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- EMDB-20651: Structure of the Human Metapneumovirus Polymerase bound to the ph... -

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Basic information

Entry
Database: EMDB / ID: EMD-20651
TitleStructure of the Human Metapneumovirus Polymerase bound to the phosphoprotein tetramer
Map dataNone
Sample
  • Complex: Human metapneumovirus CAN97-83
    • Protein or peptide: RNA-directed RNA polymerase L
    • Protein or peptide: Phosphoprotein
Keywordshuman metapneumovirus / HMPV / Polymerase / Phosphoprotein / Respiratory syncytial virus / RSV / pneumoviridae / Mononegavirales / VIRAL PROTEIN
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 CAN97-83 / Human metapneumovirus (strain CAN97-83)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsPan J / Qian X
Funding support Singapore, United States, 3 items
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, 2019-
Header (metadata) releaseSep 25, 2019-
Map releaseSep 25, 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: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6u5o
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20651.png

Downloads & links

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Map

FileDownload / File: emd_20651.map.gz / Format: CCP4 / Size: 3.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 1.24 Å
Density
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.2537981 - 0.39055532
Average (Standard dev.)0.0006125627 (±0.030375354)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions969696
Spacing969696
CellA=B=C: 119.04 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.241.241.24
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z119.040119.040119.040
α/β/γ90.00090.00090.000
start NX/NY/NZ1548552
NX/NY/NZ198170217
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS969696
D min/max/mean-0.2540.3910.001

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Supplemental data

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Sample components

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Entire : Human metapneumovirus CAN97-83

EntireName: Human metapneumovirus CAN97-83
Components
  • Complex: Human metapneumovirus CAN97-83
    • Protein or peptide: RNA-directed RNA polymerase L
    • Protein or peptide: Phosphoprotein

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Supramolecule #1: Human metapneumovirus CAN97-83

SupramoleculeName: Human metapneumovirus CAN97-83 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
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.
Source (natural)Organism: Human metapneumovirus CAN97-83
Molecular weightTheoretical: 360 KDa

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Macromolecule #1: RNA-directed RNA polymerase L

MacromoleculeName: RNA-directed RNA polymerase L / type: protein_or_peptide / ID: 1
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.
Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed RNA polymerase
Source (natural)Organism: Human metapneumovirus (strain CAN97-83) / Strain: CAN97-83
Molecular weightTheoretical: 233.889562 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGWSHPQFEK SSGVDLGTEN LYFQSMDPLN ESTVNVYLPD SYLKGVISFS ETNAIGSCLL KRPYLKNDNT AKVAIENPVI EHVRLKNAV NSKMKISDYK VVEPVNMQHE IMKNVHSCEL TLLKQFLTRS KNISTLKLNM ICDWLQLKST SDDTSILSFI D VEFIPSWV ...String:
MGWSHPQFEK SSGVDLGTEN LYFQSMDPLN ESTVNVYLPD SYLKGVISFS ETNAIGSCLL KRPYLKNDNT AKVAIENPVI EHVRLKNAV NSKMKISDYK VVEPVNMQHE IMKNVHSCEL TLLKQFLTRS KNISTLKLNM ICDWLQLKST SDDTSILSFI D VEFIPSWV SNWFSNWYNL NKLILEFRRE EVIRTGSILC RSLGKLVFIV SSYGCIVKSN KSKRVSFFTY NQLLTWKDVM LS RFNANFC IWVSNSLNEN QEGLGLRSNL QGMLTNKLYE TVDYMLSLCC NEGFSLVKEF EGFIMSEILR ITEHAQFSTR FRN TLLNGL TDQLTKLKNK NRLRVHSTVL ENNDYPMYEV VLKLLGDTLR CIKLLINKNL ENAAELYYIF RIFGHPMVDE RDAM DAVKL NNEITKILRL ESLTELRGAF ILRIIKGFVD NNKRWPKIKN LKVLSKRWTM YFKAKNYPSQ LELSEQDFLE LAAIQ FEQE FSVPEKTNLE MVLNDKAISP PKRLIWSVYP KNYLPETIKN RYLEETFNAS DSLKTRRVLE YYLKDNKFDQ KELKSY VVR QEYLNDKEHI VSLTGKEREL SVGRMFAMQP GKQRQIQILA EKLLADNIVP FFPETLTKYG DLDLQRIMEI KSELSSI KT RRNDSYNNYI ARASIVTDLS KFNQAFRYET TAICADVADE LHGTQSLFCW LHLIVPMTTM ICAYRHAPPE TKGEYDID K IEEQSGLYRY HMGGIEGWCQ KLWTMEAISL LDVVSVKTRC QMTSLLNGDN QSIDVSKPVK LSEGLDEVKA DYRLAVKML KEIRDAYRNI GHKLKEGETY ISRDLQFISK VIQSEGVMHP TPIKKVLRVG PWINTILDDI KTSAESIGSL CQELEFRGES IIVSLILRN FWLYNLYMHE SKQHPLAGKQ LFKQLNKTLT SVQRFFEIKR ENEVVDLWMN IPMQFGGGDP VVFYRSFYRR T PDFLTEAI SHVDILLKIS ANIKNETKVS FFKALLSIEK NERATLTTLM RDPQAVGSER QAKVTSDINR TAVTSILSLS PN QLFSDSA IHYSRNEEEV GIIAENITPV YPHGLRVLYE SLPFHKAEKV VNMISGTKSI TNLLQRTSAI NGEDIDRAVS MML ENLGLL SRILSVVVDS IEIPIKSNGR LICCQISRTL RETSWNNMEI VGVTSPSITT CMDVIYATSS HLKGIIIEKF STDR TTRGQ RGPKSPWVGS STQEKKLVPV YNRQILSKQQ REQLEAIGKM RWVYKGTPGL RRLLNKICLG SLGISYKCVK PLLPR FMSV NFLHRLSVSS RPMEFPASVP AYRTTNYHFD TSPINQALSE RFGNEDINLV FQNAISCGIS IMSVVEQLTG RSPKQL VLI PQLEEIDIMP PPVFQGKFNY KLVDKITSDQ HIFSPDKIDM LTLGKMLMPT IKGQKTDQFL NKRENYFHGN NLIESLS AA LACHWCGILT EQCIENNIFK KDWGDGFISD HAFMDFKIFL CVFKTKLLCS WGSQGKNIKD EDIVDESIDK LLRIDNTF W RMFSKVMFEP KVKKRIMLYD VKFLSLVGYI GFKNWFIEQL RSAELHEIPW IVNAEGDLVE IKSIKIYLQL IEQSLFLRI TVLNYTDMAH ALTRLIRKKL MCDNALLTPI SSPMVNLTQV IDPTTQLDYF PKITFERLKN YDTSSNYAKG KLTRNYMILL PWQHVNRYN FVFSSTGCKV SLKTCIGKLM KDLNPKVLYF IGEGAGNWMA RTACEYPDIK FVYRSLKDDL DHHYPLEYQR V IGELSRII DSGEGLSMET TDATQKTHWD LIHRVSKDAL LITLCDAEFK DRDDFFKMVI LWRKHVLSCR ICTTYGTDLY LF AKYHAKD CNVKLPFFVR SVATFIMQGS KLSGSECYIL LTLGHHNSLP CHGEIQNSKM KIAVCNDFYA AKKLDNKSIE ANC KSLLSG LRIPINKKEL DRQRRLLTLQ SNHSSVATVG GSKIIESKWL TNKASTIIDW LEHILNSPKG ELNYDFFEAL ENTY PNMIK LIDNLGNAEI KKLIKVTGYM LVSKK

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 metapneumovirus (strain CAN97-83) / Strain: CAN97-83
Molecular weightTheoretical: 35.685066 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGHHHHHHHH SSGVDLGTEN LYFQSMSFPE GKDILFMGNE AAKLAEAFQK SLRKPSHKRS QSIIGEKVNT VSETLELPTI SRPTKPTIL SEPKLAWTDK GGAIKTEAKQ TIKVMDPIEE EEFTEKRVLP SSDGKTPAEK KLKPSTNTKK KVSFTPNEPG K YTKLEKDA ...String:
MGHHHHHHHH SSGVDLGTEN LYFQSMSFPE GKDILFMGNE AAKLAEAFQK SLRKPSHKRS QSIIGEKVNT VSETLELPTI SRPTKPTIL SEPKLAWTDK GGAIKTEAKQ TIKVMDPIEE EEFTEKRVLP SSDGKTPAEK KLKPSTNTKK KVSFTPNEPG K YTKLEKDA LDLLSDNEEE DAESSILTFE ERDTSSLSIE ARLESIEEKL SMILGLLRTL NIATAGPTAA RDGIRDAMIG IR EELIADI IKEAKGKAAE MMEEEMNQRT KIGNGSVKLT EKAKELNKIV EDESTSGESE EEEELKDTQE NNQEDDIYQL IM

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

Concentration0.8 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4S2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
300.0 mMNaClSodium chloridesodium chloride
1.0 mMMgCl2magnesium chloride
0.5 mMC9H15O6Ptris(2-carboxyethyl)phosphine

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).
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR / Details: 20 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
Details: 3.5 uL sample (0.8 mg/mL)/grid, 4-second blotting (double-sided) at offset -2 before plunging..
Detailspurified human metapneumovirus L:P complex in a 1:4 stoichiometry.

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 3.0 µm / Calibrated defocus min: 1.5 µm / Calibrated magnification: 40323 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.26 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 31000
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
TemperatureMin: 77.0 K / Max: 90.0 K
Detailspreliminary grid screening was performed manually on a FEI Tecnai F20 microscope equipped with a Gatan K2 summit camera.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7676 pixel / Digitization - Dimensions - Height: 7420 pixel / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 7438 / Average exposure time: 0.2 sec. / Average electron dose: 1.2 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5381943
Startup modelType of model: OTHER / Details: de novo model was generated using Relion.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.3)
Software - details: initial angular assignment generated from 2D classification
Details: initial angular assignment generated from 2D classification in Relion.
Final 3D classificationNumber classes: 6 / Software - Name: RELION (ver. 3.0.4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.4) / Software - details: relion autorefinement.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.4) / Number images used: 302346
FSC plot (resolution estimation)

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