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- PDB-6u5o: Structure of the Human Metapneumovirus Polymerase bound to the ph... -

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Basic information

Entry
Database: PDB / ID: 6u5o
TitleStructure of the Human Metapneumovirus Polymerase bound to the phosphoprotein tetramer
Components
  • Phosphoprotein
  • RNA-directed RNA polymerase L
KeywordsVIRAL PROTEIN / human metapneumovirus / HMPV / Polymerase / Phosphoprotein / Respiratory syncytial virus / RSV / pneumoviridae / Mononegavirales
Function / homology
Function and homology information


NNS virus cap methyltransferase / GDP polyribonucleotidyltransferase / 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
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 ...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
RNA-directed RNA polymerase L / Phosphoprotein
Similarity search - Component
Biological speciesHuman metapneumovirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsPan, J. / Qian, X. / Lattmann, S. / Sahili, A.E. / Yeo, T.H. / Kalocsay, M. / Fearns, R. / Lescar, J.
Funding support Singapore, United States, 3items
OrganizationGrant numberCountry
Ministry of Education (MoE, Singapore)MOE2016-T3-1-003 Singapore
National Research Foundation (NRF, Singapore)NRF2016NRF-CRP001-063 Singapore
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)AI089618 United States
CitationJournal: Nature / Year: 2020
Title: Structure of the human metapneumovirus polymerase phosphoprotein complex.
Authors: Junhua Pan / Xinlei Qian / Simon Lattmann / Abbas El Sahili / Tiong Han Yeo / Huan Jia / Tessa Cressey / Barbara Ludeke / Sarah Noton / Marian Kalocsay / Rachel Fearns / Julien Lescar /
Abstract: Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) cause severe respiratory diseases in infants and elderly adults. No vaccine or effective antiviral therapy currently exists to ...Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) cause severe respiratory diseases in infants and elderly adults. No vaccine or effective antiviral therapy currently exists to control RSV or HMPV infections. During viral genome replication and transcription, the tetrameric phosphoprotein P serves as a crucial adaptor between the ribonucleoprotein template and the L protein, which has RNA-dependent RNA polymerase (RdRp), GDP polyribonucleotidyltransferase and cap-specific methyltransferase activities. How P interacts with L and mediates the association with the free form of N and with the ribonucleoprotein is not clear for HMPV or other major human pathogens, including the viruses that cause measles, Ebola and rabies. Here we report a cryo-electron microscopy reconstruction that shows the ring-shaped structure of the polymerase and capping domains of HMPV-L bound to a tetramer of P. The connector and methyltransferase domains of L are mobile with respect to the core. The putative priming loop that is important for the initiation of RNA synthesis is fully retracted, which leaves space in the active-site cavity for RNA elongation. P interacts extensively with the N-terminal region of L, burying more than 4,016 Å of the molecular surface area in the interface. Two of the four helices that form the coiled-coil tetramerization domain of P, and long C-terminal extensions projecting from these two helices, wrap around the L protein in a manner similar to tentacles. The structural versatility of the four P protomers-which are largely disordered in their free state-demonstrates an example of a 'folding-upon-partner-binding' mechanism for carrying out P adaptor functions. The structure shows that P has the potential to modulate multiple functions of L and these results should accelerate the design of specific antiviral drugs.
History
DepositionAug 28, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 25, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 4, 2019Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Jan 22, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year
Revision 1.4Mar 20, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
L: RNA-directed RNA polymerase L
P: Phosphoprotein
Q: Phosphoprotein
R: Phosphoprotein
S: Phosphoprotein


Theoretical massNumber of molelcules
Total (without water)376,6305
Polymers376,6305
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein RNA-directed RNA polymerase L / Protein L / Large structural protein / Replicase / Transcriptase


Mass: 233889.562 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: amino acid residues 1-25 is the N-terminal expression/purification tag containing a Strep II tag, a linker and a TEV protease cleavage site.
Source: (gene. exp.) Human metapneumovirus (strain CAN97-83)
Strain: CAN97-83 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): IPLB-Sf21-AE
References: UniProt: Q6WB93, RNA-directed RNA polymerase, mRNA (guanine-N7)-methyltransferase, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases, GDP polyribonucleotidyltransferase
#2: Protein
Phosphoprotein / / Protein P


Mass: 35685.066 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human metapneumovirus (strain CAN97-83)
Strain: CAN97-83 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): IPLB-Sf21-AE / References: UniProt: Q8B9Q8

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Human metapneumovirus CAN97-83 / Type: COMPLEX
Details: Human metapneumovirus L and P genes were co-expressed in sf9 insect cells using recombinant baculovirus expression system. The protein complex were affinity purified using nickel-NTA resin, ...Details: Human metapneumovirus L and P genes were co-expressed in sf9 insect cells using recombinant baculovirus expression system. The protein complex were affinity purified using nickel-NTA resin, followed by purification on a HiTrap Heparin column and a superpose 6 size exclusion column.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.36 MDa / Experimental value: YES
Source (natural)Organism: Human metapneumovirus CAN97-83
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: IPLB-Sf21-AE / Cell: sf9 / Plasmid: pFastBacDual
Natural hostOrganism: homo sa
Buffer solutionpH: 7.4
Details: sample of protein complex was in buffer containing 20 mM Na Hepes, pH 7.4, 300 mM NaCl, 1 mM MgCl2 and 0.5 mM TCEP (components as listed).
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acidC8H18N2O4S1
2300 mMsodium chlorideNaClSodium chloride1
31 mMmagnesium chlorideMgCl21
40.5 mMtris(2-carboxyethyl)phosphineC9H15O6P1
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: purified human metapneumovirus L:P complex in a 1:4 stoichiometry.
Specimen supportDetails: 20 mA / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K
Details: 3.5 uL sample (0.8 mg/mL)/grid, 4-second blotting (double-sided) at offset -2 before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Details: preliminary grid screening was performed manually on a FEI Tecnai F20 microscope equipped with a Gatan K2 summit camera.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 31000 X / Calibrated magnification: 40323 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Calibrated defocus min: 1500 nm / Calibrated defocus max: 3000 nm / Cs: 2.26 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER / Temperature (max): 90 K / Temperature (min): 77 K
Image recordingAverage exposure time: 0.2 sec. / Electron dose: 1.2 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7438
Image scansSampling size: 5 µm / Width: 7676 / Height: 7420 / Movie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2SerialEM3.7.1image acquisitionSerialEM was used to automatically collect data
4CTFFIND4.1.9CTF correction
10RELION3.0.3initial Euler assignmentinitial angular assignment generated from 2D classification
11RELION3.0.4final Euler assignmentrelion autorefinement.
12RELION3.0.4classification
13RELION3.0.43D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 5381943
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 302346 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00426524
ELECTRON MICROSCOPYf_angle_d0.71748138
ELECTRON MICROSCOPYf_dihedral_angle_d6.96410593
ELECTRON MICROSCOPYf_chiral_restr0.0452055
ELECTRON MICROSCOPYf_plane_restr0.0053810

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