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- EMDB-20407: In situ structure of BTV RNA-dependent RNA polymerase in BTV core -

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Basic information

Entry
Database: EMDB / ID: EMD-20407
TitleIn situ structure of BTV RNA-dependent RNA polymerase in BTV core
Map dataBTV RNA-dependent RNA polymerase in BTV core
Sample
  • Virus: Bluetongue virus 1
    • Protein or peptide: RNA-directed RNA polymeraseRNA-dependent RNA polymerase
    • Protein or peptide: Inner core structural protein VP3
KeywordsRNA dependent RNA polymerase / Viral protein / Transferase
Function / homology
Function and homology information


viral genome replication / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / nucleotide binding / DNA-templated transcription / structural molecule activity / RNA binding
Similarity search - Function
RNA-dependent RNA polymerase, orbiviral / Orbivirus RNA-dependent RNA polymerase (VP1) / Inner layer core protein VP3, Orbivirus / Orbivirus VP3 (T2) protein / Inner layer core protein VP3, Reovirus / RNA-directed RNA polymerase, reovirus / RdRp of Reoviridae dsRNA viruses catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Core protein VP3 / RNA-directed RNA polymerase
Similarity search - Component
Biological speciesBluetongue virus 1
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsHe Y / Shivakoti S
Funding support United States, United Kingdom, 7 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI094386 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI045000 United States
Wellcome Trust100218 United Kingdom
National Institutes of Health/Office of the Director1S10OD018111 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1U24GM116792 United States
National Science Foundation (NSF, United States)DBI-1338135 United States
National Science Foundation (NSF, United States)DMR-1548924 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2019
Title: In situ structures of RNA-dependent RNA polymerase inside bluetongue virus before and after uncoating.
Authors: Yao He / Sakar Shivakoti / Ke Ding / Yanxiang Cui / Polly Roy / Z Hong Zhou /
Abstract: Bluetongue virus (BTV), a major threat to livestock, is a multilayered, nonturreted member of the , a family of segmented dsRNA viruses characterized by endogenous RNA transcription through an RNA- ...Bluetongue virus (BTV), a major threat to livestock, is a multilayered, nonturreted member of the , a family of segmented dsRNA viruses characterized by endogenous RNA transcription through an RNA-dependent RNA polymerase (RdRp). To date, the structure of BTV RdRp has been unknown, limiting our mechanistic understanding of BTV transcription and hindering rational drug design effort targeting this essential enzyme. Here, we report the in situ structures of BTV RdRp VP1 in both the triple-layered virion and double-layered core, as determined by cryo-electron microscopy (cryoEM) and subparticle reconstruction. BTV RdRp has 2 unique motifs not found in other viral RdRps: a fingernail, attached to the conserved fingers subdomain, and a bundle of 3 helices: 1 from the palm subdomain and 2 from the N-terminal domain. BTV RdRp VP1 is anchored to the inner surface of the capsid shell via 5 asymmetrically arranged N termini of the inner capsid shell protein VP3A around the 5-fold axis. The structural changes of RdRp VP1 and associated capsid shell proteins between BTV virions and cores suggest that the detachment of the outer capsid proteins VP2 and VP5 during viral entry induces both global movements of the inner capsid shell and local conformational changes of the N-terminal latch helix (residues 34 to 51) of 1 inner capsid shell protein VP3A, priming RdRp VP1 within the capsid for transcription. Understanding this mechanism in BTV also provides general insights into RdRp activation and regulation during viral entry of other multilayered, nonturreted dsRNA viruses.
History
DepositionJul 3, 2019-
Header (metadata) releaseAug 7, 2019-
Map releaseAug 7, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
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  • Surface view colored by height
  • Surface level: 0.02
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  • Surface view with fitted model
  • Atomic models: PDB-6po2
  • Surface level: 0.02
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6po2
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20407.png

Downloads & links

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Map

FileDownload / File: emd_20407.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBTV RNA-dependent RNA polymerase in BTV core
Voxel sizeX=Y=Z: 1.36 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.044679813 - 0.07617847
Average (Standard dev.)0.001415685 (±0.0055442397)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 348.16 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.361.361.36
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z348.160348.160348.160
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ1128100
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0450.0760.001

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Supplemental data

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Sample components

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Entire : Bluetongue virus 1

EntireName: Bluetongue virus 1
Components
  • Virus: Bluetongue virus 1
    • Protein or peptide: RNA-directed RNA polymeraseRNA-dependent RNA polymerase
    • Protein or peptide: Inner core structural protein VP3

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Supramolecule #1: Bluetongue virus 1

SupramoleculeName: Bluetongue virus 1 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / NCBI-ID: 35327 / Sci species name: Bluetongue virus 1 / Virus type: VIRION / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: No

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Macromolecule #1: RNA-directed RNA polymerase

MacromoleculeName: RNA-directed RNA polymerase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed RNA polymerase
Source (natural)Organism: Bluetongue virus 1
Molecular weightTheoretical: 149.926281 KDa
SequenceString: MVAITVQGAQ LIKRVVERFY PGIAFNINEG ACYIYKFSDH IRRIRMKHGT KYRRQAEEII RNISLRKERL YGIPVLDEVE WKYVFDGQT FQSYAFEVYV NSILPWSELD PEEEFLRNYR VSREMTEVEK FIEFRAKNEM QIYGDIPIKV WCCFINELSA E LKHVPLGM ...String:
MVAITVQGAQ LIKRVVERFY PGIAFNINEG ACYIYKFSDH IRRIRMKHGT KYRRQAEEII RNISLRKERL YGIPVLDEVE WKYVFDGQT FQSYAFEVYV NSILPWSELD PEEEFLRNYR VSREMTEVEK FIEFRAKNEM QIYGDIPIKV WCCFINELSA E LKHVPLGM QVMADFVNRF DSPFHQGNRD LSNLEDFQVA YTTPLLFEMC CMESILEFNI KMRMREEEIS ALEFGDMKVD PV GLLREFF ILCLPHPKKI NNVLRAPYSW FVKMWGVGAD PIVVLQSTAG DDRNSKDVFY DKFRTEPNRY KALFRSSFYN ESR RMNEEK ILEAVKYSQK LGSHDRRLPL FEKMLKTVYT TPFYPHKSSN MILASFLLSI QTITGYGRAW VKNVSTEFDK QLKP NPSNL VQDVSDLTRE FFKQAYVEAK ERREEIVKPE DLYTSMLRLA RNTSSGFSTE IYVKKRFGPR LRDKDLIKIN SRIKA LVIF TKGHTVFTDE ELHKKYNSVE LYQTKGSRDV PIKATRTIYS INLSVLVPQL IVTLPLNEYF SRVGGITSPD YKKIGG KVI VGDLEATGSR VMDAADCFRN SADRDIFTIA IDYSEYDTHL TRHNFRTGML QGIREAMAPY RDLRYEGYTL EQIIDFG YG EGRVANTLWN GKRRLFKTTF DAYIRLDESE RDKGSFKVPK GVLPVSSVDV ANRIAVDKGF DTLIAATDGS DLALIDTH L SGENSTLIAN SMHNMAIGTL MQREVGREQP GVLTFLSEQY VGDDTLFYTK LHTTDTKVFD KVAASIFDTV AKCGHEASP SKTMMTPYSV EKTQTHAKQG CYVPQDRMMI ISSERRKDIE DVQGYVRSQV QTMITKVSRG FCHDLAQLIL MLKTTFIGAW KMKRTIKED AMYRDRKFDS NDEDGFTLIQ IRNPLALYVP IGWNGYGAHP AALNIVMTEE MYVDSIMISK LDEIMAPIRR I VHDIPPCW NETQGDKRGL ISATKMSFFS KMARPAVQAA LSDPQIINLV EELPLGEFSP GRISRTMMHS ALLKESSART LL SSGYELE YQKALNSWIT QVSMRLGEES GVISTSYAKL FDVYFEGELD GAPHMFPDQN LSPQFYIQKM MIGPRVSSRV RNS YVDRID VILRKDVVMR GFITANTILN VIEKLGTNHS VGDLVTVFTL MNIETRVAEE LAEYMTSEKI RFDALKLLKK GIAG DEFTM SLNVATQDFI DTYLAYPYQL TKTEVDAISL YCTQMIMLRA ALGLPKKKMK IVVTDDAKKR YKIRLQRFRT HVPKI KVLK KLIDPNRMTV RNLENQFV

UniProtKB: RNA-directed RNA polymerase

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Macromolecule #2: Inner core structural protein VP3

MacromoleculeName: Inner core structural protein VP3 / type: protein_or_peptide / ID: 2 / Number of copies: 10 / Enantiomer: LEVO
Source (natural)Organism: Bluetongue virus 1
Molecular weightTheoretical: 103.410508 KDa
Recombinant expressionOrganism: Bluetongue virus 1
SequenceString: MAAQNEQRPE RIKTTPYLEG DVLSSDSGPL LSVFALQEIM QKVRQVQADY MTATREVDFT VPDVQKILDD IKALAAEQVY KIVKVPSIS FRHIVMQSRD RVLRVDTYYE EMSQVGDVIT EDEPEKFYST IIKKVRFIRG KGSFILHDIP TRDHRGMEVA E PEVLGVEF ...String:
MAAQNEQRPE RIKTTPYLEG DVLSSDSGPL LSVFALQEIM QKVRQVQADY MTATREVDFT VPDVQKILDD IKALAAEQVY KIVKVPSIS FRHIVMQSRD RVLRVDTYYE EMSQVGDVIT EDEPEKFYST IIKKVRFIRG KGSFILHDIP TRDHRGMEVA E PEVLGVEF KNVLPVLTAE HRAMIQNALD GSIIENGNVA TRDVDVFIGA CSEPVYRIYN RLQGYIEAVQ LQELRNSIGW LE RLGHRKR ITYSQEVLTD FRRQDTIWVL ALQLPVNPQV VWDVPRSSIA NLIMNIATCL PTGEYIAPNP RISSITLTQR ITT TGPFAI LTGSTPTAQQ LNDVRKIYLA LMFPGQIILD LKIDPGERMD PAVRMVAGVV GHLLFTAGGR FTNLTQNMAR QLDI ALNDY LLYMYNTRVQ VNYGPTGEPL DFQIGRNQYD CNVFRADFAT GTGYNGWATI DVEYREPAPY VHAQRYIRYC GIDSR ELIN PTTYGIGMTY HCYNEMLRML VAAGKDSEAA YFRSMLPFHM VRFARINQII NEDLHSVFSL PDDMFNALLP DLIAGA HQN ADPVVLDVSW ISLWFAFNRS FEPTHRNEML EVAPLIESVY ASELSVMKVD MRHLSLMQRR FPDVLIQARP SHFWKAV LN DSPEAVKAVM NLSHSHNFIN IRDMMRWVML PSLQPSLKLA LEEEAWAAAN DFEDLMLTDQ VYMHRDMLPE PRLDDIER F RQEGFYYTNM LEAPPEIDRV VQYTYEIARL QANMGQFRAA LRRIMDDDDW VRFGGVLRTV RVKFYDARPP DDVLQGLPF SYDTNERGGL AYATIKYATE TTIFYLIYNV EFSNTPDSLV LINPTYTMTK VFINKRIVER VRVGQILAVL NRRFVAYKGK MRIMDITQS LKMGTKLAAP TV

UniProtKB: Core protein VP3

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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

BufferpH: 8.8
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 32.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

2btv

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 150346

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