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- EMDB-20536: Cryo-EM Structure of the Respiratory Syncytial Virus Polymerase (... -

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Basic information

Entry
Database: EMDB / ID: EMD-20536
TitleCryo-EM Structure of the Respiratory Syncytial Virus Polymerase (L) Protein Bound by the Tetrameric Phosphoprotein (P)
Map dataSharpened map after non-uniform refinement in cryoSPARC
Sample
  • Complex: Respiratory Syncytial Virus Polymerase (L) Protein Bound by the Tetrameric Phosphoprotein (P)
    • Protein or peptide: RNA-directed RNA polymerase L
    • Protein or peptide: Phosphoprotein
KeywordsRNA-binding protein / RSV / RdRp / RNA-dependent RNA polymerase / PRNTase / polyribonucleotidyl transferase / RNA capping / viral replication / VIRAL PROTEIN
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: 3.2 Å
AuthorsGilman MSA / McLellan JS
CitationJournal: Cell / Year: 2019
Title: Structure of the Respiratory Syncytial Virus Polymerase Complex.
Authors: Morgan S A Gilman / Cheng Liu / Amy Fung / Ishani Behera / Paul Jordan / Peter Rigaux / Nina Ysebaert / Sergey Tcherniuk / Julien Sourimant / Jean-François Eléouët / Priscila Sutto-Ortiz ...Authors: Morgan S A Gilman / Cheng Liu / Amy Fung / Ishani Behera / Paul Jordan / Peter Rigaux / Nina Ysebaert / Sergey Tcherniuk / Julien Sourimant / Jean-François Eléouët / Priscila Sutto-Ortiz / Etienne Decroly / Dirk Roymans / Zhinan Jin / Jason S McLellan /
Abstract: Numerous interventions are in clinical development for respiratory syncytial virus (RSV) infection, including small molecules that target viral transcription and replication. These processes are ...Numerous interventions are in clinical development for respiratory syncytial virus (RSV) infection, including small molecules that target viral transcription and replication. These processes are catalyzed by a complex comprising the RNA-dependent RNA polymerase (L) and the tetrameric phosphoprotein (P). RSV P recruits multiple proteins to the polymerase complex and, with the exception of its oligomerization domain, is thought to be intrinsically disordered. Despite their critical roles in RSV transcription and replication, structures of L and P have remained elusive. Here, we describe the 3.2-Å cryo-EM structure of RSV L bound to tetrameric P. The structure reveals a striking tentacular arrangement of P, with each of the four monomers adopting a distinct conformation. The structure also rationalizes inhibitor escape mutants and mutations observed in live-attenuated vaccine candidates. These results provide a framework for determining the molecular underpinnings of RSV replication and transcription and should facilitate the design of effective RSV inhibitors.
History
DepositionAug 1, 2019-
Header (metadata) releaseSep 11, 2019-
Map releaseSep 11, 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: 0.35
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.35
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6pzk
  • Surface level: 0.35
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20536.png

Downloads & links

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Map

FileDownload / File: emd_20536.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map after non-uniform refinement in cryoSPARC
Voxel sizeX=Y=Z: 1.075 Å
Density
Contour LevelBy AUTHOR: 0.35 / Movie #1: 0.35
Minimum - Maximum-2.3509736 - 3.9801807
Average (Standard dev.)0.00022415227 (±0.08759908)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 275.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0751.0751.075
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z275.200275.200275.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-2.3513.9800.000

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Supplemental data

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Additional map: Unsharpened map after non-uniform refinement in cryoSPARC

Fileemd_20536_additional.map
AnnotationUnsharpened map after non-uniform refinement in cryoSPARC
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Respiratory Syncytial Virus Polymerase (L) Protein Bound by the T...

EntireName: Respiratory Syncytial Virus Polymerase (L) Protein Bound by the Tetrameric Phosphoprotein (P)
Components
  • Complex: Respiratory Syncytial Virus Polymerase (L) Protein Bound by the Tetrameric Phosphoprotein (P)
    • Protein or peptide: RNA-directed RNA polymerase L
    • Protein or peptide: Phosphoprotein

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Supramolecule #1: Respiratory Syncytial Virus Polymerase (L) Protein Bound by the T...

SupramoleculeName: Respiratory Syncytial Virus Polymerase (L) Protein Bound by the Tetrameric Phosphoprotein (P)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Human respiratory syncytial virus A2
Molecular weightTheoretical: 370 KDa

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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 / Strain: 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 / Strain: 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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.29 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
35.0 mMNH2C(CH2OH)3-HClTris-HClTris
350.0 mMNaClSodium chloridesodium chloride
0.5 mMC9H15O6P-HClTCEP
5.0 %C3H8O3glycerol
GridMaterial: GOLD
VitrificationCryogen name: ETHANE / 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
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 48.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Ab initio model generated using cryoSPARC.
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 196720
FSC plot (resolution estimation)

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