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- EMDB-17352: Structure of hantaan orthohantavirus (HTNV) polymerase - Apo core... -

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Basic information

Entry
Database: EMDB / ID: EMD-17352
TitleStructure of hantaan orthohantavirus (HTNV) polymerase - Apo core endonuclease
Map dataGlobal sharpening
Sample
  • Complex: RNA-dependent RNA polymerase of Hantaan orthohantavirus (HTNV).
    • Protein or peptide: RNA-directed RNA polymerase L
KeywordsRNA-dependent RNA polymerase / HTNV / Hantaan orthohantavirus / Bunyavirales / Hantaviridae. / 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-directed 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 / Cap-snatching endonuclease / : / 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 orthohantavirus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.24 Å
AuthorsKeown JR / Carrique L / Grimes JM
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Wellcome Trust200835/Z/16/Z United Kingdom
Medical Research Council (MRC, United Kingdom)MR/R009945/1 United Kingdom
University of Oxford, Medical Sciences Internal Fund (MSIF)BDR00281 United Kingdom
CitationJournal: PLoS Pathog / Year: 2024
Title: Structural characterization of the full-length Hantaan virus polymerase.
Authors: Jeremy R Keown / Loïc Carrique / Benjamin E Nilsson-Payant / Ervin Fodor / Jonathan M Grimes /
Abstract: Hantaviridae are a family of segmented negative-sense RNA viruses that contain important human and animal pathogens. Hantaviridae contain a viral RNA-dependent RNA polymerase that replicates and ...Hantaviridae are a family of segmented negative-sense RNA viruses that contain important human and animal pathogens. Hantaviridae contain a viral RNA-dependent RNA polymerase that replicates and transcribes the viral genome. Here we establish the expression and purification of the polymerase from the Old World Hantaan virus and characterise the structure using Cryo-EM. We determine a series of structures at resolutions between 2.7 and 3.3 Å of RNA free polymerase comprising the core, core and endonuclease, and a full-length polymerase. The full-length polymerase structure depicts the location of the cap binding and C-terminal domains which are arranged in a conformation that is incompatible with transcription and in a novel conformation not observed in previous conformations of cap-snatching viral polymerases. We further describe structures with 5' vRNA promoter in the presence and absence of a nucleotide triphosphate. The nucleotide bound structure mimics a replication pre-initiation complex and the nucleotide stabilises the motif E in a conformation distinct from those previously observed. We observe motif E in four distinct conformations including β-sheet, two helical arrangements, and nucleotide primed arrangement. The insights gained here guide future mechanistic studies of both the transcription and replication activities of the hantavirus polymerase and for the development of therapeutic targets.
History
DepositionMay 12, 2023-
Header (metadata) releaseMay 22, 2024-
Map releaseMay 22, 2024-
UpdateJan 8, 2025-
Current statusJan 8, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17352.png

Downloads & links

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Map

FileDownload / File: emd_17352.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGlobal sharpening
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.93 Å/pix.
x 300 pix.
= 279.6 Å		0.93 Å/pix.
x 300 pix.
= 279.6 Å		0.93 Å/pix.
x 300 pix.
= 279.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.932 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.175
Minimum - Maximum-0.81550044 - 1.2722166
Average (Standard dev.)0.00001966777 (±0.037779927)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 279.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17352_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram (log scale)

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Additional map: deepEMhancer sharpening

Fileemd_17352_additional_1.map
AnnotationdeepEMhancer sharpening
Projections & Slices
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Half map: #1

Fileemd_17352_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_17352_half_map_2.map
Projections & Slices
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Sample components

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Entire : RNA-dependent RNA polymerase of Hantaan orthohantavirus (HTNV).

EntireName: RNA-dependent RNA polymerase of Hantaan orthohantavirus (HTNV).
Components
  • Complex: RNA-dependent RNA polymerase of Hantaan orthohantavirus (HTNV).
    • Protein or peptide: RNA-directed RNA polymerase L

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Supramolecule #1: RNA-dependent RNA polymerase of Hantaan orthohantavirus (HTNV).

SupramoleculeName: RNA-dependent RNA polymerase of Hantaan orthohantavirus (HTNV).
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Hantaan orthohantavirus
Molecular weightTheoretical: 247 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 orthohantavirus
Molecular weightTheoretical: 251.806016 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MWSHPQFEKG GGSGGGSGGS SAWSHPQFEK HHHHHHHHEN LYFQGMDKYR EIHNKLKEFS PGTLTAVECI DYLDRLYAVR HDIVDQMIK HDWSDNKDSE EAIGKVLLFA GVPSNIITAL EKKIIPNHPT GKSLKAFFKM TPANYKISGT TIEFVEVTVT A DVDKGIRE ...String:
MWSHPQFEKG GGSGGGSGGS SAWSHPQFEK HHHHHHHHEN LYFQGMDKYR EIHNKLKEFS PGTLTAVECI DYLDRLYAVR HDIVDQMIK HDWSDNKDSE EAIGKVLLFA GVPSNIITAL EKKIIPNHPT GKSLKAFFKM TPANYKISGT TIEFVEVTVT A DVDKGIRE KKLKYEAGLT YIEQELHKFF LKGEIPQPYK ITFNVVAVRT DGSNITTQWP SRRNDGVVQY MRLVQAEISY VR EHLIKTE ERAALEAMFN LKFNISTHKS QPYYIPDYKG MEPIGANIED LVDYSKDWLS RARNFSFFEV KGTAVFECFN SNE ANHCQR YPMSRKPRNF LLIQCSLITS YKPATTLSDQ IDSRRACSYI LNLIPDTPAS YLIHDMAYRY INLTREDMIN YYAP RIQFK QTQNVREPGT FKLTSSMLRA ESKAMLDLLN NHKSGEKHGA QIESLNIASH IVQSESVSLI TKILSDLELN ITEPS TQEY STTKHTYVDT VLDKFFQNET QKYLIDVLKK TTAWHIGHLI RDITESLIAH SGLKRSKYWS LHSYNNGNVI LFILPS KSL EVAGSFIRFI TVFRIGPGLV DKDNLDTILI DGDSQWGVSK VMSIDLNRLL ALNIAFEKAL IATATWFQYY TEDQGQF PL QYAIRSVFAN HFLLAICQKM KLCAIFDNLR YLIPAVTSLY SGFPSLIEKL FERPFKSSLE VYIYYNIKSL LVALAQNN K ARFYSKVKLL GLTVDQSTVG ASGVYPSFMS RIVYKHYRSL ISEVTTCFFL FEKGLHGNMN EEAKIHLETV EWALKFREK EEKYGESLVE NGYMMWELRA NAELAEQQLY CQDAIELAAI ELNKVLATKS SVVANSILSK NWEEPYFSQT RNISLKGMSG QVQEDGHLS SSVTIIEAIR YLSNSRHNPS LLKLYEETRE QKAMARIVRK YQRTEADRGF FITTLPTRCR LEIIEDYYDA I AKNISEEY ISYGGEKKIL AIQGALEKAL RWASGESFIE LSNHKFIRMK RKLMYVSADA TKWSPGDNSA KFRRFTSMLH NG LPNNKLK NCVIDALKQV YKTDFFMSRK LRNYIDSMES LDPHIKQFLD FFPDGHHGEV KGNWLQGNLN KCSSLFGVAM SLL FKQVWT NLFPELDCFF EFAHHSDDAL FIYGYLEPVD DGTDWFLFVS QQIQAGHLHW FSVNTEMWKS MFNLHEHILL LGSI KISPK KTTVSPTNAE FLSTFFEGCA VSIPFVKILL GSLSDLPGLG YFDDLAAAQS RCVKALDLGA SPQVAQLAVA LCTSK VERL YGTAPGMVNH PAAYLQVKHT DTPIPLGGNG AMSIMELATA GIGMSDKNLL KRALLGYSHK RQKSMLYILG LFKFLM KLS DETFQHERLG QFSFIGKVQW KIFTPKSEFE FADMYTSKFL ELWSSQHVTY DYIIPKGRDN LLIYLVRKLN DPSIVTA MT MQSPLQLRFR MQAKQHMKVC RLDGEWVTFR EVLAAANSFA ENYSATSQDM DLFQTLTSCT FSKEYAWKDF LNGIHCDV I PTKQVQRAKV ARTFTVREKD QIIQNSIPAV IGYKFAVTVE EMSDVLDTAK FPDSLSVDLK TMKDGVYREL GLDISLPDV MKRIAPMLYK SSKSRVVIVQ GNVEGTAEAI CRYWLKSMSL VKTIRVKPHK EVLQAVSIFN RKEDIGQQKD LAALKLCIEV WRWCKANSA PYRDWFQALW FEDKTFSEWL DRFCRVGVPP IDPEIQCAAL MIADIKGDYS VLQLQANRRA YSGKQYDAYC V QTYNEVTK LYEGDLRVTF NFGLDCARLE IFWDKKAYIL ETSITQKHVL KIMMDEVSKE LIKCGMRFNT EQVQGVRHMV LF KTESGFE WGKPNIPCIV YKNCVLRTSL RTTQAINHKF MITIKDDGLR AIAQHDEDSP RFLLAHAFHT IRDIRYQAVD AVS NVWFIH KGVKLYLNPI ISSGLLENFM KNLPAAIPPA AYSLIMNRAK ISVDLFMFND LLKLINPRNT LDLSGLETTG DEFS TVSSM SSRLWSEEMS LVDDDEELDD EFTIDLQDVD FENIDIEADI EHFLQDESSY TGDLLISTEE TESKKMRGIV KILEP VRLI KSWVSRGLSI EKVYSPVNII LMSRYISKTF NLSTKQVSLL DPYDLTELES IVRGWGECVI DQFESLDREA QNMVVN KGI 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

Concentration0.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMHEPES2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
500.0 mMNaClsodium chloride
1.0 mMDTTdithiothreitol
5.0 %GlycerolGlycerol
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.4000000000000001 µm
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

Startup modelType of model: INSILICO MODEL / In silico model: Alphafold2 model
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.24 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0) / Number images used: 50000
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.0)
Final 3D classificationNumber classes: 8 / Software - Name: cryoSPARC (ver. 4.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-8p1k:
Structure of hantaan orthohantavirus (HTNV) polymerase - Apo core endonuclease

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