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- EMDB-31077: Structure of Rift Valley fever virus RNA-dependent RNA polymerase -

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Basic information

Entry
Database: EMDB / ID: EMD-31077
TitleStructure of Rift Valley fever virus RNA-dependent RNA polymerase
Map data
Sample
  • Complex: Structural insights into Rift Valley fever virus replication machinery
    • Protein or peptide: ReplicaseRNA-dependent RNA polymerase
Function / homology
Function and homology information


nucleoside binding / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / DNA-templated transcription
Similarity search - Function
RNA-directed RNA polymerase, phlebovirus / RNA-directed RNA polymerase L, PA-C-like domain / RNA-directed RNA polymerase L, PA-C-like domain / RNA-directed RNA polymerase L, N-terminal / L protein N-terminus / 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
Biological speciesRift Valley fever virus / Rift valley fever virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsWang X / Hu CX
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: J Virol / Year: 2022
Title: Structure of Rift Valley Fever Virus RNA-Dependent RNA Polymerase.
Authors: Xue Wang / Cuixia Hu / Wei Ye / Jia Wang / Xiaofei Dong / Jie Xu / Xiaorong Li / Manfeng Zhang / Hongyun Lu / Fanglin Zhang / Wei Wu / Shaodong Dai / Hong-Wei Wang / Zhongzhou Chen /
Abstract: Rift Valley fever virus (RVFV) belongs to the order and is the type species of genus , which accounts for over 50% of family species. RVFV is mosquito-borne and causes severe diseases in both ...Rift Valley fever virus (RVFV) belongs to the order and is the type species of genus , which accounts for over 50% of family species. RVFV is mosquito-borne and causes severe diseases in both humans and livestock, and consists of three segments (S, M, L) in the genome. The L segment encodes an RNA-dependent RNA polymerase (RdRp, L protein) that is responsible for facilitating the replication and transcription of the virus. It is essential for the virus and has multiple drug targets. Here, we established an expression system and purification procedures for full-length L protein, which is composed of an endonuclease domain, RdRp domain, and cap-binding domain. A cryo-EM L protein structure was reported at 3.6 Å resolution. In this first L protein structure of genus , the priming loop of RVFV L protein is distinctly different from those of other L proteins and undergoes large movements related to its replication role. Structural and biochemical analyses indicate that a single template can induce initiation of RNA synthesis, which is notably enhanced by 5' viral RNA. These findings help advance our understanding of the mechanism of RNA synthesis and provide an important basis for developing antiviral inhibitors. The zoonosis RVF virus (RVFV) is one of the most serious arbovirus threats to both human and animal health. RNA-dependent RNA polymerase (RdRp) is a multifunctional enzyme catalyzing genome replication as well as viral transcription, so the RdRp is essential for studying the virus and has multiple drug targets. In our study, we report the structure of RVFV L protein at 3.6 Å resolution by cryo-EM. This is the first L protein structure of genus . Strikingly, a single template can initiate RNA replication. The structure and assays provide a comprehensive and in-depth understanding of the catalytic and substrate recognition mechanism of RdRp.
History
DepositionMar 18, 2021-
Header (metadata) releaseNov 17, 2021-
Map releaseNov 17, 2021-
UpdateMar 16, 2022-
Current statusMar 16, 2022Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7eei
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_31077.png

Downloads & links

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Map

FileDownload / File: emd_31077.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.018 / Movie #1: 0.018
Minimum - Maximum-0.05007629 - 0.097795956
Average (Standard dev.)0.0005041874 (±0.0043277643)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 172.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z172.800172.800172.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ256256256
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.0500.0980.001

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Supplemental data

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Sample components

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Entire : Structural insights into Rift Valley fever virus replication machinery

EntireName: Structural insights into Rift Valley fever virus replication machinery
Components
  • Complex: Structural insights into Rift Valley fever virus replication machinery
    • Protein or peptide: ReplicaseRNA-dependent RNA polymerase

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Supramolecule #1: Structural insights into Rift Valley fever virus replication machinery

SupramoleculeName: Structural insights into Rift Valley fever virus replication machinery
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Rift Valley fever virus
Recombinant expressionOrganism: Pichia aff. alni PL5W1 (fungus)
Molecular weightExperimental: 238 KDa

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Macromolecule #1: Replicase

MacromoleculeName: Replicase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed RNA polymerase
Source (natural)Organism: Rift valley fever virus
Molecular weightTheoretical: 202.260734 KDa
Recombinant expressionOrganism: Pichia aff. alni PL5W1 (fungus)
SequenceString: GKTERELLAM VSSIQINWSV TESVFPPFSR EMFDRFRSSP PDSEYITRIV SRCLINSQEK LINSSFFAEG NDKALRFSKN AEECSLAVE RALNQYRAED NLRDLNDHSS TIQLPPWLSY HDVDGKDLCP LQGLDVRGDH PMCNLWREVV TSANLEEIER M HDDAAAEL ...String:
GKTERELLAM VSSIQINWSV TESVFPPFSR EMFDRFRSSP PDSEYITRIV SRCLINSQEK LINSSFFAEG NDKALRFSKN AEECSLAVE RALNQYRAED NLRDLNDHSS TIQLPPWLSY HDVDGKDLCP LQGLDVRGDH PMCNLWREVV TSANLEEIER M HDDAAAEL EFALSGVKDR PDERNRYHRV HLNMGSDDSV YIAALGVNGK KHKADTLVQQ MRDRSKQPFS PDHDVDHISE FL SACSSDL WATDEDLYNP LSCDKELRLA AQRIHQPSLS ERGFNEIITE HYKFMGSRIG SWCQMVSLIG AELSASVKQH VKP NYFVIK RLLGSGIFLL IKPTSSKSHI FVSFAIKRSC WAFDLSTSRV FKPYIDAGDL LVTDFVSYKL SKLTNLCKCV SLME SSFSF WAEAFGIPSW NFVGDLFRSS DSAAMDASYM GKLSLLTLLE DKAATEELQT IARYIIMEGF VSPPEIPKPH KMTSK FPKV LRSELQVYLL NCLCRTIQRI AGEPFILKKK DGSISWGGMF NPFSGRPLLD MQPLISCCYN GYFKNKEEET EPSSLS GMY KKIIELEHLR PQSDAFLGYK DPELPRMHEF SVSYLKEACN HAKLVLRSLY GQNFMEQIDN QIIRELSGLT LERLATL KA TSNFNENWYV YKDVADKNYT RDKLLVKMSK YASEGKSLAI QKFEDCMRQI ESQGCMHICL FKKQQHGGLR EIYVMGAE E RIVQSVVETI ARSIGKFFAS DTLCNPPNKV KIPETHGIRA RKQCKGPVWT CATSDDARKW NQGHFVTKFA LMLCEFTSP KWWPLIIRGC SMFTRKRMMM NLNYLKILDG HRELDIRDDF VMDLFKAYHG EAEVPWAFKG KTYLETTTGM MQGILHYTSS LLHTIHQEY IRSLSFKIFN LKVAPEMSKG LVCDMMQGSD DSSMLISFPA DDEKVLTRCK VAAAICFRMK KELGVYLAIY P SEKSTANT DFVMEYNSEF YFHTQHVRPT IRWIAACCSL PEVETLVARQ EEASNLMTSV TEGGGSFSLA AMIQQAQCTL HY MLMGMGV SELFLEYKKA VLKWNDPGLG FFLLDNPYAC GLGGFRFNLF KAITRTDLQK LYAFFMKKVK GSAARDWADE DVT IPETCS VSPGGALILS SSLKWGSRKK FQKLRDRLNI PENWIELINE NPEVLYRAPR TGPEILLRIA EKVHSPGVVS SLSS GNAVC KVMASAVYFL SATIFEDTGR PEFNFLEDSK YSLLQKMAAY SGFHGFNDME PEDILFLFPN IEELESLDSI VYNKG EIDI IPRVNIRDAT QTRVTIFNEQ KTLRTSPEKL VSDKWFGTQK SRIGKTTFLA EWEKLKKIVK WLEDTPEATL AHTPLN NHI QVRNFFARME SKPRTVRITG APVKKRSGVS KIAMVIRDNF SRMGHLRGVE DLAGFTRSVS AEILKHFLFC ILQGPYS ES YKLQLIYRVL SSVSNVEIKE SDGKTKTNLI GILQRFLDGD HVVPIIEEMG AGTVGGFIKR QQSKVVQNKV VYYGVGIW R GFMDGYQVHL EIENDIGQPP RLRNVTTNCQ SSPWDLSIPI RQWAEDMGVT NNQDYSSKSS RGARYWMHSF RMQGPSKPF GCPVYIIKGD MSDVIRLRKE EVEMKVRGST LNLYTKHHSH QDLHILSYTA SDNDLSPGIF KSISDEGVAQ ALQLFEREPS NCWVRCESV APKFISAILE ICEGKRQIRG INRTRLSEIV RICSESSLRS KVGSMFSFVA NVEEAHDVDY DALMDLMIED A KNNAFSHV VDCIELDV

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation state3D array

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 8.5
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
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 554102
FSC plot (resolution estimation)

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