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- EMDB-20086: In situ structure of rotavirus VP1 RNA-dependent RNA polymerase (TLP) -

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Entry
Database: EMDB / ID: EMD-20086
TitleIn situ structure of rotavirus VP1 RNA-dependent RNA polymerase (TLP)
Map datafiltered, B-sharpened, masked
SampleRhesus rotavirus != Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])

Rhesus rotavirus

  • Virus: Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])
    • Protein or peptide: Inner capsid protein VP2
    • Protein or peptide: RNA-directed RNA polymerase
KeywordsRotavirus / RNA-dependent RNA polymerase / VP1 / VP2 / VIRAL PROTEIN-TRANSFERASE 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 (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])
Methodsingle particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsJenni S / Salgado EN
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)CA-13202 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: J Mol Biol / Year: 2019
Title: In situ Structure of Rotavirus VP1 RNA-Dependent RNA Polymerase.
Authors: Simon Jenni / Eric N Salgado / Tobias Herrmann / Zongli Li / Timothy Grant / Nikolaus Grigorieff / Stefano Trapani / Leandro F Estrozi / Stephen C Harrison /
Abstract: Rotaviruses, like other non-enveloped, double-strand RNA viruses, package an RNA-dependent RNA polymerase (RdRp) with each duplex of their segmented genomes. Rotavirus cell entry results in loss of ...Rotaviruses, like other non-enveloped, double-strand RNA viruses, package an RNA-dependent RNA polymerase (RdRp) with each duplex of their segmented genomes. Rotavirus cell entry results in loss of an outer protein layer and delivery into the cytosol of an intact, inner capsid particle (the "double-layer particle," or DLP). The RdRp, designated VP1, is active inside the DLP; each VP1 achieves many rounds of mRNA transcription from its associated genome segment. Previous work has shown that one VP1 molecule lies close to each 5-fold axis of the icosahedrally symmetric DLP, just beneath the inner surface of its protein shell, embedded in tightly packed RNA. We have determined a high-resolution structure for the rotavirus VP1 RdRp in situ, by local reconstruction of density around individual 5-fold positions. We have analyzed intact virions ("triple-layer particles"), non-transcribing DLPs and transcribing DLPs. Outer layer dissociation enables the DLP to synthesize RNA, in vitro as well as in vivo, but appears not to induce any detectable structural change in the RdRp. Addition of NTPs, Mg, and S-adenosylmethionine, which allows active transcription, results in conformational rearrangements, in both VP1 and the DLP capsid shell protein, that allow a transcript to exit the polymerase and the particle. The position of VP1 (among the five symmetrically related alternatives) at one vertex does not correlate with its position at other vertices. This stochastic distribution of site occupancies limits long-range order in the 11-segment, double-strand RNA genome.
History
DepositionApr 10, 2019-
Header (metadata) releaseApr 24, 2019-
Map releaseApr 24, 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: 2
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6oj3
  • Surface level: 2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20086.png

Downloads & links

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Map

FileDownload / File: emd_20086.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfiltered, B-sharpened, masked
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.23 Å/pix.
x 300 pix.
= 369. Å		1.23 Å/pix.
x 300 pix.
= 369. Å		1.23 Å/pix.
x 300 pix.
= 369. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.23 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.0 / Movie #1: 2
Minimum - Maximum-6.1107807 - 9.959702500000001
Average (Standard dev.)0.018543862 (±0.42538026)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 369.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.231.231.23
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z369.000369.000369.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-420-29
NX/NY/NZ887886
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-6.1119.9600.019

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Supplemental data

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Mask #1

Fileemd_20086_msk_1.map
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Additional map: filtered, B-sharpened

Fileemd_20086_additional_1.map
Annotationfiltered, B-sharpened
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Additional map: filtered

Fileemd_20086_additional_2.map
Annotationfiltered
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Half map: half-map 1, unmodified

Fileemd_20086_half_map_1.map
Annotationhalf-map 1, unmodified
Projections & Slices
AxesZYX

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Histogram

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Half map: half-map 2, unmodified

Fileemd_20086_half_map_2.map
Annotationhalf-map 2, unmodified
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Sample components

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Entire : Rhesus rotavirus

EntireName: Rhesus rotavirus
Components
  • Virus: Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])
    • Protein or peptide: Inner capsid protein VP2
    • Protein or peptide: RNA-directed RNA polymerase

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Supramolecule #1: Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])

SupramoleculeName: Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])
type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / Details: TLP / NCBI-ID: 444185
Sci species name: Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])
Sci species strain: RVA/Monkey/United States/RRV/1975/G3P5B[3]
Virus type: VIRION / Virus isolate: SEROTYPE / Virus enveloped: No / Virus empty: No

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Macromolecule #1: Inner capsid protein VP2

MacromoleculeName: Inner capsid protein VP2 / type: protein_or_peptide / ID: 1 / Number of copies: 10 / Enantiomer: LEVO
Source (natural)Organism: Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])
Strain: RVA/Monkey/United States/RRV/1975/G3P5B[3]
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 #2: RNA-directed RNA polymerase

MacromoleculeName: RNA-directed RNA polymerase / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed RNA polymerase
Source (natural)Organism: Rotavirus A (strain RVA/Monkey/United States/RRV/1975/G3P5B[3])
Strain: RVA/Monkey/United States/RRV/1975/G3P5B[3]
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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI POLARA 300
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 64.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.01) / Software - details: FrealignX / Number images used: 30606
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-6oj3:
In situ structure of rotavirus VP1 RNA-dependent RNA polymerase (TLP)

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