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- EMDB-0706: Structure of apo Lassa virus polymerase -

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Basic information

Entry
Database: EMDB / ID: EMD-0706
TitleStructure of apo Lassa virus polymerase
Map dataapo lassa virus polymerase
Sample
  • Complex: Lassa virus polymerase
    • Protein or peptide: RNA-directed RNA polymerase L
  • Ligand: MANGANESE (II) ION
Keywordspolymerase / RNA virus / 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: 3.9 Å
AuthorsPeng R / Xu X
CitationJournal: Nature / Year: 2020
Title: Structural insight into arenavirus replication machinery.
Authors: Ruchao Peng / Xin Xu / Jiamei Jing / Min Wang / Qi Peng / Sheng Liu / Ying Wu / Xichen Bao / Peiyi Wang / Jianxun Qi / George F Gao / Yi Shi /
Abstract: Arenaviruses can cause severe haemorrhagic fever and neurological diseases in humans and other animals, exemplified by Lassa mammarenavirus, Machupo mammarenavirus and lymphocytic choriomeningitis ...Arenaviruses can cause severe haemorrhagic fever and neurological diseases in humans and other animals, exemplified by Lassa mammarenavirus, Machupo mammarenavirus and lymphocytic choriomeningitis virus, posing great threats to public health. These viruses encode a large multi-domain RNA-dependent RNA polymerase for transcription and replication of the viral genome. Viral polymerases are one of the leading antiviral therapeutic targets. However, the structure of arenavirus polymerase is not yet known. Here we report the near-atomic resolution structures of Lassa and Machupo virus polymerases in both apo and promoter-bound forms. These structures display a similar overall architecture to influenza virus and bunyavirus polymerases but possess unique local features, including an arenavirus-specific insertion domain that regulates the polymerase activity. Notably, the ordered active site of arenavirus polymerase is inherently switched on, without the requirement for allosteric activation by 5'-viral RNA, which is a necessity for both influenza virus and bunyavirus polymerases. Moreover, dimerization could facilitate the polymerase activity. These findings advance our understanding of the mechanism of arenavirus replication and provide an important basis for developing antiviral therapeutics.
History
DepositionJul 30, 2019-
Header (metadata) releaseMar 18, 2020-
Map releaseMar 18, 2020-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6klc
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0706.png

Downloads & links

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Map

FileDownload / File: emd_0706.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationapo lassa virus polymerase
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.36 Å/pix.
x 160 pix.
= 217.6 Å		1.36 Å/pix.
x 160 pix.
= 217.6 Å		1.36 Å/pix.
x 160 pix.
= 217.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.36 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.05
Minimum - Maximum-0.25354466 - 0.39067975
Average (Standard dev.)0.0008273634 (±0.011564622)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 217.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.361.361.36
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z217.600217.600217.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.2540.3910.001

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Supplemental data

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Sample components

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Entire : Lassa virus polymerase

EntireName: Lassa virus polymerase
Components
  • Complex: Lassa virus polymerase
    • Protein or peptide: RNA-directed RNA polymerase L
  • Ligand: MANGANESE (II) ION

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Supramolecule #1: Lassa virus polymerase

SupramoleculeName: Lassa virus polymerase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Lassa mammarenavirus
Molecular weightTheoretical: 250 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: 253.505828 KDa
Recombinant expressionOrganism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
SequenceString: MEEDIACVKD LVSKYLANNE RLSRQKLAFL VQTEPRMLLM EGLKLLSLCI EVDSCNANGC EHNSEDKSVE RILHDHGVLT PSLCFVVPD GYKLTGNVLI LLECFVRSSP ANFEQKYVED FKKLEQLKED LKSVDINLIP LIDGRTSFYN EQIPDWVNDK L RDTLFSLL ...String:
MEEDIACVKD LVSKYLANNE RLSRQKLAFL VQTEPRMLLM EGLKLLSLCI EVDSCNANGC EHNSEDKSVE RILHDHGVLT PSLCFVVPD GYKLTGNVLI LLECFVRSSP ANFEQKYVED FKKLEQLKED LKSVDINLIP LIDGRTSFYN EQIPDWVNDK L RDTLFSLL KYAQESNSLF EESEYSRLCE SLSMTSGRLS GVESLNALLD NRSNHYEEVI ASCHQGINNK LTAHEVKLQI EE EYQVFRN RLRKGEIEGQ FLKVEKNQLL NEFNNLYADK VAEKDSVEHL THQFKRASPI LRFLYANISK GDNGEGNLII GEC QMQCWR SFLNKVKSMR ILNTRRKLLL IFDALILLAS KHDQVRKKPL RGWLGTCFVS VNDRLVSLES TKKDLKKWVE RRQQ VERSR TMQSFQCPSK NQILNSIFQK TISKATTALR DVGISVDHYK IDMEVICPDG YDLIMDFDVS GVTPTISYQR SEEEA FPYI MGDVDLLKTT DLERLSSLSL ALVNSMKTSS TVKLRQNEFG PARYQVVKCK EAYCQEFSLG ETEFQLIYQK TGECSK CYA INDNRVGEVC SFYADPKRYF PAIFSAEVLQ TTVSTMISWI EDCNELEEQL DKIRSLTKMI LILILAHPSK RSQKLLQ NL RYFIMAYVSD YYHKDLIDKV REELITDVEF LLYRLLRTLM GLVLSEDVKS MMTNRFKFIL NISYMCHFIT KETPDRLT D QIKCFEKFLE PKVKFGHVSI NPADTATEEE LDDMVYNAKK FLSKGGCTSA KGPSYKKPGV SKKYLSLLTS SFNNGSLFK EREVKKEIKD PLITSGCATA LDLASNKSVV VNKYTDGSRV LNYDFNKLTA LAVSQLTEVF SRKGKHLLNK QDYEYKVQQA MSNLVLGSK QHKGDADEAD LDEILLDGGA STYFNQLKET VEKIVDQYRE PVKMGSGSND GDQPSINDLD EIVSNKFYIR L IKGELSNH MVEDFDHDVL PDKFYEEFCD AVYENSKLKE KYFYCGHMSQ CPIGELTKAV STRTYLDHEY FQCFKSILLI MN ANALMGK YTHYKSRNLN FKFDMGKLSD DARISERESN SEALSKALSL TNCTTAMLKN LCFYSQESPQ SYNSVGPDTG RLK FSLSYK EQVGGNRELY IGDLRTKMFT RLIEDYFEAL SSQLSGSCLN NEKEFENAIL SMKLNVSMAH VSYSMDHSKW GPMM CPFLF LTVLQNLIFL SKDLQADIKG RDYLSTLLMW HMHKMVEIPF NVVSAMMKSF IKAQLGLRKK TKQSITEDFF YSNFQ IGVV PSHISSILDM GQGILHNTSD FYALITERFI NYAISCVCGG TIDAYTSSDD QISLFDQTLT ELLHRDPEEF RALMEF HYY MSDQLNKFVS PKSVIGRFVA EFKSRFFVWG DEVPLLTKFV AAALHNIKCK EPHQLAETID TIVDQSVANG VPVHLCN LI QIRTLSLLQY ARYPIDPFLL NCETDVRDWV DGNRSYRIMR QIEGLIPDAC SKIRSMLRRL YNRLKTGQLH EEFTTNYL S SEHLSSLKNL CELLGVEPPS ESDLEYSWLN LAAHHPLRMV LRQKIIYSGA VNLDDEKIPT IVKTIQNKLS STFTRGAQK LLSEAINKSA FQSSIASGFV GLCRTLGSKC VRGPNKENLY IKSIQSLITG TQGIELLTNS IGVQYWRVPL GLRNKSESVV SYFRPLLWD YMCISLSTAI ELGAWVLGDP KTTKALDFFK HNPCDYFPLK PTASKLLEDR VGLNHIIHSL RRLYPSVFEK H ILPFMSDL ASTKMKWSPR IKFLDLCVAL DVNCEALSLV SHIVKWKREE HYIVLSSELR FSHTRTHEPM VEERVVSTSD AV DNFMRQI YFESYVRPFV ATTRTLGSFT WFPHRTSIPE GEGLHRLGPF SSFVEKVIHK GVERPMFKHD LMMGYAWIDF DIE PARFNQ NQLIASGLVD SKFDSLEDFF DAVASLPTGS AKLSQTVRFR IKSQDASFRE SFAIHLDYIG SMNQQAKYLV HDVT AMYSG AVSPCVLSDC WRLVLSGPTF KGKPVWYVDT EVINEFLVDT NQLGHVTPVE VVVDMEKLQF AEYDFMLVGP CAEPV PLVV RRGGLWECEK KLASFTPVIQ DQDLEMFVRE VGDTSSDLLI RALSDMITDR LGLRMQWSGV DIVSTLRAAA PGNAEV LSA VLEVVDNWVE FKGYALCYSK SRGRVMVQSS SGKLRLKGRT CEELTEGGEH VEDIE

UniProtKB: RNA-directed RNA polymerase L

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Macromolecule #2: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: MN
Molecular weightTheoretical: 54.938 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

Concentration1.0 mg/mL
BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1500000
Details: approximately 1,500,000 particles were autopicked from ~2700 micrographs
Startup modelType of model: EMDB MAP
EMDB ID:

2930

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 295000
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final 3D classificationSoftware - Name: RELION (ver. 2.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Target criteria: Correlation coefficient
Output model

PDB-6klc:
Structure of apo Lassa virus polymerase

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