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- EMDB-0708: Apo Machupo virus polymerase homodimer -

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Basic information

Entry
Database: EMDB / ID: EMD-0708
TitleApo Machupo virus polymerase homodimer
Map dataapo Machupo virus polymerase homodimer
Sample
  • Complex: Machupo virus polymerase
    • Protein or peptide: Machupo virus polymerase
Biological speciesMachupo mammarenavirus
Methodsingle particle reconstruction / cryo EM / Resolution: 7.4 Å
AuthorsPeng R / Xu X / Jing J / Peng Q / Gao GF / Shi Y
Funding support China, 1 items
OrganizationGrant numberCountry
National Science Foundation (China)81622031 China
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-
UpdateApr 8, 2020-
Current statusApr 8, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.09
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.09
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0708.png

Downloads & links

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Map

FileDownload / File: emd_0708.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationapo Machupo virus polymerase homodimer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.72 Å/pix.
x 150 pix.
= 408. Å		2.72 Å/pix.
x 150 pix.
= 408. Å		2.72 Å/pix.
x 150 pix.
= 408. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.72 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.09 / Movie #1: 0.09
Minimum - Maximum-0.21302661 - 0.5085232
Average (Standard dev.)0.0011143158 (±0.014782983)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions150150150
Spacing150150150
CellA=B=C: 408.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.722.722.72
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z408.000408.000408.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS150150150
D min/max/mean-0.2130.5090.001

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Supplemental data

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Sample components

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Entire : Machupo virus polymerase

EntireName: Machupo virus polymerase
Components
  • Complex: Machupo virus polymerase
    • Protein or peptide: Machupo virus polymerase

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Supramolecule #1: Machupo virus polymerase

SupramoleculeName: Machupo virus polymerase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Machupo mammarenavirus
Recombinant expressionOrganism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
Recombinant cell: HiFive
Molecular weightExperimental: 500 KDa

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Macromolecule #1: Machupo virus polymerase

MacromoleculeName: Machupo virus polymerase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Machupo mammarenavirus
Recombinant expressionOrganism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
SequenceString: MDEYVQELKG LIRKHIPERC EFGHQKVTFL SQVHPSPLLT EGFKLLSSLV ELESCEAHAC QANTDQRFVD VILSDNGILC PTLPKVIPDG FKLTGKTLIL LETFVRVNPD EFEKKWKADM SKLLNLKHDL QKSGVTLVPI VDGRSNYNNR FVADWVIERI RWLLIEILKA ...String:
MDEYVQELKG LIRKHIPERC EFGHQKVTFL SQVHPSPLLT EGFKLLSSLV ELESCEAHAC QANTDQRFVD VILSDNGILC PTLPKVIPDG FKLTGKTLIL LETFVRVNPD EFEKKWKADM SKLLNLKHDL QKSGVTLVPI VDGRSNYNNR FVADWVIERI RWLLIEILKA SKSMLEIDIE DQEYQRLIHS LSNVKNQSLG LENLEHLKRN SLDYDERLNE SLFIGLKGDI RESTVREELI KLKLWFKDEV FSKGLGKFKL TDRRELLESL SSLGAHLDSD VSSCPFCNNK LMEIVYNVTF SCVERTDGVA TVDQQFSTTH SNIEKHYLSV LSLCNKIKGL KVFNTRRNTL LFLDLIMVNL MVDISDSCQD AIESLRKSGL IVGQMVMLVN DRVLDILEAV KLIRKKIGTN PNWVKNCSKI LERSHPEIWH HLSTLIKQPD FNSLISIAQH LVSDRPIMRY SVERGSDKIC RHKLFQEMSS FEQMRLFKTL SSISLSLINS MKTSFSSRLL VNEREFSKYF GNVRLRECYA QRFYLAESLV GFLFYQKTGE RSRCYSVYLS DNGVMSEQGS FYCDPKRFFL PVFSDEVLAG MCEEMTSWLD FDTGLMNDTG PILRLLVLAI LCSPSKRNQT FLQGLRYFLM AFANQIHHID LTSKLVVECK SSSEVVVQRL AVGLFIRLLS GESDASLFFS RRFKYLLNVS YLCHLITKET PDRLTDQIKC FEKFIEPKVK FGCAVVNPSL NGKLTVDQED IMINGLKKFF SKSLRDTEDV QTPGVCKELL NYCVSLFNRG KLKVSGELKN NPFRPNITST ALDLSSNKSV VIPKLDELGN ILSTYDKEKL VSACVSSMAE RFKTKGRYNL DPDSTDYLIL KNLTGLVSAG PKAKSTQEEL SLMYEALTEE QVESFNEIKH DVQVALAKMA DNSVNTRTKN LGRADNSVKN GNNPLDNLWS PFGVMKEIRA EVSLHEVKDF DPDVLPPEVY KELCDAVYKS SEKCNFFLEG VLDVCPLGLL LKNLTTSSYV DEEYFMCFKY LLIQGHFDQK LGSYEHKSRS RLGFTDETLR LKDEVRLSIR ESNSEAIADK LDKSYFTNAA LRNLCFYSED SPTEFTSISS NSGNLKFGLS YKEQVGSNRE LYVGDLNTKL MTRLVEDFSE AVGNSMKYTC LNSEKEFERA ICDMKMAVNN GDLSCSYDHS KWGPTMSPAL FLALLQMLEL RTPVDRSKID LDSVKSILKW HLHKVVEVPI NVAEAYCIGK LKRSLGLMGC GSTSLSEEFF HQTMQLNGQI PSHIMSVLDM GQGILHNTSD LYGLITEQFL CYALDLLYDV IPVSYTSSDD QITLIKTPSL DIEGGSDAAE WLEMICFHEF LSSKLNKFVS PKSVIGTFVA EFKSRFFVMG EETPLLTKFV AAALHNVKCK TPTQLSETID TICDQCIANG VSTKIVTRIS KRVNQLIRYS GYGETPFGAI EDQDVKDWVD GSRGYRLQRK IEAIFHDDKE TSFIRNCARK VFNDIKRGRI FEENLINLIG RGGDEALTGF LQYAGCSEQE VNRVLNYRWV NLSSFGDLRL VLRTKLMTSR RVLEREEVPT LIKTLQSKLS RNFTKGVKKI LAESINKSAF QSSVASGFIG FCKSMGSKCV RDGKGGFLYI KEVYSGVSAC TCEICALKPK IIYCNNSLNK VSQFSKPILW DYFSLVLTNA CELGEWVFST VKEPQKPLVL NNQNFFWAVK PKVVRQIEDQ LGMNHVLQSI RRNYPVLFDE HLTPFMNDLQ VSRTMDSGRL KFLDVCIALD MMNENLGIIS HLLKTRDNSV YIVKQSDCAL AHIRQSSYTD WELGLSPQQI CTNFKTQLVL SSMVNPLVLS TSCLKSFFWF NEVLELEDDS QIELAELTDF ALMVKNQNVS RAMFVEDIAM GYVVSNFEGV RISLSNVMVD GVQLPPQEKA PDIGELFGLK AENVIVGLVV QIDHVRMSTK FKLKRKMVYS FSLECIMDVG EIQNKEVILK VVAVDQSVSG SGGNHMLLDG VSVVASLPLF TGQASFDLAA MLIESNLAGS NDNFLMRNVT LDLGGFSPEL SDKYSYRLSG PENQEDPLVL KDGAFYVGGE RLSTYKVEFT GDLVVKALGA LEDDESVVSM LHQLWPYLKA TSQVILFQQE DFTIVHDLYK KQLTKSIESF GEWIEFTNFK VAYSKSLKEL VISDTQGSFR LKGVMCRPLA STPQVEDIE

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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 - 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: 1000000
CTF correctionSoftware - Name: Gctf (ver. 0.50)
Startup modelType of model: EMDB MAP
EMDB ID:

0707

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 7.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 25000
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: FLEXIBLE FIT / Target criteria: Correlation coefficient

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