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- EMDB-11107: Masked cryo-EM map of La Crosse virus polymerase zinc-binding dom... -

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Basic information

Entry
Database: EMDB / ID: EMD-11107
TitleMasked cryo-EM map of La Crosse virus polymerase zinc-binding domain and mid domain
Map dataMasked cryo-EM map of La Crosse virus polymerase zinc-binding domain and mid domain
Sample
  • Complex: La Crosse virus polymerase mid domain and zinc-binding domain
    • Protein or peptide: La Crosse virus polymerase (expression and purification: full length polymerase, masked map: zinc-binding domain and mid domain only)
Biological speciesLa Crosse orthobunyavirus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.49 Å
AuthorsArragain B / Effantin G / 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 29, 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: 0.016
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.016
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11107.png

Downloads & links

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Map

FileDownload / File: emd_11107.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMasked cryo-EM map of La Crosse virus polymerase zinc-binding domain and mid domain
Voxel sizeX=Y=Z: 0.826 Å
Density
Contour LevelBy AUTHOR: 0.016 / Movie #1: 0.016
Minimum - Maximum-0.037164014 - 0.07140159
Average (Standard dev.)0.0000521846 (±0.0011921142)
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-0.0370.0710.000

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Supplemental data

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Sample components

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Entire : La Crosse virus polymerase mid domain and zinc-binding domain

EntireName: La Crosse virus polymerase mid domain and zinc-binding domain
Components
  • Complex: La Crosse virus polymerase mid domain and zinc-binding domain
    • Protein or peptide: La Crosse virus polymerase (expression and purification: full length polymerase, masked map: zinc-binding domain and mid domain only)

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Supramolecule #1: La Crosse virus polymerase mid domain and zinc-binding domain

SupramoleculeName: La Crosse virus polymerase mid domain and zinc-binding domain
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Masked cryo-EM map of La Crosse virus full-length polymerase, displaying only the mid domain and the zinc-binding domain. Masking, 3D classification and refinement enable to obtain a ...Details: Masked cryo-EM map of La Crosse virus full-length polymerase, displaying only the mid domain and the zinc-binding domain. Masking, 3D classification and refinement enable to obtain a structure of the zinc-binding domain.
Molecular weightTheoretical: 265 KDa

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Macromolecule #1: La Crosse virus polymerase (expression and purification: full len...

MacromoleculeName: La Crosse virus polymerase (expression and purification: full length polymerase, masked map: zinc-binding domain and mid domain only)
type: protein_or_peptide / ID: 1
Details: La Crosse virus polymerase expression and purification: full length polymerase with an uncleaved N-terminal His-tag masked map: zinc-binding domain and mid domain only (residues 1752-1841 and 1978-2263)
Enantiomer: LEVO / EC number: RNA-directed RNA polymerase
Source (natural)Organism: La Crosse orthobunyavirus
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGHHHHHHDY DIPTTENLYF QGMDYQEYQQ FLARINTARD ACVAKDIDVD LLMARHDYFG RELCKSLNIE YRNDVPFIDI ILDIRPEVDP LTIDAPHITP DNYLYINNVL YIIDYKVSVS NESSVITYDK YYELTRDISD RLSIPIEIVI IRIDPVSRDL HINSDRFKEL ...String:
MGHHHHHHDY DIPTTENLYF QGMDYQEYQQ FLARINTARD ACVAKDIDVD LLMARHDYFG RELCKSLNIE YRNDVPFIDI ILDIRPEVDP LTIDAPHITP DNYLYINNVL YIIDYKVSVS NESSVITYDK YYELTRDISD RLSIPIEIVI IRIDPVSRDL HINSDRFKEL YPTIVVDINF NQFFDLKQLL YEKFGDDEEF LLKVAHGDFT LTAPWCKTGC PEFWKHPIYK EFKMSMPVPE RRLFEESVKF NAYESERWNT NLVKIREYTK KDYSEHISKS AKNIFLASGF YKQPNKNEIS EGWTLMVERV QDQREISKSL HDQKPSIHFI WGAHNPGNSN NATFKLILLS KSLQSIKGIS TYTEAFKSLG KMMDIGDKAI EYEEFCMSLK SKARSSWKQI MNKKLEPKQI NNALVLWEQQ FMINNDLIDK SEKLKLFKNF CGIGKHKQFK NKMLEDLEVS KPKILDFDDA NMYLASLTMM EQSKKILSKS NGLKPDNFIL NEFGSRIKDA NKETYDNMHK IFETGYWQCI SDFSTLMKNI LSVSQYNRHN TFRIAMCANN NVFAIVFPSA DIKTKKATVV YSIIVLHKEE ENIFNPGCLH GTFKCMNGYI SISRAIRLDK ERCQRIVSSP GLFLTTCLLF KHDNPTLVMS DIMNFSIYTS LSITKSVLSL TEPARYMIMN SLAISSNVKD YIAEKFSPYT KTLFSVYMTR LIKNACFDAY DQRQRVQLRD IYLSDYDITQ KGIKDNRELT SIWFPGSVTL KEYLTQIYLP FYFNAKGLHE KHHVMVDLAK TILEIECEQR ENIKEIWSTN CTKQTVNLKI LIHSLCKNLL ADTSRHNHLR NRIENRNNFR RSITTISTFT SSKSCLKIGD FRKEKELQSV KQKKILEVQS RKMRLANPMF VTDEQVCLEV GHCNYEMLRN AMPNYTDYIS TKVFDRLYEL LDKKVLTDKP VIEQIMDMMI DHKKFYFTFF NKGQKTSKDR EIFVGEYEAK MCMYAVERIA KERCKLNPDE MISEPGDGKL KVLEQKSEQE IRFLVETTRQ KNREIDEAIE ALATEGYESN LGKIEKLSLG KAKGLKMEIN ADMSKWSAQD VFYKYFWLIA LDPILYPQEK ERILYFMCNY MDKELILPDE LLFNLLDQKV AYQNDIIATM TNQLNSNTVL IKRNWLQGNF NYTSSYVHSC AMSVYKEILK EAITLLDGSI LVNSLVHSDD NQTSITIVQD KMENDKIIDF AMKEFERACL TFGCQANMKK TYVTNCIKEF VSLFNLYGEP FSIYGRFLLT SVGDCAYIGP YEDLASRISS AQTAIKHGCP PSLAWVSIAI SHWMTSLTYN MLPGQSNDPI DYFPAENRKD IPIELNGVLD APLSMISTVG LESGNLYFLI KLLSKYTPVM QKRESVVNQI AEVKNWKVED LTDNEIFRLK ILRYLVLDAE MDPSDIMGET SDMRGRSILT PRKFTTAGSL RKLYSFSKYQ DRLSSPGGMV ELFTYLLEKP ELLVTKGEDM KDYMESVIFR YNSKRFKESL SIQNPAQLFI EQILFSHKPV IDFSGIRDKY INLHDSRALE KEPDILGKVT FTEAYRLLMR DLSSLELTND DIQVIYSYII LNDPMMITIA NTHILSIYGS PQRRMGMSCS TMPEFRNLKL IHHSPALVLR AYSKNNPDIQ GADPTEMARD LVHLKEFVEN TNLEEKMKVR IAMNEAEKGQ RDIVFELKEM TRFYQVCYEY VKSTEHKIKV FILPAKSYTT TDFCSLMQGN LIKDKEWYTV HYLKQILSGG HKAIMQHNAT SEQNIAFECF KLITHFADSF IDSLSRSAFL QLIIDEFSYK DVKVSKLYDI IKNGYNRTDF IPLLFRTGDL RQADLDKYDA MKSHERVTWN DWQTSRHLDM GSINLTITGY NRSITIIGED NKLTYAELCL TRKTPENITI SGRKLLGSRH GLKFENMSKI QTYPGNYYIT YRKKDRHQFV YQIHSHESIT RRNEEHMAIR TRIYNEITPV CVVNVAEVDG DQRILIRSLD YLNNDIFSLS RIKVGLDEFA TIKKAHFSKM VSFEGPPIKT GLLDLTELMK SQDLLNLNYD NIRNSNLISF SKLICCEGSD NINDGLEFLS DDPMNFTEGE AIHSTPIFNI YYSKRGERHM TYRNAIKLLI ERETKIFEEA FTFSENGFIS PENLGCLEAV VSLIKLLKTN EWSTVIDKCI HICLIKNGMD HMYHSFDVPK CFMGNPITRD INWVMFREFI NSLPGTDIPP WNVMTENFKK KCIALINSKF ETQRDFSEFT KLMKKEGGRS NIEFD

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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: 30 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 90 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)
Details: Masked 3D classification with the mid domain and the zinc-binding domain
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: 3.49 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 51842
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 84.74 / Target criteria: Cross-correlation

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