stage
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: La Crosse virus polymerase at pre-initiation stage

SupramoleculeName: La Crosse virus polymerase at pre-initiation stage / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Molecular weightTheoretical: 265 KDa

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Supramolecule #2: La Crosse virus polymerase at pre-initiation stage

SupramoleculeName: La Crosse virus polymerase at pre-initiation stage / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#4
Source (natural)Organism: Bunyavirus La Crosse
Recombinant expressionOrganism: cabbage looper (cabbage looper)

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Supramolecule #3: La Crosse virus polymerase at pre-initiation stage

SupramoleculeName: La Crosse virus polymerase at pre-initiation stage / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1-#3
Source (natural)Organism: La Crosse orthobunyavirus
Recombinant expressionOrganism: synthetic construct (others)

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Macromolecule #1: 5'vRNA 1-10

MacromoleculeName: 5'vRNA 1-10 / type: rna / ID: 1 / Details: 5'vRNA 1-10 / Number of copies: 1
Source (natural)Organism: La Crosse orthobunyavirus
Molecular weightTheoretical: 3.217956 KDa
SequenceString:
AGUAGUGUGC

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Macromolecule #2: 5'vRNA 9-16

MacromoleculeName: 5'vRNA 9-16 / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: La Crosse orthobunyavirus
Molecular weightTheoretical: 2.509577 KDa
SequenceString:
GCUACCAA

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Macromolecule #3: 3'vRNA 1-16

MacromoleculeName: 3'vRNA 1-16 / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: La Crosse orthobunyavirus
Molecular weightTheoretical: 5.060023 KDa
SequenceString:
UUGGUAGUAC ACUACU

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Macromolecule #4: RNA-directed RNA polymerase L

MacromoleculeName: RNA-directed RNA polymerase L / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed RNA polymerase
Source (natural)Organism: Bunyavirus La Crosse
Molecular weightTheoretical: 266.047469 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGHHHHHHDY DIPTTENLYF QGMDYQEYQQ FLARINTARD ACVAKDIDVD LLMARHDYFG RELCKSLNIE YRNDVPFIDI ILDIRPEVD PLTIDAPHIT PDNYLYINNV LYIIDYKVSV SNESSVITYD KYYELTRDIS DRLSIPIEIV IIRIDPVSRD L HINSDRFK ...String:
MGHHHHHHDY DIPTTENLYF QGMDYQEYQQ FLARINTARD ACVAKDIDVD LLMARHDYFG RELCKSLNIE YRNDVPFIDI ILDIRPEVD PLTIDAPHIT PDNYLYINNV LYIIDYKVSV SNESSVITYD KYYELTRDIS DRLSIPIEIV IIRIDPVSRD L HINSDRFK ELYPTIVVDI NFNQFFDLKQ LLYEKFGDDE EFLLKVAHGD FTLTAPWCKT GCPEFWKHPI YKEFKMSMPV PE RRLFEES VKFNAYESER WNTNLVKIRE YTKKDYSEHI SKSAKNIFLA SGFYKQPNKN EISEGWTLMV ERVQDQREIS KSL HDQKPS IHFIWGAHNP GNSNNATFKL ILLSKSLQSI KGISTYTEAF KSLGKMMDIG DKAIEYEEFC MSLKSKARSS WKQI MNKKL EPKQINNALV LWEQQFMINN DLIDKSEKLK LFKNFCGIGK HKQFKNKMLE DLEVSKPKIL DFDDANMYLA SLTMM EQSK KILSKSNGLK PDNFILNEFG SRIKDANKET YDNMHKIFET GYWQCISDFS TLMKNILSVS QYNRHNTFRI AMCANN NVF AIVFPSADIK TKKATVVYSI IVLHKEEENI FNPGCLHGTF KCMNGYISIS RAIRLDKERC QRIVSSPGLF LTTCLLF KH DNPTLVMSDI MNFSIYTSLS ITKSVLSLTE PARYMIMNSL AISSNVKDYI AEKFSPYTKT LFSVYMTRLI KNACFDAY D QRQRVQLRDI YLSDYDITQK GIKDNRELTS IWFPGSVTLK EYLTQIYLPF YFNAKGLHEK HHVMVDLAKT ILEIECEQR ENIKEIWSTN CTKQTVNLKI LIHSLCKNLL ADTSRHNHLR NRIENRNNFR RSITTISTFT SSKSCLKIGD FRKEKELQSV KQKKILEVQ SRKMRLANPM FVTDEQVCLE VGHCNYEMLR NAMPNYTDYI STKVFDRLYE LLDKKVLTDK PVIEQIMDMM I DHKKFYFT FFNKGQKTSK DREIFVGEYE AKMCMYAVER IAKERCKLNP DEMISEPGDG KLKVLEQKSE QEIRFLVETT RQ KNREIDE AIEALATEGY ESNLGKIEKL SLGKAKGLKM EINADMSKWS AQDVFYKYFW LIALDPILYP QEKERILYFM CNY MDKELI LPDELLFNLL DQKVAYQNDI IATMTNQLNS NTVLIKRNWL QGNFNYTSSY VHSCAMSVYK EILKEAITLL DGSI LVNSL VHSDDNQTSI TIVQDKMEND KIIDFAMKEF ERACLTFGCQ ANMKKTYVTN CIKEFVSLFN LYGEPFSIYG RFLLT SVGD CAYIGPYEDL ASRISSAQTA IKHGCPPSLA WVSIAISHWM TSLTYNMLPG QSNDPIDYFP AENRKDIPIE LNGVLD APL SMISTVGLES GNLYFLIKLL SKYTPVMQKR ESVVNQIAEV KNWKVEDLTD NEIFRLKILR YLVLDAEMDP SDIMGET SD MRGRSILTPR KFTTAGSLRK LYSFSKYQDR LSSPGGMVEL FTYLLEKPEL LVTKGEDMKD YMESVIFRYN SKRFKESL S IQNPAQLFIE QILFSHKPVI DFSGIRDKYI NLHDSRALEK EPDILGKVTF TEAYRLLMRD LSSLELTNDD IQVIYSYII LNDPMMITIA NTHILSIYGS PQRRMGMSCS TMPEFRNLKL IHHSPALVLR AYSKNNPDIQ GADPTEMARD LVHLKEFVEN TNLEEKMKV RIAMNEAEKG QRDIVFELKE MTRFYQVCYE YVKSTEHKIK VFILPAKSYT TTDFCSLMQG NLIKDKEWYT V HYLKQILS GGHKAIMQHN ATSEQNIAFE CFKLITHFAD SFIDSLSRSA FLQLIIDEFS YKDVKVSKLY DIIKNGYNRT DF IPLLFRT GDLRQADLDK YDAMKSHERV TWNDWQTSRH LDMGSINLTI TGYNRSITII GEDNKLTYAE LCLTRKTPEN ITI SGRKLL GSRHGLKFEN MSKIQTYPGN YYITYRKKDR HQFVYQIHSH ESITRRNEEH MAIRTRIYNE ITPVCVVNVA EVDG DQRIL IRSLDYLNND IFSLSRIKVG LDEFATIKKA HFSKMVSFEG PPIKTGLLDL TELMKSQDLL NLNYDNIRNS NLISF SKLI CCEGSDNIND GLEFLSDDPM NFTEGEAIHS TPIFNIYYSK RGERHMTYRN AIKLLIERET KIFEEAFTFS ENGFIS PEN LGCLEAVVSL IKLLKTNEWS TVIDKCIHIC LIKNGMDHMY HSFDVPKCFM GNPITRDINW VMFREFINSL PGTDIPP WN VMTENFKKKC IALINSKFET QRDFSEFTKL MKKEGGRSNI EFD

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / 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

Concentration0.2 mg/mL
BufferpH: 8
Component:
ConcentrationFormula
20.0 mMTris-HClTris
150.0 mMNaClSodium chloride
2.0 mMTCEP

Details: 20 mM Tris-HCl pH 8, 150 mM NaCl, 2 mM TCEP
GridModel: UltrAuFoil / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Details: 30mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV / Details: blot time: 2 sec, blot force: 1.

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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: -3.5 µm / Nominal defocus min: -0.8 µm / Nominal magnification: 165000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 90.0 K / Max: 90.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 2 / Number real images: 16498 / Average electron dose: 1.25 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4065475
CTF correctionSoftware - Name: cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationNumber classes: 10 / Avg.num./class: 37000 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 57660
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5amq


Chain - Chain ID: A
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-6z6g:
Cryo-EM structure of La Crosse virus polymerase at pre-initiation stage

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