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- EMDB-20059: In situ structure of Rotavirus RNA-dependent RNA polymerase at du... -

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Basic information

Entry
Database: EMDB / ID: EMD-20059
TitleIn situ structure of Rotavirus RNA-dependent RNA polymerase at duplex-open state
Map dataem-volume_P1
Sample
  • Virus: Rotavirus A
    • Protein or peptide: RNA-dependent RNA polymerase of rotavirus A
    • Protein or peptide: Inner capsid protein VP2
    • Other: DNA/RNA (5'-D(*(GTG))-R(P*GP*C)-3')
    • RNA: RNA (5'-R(P*AP*GP*CP*C)-3')
KeywordsRNA-dependent RNA polymerase / capsid shell protein / transcription / in situ structure / rotavirus / transcriptional factors / reovirus / VIRUS / viral protein-transferase-rna complex
Function / homology
Function and homology information


T=2 icosahedral viral capsid / viral inner capsid / viral genome replication / virion component / viral nucleocapsid / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / nucleotide binding / DNA-templated transcription / RNA binding
Similarity search - Function
Rotavirus VP1 RNA-directed RNA polymerase, C-terminal / Viral RNA-directed RNA polymerase, 4-helical domain / Rotavirus VP1 C-terminal domain / RNA-directed RNA polymerase, luteovirus / Rotavirus VP2 / Viral RNA-directed RNA-polymerase / Rotavirus VP2 protein / RNA-directed RNA polymerase, reovirus / RdRp of Reoviridae dsRNA viruses catalytic domain profile. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
RNA-directed RNA polymerase / Inner capsid protein VP2
Similarity search - Component
Biological speciesRotavirus A
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsDing K / Chang T
Funding support United States, 8 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 General Medical Sciences (NIH/NIGMS)GM071940 United States
National Institutes of Health/National Institute of Dental and Craniofacial Research (NIH/NIDCR)DE025567 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)1S10RR23057 United States
National Institutes of Health/Office of the Director1S10OD018111 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM116792 United States
National Science Foundation (NSF, United States)DBI-1338135 United States
National Science Foundation (NSF, United States)DMR-1548924 United States
CitationJournal: Nat Commun / Year: 2019
Title: In situ structures of rotavirus polymerase in action and mechanism of mRNA transcription and release.
Authors: Ke Ding / Cristina C Celma / Xing Zhang / Thomas Chang / Wesley Shen / Ivo Atanasov / Polly Roy / Z Hong Zhou /
Abstract: Transcribing and replicating a double-stranded genome require protein modules to unwind, transcribe/replicate nucleic acid substrates, and release products. Here we present in situ cryo-electron ...Transcribing and replicating a double-stranded genome require protein modules to unwind, transcribe/replicate nucleic acid substrates, and release products. Here we present in situ cryo-electron microscopy structures of rotavirus dsRNA-dependent RNA polymerase (RdRp) in two states pertaining to transcription. In addition to the previously discovered universal "hand-shaped" polymerase core domain shared by DNA polymerases and telomerases, our results show the function of N- and C-terminal domains of RdRp: the former opens the genome duplex to isolate the template strand; the latter splits the emerging template-transcript hybrid, guides genome reannealing to form a transcription bubble, and opens a capsid shell protein (CSP) to release the transcript. These two "helicase" domains also extensively interact with CSP, which has a switchable N-terminal helix that, like cellular transcriptional factors, either inhibits or promotes RdRp activity. The in situ structures of RdRp, CSP, and RNA in action inform mechanisms of not only transcription, but also replication.
History
DepositionApr 3, 2019-
Header (metadata) releaseApr 10, 2019-
Map releaseMay 22, 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: 0.0311
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0311
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6ogy
  • Surface level: 0.0311
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6ogy
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20059.png

Downloads & links

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Map

FileDownload / File: emd_20059.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationem-volume_P1
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 320 pix.
= 342.4 Å		1.07 Å/pix.
x 320 pix.
= 342.4 Å		1.07 Å/pix.
x 320 pix.
= 342.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0311 / Movie #1: 0.0311
Minimum - Maximum-0.16386285 - 0.23116834
Average (Standard dev.)0.003768775 (±0.017904727)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 342.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z342.400342.400342.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ513513513
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.1640.2310.004

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Supplemental data

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Sample components

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Entire : Rotavirus A

EntireName: Rotavirus A
Components
  • Virus: Rotavirus A
    • Protein or peptide: RNA-dependent RNA polymerase of rotavirus A
    • Protein or peptide: Inner capsid protein VP2
    • Other: DNA/RNA (5'-D(*(GTG))-R(P*GP*C)-3')
    • RNA: RNA (5'-R(P*AP*GP*CP*C)-3')

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Supramolecule #1: Rotavirus A

SupramoleculeName: Rotavirus A / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / NCBI-ID: 28875 / Sci species name: Rotavirus A / Virus type: VIRION / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: No

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Macromolecule #1: RNA-dependent RNA polymerase of rotavirus A

MacromoleculeName: RNA-dependent RNA polymerase of rotavirus A / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed RNA polymerase
Source (natural)Organism: Rotavirus A
Molecular weightTheoretical: 125.276305 KDa
SequenceString: MGKYNLILSE YLSFIYNSQS AVQIPIYYSS NSELENRCIE FHSKCLENSK NGLSLKKLFV EYSDVIENAT LLSILSYSYD KYNAVERKL VKYAKGKPLE ADLTVNELDY ENNKITSELF PTAEEYTDLL MDPAILTSLS SNLNAVMFWL EKHENDVAEK L KIYKRRLD ...String:
MGKYNLILSE YLSFIYNSQS AVQIPIYYSS NSELENRCIE FHSKCLENSK NGLSLKKLFV EYSDVIENAT LLSILSYSYD KYNAVERKL VKYAKGKPLE ADLTVNELDY ENNKITSELF PTAEEYTDLL MDPAILTSLS SNLNAVMFWL EKHENDVAEK L KIYKRRLD LFTIVASTVN KYGVPRHNAK YRYEYEVMKD KPYYLVTWAN SSIEMLMSVF SHEDYLIARE LIVLSYSNRS TL AKLVSSP MSILVALVDI NGTFITNEEL ELEFSNKYVR AIVPDQTFDE LKQMLDNMRK AGLTDIPKMI QDWLVDCSIE KFP LMAKIY SWSFHVGFRK QKMLDAALDQ LKTEYTEDVD DEMYREYTML IRDEVVKMLE EPVKHDDHLL QDSELAGLLS MSSA SNGES RQLKFGRKTI FSTKKNMHVM DDMANGRYTP GIIPPVNVDK PIPLGRRDVP GRRTRIIFIL PYEYFIAQHA VVEKM LIYA KHTREYAEFY SQSNQLLSYG DVTRFLSNNS MVLYTDVSQW DSSQHNTQPF RKGIIMGLDM LANMTNDARV IQTLNL YKQ TQINLMDSYV QIPDGNVIKK IQYGAVASGE KQTKAANSIA NLALIKTVLS RISNKYSFAT KIIRVDGDDN YAVLQFN TE VTKQMVQDVS NDVRETYARM NTKVKALVST VGIEIAKRYI AGGKIFFRAG INLLNNEKKG QSTQWDQAAV LYSNYIVN R LRGFETDREF ILTKIMQMTS VAITGSLRLF PSERVLTTNS TFKVFDSEDF IIEYGTTDDE VYIQRAFMSL SSQKSGIAD EIAASSTFKN YVSRLSEQLL FSKNNIVSRG IALTEKAKLN SYAPISLEKR RAQISALLTM LQKPVTFKSS KITINDILRD IKPFFTVNE AHLPIQYQKF MPTLPDNVQY IIQCIGSRTY QIEDDGSKSA ISRLISKYSV YKPSIEELYK VISLHENEIQ L YLISLGIP KIDADTYVGS KIYSQDKYRI LESYVYNLLS INYGCYQLFD FNSPDLEKLI RIPFKGKIPA VTFILHLYAK LE VINHAIK NGSWISLFCN YPKSEMIKLW KKMWNITSLR SPYTNANFFQ D

UniProtKB: RNA-directed RNA polymerase

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Macromolecule #2: Inner capsid protein VP2

MacromoleculeName: Inner capsid protein VP2 / type: protein_or_peptide / ID: 2 / Number of copies: 10 / Enantiomer: LEVO
Source (natural)Organism: Rotavirus A
Molecular weightTheoretical: 103.425992 KDa
SequenceString: MAYRKRGARR ETNLKQDDRM QEKEENKNVN TNSENKNATK PQLSEKVLSQ KEEVITDNQE EIKIADEVKK SNKEESKQLL EVLKTKEEH QKEVQYEILQ KTIPTFEPKE SILKKLEDIK PEQVKKQTKL FRIFEPRQLP VYRANGEKEL RNRWYWKLKR D TLPDGDYD ...String:
MAYRKRGARR ETNLKQDDRM QEKEENKNVN TNSENKNATK PQLSEKVLSQ KEEVITDNQE EIKIADEVKK SNKEESKQLL EVLKTKEEH QKEVQYEILQ KTIPTFEPKE SILKKLEDIK PEQVKKQTKL FRIFEPRQLP VYRANGEKEL RNRWYWKLKR D TLPDGDYD VREYFLNLYD QVLTEMPDYL LLKDMAVENK NSRDAGKVVD SETAAICDAI FQDEETEGVV RRFIAEMRQR VQ ADRNVVN YPSILHPIDH AFNEYFLQHQ LVEPLNNDII FNYIPERIRN DVNYILNMDR NLPSTARYIR PNLLQDRLNL HDN FESLWD TITTSNYILA RSVVPDLKEL VSTEAQIQKM SQDLQLEALT IQSETQFLTG INSQAANDCF KTLIAAMLSQ RTMS LDFVT TNYMSLISGM WLLTVVPNDM FIRESLVACQ LAIINTIIYP AFGMQRMHYR NGDPQTPFQI AEQQIQNFQV ANWLH FVNN NQFRQVVIDG VLNQVLNDNI RNGHVVNQLM EALMQLSRQQ FPTMPVDYKR SIQRGILLLS NRLGQLVDLT RLLAYN YET LMACITMNMQ HVQTLTTEKL QLTSVTSLCM LIGNATVIPS PQTLFHYYNV NVNFHSNYNE RINDAVAIIT AANRLNL YQ KKMKSIVEDF LKRLQIFDIS RVPDDQMYRL RDRLRLLPVE IRRLDIFNLI LMNMEQIERA SDKIAQGVII AYRDMQLE R DEMYGYVNIA RNLDGFQQIN LEELMRTGDY AQITNMLLNN QPVALVGALP FITDSSVISL VAKLDATVFA QIVKLRKVD TLKPILYKIN SDSNDFYLVA NYDWVPTSTT KVYKQIPQQF DFRASMHMLT SNLTFTVYSD LLAFVSADTV EPINAVAFDN MRIMNEL

UniProtKB: Inner capsid protein VP2

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Macromolecule #3: DNA/RNA (5'-D(*(GTG))-R(P*GP*C)-3')

MacromoleculeName: DNA/RNA (5'-D(*(GTG))-R(P*GP*C)-3') / type: other / ID: 3 / Number of copies: 1
Classification: polydeoxyribonucleotide/polyribonucleotide hybrid
Source (natural)Organism: Rotavirus A
Molecular weightTheoretical: 1.390855 KDa
SequenceString:
(GTG)GC

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

MacromoleculeName: RNA (5'-R(P*AP*GP*CP*C)-3') / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Rotavirus A
Molecular weightTheoretical: 1.239818 KDa
SequenceString:
AGCC

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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: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Average electron dose: 22.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Gaussian Ball
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 798260
Initial angle assignmentType: PROJECTION MATCHING / 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)

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