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- EMDB-40642: Cryo-EM structure of the respiratory syncytial virus polymerase (... -

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Basic information

Entry
Database: EMDB / ID: EMD-40642
TitleCryo-EM structure of the respiratory syncytial virus polymerase (L:P) bound to the trailer complementary promoter
Map data
Sample
  • Complex: The respiratory syncytial virus polymerase (L:P) bound to the trailer complementary promoter
    • Protein or peptide: RNA-directed RNA polymerase L
    • Protein or peptide: Phosphoprotein
    • RNA: RNA (5'-R(*UP*UP*UP*UP*UP*CP*UP*CP*GP*U)-3')
KeywordsRespiratory syncytial virus / RNA-dependent RNA polymerase / VIRAL PROTEIN-RNA complex
Function / homology
Function and homology information


NNS virus cap methyltransferase / GDP polyribonucleotidyltransferase / negative stranded viral RNA replication / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / virion component / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / host cell cytoplasm / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / GTPase activity ...NNS virus cap methyltransferase / GDP polyribonucleotidyltransferase / negative stranded viral RNA replication / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / virion component / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / host cell cytoplasm / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / GTPase activity / ATP binding / metal ion binding
Similarity search - Function
RNA-directed RNA polymerase, paramyxovirus / Phosphoprotein, pneumoviral / Pneumovirus phosphoprotein / RNA-directed RNA polymerase L methyltransferase domain, rhabdovirus / Virus-capping methyltransferase, MT domain / Mononegavirales RNA-directed RNA polymerase catalytic domain / Mononegavirus L protein 2-O-ribose methyltransferase / Mononegavirales mRNA-capping domain V / RNA-directed RNA polymerase L, C-terminal / Mononegavirales RNA dependent RNA polymerase ...RNA-directed RNA polymerase, paramyxovirus / Phosphoprotein, pneumoviral / Pneumovirus phosphoprotein / RNA-directed RNA polymerase L methyltransferase domain, rhabdovirus / Virus-capping methyltransferase, MT domain / Mononegavirales RNA-directed RNA polymerase catalytic domain / Mononegavirus L protein 2-O-ribose methyltransferase / Mononegavirales mRNA-capping domain V / RNA-directed RNA polymerase L, C-terminal / Mononegavirales RNA dependent RNA polymerase / Mononegavirales mRNA-capping region V / RdRp of negative ssRNA viruses with non-segmented genomes catalytic domain profile. / Mononegavirus L protein 2'-O-ribose methyltransferase domain profile.
Similarity search - Domain/homology
Phosphoprotein / RNA-directed RNA polymerase L
Similarity search - Component
Biological speciesRespiratory syncytial virus A2
Methodsingle particle reconstruction / cryo EM / Resolution: 3.41 Å
AuthorsCao D / Gao Y / Chen Z / Gooneratne I / Roesler C / Mera C / Liang B
Funding support United States, 5 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM130950 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM116788 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM116789 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM129539 United States
Simons FoundationSF349247 United States
CitationJournal: Nature / Year: 2024
Title: Structures of the promoter-bound respiratory syncytial virus polymerase.
Authors: Dongdong Cao / Yunrong Gao / Zhenhang Chen / Inesh Gooneratne / Claire Roesler / Cristopher Mera / Paul D'Cunha / Anna Antonova / Deepak Katta / Sarah Romanelli / Qi Wang / Samantha Rice / ...Authors: Dongdong Cao / Yunrong Gao / Zhenhang Chen / Inesh Gooneratne / Claire Roesler / Cristopher Mera / Paul D'Cunha / Anna Antonova / Deepak Katta / Sarah Romanelli / Qi Wang / Samantha Rice / Wesley Lemons / Anita Ramanathan / Bo Liang /
Abstract: The respiratory syncytial virus (RSV) polymerase is a multifunctional RNA-dependent RNA polymerase composed of the large (L) protein and the phosphoprotein (P). It transcribes the RNA genome into ten ...The respiratory syncytial virus (RSV) polymerase is a multifunctional RNA-dependent RNA polymerase composed of the large (L) protein and the phosphoprotein (P). It transcribes the RNA genome into ten viral mRNAs and replicates full-length viral genomic and antigenomic RNAs. The RSV polymerase initiates RNA synthesis by binding to the conserved 3'-terminal RNA promoters of the genome or antigenome. However, the lack of a structure of the RSV polymerase bound to the RNA promoter has impeded the mechanistic understanding of RSV RNA synthesis. Here we report cryogenic electron microscopy structures of the RSV polymerase bound to its genomic and antigenomic viral RNA promoters, representing two of the first structures of an RNA-dependent RNA polymerase in complex with its RNA promoters in non-segmented negative-sense RNA viruses. The overall structures of the promoter-bound RSV polymerases are similar to that of the unbound (apo) polymerase. Our structures illustrate the interactions between the RSV polymerase and the RNA promoters and provide the structural basis for the initiation of RNA synthesis at positions 1 and 3 of the RSV promoters. These structures offer a deeper understanding of the pre-initiation state of the RSV polymerase and could aid in antiviral research against RSV.
History
DepositionApr 28, 2023-
Header (metadata) releaseDec 20, 2023-
Map releaseDec 20, 2023-
UpdateJan 31, 2024-
Current statusJan 31, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40642.png

Downloads & links

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Map

FileDownload / File: emd_40642.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.058 Å
Density
Contour LevelBy AUTHOR: 0.0155
Minimum - Maximum-0.05825679 - 0.09829023
Average (Standard dev.)0.00015834754 (±0.004962307)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 211.60063 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_40642_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_40642_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : The respiratory syncytial virus polymerase (L:P) bound to the tra...

EntireName: The respiratory syncytial virus polymerase (L:P) bound to the trailer complementary promoter
Components
  • Complex: The respiratory syncytial virus polymerase (L:P) bound to the trailer complementary promoter
    • Protein or peptide: RNA-directed RNA polymerase L
    • Protein or peptide: Phosphoprotein
    • RNA: RNA (5'-R(*UP*UP*UP*UP*UP*CP*UP*CP*GP*U)-3')

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Supramolecule #1: The respiratory syncytial virus polymerase (L:P) bound to the tra...

SupramoleculeName: The respiratory syncytial virus polymerase (L:P) bound to the trailer complementary promoter
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Respiratory syncytial virus A2

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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: Respiratory syncytial virus A2
Molecular weightTheoretical: 250.704484 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDPIINGNSA NVYLTDSYLK GVISFSECNA LGSYIFNGPY LKNDYTNLIS RQNPLIEHMN LKKLNITQSL ISKYHKGEIK LEEPTYFQS LLMTYKSMTS SEQIATTNLL KKIIRRAIEI SDVKVYAILN KLGLKEKDKI KSNNGQDEDN SVITTIIKDD I LSAVKDNQ ...String:
MDPIINGNSA NVYLTDSYLK GVISFSECNA LGSYIFNGPY LKNDYTNLIS RQNPLIEHMN LKKLNITQSL ISKYHKGEIK LEEPTYFQS LLMTYKSMTS SEQIATTNLL KKIIRRAIEI SDVKVYAILN KLGLKEKDKI KSNNGQDEDN SVITTIIKDD I LSAVKDNQ SHLKADKNHS TKQKDTIKTT LLKKLMCSMQ HPPSWLIHWF NLYTKLNNIL TQYRSNEVKN HGFTLIDNQT LS GFQFILN QYGCIVYHKE LKRITVTTYN QFLTWKDISL SRLNVCLITW ISNCLNTLNK SLGLRCGFNN VILTQLFLYG DCI LKLFHN EGFYIIKEVE GFIMSLILNI TEEDQFRKRF YNSMLNNITD AANKAQKNLL SRVCHTLLDK TVSDNIINGR WIIL LSKFL KLIKLAGDNN LNNLSELYFL FRIFGHPMVD ERQAMDAVKI NCNETKFYLL SSLSMLRGAF IYRIIKGFVN NYNRW PTLR NAIVLPLRWL TYYKLNTYPS LLELTERDLI VLSGLRFYRE FRLPKKVDLE MIINDKAISP PKNLIWTSFP RNYMPS HIQ NYIEHEKLKF SESDKSRRVL EYYLRDNKFN ECDLYNCVVN QSYLNNPNHV VSLTGKEREL SVGRMFAMQP GMFRQVQ IL AEKMIAENIL QFFPESLTRY GDLELQKILE LKAGISNKSN RYNDNYNNYI SKCSIITDLS KFNQAFRYET SCICSDVL D ELHGVQSLFS WLHLTIPHVT IICTYRHAPP YIGDHIVDLN NVDEQSGLYR YHMGGIEGWC QKLWTIEAIS LLDLISLKG KFSITALING DNQSIDISKP IRLMEGQTHA QADYLLALNS LKLLYKEYAG IGHKLKGTET YISRDMQFMS KTIQHNGVYY PASIKKVLR VGPWINTILD DFKVSLESIG SLTQELEYRG ESLLCSLIFR NVWLYNQIAL QLKNHALCNN KLYLDILKVL K HLKTFFNL DNIDTALTLY MNLPMLFGGG DPNLLYRSFY RRTPDFLTEA IVHSVFILSY YTNHDLKDKL QDLSDDRLNK FL TCIITFD KNPNAEFVTL MRDPQALGSE RQAKITSEIN RLAVTEVLST APNKIFSKSA QHYTTTEIDL NDIMQNIEPT YPH GLRVVY ESLPFYKAEK IVNLISGTKS ITNILEKTSA IDLTDIDRAT EMMRKNITLL IRILPLDCNR DKREILSMEN LSIT ELSKY VRERSWSLSN IVGVTSPSIM YTMDIKYTTS TISSGIIIEK YNVNSLTRGE RGPTKPWVGS STQEKKTMPV YNRQV LTKK QRDQIDLLAK LDWVYASIDN KDEFMEELSI GTLGLTYEKA KKLFPQYLSV NYLHRLTVSS RPCEFPASIP AYRTTN YHF DTSPINRILT EKYGDEDIDI VFQNCISFGL SLMSVVEQFT NVCPNRIILI PKLNEIHLMK PPIFTGDVDI HKLKQVI QK QHMFLPDKIS LTQYVELFLS NKTLKSGSHV NSNLILAHKI SDYFHNTYIL STNLAGHWIL IIQLMKDSKG IFEKDWGE G YITDHMFINL KVFFNAYKTY LLCFHKGYGK AKLECDMNTS DLLCVLELID SSYWKSMSKV FLEQKVIKYI LSQDASLHR VKGCHSFKLW FLKRLNVAEF TVCPWVVNID YHPTHMKAIL TYIDLVRMGL INIDRIHIKN KHKFNDEFYT SNLFYINYNF SDNTHLLTK HIRIANSELE NNYNKLYHPT PETLENILAN PIKSNDKKTL NDYCIGKNVD SIMLPLLSNK KLIKSSAMIR T NYSKQDLY NLFPMVVIDR IIDHSGNTAK SNQLYTTTSH QISLVHNSTS LYCMLPWHHI NRFNFVFSST GCKISIEYIL KD LKIKDPN CIAFIGEGAG NLLLRTVVEL HPDIRYIYRS LKDCNDHSLP IEFLRLYNGH INIDYGENLT IPATDATNNI HWS YLHIKF AEPISLFVCD AELSVTVNWS KIIIEWSKHV RKCKYCSSVN KCMLIVKYHA QDDIDFKLDN ITILKTYVCL GSKL KGSEV YLVLTIGPAN IFPVFNVVQN AKLILSRTKN FIMPKKADKE SIDANIKSLI PFLCYPITKK GINTALSKLK SVVSG DILS YSIAGRNEVF SNKLINHKHM NILKWFNHVL NFRSTELNYN HLYMVESTYP YLSELLNSLT TNELKKLIKI TGSLLY NFH NE

UniProtKB: RNA-directed RNA polymerase L

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Macromolecule #2: Phosphoprotein

MacromoleculeName: Phosphoprotein / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Respiratory syncytial virus A2
Molecular weightTheoretical: 27.165838 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MEKFAPEFHG EDANNRATKF LESIKGKFTS PKDPKKKDSI ISVNSIDIEV TKESPITSNS TIINPTNETD DTAGNKPNYQ RKPLVSFKE DPTPSDNPFS KLYKETIETF DNNEEESSYS YEEINDQTND NITARLDRID EKLSEILGML HTLVVASAGP T SARDGIRD ...String:
MEKFAPEFHG EDANNRATKF LESIKGKFTS PKDPKKKDSI ISVNSIDIEV TKESPITSNS TIINPTNETD DTAGNKPNYQ RKPLVSFKE DPTPSDNPFS KLYKETIETF DNNEEESSYS YEEINDQTND NITARLDRID EKLSEILGML HTLVVASAGP T SARDGIRD AMVGLREEMI EKIRTEALMT NDRLEAMARL RNEESEKMAK DTSDEVSLNP TSEKLNNLLE GNDSDNDLSL ED F

UniProtKB: Phosphoprotein

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Macromolecule #3: RNA (5'-R(*UP*UP*UP*UP*UP*CP*UP*CP*GP*U)-3')

MacromoleculeName: RNA (5'-R(*UP*UP*UP*UP*UP*CP*UP*CP*GP*U)-3') / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Respiratory syncytial virus A2
Molecular weightTheoretical: 3.053772 KDa
SequenceString:
UUUUUCUCGU

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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

Concentration0.35 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293.15 K / Instrument: FEI VITROBOT MARK IV

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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 / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 56.86 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:

6uen

Initial angle assignmentType: COMMON LINE
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.41 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 197859
FSC plot (resolution estimation)

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