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- EMDB-18394: Focused map of Hantaan virus polymerase dimer used to refine the ... -

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ID or keywords:

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Basic information

Entry
Database: EMDB / ID: EMD-18394
TitleFocused map of Hantaan virus polymerase dimer used to refine the cap-binding domain
Map dataHTNV-L Apo Dimer focused on CBD
Sample
  • Complex: Hantaan virus polymerase
    • Protein or peptide: Hantaan virus polymerase
KeywordsBunyavirus / Hantaan virus / polymerase / dimer / VIRAL PROTEIN
Function / homology
Function and homology information


RNA-templated viral transcription / negative stranded viral RNA replication / cap snatching / endonuclease activity / Hydrolases; Acting on ester bonds / host cell perinuclear region of cytoplasm / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / nucleotide binding / DNA-templated transcription / metal ion binding
Similarity search - Function
RNA-directed RNA polymerase, hantavirus / RNA-directed RNA polymerase, hantavirus, N-terminal / RNA dependent RNA polymerase / : / 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 speciesHantaan virus 76-118
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsDurieux Trouilleton Q / Arragain B / Malet H
Funding support France, 1 items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-19-CE11-0024 France
CitationJournal: Nat Commun / Year: 2024
Title: Structural characterization of the oligomerization of full-length Hantaan virus polymerase into symmetric dimers and hexamers.
Authors: Quentin Durieux Trouilleton / Dominique Housset / Paco Tarillon / Benoît Arragain / Hélène Malet /
Abstract: Hantaan virus is a dangerous human pathogen whose segmented negative-stranded RNA genome is replicated and transcribed by a virally-encoded multi-functional polymerase. Here we describe the complete ...Hantaan virus is a dangerous human pathogen whose segmented negative-stranded RNA genome is replicated and transcribed by a virally-encoded multi-functional polymerase. Here we describe the complete cryo-electron microscopy structure of Hantaan virus polymerase in several oligomeric forms. Apo polymerase protomers can adopt two drastically different conformations, which assemble into two distinct symmetric homodimers, that can themselves gather to form hexamers. Polymerase dimerization induces the stabilization of most polymerase domains, including the C-terminal domain that contributes the most to dimer's interface, along with a lariat region that participates to the polymerase steadying. Binding to viral RNA induces significant conformational changes resulting in symmetric oligomer disruption and polymerase activation, suggesting the possible involvement of apo multimers as protecting systems that would stabilize the otherwise flexible C-terminal domains. Overall, these results provide insights into the multimerization capability of Hantavirus polymerase and may help to define antiviral compounds to counteract these life-threatening viruses.
History
DepositionSep 6, 2023-
Header (metadata) releaseMar 27, 2024-
Map releaseMar 27, 2024-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18394.png

Downloads & links

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Map

FileDownload / File: emd_18394.map.gz / Format: CCP4 / Size: 266.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHTNV-L Apo Dimer focused on CBD
Voxel sizeX=Y=Z: 0.839 Å
Density
Contour LevelBy AUTHOR: 0.221
Minimum - Maximum-1.6230264 - 2.6891482
Average (Standard dev.)0.0015044407 (±0.035871763)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions412412412
Spacing412412412
CellA=B=C: 345.668 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: HTNV-L Apo Dimer focused on CBD half map A

Fileemd_18394_half_map_1.map
AnnotationHTNV-L Apo Dimer focused on CBD half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: HTNV-L Apo Dimer focused on CBD half map B

Fileemd_18394_half_map_2.map
AnnotationHTNV-L Apo Dimer focused on CBD half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Hantaan virus polymerase

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

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Supramolecule #1: Hantaan virus polymerase

SupramoleculeName: Hantaan virus polymerase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Hantaan virus 76-118
Molecular weightTheoretical: 500 KDa

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Macromolecule #1: Hantaan virus polymerase

MacromoleculeName: Hantaan virus polymerase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Hantaan virus 76-118
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGHHHHHHDY DIPTTENLYF QGMDKYREIH NKLKEFSPGT LTAVECIDYL DRLYAVRHDI VDQMIKHDWS DNKDSEEAIG KVLLFAGVPS NIITALEKKI IPNHPTGKSL KAFFKMTPDN YKISGTTIEF VEVTVTADVD KGIREKKLKY EAGLTYIEQE LHKFFLKGEI ...String:
MGHHHHHHDY DIPTTENLYF QGMDKYREIH NKLKEFSPGT LTAVECIDYL DRLYAVRHDI VDQMIKHDWS DNKDSEEAIG KVLLFAGVPS NIITALEKKI IPNHPTGKSL KAFFKMTPDN YKISGTTIEF VEVTVTADVD KGIREKKLKY EAGLTYIEQE LHKFFLKGEI PQPYKITFNV VAVRTDGSNI TTQWPSRRND GVVQYMRLVQ AEISYVREHL IKTEERAALE AMFNLKFNIS THKSQPYYIP DYKGMEPIGA NIEDLVDYSK DWLSRARNFS FFEVKGTAVF ECFNSNEANH CQRYPMSRKP RNFLLIQCSL ITSYKPATTL SDQIDSRRAC SYILNLIPDT PASYLIHDMA YRYINLTRED MINYYAPRIQ FKQTQNVREP GTFKLTSSML RAESKAMLDL LNNHKSGEKH GAQIESLNIA SHIVQSESVS LITKILSDLE LNITEPSTQE YSTTKHTYVD TVLDKFFQNE TQKYLIDVLK KTTAWHIGHL IRDITESLIA HSGLKRSKYW SLHSYNNGNV ILFILPSKSL EVAGSFIRFI TVFRIGPGLV DKDNLDTILI DGDSQWGVSK VMSIDLNRLL ALNIAFEKAL IATATWFQYY TEDQGQFPLQ YAIRSVFANH FLLAICQKMK LCAIFDNLRY LIPAVTSLYS GFPSLIEKLF ERPFKSSLEV YIYYNIKSLL VALAQNNKAR FYSKVKLLGL TVDQSTVGAS GVYPSFMSRI VYKHYRSLIS EVTTCFFLFE KGLHGNMNEE AKIHLETVEW ALKFREKEEK YGESLVENGY MMWELRANAE LAEQQLYCQD AIELAAIELN KVLATKSSVV ANSILSKNWE EPYFSQTRNI SLKGMSGQVQ EDGHLSSSVT IIEAIRYLSN SRHNPSLLKL YEETREQKAM ARIVRKYQRT EADRGFFITT LPTRCRLEII EDYYDAIAKN ISEEYISYGG EKKILAIQGA LEKALRWASG ESFIELSNHK FIRMKRKLMY VSADATKWSP GDNSAKFRRF TSMLHNGLPN NKLKNCVIDA LKQVYKTDFF MSRKLRNYID SMESLDPHIK QFLDFFPDGH HGEVKGNWLQ GNLNKCSSLF GVAMSLLFKQ VWTNLFPELD CFFEFAHHSD DALFIYGYLE PVDDGTDWFL FVSQQIQAGH LHWFSVNTEM WKSMFNLHEH ILLLGSIKIS PKKTTVSPTN AEFLSTFFEG CAVSIPFVKI LLGSLSDLPG LGYFDDLAAA QSRCVKALDL GASPQVAQLA VALCTSKVER LYGTAPGMVN HPAAYLQVKH TDTPIPLGGN GAMSIMELAT AGIGMSDKNL LKRALLGYSH KRQKSMLYIL GLFKFLMKLS DETFQHERLG QFSFIGKVQW KIFTPKSEFE FADMYTSKFL ELWSSQHVTY DYIIPKGRDN LLIYLVRKLN DPSIVTAMTM QSPLQLRFRM QAKQHMKVCR LDGEWVTFRE VLAAANSFAE NYSATSQDMD LFQTLTSCTF SKEYAWKDFL NGIHCDVIPT KQVQRAKVAR TFTVREKDQI IQNSIPAVIG YKFAVTVEEM SDVLDTAKFP DSLSVDLKTM KDGVYRELGL DISLPDVMKR IAPMLYKSSK SRVVIVQGNV EGTAEAICRY WLKSMSLVKT IRVKPHKEVL QAVSIFNRKE DIGQQKDLAA LKLCIEVWRW CKANSAPYRD WFQALWFEDK TFSEWLDRFC RVGVPPIDPE IQCAALMIAD IKGDYSVLQL QANRRAYSGK QYDAYCVQTY NEVTKLYEGD LRVTFNFGLD CARLEIFWDK KAYILETSIT QKHVLKIMMD EVSKELIKCG MRFNTEQVQG VRHMVLFKTE SGFEWGKPNI PCIVYKNCVL RTSLRTTQAI NHKFMITIKD DGLRAIAQHD EDSPRFLLAH AFHTIRDIRY QAVDAVSNVW FIHKGVKLYL NPIISSGLLE NFMKNLPAAI PPAAYSLIMN RAKISVDLFM FNDLLKLINP RNTLDLSGLE TTGDEFSTVS SMSSRLWSEE MSLVDDDEEL DDEFTIDLQD VDFENIDIEA DIEHFLQDES SYTGDLLIST EETESKKMRG IVKILEPVRL IKSWVSRGLS IEKVYSPVNI ILMSRYISKT FNLSTKQVSL LDPYDLTELE SIVRGWGECV IDQFESLDRE AQNMVVNKGI CPEDVIPDSL FSFRHTMVLL RRLFPQDSIS SFY

UniProtKB: RNA-directed RNA polymerase L

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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

Concentration0.25 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
30.0 mMC8H18N2O4SHEPES
250.0 mMNaClSodium chlorideNaClSodium chloride
10.0 mMC9H15O6PTCEP
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 50 sec. / Pretreatment - Atmosphere: AIR / Details: 25 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 77 K / Instrument: FEI VITROBOT MARK IV / Details: blot force 1 3s.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.0 µm / Calibrated defocus min: 0.8 µm / Calibrated magnification: 105000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
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
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 14650 / Average exposure time: 1.45 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3354153
Startup modelType of model: INSILICO MODEL / In silico model: Ab-initio reconstruction cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC (ver. 4.2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Number images used: 53748
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8c4s


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: AB INITIO MODEL

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