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- EMDB-12953: Lassa virus L protein with endonuclease and C-terminal domains in... -

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Basic information

Entry
Database: EMDB / ID: EMD-12953
TitleLassa virus L protein with endonuclease and C-terminal domains in close proximity [MID-LINK]
Map dataLassa virus L protein with endonuclease and C-terminal domains in close proximity [MID-LINK]
Sample
  • Complex: RNA-directed RNA polymerase L Lassa mammarenavirus
    • Protein or peptide: RNA-directed RNA polymerase L
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
KeywordsLassa virus RNA-dependent RNA polymerase viral RNA / VIRAL PROTEIN
Function / homology
Function and homology information


negative stranded viral RNA replication / cap snatching / virion component / host cell cytoplasm / Hydrolases; Acting on ester bonds / hydrolase activity / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / nucleotide binding / metal ion binding
Similarity search - Function
RNA polymerase, arenaviral / RNA endonuclease, cap-snatching / Arenavirus RNA polymerase / Arenavirus cap snatching domain / : / 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 speciesLassa mammarenavirus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsKouba T / Vogel D
Funding support Germany, 2 items
OrganizationGrant numberCountry
Leibniz AssociationK27/2017 Germany
German Research Foundation (DFG)INST 152/777-1 FUGG Germany
CitationJournal: Nat Commun / Year: 2021
Title: Conformational changes in Lassa virus L protein associated with promoter binding and RNA synthesis activity.
Authors: Tomas Kouba / Dominik Vogel / Sigurdur R Thorkelsson / Emmanuelle R J Quemin / Harry M Williams / Morlin Milewski / Carola Busch / Stephan Günther / Kay Grünewald / Maria Rosenthal / Stephen Cusack /
Abstract: Lassa virus is endemic in West Africa and can cause severe hemorrhagic fever. The viral L protein transcribes and replicates the RNA genome via its RNA-dependent RNA polymerase activity. Here, we ...Lassa virus is endemic in West Africa and can cause severe hemorrhagic fever. The viral L protein transcribes and replicates the RNA genome via its RNA-dependent RNA polymerase activity. Here, we present nine cryo-EM structures of the L protein in the apo-, promoter-bound pre-initiation and active RNA synthesis states. We characterize distinct binding pockets for the conserved 3' and 5' promoter RNAs and show how full-promoter binding induces a distinct pre-initiation conformation. In the apo- and early elongation states, the endonuclease is inhibited by two distinct L protein peptides, whereas in the pre-initiation state it is uninhibited. In the early elongation state, a template-product duplex is bound in the active site cavity together with an incoming non-hydrolysable nucleotide and the full C-terminal region of the L protein, including the putative cap-binding domain, is well-ordered. These data advance our mechanistic understanding of how this flexible and multifunctional molecular machine is activated.
History
DepositionMay 16, 2021-
Header (metadata) releaseDec 1, 2021-
Map releaseDec 1, 2021-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.13
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.13
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ojj
  • Surface level: 0.13
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12953.png

Downloads & links

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Map

FileDownload / File: emd_12953.map.gz / Format: CCP4 / Size: 386 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLassa virus L protein with endonuclease and C-terminal domains in close proximity [MID-LINK]
Voxel sizeX=Y=Z: 0.87 Å
Density
Contour LevelBy AUTHOR: 0.13 / Movie #1: 0.13
Minimum - Maximum-0.17441256 - 0.53573984
Average (Standard dev.)0.0005637909 (±0.009890251)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions466466466
Spacing466466466
CellA=B=C: 405.42 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.870.870.87
M x/y/z466466466
origin x/y/z0.0000.0000.000
length x/y/z405.420405.420405.420
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ384384384
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS466466466
D min/max/mean-0.1740.5360.001

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Supplemental data

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Additional map: #1

Fileemd_12953_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_12953_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_12953_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 : RNA-directed RNA polymerase L Lassa mammarenavirus

EntireName: RNA-directed RNA polymerase L Lassa mammarenavirus
Components
  • Complex: RNA-directed RNA polymerase L Lassa mammarenavirus
    • Protein or peptide: RNA-directed RNA polymerase L
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: RNA-directed RNA polymerase L Lassa mammarenavirus

SupramoleculeName: RNA-directed RNA polymerase L Lassa mammarenavirus / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Lassa mammarenavirus
Molecular weightTheoretical: 253.346 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 / EC number: RNA-directed RNA polymerase
Source (natural)Organism: Lassa mammarenavirus
Molecular weightTheoretical: 253.656938 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MEDDMACVKD LVSKYLADNE RLSRQKLAFL VQTEPRMLLM EGLKLLSLCI EIDSCNANGC EHNSEDKSVE RILHDHGILT PSLCFVVPD GYKLTGNVLI LLECFVRSSP ANFEQKYIED FKKLEQLKED LKSVDINLIP LIDGRTSFYN EQIPDWVNDK L RDTLFSLL ...String:
MEDDMACVKD LVSKYLADNE RLSRQKLAFL VQTEPRMLLM EGLKLLSLCI EIDSCNANGC EHNSEDKSVE RILHDHGILT PSLCFVVPD GYKLTGNVLI LLECFVRSSP ANFEQKYIED FKKLEQLKED LKSVDINLIP LIDGRTSFYN EQIPDWVNDK L RDTLFSLL RYAQESNSLF EESEYSRLCE SLSMTSGRLS GVESLNVLLD NRSSHYEEII ASCHQGINNK LTAHEVKLQI EE EYQVFRN RLRKGEITGQ FLKVDKSRLL NDFNNLYVDE VTATKDNIEH LIYQFKRASP ILRFLYANIG EGNGEERHHT IKE CQMQYW RSFLNKVKSL RILNTRRKLL LIFDALILLA SIHDQTRHKC SKGWLGSCFI SVNDRLVSLE STKRDLEKWV GRRQ QSERS NTIQPPDKNQ ILISMFQKTI LKATAALKDV GISVEHYKIN MEVICPDSYD LILNFDVSGV VPTISYQRTE DEKFP FIMG GVELLESTDL ERLSSLSLAL VNSMKTSSTV KLRQNEFGPA RYQVVRCKEA YCQEFLLSGA EFQLIYQKTG ECSKCY AIN DNRVGEICSF YADPKRYFPA IFSAEVLQTT VSTMISWVKD CSELEEQLCN INSLTKMILV LILAHPSKRS QKLLQNL RY FIMAYVSDYH HKDLIDKLRE ELITDVEFLL YRLVRALVNL ILSEDVKSMM TNRFKFILNI SYMCHFITKE TPDRLTDQ I KCFEKFLEPK LEFGHVSINP ADVATEEELD DMVYNAKKFL SKEGCTSIKG PDYKKPGVSK RFLSLLTSSF NNGSLFKES EVKREIKDPL VTSGCATALD LASNKSVVVN KYTDGSRVLN YDFNKLTALA VSQLTEVFSR KGKHLLNKQD YDYKVQQAMS NLVLGPRQN KVGADEADLD EILLDGGASV YFDQLKETVE RIIDQYREPV KPGSNPNGGD QPSVNDLDEV VPNKFYIRLI K GELSNHMV EDFDYDVLPG NFYEEFCDAV YKNNKLKERY FYCGQMSQCP IGELTKAVAT RTYFDQEYFQ CFKSILLIMN AN TLMGRYT HYKSRNLNFK FDMGRLSDDV RISERESNSE ALSKALSLTN CTTAMLKNLC FYSQESPQSY SSTGPDTGRL KFS LSYKEQ VGGNRELYIG DLRTKMFTRL IEDYFEALSL QLSGSCLNNE REFENAILSM KLNVSLAHVS YSMDHSKWGP MMCP FLFLA TLQNLIFLSK DLQADIKGRD YLSTLLTWHM HKMVEIPFNV VSAMMKSFIK AQLGLKKKTT QSITEDFFYS NFQIG VVPS HVSSILDMGQ GILHNTSDFY ALISERFINY AISCICGGTI DAYTSSDDQI SLFDQVLTEL MQRDPEEFKT LIEFHY YMS DQLNKFVSPK SVIGRFVAEF KSRFYVWGDE VPLLTKFVAA ALHNIKCKEP HQLAETIDTI IDQSVANGVP VHLCNLI QK RTLSLLQYAR YPIDPFLLNC ETDVRDWVDG NRSYRIMRQI ERLIPDACGR IRSMLRKLYN KLKTGQLHEE FTTNYLSS E HLSSLSNLCE LLGVEPPSES DLEFSWLNLA AHHPLRMVLR QKIIYSGAVN LDDEKVPTIV KTIQNKLSST FTRGAQKLL SEAINKSAFQ SSIASGFVGL CRTLGSKCVR GPNKESLYIK SIQSLISDIQ GIEPLIDSHG VQYWRVPLNI RDGNEGVISY FRPLLWDYM CISLSTAIEL GAWVLGEPKK VRVLEFFKHN PCDYFPLKPA ASKLLEDRVG LNHIIHSLRR LYPSVFEKHI L PFMSDLAS TKMKWSPRIK FLDLCVALDV NCEALSLVSH IVKWKREEHY IVLSSELRLS HTRTHEPMVE ERVVSTSDAV DN FMRQIYF ESYVRSFVAT TRTLGSFTWF PHKTSVPEGE GLQRLGPFSS FVEKAIHKGI ERPMFKHDLM MGYAWIDFDI EPA RFNHNQ LIASGLVGPR FDSLEDFFDA VESLPPGSAK LSQTVRFRIK SQDASFKESF AIHLDYTGSI NQQTKYLVHE VSAM YSGAV SPCVLSDCWR LVLSGPTFKG KSAWYVDTEI VNEFLTDTNQ LGHVTPVEIV VDMEKLQFTE YDFVLVGPCV EPVPL VVHR GGLWECDKKL ASFTPVVQDQ DLEMFVKEVG DSSLDLLIGA LSAMILDRLK LRMQWSEVDI VSMLKAAMPS NSVKVL NAV LEAVDDWVDF KGYALCYSKS RKKVMVHSSG GKLRLKGRTC EELVKEDEGI EDIE

UniProtKB: RNA-directed RNA polymerase L

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Macromolecule #2: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / 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

BufferpH: 7
VitrificationCryogen name: ETHANE

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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
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 49.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 47000
FSC plot (resolution estimation)

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