+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-25898 | |||||||||||||||
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Title | SARS-CoV-2 nsp12/7/8 complex with a native N-terminus nsp9 | |||||||||||||||
Map data | SARS-CoV-2 nsp12/7/8 complex with a native N-terminus nsp9 | |||||||||||||||
Sample |
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Keywords | polymerase / NiRAN / capping / nsp9 / VIRAL PROTEIN | |||||||||||||||
Function / homology | Function and homology information protein guanylyltransferase activity / RNA endonuclease activity, producing 3'-phosphomonoesters / mRNA guanylyltransferase activity / 5'-3' RNA helicase activity / Lyases; Phosphorus-oxygen lyases / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / ISG15-specific peptidase activity / Transcription of SARS-CoV-2 sgRNAs ...protein guanylyltransferase activity / RNA endonuclease activity, producing 3'-phosphomonoesters / mRNA guanylyltransferase activity / 5'-3' RNA helicase activity / Lyases; Phosphorus-oxygen lyases / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / ISG15-specific peptidase activity / Transcription of SARS-CoV-2 sgRNAs / Translation of Replicase and Assembly of the Replication Transcription Complex / TRAF3-dependent IRF activation pathway / Replication of the SARS-CoV-2 genome / snRNP Assembly / double membrane vesicle viral factory outer membrane / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / 5'-3' DNA helicase activity / SARS coronavirus main proteinase / 3'-5'-RNA exonuclease activity / host cell endoplasmic reticulum-Golgi intermediate compartment / host cell endosome / symbiont-mediated suppression of host toll-like receptor signaling pathway / symbiont-mediated degradation of host mRNA / mRNA guanylyltransferase / symbiont-mediated suppression of host ISG15-protein conjugation / G-quadruplex RNA binding / omega peptidase activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / SARS-CoV-2 modulates host translation machinery / mRNA (guanine-N7)-methyltransferase / host cell Golgi apparatus / methyltransferase cap1 / symbiont-mediated perturbation of host ubiquitin-like protein modification / DNA helicase / mRNA (nucleoside-2'-O-)-methyltransferase activity / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / single-stranded RNA binding / host cell perinuclear region of cytoplasm / host cell endoplasmic reticulum membrane / viral protein processing / lyase activity / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / RNA helicase / induction by virus of host autophagy / copper ion binding / RNA-directed RNA polymerase / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / virus-mediated perturbation of host defense response / DNA-templated transcription / lipid binding / host cell nucleus / SARS-CoV-2 activates/modulates innate and adaptive immune responses / ATP hydrolysis activity / proteolysis / RNA binding / zinc ion binding / ATP binding / membrane Similarity search - Function | |||||||||||||||
Biological species | Severe acute respiratory syndrome coronavirus 2 | |||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.18 Å | |||||||||||||||
Authors | Osinski A / Tagliabracci VS | |||||||||||||||
Funding support | United States, 4 items
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Citation | Journal: Res Sq / Year: 2022 Title: The mechanism of RNA capping by SARS-CoV-2. Authors: Gina J Park / Adam Osinski / Genaro Hernandez / Jennifer L Eitson / Abir Majumdar / Marco Tonelli / Katie Henzler-Wildman / Krzysztof Pawłowski / Zhe Chen / Yang Li / John W Schoggins / ...Authors: Gina J Park / Adam Osinski / Genaro Hernandez / Jennifer L Eitson / Abir Majumdar / Marco Tonelli / Katie Henzler-Wildman / Krzysztof Pawłowski / Zhe Chen / Yang Li / John W Schoggins / Vincent S Tagliabracci / Abstract: The SARS-CoV-2 RNA genome contains a 5'-cap that facilitates translation of viral proteins, protection from exonucleases and evasion of the host immune response1-4. How this cap is made is not ...The SARS-CoV-2 RNA genome contains a 5'-cap that facilitates translation of viral proteins, protection from exonucleases and evasion of the host immune response1-4. How this cap is made is not completely understood. Here, we reconstitute the SARS-CoV-2 7MeGpppA2'-O-Me-RNA cap using virally encoded non-structural proteins (nsps). We show that the kinase-like NiRAN domain5 of nsp12 transfers RNA to the amino terminus of nsp9, forming a covalent RNA-protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers RNA to GDP, forming the cap core structure GpppA-RNA. The nsp146 and nsp167 methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replication-transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system8 that the N-terminus of nsp9 and the kinase-like active site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19. | |||||||||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_25898.map.gz | 118.1 MB | EMDB map data format | |
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Header (meta data) | emd-25898-v30.xml emd-25898.xml | 18.5 KB 18.5 KB | Display Display | EMDB header |
Images | emd_25898.png | 73.9 KB | ||
Filedesc metadata | emd-25898.cif.gz | 6.7 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-25898 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-25898 | HTTPS FTP |
-Related structure data
Related structure data | 7thmMC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_25898.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | SARS-CoV-2 nsp12/7/8 complex with a native N-terminus nsp9 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.094 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : SARS-CoV-2 replication-transcription complex with a native N-term...
Entire | Name: SARS-CoV-2 replication-transcription complex with a native N-terminus nsp9 bound to NiRAN domain of nsp12. |
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Components |
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-Supramolecule #1: SARS-CoV-2 replication-transcription complex with a native N-term...
Supramolecule | Name: SARS-CoV-2 replication-transcription complex with a native N-terminus nsp9 bound to NiRAN domain of nsp12. type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 |
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Source (natural) | Organism: Severe acute respiratory syndrome coronavirus 2 |
Molecular weight | Theoretical: 172.03949 KDa |
-Macromolecule #1: RNA-directed RNA polymerase
Macromolecule | Name: RNA-directed RNA polymerase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed RNA polymerase |
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Source (natural) | Organism: Severe acute respiratory syndrome coronavirus 2 |
Molecular weight | Theoretical: 106.780977 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: SADAQSFLNR VCGVSAARLT PCGTGTSTDV VYRAFDIYND KVAGFAKFLK TNCCRFQEKD EDDNLIDSYF VVKRHTFSNY QHEETIYNL LKDCPAVAKH DFFKFRIDGD MVPHISRQRL TKYTMADLVY ALRHFDEGNC DTLKEILVTY NCCDDDYFNK K DWYDFVEN ...String: SADAQSFLNR VCGVSAARLT PCGTGTSTDV VYRAFDIYND KVAGFAKFLK TNCCRFQEKD EDDNLIDSYF VVKRHTFSNY QHEETIYNL LKDCPAVAKH DFFKFRIDGD MVPHISRQRL TKYTMADLVY ALRHFDEGNC DTLKEILVTY NCCDDDYFNK K DWYDFVEN PDILRVYANL GERVRQALLK TVQFCDAMRN AGIVGVLTLD NQDLNGNWYD FGDFIQTTPG SGVPVVDSYY SL LMPILTL TRALTAESHV DTDLTKPYIK WDLLKYDFTE ERLKLFDRYF KYWDQTYHPN CVNCLDDRCI LHCANFNVLF STV FPPTSF GPLVRKIFVD GVPFVVSTGY HFRELGVVHN QDVNLHSSRL SFKELLVYAA DPAMHAASGN LLLDKRTTCF SVAA LTNNV AFQTVKPGNF NKDFYDFAVS KGFFKEGSSV ELKHFFFAQD GNAAISDYDY YRYNLPTMCD IRQLLFVVEV VDKYF DCYD GGCINANQVI VNNLDKSAGF PFNKWGKARL YYDSMSYEDQ DALFAYTKRN VIPTITQMNL KYAISAKNRA RTVAGV SIC STMTNRQFHQ KLLKSIAATR GATVVIGTSK FYGGWHNMLK TVYSDVENPH LMGWDYPKCD RAMPNMLRIM ASLVLAR KH TTCCSLSHRF YRLANECAQV LSEMVMCGGS LYVKPGGTSS GDATTAYANS VFNICQAVTA NVNALLSTDG NKIADKYV R NLQHRLYECL YRNRDVDTDF VNEFYAYLRK HFSMMILSDD AVVCFNSTYA SQGLVASIKN FKSVLYYQNN VFMSEAKCW TETDLTKGPH EFCSQHTMLV KQGDDYVYLP YPDPSRILGA GCFVDDIVKT DGTLMIERFV SLAIDAYPLT KHPNQEYADV FHLYLQYIR KLHDELTGHM LDMYSVMLTN DNTSRYWEPE FYEAMYTPHT VLQ UniProtKB: Replicase polyprotein 1ab |
-Macromolecule #2: Non-structural protein 8
Macromolecule | Name: Non-structural protein 8 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Severe acute respiratory syndrome coronavirus 2 |
Molecular weight | Theoretical: 21.903047 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: AIASEFSSLP SYAAFATAQE AYEQAVANGD SEVVLKKLKK SLNVAKSEFD RDAAMQRKLE KMADQAMTQM YKQARSEDKR AKVTSAMQT MLFTMLRKLD NDALNNIINN ARDGCVPLNI IPLTTAAKLM VVIPDYNTYK NTCDGTTFTY ASALWEIQQV V DADSKIVQ ...String: AIASEFSSLP SYAAFATAQE AYEQAVANGD SEVVLKKLKK SLNVAKSEFD RDAAMQRKLE KMADQAMTQM YKQARSEDKR AKVTSAMQT MLFTMLRKLD NDALNNIINN ARDGCVPLNI IPLTTAAKLM VVIPDYNTYK NTCDGTTFTY ASALWEIQQV V DADSKIVQ LSEISMDNSP NLAWPLIVTA LRANSAVKLQ UniProtKB: Replicase polyprotein 1ab |
-Macromolecule #3: Non-structural protein 7
Macromolecule | Name: Non-structural protein 7 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Severe acute respiratory syndrome coronavirus 2 |
Molecular weight | Theoretical: 9.248804 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: SKMSDVKCTS VVLLSVLQQL RVESSSKLWA QCVQLHNDIL LAKDTTEAFE KMVSLLSVLL SMQGAVDINK LCEEMLDNRA TLQ UniProtKB: Replicase polyprotein 1ab |
-Macromolecule #4: Non-structural protein 9
Macromolecule | Name: Non-structural protein 9 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Severe acute respiratory syndrome coronavirus 2 |
Molecular weight | Theoretical: 12.391171 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: NNELSPVALR QMSCAAGTTQ TACTDDNALA YYNTTKGGRF VLALLSDLQD LKWARFPKSD GTGTIYTELE PPCRFVTDTP KGPKVKYLY FIKGLNNLNR GMVLGSLAAT VRLQ UniProtKB: Replicase polyprotein 1ab |
-Macromolecule #5: ZINC ION
Macromolecule | Name: ZINC ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: ZN |
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Molecular weight | Theoretical: 65.409 Da |
-Macromolecule #6: MANGANESE (II) ION
Macromolecule | Name: MANGANESE (II) ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: MN |
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Molecular weight | Theoretical: 54.938 Da |
-Macromolecule #7: PYROPHOSPHATE 2-
Macromolecule | Name: PYROPHOSPHATE 2- / type: ligand / ID: 7 / Number of copies: 1 / Formula: POP |
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Molecular weight | Theoretical: 175.959 Da |
Chemical component information | ChemComp-POP: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 2 mg/mL | |||||||||||||||
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Buffer | pH: 7.5 Component:
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Vitrification | Cryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | TFS KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 54.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Startup model | Type of model: OTHER |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.18 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 39985 |
Initial angle assignment | Type: ANGULAR RECONSTITUTION |
Final angle assignment | Type: ANGULAR RECONSTITUTION |