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- EMDB-29365: Co-structure of the Respiratory Syncytial Virus RNA-dependent RNA... -

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Basic information

Entry
Database: EMDB / ID: EMD-29365
TitleCo-structure of the Respiratory Syncytial Virus RNA-dependent RNA polymerase with MRK-1
Map datasharpened map used for model building and real space refinement
Sample
  • Complex: RESPIRATORY SYNCYTIAL VIRUS POLYMERASE (L) PROTEIN BOUND BY THE TETRAMERIC PHOSPHOPROTEIN (P) AND COMPLEXED WITH MRK-1
    • Protein or peptide: Phosphoprotein
  • Protein or peptide: RNA-directed RNA polymerase L
  • Ligand: 4-(2-aminopropan-2-yl)-N'-[4-(cyclopropyloxy)-3-methoxybenzoyl]-6-(4-fluorophenyl)pyridine-2-carbohydrazide
KeywordsRNA-BINDING PROTEIN / RSV / RDRP / RNA-DEPENDENT RNA POLYMERASE / PRNTASE / POLYRIBONUCLEOTIDYL TRANSFERASE / RNA CAPPING / VIRAL REPLICATION / VIRAL PROTEIN / REPLICATION
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 orthopneumovirus / Human respiratory syncytial virus A2
Methodsingle particle reconstruction / cryo EM / Resolution: 2.52 Å
AuthorsFischmann TO
Funding support United States, 1 items
OrganizationGrant numberCountry
Other private United States
CitationJournal: Commun Biol / Year: 2023
Title: Conserved allosteric inhibitory site on the respiratory syncytial virus and human metapneumovirus RNA-dependent RNA polymerases.
Authors: Victoria A Kleiner / Thierry O Fischmann / John A Howe / Douglas C Beshore / Michael J Eddins / Yan Hou / Todd Mayhood / Daniel Klein / Debbie D Nahas / Bob J Lucas / He Xi / Edward Murray / ...Authors: Victoria A Kleiner / Thierry O Fischmann / John A Howe / Douglas C Beshore / Michael J Eddins / Yan Hou / Todd Mayhood / Daniel Klein / Debbie D Nahas / Bob J Lucas / He Xi / Edward Murray / Daphne Y Ma / Krista Getty / Rachel Fearns /
Abstract: Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are related RNA viruses responsible for severe respiratory infections and resulting disease in infants, elderly, and ...Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are related RNA viruses responsible for severe respiratory infections and resulting disease in infants, elderly, and immunocompromised adults. Therapeutic small molecule inhibitors that bind to the RSV polymerase and inhibit viral replication are being developed, but their binding sites and molecular mechanisms of action remain largely unknown. Here we report a conserved allosteric inhibitory site identified on the L polymerase proteins of RSV and HMPV that can be targeted by a dual-specificity, non-nucleoside inhibitor, termed MRK-1. Cryo-EM structures of the inhibitor in complexes with truncated RSV and full-length HMPV polymerase proteins provide a structural understanding of how MRK-1 is active against both viruses. Functional analyses indicate that MRK-1 inhibits conformational changes necessary for the polymerase to engage in RNA synthesis initiation and to transition into an elongation mode. Competition studies reveal that the MRK-1 binding pocket is distinct from that of a capping inhibitor with an overlapping resistance profile, suggesting that the polymerase conformation bound by MRK-1 may be distinct from that involved in mRNA capping. These findings should facilitate optimization of dual RSV and HMPV replication inhibitors and provide insights into the molecular mechanisms underlying their polymerase activities.
History
DepositionJan 4, 2023-
Header (metadata) releaseJun 14, 2023-
Map releaseJun 14, 2023-
UpdateJul 5, 2023-
Current statusJul 5, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29365.png

Downloads & links

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Map

FileDownload / File: emd_29365.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map used for model building and real space refinement
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-3.718707 - 5.608296
Average (Standard dev.)0.0010533391 (±0.17637102)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 211.99998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map

Fileemd_29365_half_map_1.map
Annotationhalf map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map

Fileemd_29365_half_map_2.map
Annotationhalf 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 : RESPIRATORY SYNCYTIAL VIRUS POLYMERASE (L) PROTEIN BOUND BY THE T...

EntireName: RESPIRATORY SYNCYTIAL VIRUS POLYMERASE (L) PROTEIN BOUND BY THE TETRAMERIC PHOSPHOPROTEIN (P) AND COMPLEXED WITH MRK-1
Components
  • Complex: RESPIRATORY SYNCYTIAL VIRUS POLYMERASE (L) PROTEIN BOUND BY THE TETRAMERIC PHOSPHOPROTEIN (P) AND COMPLEXED WITH MRK-1
    • Protein or peptide: Phosphoprotein
  • Protein or peptide: RNA-directed RNA polymerase L
  • Ligand: 4-(2-aminopropan-2-yl)-N'-[4-(cyclopropyloxy)-3-methoxybenzoyl]-6-(4-fluorophenyl)pyridine-2-carbohydrazide

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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) AND COMPLEXED WITH MRK-1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2
Source (natural)Organism: Human orthopneumovirus

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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: 173.267922 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGSDYKDHDG DYKDHDIDYK DDDDKGSGSL EVLFQGPMDP IINGNSANVY LTDSYLKGVI SFSECNALGS YIFNGPYLKN DYTNLISRQ NPLIEHMNLK KLNITQSLIS KYHKGEIKLE EPTYFQSLLM TYKSMTSSEQ IATTNLLKKI IRRAIEISDV K VYAILNKL ...String:
MGSDYKDHDG DYKDHDIDYK DDDDKGSGSL EVLFQGPMDP IINGNSANVY LTDSYLKGVI SFSECNALGS YIFNGPYLKN DYTNLISRQ NPLIEHMNLK KLNITQSLIS KYHKGEIKLE EPTYFQSLLM TYKSMTSSEQ IATTNLLKKI IRRAIEISDV K VYAILNKL GLKEKDKIKS NNGQDEDNSV ITTIIKDDIL SAVKDNQSHL KADKNHSTKQ KDTIKTTLLK KLMCSMQHPP SW LIHWFNL YTKLNNILTQ YRSNEVKNHG FTLIDNQTLS GFQFILNQYG CIVYHKELKR ITVTTYNQFL TWKDISLSRL NVC LITWIS NCLNTLNKSL GLRCGFNNVI LTQLFLYGDC ILKLFHNEGF YIIKEVEGFI MSLILNITEE DQFRKRFYNS MLNN ITDAA NKAQKNLLSR VCHTLLDKTV SDNIINGRWI ILLSKFLKLI KLAGDNNLNN LSELYFLFRI FGHPMVDERQ AMDAV KINC NETKFYLLSS LSMLRGAFIY RIIKGFVNNY NRWPTLRNAI VLPLRWLTYY KLNTYPSLLE LTERDLIVLS GLRFYR EFR LPKKVDLEMI INDKAISPPK NLIWTSFPRN YMPSHIQNYI EHEKLKFSES DKSRRVLEYY LRDNKFNECD LYNCVVN QS YLNNPNHVVS LTGKERELSV GRMFAMQPGM FRQVQILAEK MIAENILQFF PESLTRYGDL ELQKILELKA GISNKSNR Y NDNYNNYISK CSIITDLSKF NQAFRYETSC ICSDVLDELH GVQSLFSWLH LTIPHVTIIC TYRHAPPYIG DHIVDLNNV DEQSGLYRYH MGGIEGWCQK LWTIEAISLL DLISLKGKFS ITALINGDNQ SIDISKPIRL MEGQTHAQAD YLLALNSLKL LYKEYAGIG HKLKGTETYI SRDMQFMSKT IQHNGVYYPA SIKKVLRVGP WINTILDDFK VSLESIGSLT QELEYRGESL L CSLIFRNV WLYNQIALQL KNHALCNNKL YLDILKVLKH LKTFFNLDNI DTALTLYMNL PMLFGGGDPN LLYRSFYRRT PD FLTEAIV HSVFILSYYT NHDLKDKLQD LSDDRLNKFL TCIITFDKNP NAEFVTLMRD PQALGSERQA KITSEINRLA VTE VLSTAP NKIFSKSAQH YTTTEIDLND IMQNIEPTYP HGLRVVYESL PFYKAEKIVN LISGTKSITN ILEKTSAIDL TDID RATEM MRKNITLLIR ILPLDCNRDK REILSMENLS ITELSKYVRE RSWSLSNIVG VTSPSIMYTM DIKYTTSTIS SGIII EKYN VNSLTRGERG PTKPWVGSST QEKKTMPVYN RQVLTKKQRD QIDLLAKLDW VYASIDNKDE FMEELSIGTL GLTYEK AKK LFPQYLSVNY LHRLTVSSRP CEFPASIPAY RTTNYHFDTS PINRILTEKY GDEDIDIVFQ NCISFGLSLM SVVEQFT NV CPNRIILIPK LNEIHLMKPP IFTGDVDIHK LKQVIQKQHM FLPDKISLTQ YVELF

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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

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Macromolecule #3: 4-(2-aminopropan-2-yl)-N'-[4-(cyclopropyloxy)-3-methoxybenzoyl]-6...

MacromoleculeName: 4-(2-aminopropan-2-yl)-N'-[4-(cyclopropyloxy)-3-methoxybenzoyl]-6-(4-fluorophenyl)pyridine-2-carbohydrazide
type: ligand / ID: 3 / Number of copies: 1 / Formula: Y6L
Molecular weightTheoretical: 478.515 Da
Chemical component information

ChemComp-Y6L:
4-(2-aminopropan-2-yl)-N'-[4-(cyclopropyloxy)-3-methoxybenzoyl]-6-(4-fluorophenyl)pyridine-2-carbohydrazide

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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

Concentration0.6 mg/mL
BufferpH: 7.4
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 / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 34.5 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:

6pzk

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionNumber classes used: 13 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.52 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 589457
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-8fpi:
Co-structure of the Respiratory Syncytial Virus RNA-dependent RNA polymerase with MRK-1

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