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- EMDB-16430: Hantaan virus polymerase in replication elongation state -

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Basic information

Entry
Database: EMDB / ID: EMD-16430
TitleHantaan virus polymerase in replication elongation state
Map dataHantaan virus polymerase cryo-EM map in replication elongation state Local resolution filtered map
Sample
  • Complex: Hantaan virus polymerase in replication elongation state
    • Protein or peptide: RNA-directed RNA polymerase L
    • RNA: RNA (5'-R(P*AP*GP*GP*AP*GP*UP*AP*UP*CP*CP*AP*CP*CP*GP*CP*AP*AP*GP*A)-3')
    • RNA: RNA (5'-R(P*AP*CP*UP*AP*CP*UP*A)-3')
    • RNA: RNA (5'-R(P*AP*GP*UP*AP*GP*UP*AP*GP*A)-3')
  • Ligand: MAGNESIUM ION
KeywordsPolymerase / replication / bunyavirus / 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: 3.14 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: 2023
Title: Structures of active Hantaan virus polymerase uncover the mechanisms of Hantaviridae genome replication.
Authors: Quentin Durieux Trouilleton / Sergio Barata-García / Benoît Arragain / Juan Reguera / Hélène Malet /
Abstract: Hantaviruses are causing life-threatening zoonotic infections in humans. Their tripartite negative-stranded RNA genome is replicated by the multi-functional viral RNA-dependent RNA-polymerase. Here ...Hantaviruses are causing life-threatening zoonotic infections in humans. Their tripartite negative-stranded RNA genome is replicated by the multi-functional viral RNA-dependent RNA-polymerase. Here we describe the structure of the Hantaan virus polymerase core and establish conditions for in vitro replication activity. The apo structure adopts an inactive conformation that involves substantial folding rearrangement of polymerase motifs. Binding of the 5' viral RNA promoter triggers Hantaan virus polymerase reorganization and activation. It induces the recruitment of the 3' viral RNA towards the polymerase active site for prime-and-realign initiation. The elongation structure reveals the formation of a template/product duplex in the active site cavity concomitant with polymerase core widening and the opening of a 3' viral RNA secondary binding site. Altogether, these elements reveal the molecular specificities of Hantaviridae polymerase structure and uncover the mechanisms underlying replication. They provide a solid framework for future development of antivirals against this group of emerging pathogens.
History
DepositionJan 4, 2023-
Header (metadata) releaseJun 7, 2023-
Map releaseJun 7, 2023-
UpdateJun 7, 2023-
Current statusJun 7, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_16430.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHantaan virus polymerase cryo-EM map in replication elongation state Local resolution filtered map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 260 pix.
= 286. Å
1.1 Å/pix.
x 260 pix.
= 286. Å
1.1 Å/pix.
x 260 pix.
= 286. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.0271
Minimum - Maximum-0.18631348 - 0.28077945
Average (Standard dev.)0.00006250061 (±0.0049033165)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions260260260
Spacing260260260
CellA=B=C: 286.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Hantaan virus polymerase cryo-EM map in replication elongation...

Fileemd_16430_additional_1.map
AnnotationHantaan virus polymerase cryo-EM map in replication elongation state Post-processed map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Hantaan virus polymerase cryo-EM map in replication elongation...

Fileemd_16430_half_map_1.map
AnnotationHantaan virus polymerase cryo-EM map in replication elongation state Half-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Hantaan virus polymerase cryo-EM map in replication elongation...

Fileemd_16430_half_map_2.map
AnnotationHantaan virus polymerase cryo-EM map in replication elongation state Half-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : Hantaan virus polymerase in replication elongation state

EntireName: Hantaan virus polymerase in replication elongation state
Components
  • Complex: Hantaan virus polymerase in replication elongation state
    • Protein or peptide: RNA-directed RNA polymerase L
    • RNA: RNA (5'-R(P*AP*GP*GP*AP*GP*UP*AP*UP*CP*CP*AP*CP*CP*GP*CP*AP*AP*GP*A)-3')
    • RNA: RNA (5'-R(P*AP*CP*UP*AP*CP*UP*A)-3')
    • RNA: RNA (5'-R(P*AP*GP*UP*AP*GP*UP*AP*GP*A)-3')
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Hantaan virus polymerase in replication elongation state

SupramoleculeName: Hantaan virus polymerase in replication elongation state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Hantaan virus 76-118 / Strain: 76-118
Molecular weightTheoretical: 246 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: Hantaan virus 76-118 / Strain: 76-118
Molecular weightTheoretical: 249.432484 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGHHHHHHDY DIPTTENLYF QGMDKYREIH NKLKEFSPGT LTAVECIDYL DRLYAVRHDI VDQMIKHDWS DNKDSEEAIG KVLLFAGVP SNIITALEKK IIPNHPTGKS LKAFFKMTPA NYKISGTTIE FVEVTVTADV DKGIREKKLK YEAGLTYIEQ E LHKFFLKG ...String:
MGHHHHHHDY DIPTTENLYF QGMDKYREIH NKLKEFSPGT LTAVECIDYL DRLYAVRHDI VDQMIKHDWS DNKDSEEAIG KVLLFAGVP SNIITALEKK IIPNHPTGKS LKAFFKMTPA NYKISGTTIE FVEVTVTADV DKGIREKKLK YEAGLTYIEQ E LHKFFLKG EIPQPYKITF NVVAVRTDGS NITTQWPSRR NDGVVQYMRL VQAEISYVRE HLIKTEERAA LEAMFNLKFN IS THKSQPY YIPDYKGMEP IGANIEDLVD YSKDWLSRAR NFSFFEVKGT AVFECFNSNE ANHCQRYPMS RKPRNFLLIQ CSL ITSYKP ATTLSDQIDS RRACSYILNL IPDTPASYLI HDMAYRYINL TREDMINYYA PRIQFKQTQN VREPGTFKLT SSML RAESK AMLDLLNNHK SGEKHGAQIE SLNIASHIVQ SESVSLITKI LSDLELNITE PSTQEYSTTK HTYVDTVLDK FFQNE TQKY LIDVLKKTTA WHIGHLIRDI TESLIAHSGL KRSKYWSLHS YNNGNVILFI LPSKSLEVAG SFIRFITVFR IGPGLV DKD NLDTILIDGD SQWGVSKVMS IDLNRLLALN IAFEKALIAT ATWFQYYTED QGQFPLQYAI RSVFANHFLL AICQKMK LC AIFDNLRYLI PAVTSLYSGF PSLIEKLFER PFKSSLEVYI YYNIKSLLVA LAQNNKARFY SKVKLLGLTV DQSTVGAS G VYPSFMSRIV YKHYRSLISE VTTCFFLFEK GLHGNMNEEA KIHLETVEWA LKFREKEEKY GESLVENGYM MWELRANAE LAEQQLYCQD AIELAAIELN KVLATKSSVV ANSILSKNWE EPYFSQTRNI SLKGMSGQVQ EDGHLSSSVT IIEAIRYLSN SRHNPSLLK LYEETREQKA MARIVRKYQR TEADRGFFIT TLPTRCRLEI IEDYYDAIAK NISEEYISYG GEKKILAIQG A LEKALRWA SGESFIELSN HKFIRMKRKL MYVSADATKW SPGDNSAKFR RFTSMLHNGL PNNKLKNCVI DALKQVYKTD FF MSRKLRN YIDSMESLDP HIKQFLDFFP DGHHGEVKGN WLQGNLNKCS SLFGVAMSLL FKQVWTNLFP ELDCFFEFAH HSD DALFIY GYLEPVDDGT DWFLFVSQQI QAGHLHWFSV NTEMWKSMFN LHEHILLLGS IKISPKKTTV SPTNAEFLST FFEG CAVSI PFVKILLGSL SDLPGLGYFD DLAAAQSRCV KALDLGASPQ VAQLAVALCT SKVERLYGTA PGMVNHPAAY LQVKH TDTP IPLGGNGAMS IMELATAGIG MSDKNLLKRA LLGYSHKRQK SMLYILGLFK FLMKLSDETF QHERLGQFSF IGKVQW KIF TPKSEFEFAD MYTSKFLELW SSQHVTYDYI IPKGRDNLLI YLVRKLNDPS IVTAMTMQSP LQLRFRMQAK QHMKVCR LD GEWVTFREVL AAANSFAENY SATSQDMDLF QTLTSCTFSK EYAWKDFLNG IHCDVIPTKQ VQRAKVARTF TVREKDQI I QNSIPAVIGY KFAVTVEEMS DVLDTAKFPD SLSVDLKTMK DGVYRELGLD ISLPDVMKRI APMLYKSSKS RVVIVQGNV EGTAEAICRY WLKSMSLVKT IRVKPHKEVL QAVSIFNRKE DIGQQKDLAA LKLCIEVWRW CKANSAPYRD WFQALWFEDK TFSEWLDRF CRVGVPPIDP EIQCAALMIA DIKGDYSVLQ LQANRRAYSG KQYDAYCVQT YNEVTKLYEG DLRVTFNFGL D CARLEIFW DKKAYILETS ITQKHVLKIM MDEVSKELIK CGMRFNTEQV QGVRHMVLFK TESGFEWGKP NIPCIVYKNC VL RTSLRTT QAINHKFMIT IKDDGLRAIA QHDEDSPRFL LAHAFHTIRD IRYQAVDAVS NVWFIHKGVK LYLNPIISSG LLE NFMKNL PAAIPPAAYS LIMNRAKISV DLFMFNDLLK LINPRNTLDL SGLETTGDEF STVSSMSSRL WSEEMSLVDD DEEL DDEFT IDLQDVDFEN IDIEADIEHF LQDESSYTGD LLISTEETES KKMRGIVKIL EPVRLIKSWV SRGLSIEKVY SPVNI ILMS RYISKTFNLS TKQVSLLDPY DLTELESIVR GWGECVIDQF ESLDREAQNM VVNKGICPED VIPDSLFSFR HTMVLL RRL FPQDSISSFY

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Macromolecule #2: RNA (5'-R(P*AP*GP*GP*AP*GP*UP*AP*UP*CP*CP*AP*CP*CP*GP*CP*AP*AP*GP...

MacromoleculeName: RNA (5'-R(P*AP*GP*GP*AP*GP*UP*AP*UP*CP*CP*AP*CP*CP*GP*CP*AP*AP*GP*A)-3')
type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Hantaan virus 76-118
Molecular weightTheoretical: 6.429919 KDa
SequenceString:
UAGGAGUAUC CACCGCAAGA

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Macromolecule #3: RNA (5'-R(P*AP*CP*UP*AP*CP*UP*A)-3')

MacromoleculeName: RNA (5'-R(P*AP*CP*UP*AP*CP*UP*A)-3') / type: rna / ID: 3 / Number of copies: 3
Source (natural)Organism: Hantaan virus 76-118
Molecular weightTheoretical: 7.851604 KDa
SequenceString:
CUUUCUUUUG CGGAGUCUAC UACUA

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Macromolecule #4: RNA (5'-R(P*AP*GP*UP*AP*GP*UP*AP*GP*A)-3')

MacromoleculeName: RNA (5'-R(P*AP*GP*UP*AP*GP*UP*AP*GP*A)-3') / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Hantaan virus 76-118
Molecular weightTheoretical: 8.092932 KDa
SequenceString:
UAGUAGUAGA CUCCGCAAAA GAAAG

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / 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

Concentration0.35 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
30.0 mMC8H18N2O4SHEPES
250.0 mMNaClSodium chlorideSodium Chloride
5.0 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: 45 sec. / Details: 25 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 276 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 2.2 µm / Calibrated defocus min: 2.2 µm / Calibrated magnification: 45454 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 0.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 36000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 77.0 K / Max: 77.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3840 pixel / Digitization - Dimensions - Height: 3712 pixel / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Number real images: 3785 / Average exposure time: 5.5 sec. / Average electron dose: 50.0 e/Å2

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Image processing

Particle selectionNumber selected: 1063763
Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2) / Software - details: Non homogeneous refinement
Final 3D classificationNumber classes: 10 / Avg.num./class: 55000 / Software - Name: RELION (ver. 4.0) / Software - details: 3D classification
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0) / Software - details: 3D auto-refine
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.14 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number images used: 159681
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 34.7
Output model

PDB-8c4v:
Hantaan virus polymerase in replication elongation state

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