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- EMDB-29452: Structure Of Respiratory Syncytial Virus Polymerase with Novel No... -

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Basic information

Entry
Database: EMDB / ID: EMD-29452
TitleStructure Of Respiratory Syncytial Virus Polymerase with Novel Non-Nucleoside Inhibitor
Map data
Sample
  • Complex: Structure Of Respiratory Syncytial Virus Polymerase with Novel Non-Nucleoside Inhibitor
    • Protein or peptide: RNA-directed RNA polymerase L
    • Protein or peptide: Phosphoprotein
  • Ligand: 8-methoxy-3-methyl-N-{(2S)-3,3,3-trifluoro-2-[5-fluoro-6-(4-fluorophenyl)-4-(2-hydroxypropan-2-yl)pyridin-2-yl]-2-hydroxypropyl}cinnoline-6-carboxamide
KeywordsRSV / Polymerase / Non-Nucleoside Inhibitor / VIRAL PROTEIN / TRANSFERASE
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 / viral life cycle / virion component / : / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / host cell cytoplasm / RNA-directed RNA polymerase ...NNS virus cap methyltransferase / GDP polyribonucleotidyltransferase / negative stranded viral RNA replication / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / viral life cycle / 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 speciesHuman respiratory syncytial virus A2
Methodsingle particle reconstruction / cryo EM / Resolution: 2.88 Å
AuthorsYu X / Abeywickrema P / Bonneux B / Behera I / Jacoby E / Fung A / Adhikary S / Bhaumik A / Carbajo RJ / Bruyn SD ...Yu X / Abeywickrema P / Bonneux B / Behera I / Jacoby E / Fung A / Adhikary S / Bhaumik A / Carbajo RJ / Bruyn SD / Miller R / Patrick A / Pham Q / Piassek M / Verheyen N / Shareef A / Sutto-Ortiz P / Ysebaert N / Vlijmen HV / Jonckers THM / Herschke F / McLellan JS / Decroly E / Fearns R / Grosse S / Roymans D / Sharma S / Rigaux P / Jin Z
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Commun Biol / Year: 2023
Title: Structural and mechanistic insights into the inhibition of respiratory syncytial virus polymerase by a non-nucleoside inhibitor.
Authors: Xiaodi Yu / Pravien Abeywickrema / Brecht Bonneux / Ishani Behera / Brandon Anson / Edgar Jacoby / Amy Fung / Suraj Adhikary / Anusarka Bhaumik / Rodrigo J Carbajo / Suzanne De Bruyn / Robyn ...Authors: Xiaodi Yu / Pravien Abeywickrema / Brecht Bonneux / Ishani Behera / Brandon Anson / Edgar Jacoby / Amy Fung / Suraj Adhikary / Anusarka Bhaumik / Rodrigo J Carbajo / Suzanne De Bruyn / Robyn Miller / Aaron Patrick / Quyen Pham / Madison Piassek / Nick Verheyen / Afzaal Shareef / Priscila Sutto-Ortiz / Nina Ysebaert / Herman Van Vlijmen / Tim H M Jonckers / Florence Herschke / Jason S McLellan / Etienne Decroly / Rachel Fearns / Sandrine Grosse / Dirk Roymans / Sujata Sharma / Peter Rigaux / Zhinan Jin /
Abstract: The respiratory syncytial virus polymerase complex, consisting of the polymerase (L) and phosphoprotein (P), catalyzes nucleotide polymerization, cap addition, and cap methylation via the RNA ...The respiratory syncytial virus polymerase complex, consisting of the polymerase (L) and phosphoprotein (P), catalyzes nucleotide polymerization, cap addition, and cap methylation via the RNA dependent RNA polymerase, capping, and Methyltransferase domains on L. Several nucleoside and non-nucleoside inhibitors have been reported to inhibit this polymerase complex, but the structural details of the exact inhibitor-polymerase interactions have been lacking. Here, we report a non-nucleoside inhibitor JNJ-8003 with sub-nanomolar inhibition potency in both antiviral and polymerase assays. Our 2.9 Å resolution cryo-EM structure revealed that JNJ-8003 binds to an induced-fit pocket on the capping domain, with multiple interactions consistent with its tight binding and resistance mutation profile. The minigenome and gel-based de novo RNA synthesis and primer extension assays demonstrated that JNJ-8003 inhibited nucleotide polymerization at the early stages of RNA transcription and replication. Our results support that JNJ-8003 binding modulates a functional interplay between the capping and RdRp domains, and this molecular insight could accelerate the design of broad-spectrum antiviral drugs.
History
DepositionJan 16, 2023-
Header (metadata) releaseNov 1, 2023-
Map releaseNov 1, 2023-
UpdateNov 1, 2023-
Current statusNov 1, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29452.png

Downloads & links

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Map

FileDownload / File: emd_29452.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.82 Å
Density
Contour LevelBy EMDB: 0.02
Minimum - Maximum-0.092405625 - 0.15697375
Average (Standard dev.)0.0001585293 (±0.005537878)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 229.59999 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_29452_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_29452_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Structure Of Respiratory Syncytial Virus Polymerase with Novel No...

EntireName: Structure Of Respiratory Syncytial Virus Polymerase with Novel Non-Nucleoside Inhibitor
Components
  • Complex: Structure Of Respiratory Syncytial Virus Polymerase with Novel Non-Nucleoside Inhibitor
    • Protein or peptide: RNA-directed RNA polymerase L
    • Protein or peptide: Phosphoprotein
  • Ligand: 8-methoxy-3-methyl-N-{(2S)-3,3,3-trifluoro-2-[5-fluoro-6-(4-fluorophenyl)-4-(2-hydroxypropan-2-yl)pyridin-2-yl]-2-hydroxypropyl}cinnoline-6-carboxamide

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Supramolecule #1: Structure Of Respiratory Syncytial Virus Polymerase with Novel No...

SupramoleculeName: Structure Of Respiratory Syncytial Virus Polymerase with Novel Non-Nucleoside Inhibitor
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Human 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: Human respiratory syncytial virus A2
Molecular weightTheoretical: 254.48275 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGSWSHPQFE KGSGSGSSWS HPQFEKGSGS LVPRGSMDPI INGNSANVYL TDSYLKGVIS FSECNALGSY IFNGPYLKND YTNLISRQN PLIEHMNLKK LNITQSLISK YHKGEIKLEE PTYFQSLLMT YKSMTSSEQI ATTNLLKKII RRAIEISDVK V YAILNKLG ...String:
MGSWSHPQFE KGSGSGSSWS HPQFEKGSGS LVPRGSMDPI INGNSANVYL TDSYLKGVIS FSECNALGSY IFNGPYLKND YTNLISRQN PLIEHMNLKK LNITQSLISK YHKGEIKLEE PTYFQSLLMT YKSMTSSEQI ATTNLLKKII RRAIEISDVK V YAILNKLG LKEKDKIKSN NGQDEDNSVI TTIIKDDILS AVKDNQSHLK ADKNHSTKQK DTIKTTLLKK LMCSMQHPPS WL IHWFNLY TKLNNILTQY RSNEVKNHGF TLIDNQTLSG FQFILNQYGC IVYHKELKRI TVTTYNQFLT WKDISLSRLN VCL ITWISN CLNTLNKSLG LRCGFNNVIL TQLFLYGDCI LKLFHNEGFY IIKEVEGFIM SLILNITEED QFRKRFYNSM LNNI TDAAN KAQKNLLSRV CHTLLDKTVS DNIINGRWII LLSKFLKLIK LAGDNNLNNL SELYFLFRIF GHPMVDERQA MDAVK INCN ETKFYLLSSL SMLRGAFIYR IIKGFVNNYN RWPTLRNAIV LPLRWLTYYK LNTYPSLLEL TERDLIVLSG LRFYRE FRL PKKVDLEMII NDKAISPPKN LIWTSFPRNY MPSHIQNYIE HEKLKFSESD KSRRVLEYYL RDNKFNECDL YNCVVNQ SY LNNPNHVVSL TGKERELSVG RMFAMQPGMF RQVQILAEKM IAENILQFFP ESLTRYGDLE LQKILELKAG ISNKSNRY N DNYNNYISKC SIITDLSKFN QAFRYETSCI CSDVLDELHG VQSLFSWLHL TIPHVTIICT YRHAPPYIGD HIVDLNNVD EQSGLYRYHM GGIEGWCQKL WTIEAISLLD LISLKGKFSI TALINGDNQS IDISKPIRLM EGQTHAQADY LLALNSLKLL YKEYAGIGH KLKGTETYIS RDMQFMSKTI QHNGVYYPAS IKKVLRVGPW INTILDDFKV SLESIGSLTQ ELEYRGESLL C SLIFRNVW LYNQIALQLK NHALCNNKLY LDILKVLKHL KTFFNLDNID TALTLYMNLP MLFGGGDPNL LYRSFYRRTP DF LTEAIVH SVFILSYYTN HDLKDKLQDL SDDRLNKFLT CIITFDKNPN AEFVTLMRDP QALGSERQAK ITSEINRLAV TEV LSTAPN KIFSKSAQHY TTTEIDLNDI MQNIEPTYPH GLRVVYESLP FYKAEKIVNL ISGTKSITNI LEKTSAIDLT DIDR ATEMM RKNITLLIRI LPLDCNRDKR EILSMENLSI TELSKYVRER SWSLSNIVGV TSPSIMYTMD IKYTTSTISS GIIIE KYNV NSLTRGERGP TKPWVGSSTQ EKKTMPVYNR QVLTKKQRDQ IDLLAKLDWV YASIDNKDEF MEELSIGTLG LTYEKA KKL FPQYLSVNYL HRLTVSSRPC EFPASIPAYR TTNYHFDTSP INRILTEKYG DEDIDIVFQN CISFGLSLMS VVEQFTN VC PNRIILIPKL NEIHLMKPPI FTGDVDIHKL KQVIQKQHMF LPDKISLTQY VELFLSNKTL KSGSHVNSNL ILAHKISD Y FHNTYILSTN LAGHWILIIQ LMKDSKGIFE KDWGEGYITD HMFINLKVFF NAYKTYLLCF HKGYGKAKLE CDMNTSDLL CVLELIDSSY WKSMSKVFLE QKVIKYILSQ DASLHRVKGC HSFKLWFLKR LNVAEFTVCP WVVNIDYHPT HMKAILTYID LVRMGLINI DRIHIKNKHK FNDEFYTSNL FYINYNFSDN THLLTKHIRI ANSELENNYN KLYHPTPETL ENILANPIKS N DKKTLNDY CIGKNVDSIM LPLLSNKKLI KSSAMIRTNY SKQDLYNLFP MVVIDRIIDH SGNTAKSNQL YTTTSHQISL VH NSTSLYC MLPWHHINRF NFVFSSTGCK ISIEYILKDL KIKDPNCIAF IGEGAGNLLL RTVVELHPDI RYIYRSLKDC NDH SLPIEF LRLYNGHINI DYGENLTIPA TDATNNIHWS YLHIKFAEPI SLFVCDAELS VTVNWSKIII EWSKHVRKCK YCSS VNKCM LIVKYHAQDD IDFKLDNITI LKTYVCLGSK LKGSEVYLVL TIGPANIFPV FNVVQNAKLI LSRTKNFIMP KKADK ESID ANIKSLIPFL CYPITKKGIN TALSKLKSVV SGDILSYSIA GRNEVFSNKL INHKHMNILK WFNHVLNFRS TELNYN HLY MVESTYPYLS ELLNSLTTNE LKKLIKITGS LLYNFHNE

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: Human respiratory syncytial virus A2
Molecular weightTheoretical: 29.062895 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 AMIGLREEMI EKIRTEALMT NDRLEAMARL RNEESEKMAK DTSDEVSLNP TSEKLNNLLE GNDSDNDLSL ED FKGENKY FQGHHHHHH

UniProtKB: Phosphoprotein

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Macromolecule #3: 8-methoxy-3-methyl-N-{(2S)-3,3,3-trifluoro-2-[5-fluoro-6-(4-fluor...

MacromoleculeName: 8-methoxy-3-methyl-N-{(2S)-3,3,3-trifluoro-2-[5-fluoro-6-(4-fluorophenyl)-4-(2-hydroxypropan-2-yl)pyridin-2-yl]-2-hydroxypropyl}cinnoline-6-carboxamide
type: ligand / ID: 3 / Number of copies: 1 / Formula: YBK
Molecular weightTheoretical: 576.515 Da
Chemical component information

ChemComp-YBK:
8-methoxy-3-methyl-N-{(2S)-3,3,3-trifluoro-2-[5-fluoro-6-(4-fluorophenyl)-4-(2-hydroxypropan-2-yl)pyridin-2-yl]-2-hydroxypropyl}cinnoline-6-carboxamide

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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
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 / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 52.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.88 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 519118
FSC plot (resolution estimation)

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