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- EMDB-28162: SARS-CoV-2 polyprotein substrate regulates the stepwise Mpro clea... -

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Entry
Database: EMDB / ID: EMD-28162
TitleSARS-CoV-2 polyprotein substrate regulates the stepwise Mpro cleavage reaction
Map data
Sample
  • Complex: SARS COV-2 Mpro in complex with polyprotein substrate
    • Protein or peptide: 3C-like proteinase nsp5
  • Protein or peptide: nsp7-nsp10 of Replicase polyprotein 1a
KeywordsSARS COV-2 Main protease (Mpro) in complex with the polyprotein substrate / VIRAL PROTEIN
Function / homology
Function and homology information


viral genome replication / methyltransferase activity / protein guanylyltransferase activity / RNA endonuclease activity producing 3'-phosphomonoesters, hydrolytic mechanism / 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 ...viral genome replication / methyltransferase activity / protein guanylyltransferase activity / RNA endonuclease activity producing 3'-phosphomonoesters, hydrolytic mechanism / 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 / TRAF3-dependent IRF activation pathway / Translation of Replicase and Assembly of the Replication Transcription Complex / Replication of the SARS-CoV-2 genome / snRNP Assembly / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / double membrane vesicle viral factory outer membrane / host cell endoplasmic reticulum-Golgi intermediate compartment / SARS coronavirus main proteinase / 3'-5'-RNA exonuclease activity / 5'-3' DNA helicase activity / host cell endosome / symbiont-mediated degradation of host mRNA / mRNA guanylyltransferase / symbiont-mediated suppression of host toll-like receptor signaling pathway / 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 / methyltransferase cap1 / host cell Golgi apparatus / symbiont-mediated perturbation of host ubiquitin-like protein modification / symbiont-mediated suppression of host NF-kappaB cascade / endonuclease activity / DNA helicase / methylation / methyltransferase cap1 activity / ubiquitinyl hydrolase 1 / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / cysteine-type deubiquitinase activity / forked DNA-dependent helicase activity / single-stranded 3'-5' DNA helicase activity / four-way junction helicase activity / double-stranded DNA helicase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / single-stranded RNA binding / host cell perinuclear region of cytoplasm / viral protein processing / lyase activity / host cell endoplasmic reticulum membrane / RNA helicase / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / copper ion binding / symbiont-mediated activation of host autophagy / viral translational frameshifting / RNA-directed RNA polymerase / chromatin extrusion motor activity / ATP-dependent H2AZ histone chaperone activity / ATP-dependent H3-H4 histone complex chaperone activity / cohesin loader activity / cysteine-type endopeptidase activity / viral RNA genome replication / DNA clamp loader activity / symbiont-mediated suppression of host gene expression / RNA-directed RNA polymerase activity / 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
RNA-dependent RNA polymerase, SARS-CoV-like / Nonstructural protein 14, betacoronavirus / NSP15, NendoU domain, coronavirus / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nonstructural protein 15, N-terminal domain, alpha/beta-coronavirus / NSP14, guanine-N7-methyltransferase domain, coronavirus / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. / NSP12 RNA-dependent RNA polymerase, coronavirus / : ...RNA-dependent RNA polymerase, SARS-CoV-like / Nonstructural protein 14, betacoronavirus / NSP15, NendoU domain, coronavirus / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nonstructural protein 15, N-terminal domain, alpha/beta-coronavirus / NSP14, guanine-N7-methyltransferase domain, coronavirus / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. / NSP12 RNA-dependent RNA polymerase, coronavirus / : / : / : / Coronavirus Nonstructural protein 13, 1B domain / Coronavirus Non-structural protein 13, zinc-binding domain / Coronavirus Nonstructural protein 13, stalk domain / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) domain profile. / Coronavirus Nsp12 Interface domain profile. / Non-structural protein NSP15, N-terminal domain superfamily, coronavirus / Non-structural protein NSP15, middle domain superfamily / Coronavirus replicase NSP15, N-terminal oligomerization / Nonstructural protein 15, middle domain, coronavirus / Nonstructural protein 13, 1B domain, coronavirus / Nidovirus 2-O-methyltransferase / Coronavirus replicase NSP15, middle domain / Coronavirus replicase NSP15, N-terminal oligomerisation / Nidovirus 2'-O-methyltransferase (2'-O-MTase) domain profile. / Non-structural protein NSP16, coronavirus-like / Non-structural protein 14, coronavirus / RNA polymerase, N-terminal, coronavirus / Nidovirus 3'-5' exoribonuclease domain / Coronavirus 2'-O-methyltransferase / Coronavirus proofreading exoribonuclease / Coronavirus RNA-dependent RNA polymerase, N-terminal / Nidovirus 3'-5' exoribonuclease (ExoN) domain profile. / Nonstructural protein 13, zinc-binding domain, coronavirus-like / Coronaviridae zinc-binding (CV ZBD) domain profile. / Arterivirus Nsp11 N-terminal/Coronavirus NSP15 middle domain / Arterivirus Nsp11 N-terminal/coronavirus NSP15 middle (AV-Nsp11N/CoV-Nsp15M) domain profile. / Nidoviral uridylate-specific endoribonuclease (NendoU) domain profile. / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain profile. / Endoribonuclease EndoU-like / NendoU domain, nidovirus / Coronavirus replicase NSP15, uridylate-specific endoribonuclease / : / Lipocalin signature. / DNA2/NAM7 helicase-like, C-terminal / AAA domain / (+) RNA virus helicase core domain / (+)RNA virus helicase core domain profile. / Non-structural protein NSP3, SUD-N (Mac2) domain, betacoronavirus / Sarbecovirus Nsp3c-N domain profile. / Non-structural protein NSP3, N-terminal, betacoronavirus / Polyprotein cleavage domain PL2pro superfamily, betacoronavirus / Non-structural protein NSP3, SUD-N (Mac2) domain superfamily, betacoronavirus / Betacoronavirus SUD-C domain / Betacoronavirus replicase NSP3, N-terminal / NSP1 globular domain superfamily, betacoronavirus / Non-structural protein 2, SARS-CoV-like / Carbamoyl-phosphate synthase subdomain signature 2. / Betacoronavirus Nsp3c-M domain profile. / NSP1, globular domain, betacoronavirus / Non-structural protein NSP3, SUD-M domain, betacoronavirus / Non-structural protein NSP3, SUD-M domain superfamily, betacoronavirus / Betacoronavirus replicase NSP1 / Betacoronavirus single-stranded poly(A) binding domain / NSP1, C-terminal domain, betacoronavirus / : / Betacoronavirus (BetaCoV) Nsp1 C-terminal domain profile. / Betacoronavirus Nsp3e group 2-specific marker (G2M) domain profile. / Betacoronavirus Nsp3c-C domain profile. / Betacoronavirus Nsp3e nucleic acid-binding (NAB) domain profile. / DPUP/SUD, C-terminal, betacoronavirus / Non-structural protein NSP3, nucleic acid-binding domain, betacoronavirus / Non-structural protein NSP3A domain-like superfamily / Non-structural protein NSP3, nucleic acid-binding domain superfamily, betacoronavirus / Non-structural protein 6, betacoronavirus / Betacoronavirus nucleic acid-binding (NAB) / Papain-like viral protease, palm and finger domains, coronavirus / Papain-like protease, N-terminal domain superfamily, coronavirus / Coronavirus replicase NSP2, N-terminal / : / Coronavirus replicase NSP2, C-terminal / NSP1, globular domain, alpha/betacoronavirus / Coronavirus (CoV) Nsp2 middle domain profile. / Coronavirus (CoV) Nsp1 globular domain profile. / Coronavirus (CoV) Nsp2 N-terminal domain profile. / Coronavirus (CoV) Nsp2 C-terminal domain profile. / Nonstructural protein 2, N-terminal domain, coronavirus / Non-structural protein 2, C-terminal domain, coronavirus / NSP3, second ubiquitin-like (Ubl) domain, coronavirus / Coronavirus Nsp3a Ubl domain profile. / Coronavirus Nsp3d Ubl domain profile. / NSP3, first ubiquitin-like (Ubl) domain, coronavirus / : / : / Coronavirus replicase NSP7 / Peptidase family C16 domain profile. / Coronavirus 3Ecto domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp7 cofactor domain profile.
Similarity search - Domain/homology
Replicase polyprotein 1a / Replicase polyprotein 1ab
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 2.49 Å
AuthorsNarwal M / Edwards T / Armache JP / Murakami KS
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R35 GM131860 United States
CitationJournal: J Biol Chem / Year: 2023
Title: SARS-CoV-2 polyprotein substrate regulates the stepwise M cleavage reaction.
Authors: Manju Narwal / Jean-Paul Armache / Thomas J Edwards / Katsuhiko S Murakami /
Abstract: The processing of the Coronavirus polyproteins pp1a and pp1ab by the main protease M to produce mature proteins is a crucial event in virus replication and a promising target for antiviral drug ...The processing of the Coronavirus polyproteins pp1a and pp1ab by the main protease M to produce mature proteins is a crucial event in virus replication and a promising target for antiviral drug development. M cleaves polyproteins in a defined order, but how M and/or the polyproteins determine the order of cleavage remains enigmatic due to a lack of structural information about polyprotein-bound M. Here, we present the cryo-EM structures of SARS-CoV-2 M in an apo form and in complex with the nsp7-10 region of the pp1a polyprotein. The complex structure shows that M interacts with only the recognition site residues between nsp9 and nsp10, without any association with the rest of the polyprotein. Comparison between the apo form and polyprotein-bound structures of M highlights the flexible nature of the active site region of M, which allows it to accommodate ten recognition sites found in the polyprotein. These observations suggest that the role of M in selecting a preferred cleavage site is limited and underscores the roles of the structure, conformation, and/or dynamics of the polyproteins in determining the sequence of polyprotein cleavage by M.
History
DepositionSep 15, 2022-
Header (metadata) releaseApr 26, 2023-
Map releaseApr 26, 2023-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28162.png

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Map

FileDownload / File: emd_28162.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesX (Sec.)Y (Row.)Z (Col.)
0.86 Å/pix.
x 320 pix.
= 273.6 Å		0.86 Å/pix.
x 320 pix.
= 273.6 Å		0.86 Å/pix.
x 320 pix.
= 273.6 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.855 Å
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Histogram (log scale)
Contour LevelBy EMDB: 0.2
Minimum - Maximum-0.81612325 - 2.118728
Average (Standard dev.)0.00026679935 (±0.036538865)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 273.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_28162_half_map_1.map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_28162_half_map_2.map
Projections & Slices
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Sample components

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Entire : SARS COV-2 Mpro in complex with polyprotein substrate

EntireName: SARS COV-2 Mpro in complex with polyprotein substrate
Components
  • Complex: SARS COV-2 Mpro in complex with polyprotein substrate
    • Protein or peptide: 3C-like proteinase nsp5
  • Protein or peptide: nsp7-nsp10 of Replicase polyprotein 1a

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Supramolecule #1: SARS COV-2 Mpro in complex with polyprotein substrate

SupramoleculeName: SARS COV-2 Mpro in complex with polyprotein substrate / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2

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Macromolecule #1: 3C-like proteinase nsp5

MacromoleculeName: 3C-like proteinase nsp5 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: SARS coronavirus main proteinase
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 33.79348 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SGFRKMAFPS GKVEGCMVQV TCGTTTLNGL WLDDVVYCPR HVICTSEDML NPNYEDLLIR KSNHNFLVQA GNVQLRVIGH SMQNCVLKL KVDTANPKTP KYKFVRIQPG QTFSVLACYN GSPSGVYQCA MRPNFTIKGS FLNGSAGSVG FNIDYDCVSF C YMHHMELP ...String:
SGFRKMAFPS GKVEGCMVQV TCGTTTLNGL WLDDVVYCPR HVICTSEDML NPNYEDLLIR KSNHNFLVQA GNVQLRVIGH SMQNCVLKL KVDTANPKTP KYKFVRIQPG QTFSVLACYN GSPSGVYQCA MRPNFTIKGS FLNGSAGSVG FNIDYDCVSF C YMHHMELP TGVHAGTDLE GNFYGPFVDR QTAQAAGTDT TITVNVLAWL YAAVINGDRW FLNRFTTTLN DFNLVAMKYN YE PLTQDHV DILGPLSAQT GIAVLDMCAS LKELLQNGMN GRTILGSALL EDEFTPFDVV RQCSGVTFQ

UniProtKB: Replicase polyprotein 1ab

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Macromolecule #2: nsp7-nsp10 of Replicase polyprotein 1a

MacromoleculeName: nsp7-nsp10 of Replicase polyprotein 1a / type: protein_or_peptide / ID: 2
Details: nsp9/10 cleavage site is the portion visible in the map
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 58.29175 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SKMSDVKCTS VVLLSVLQQL RVESSSKLWA QCVQLHNDIL LAKDTTEAFE KMVSLLSVLL SMQGAVDINK LCEEMLDNRA TLQAIASEF SSLPSYAAFA TAQEAYEQAV ANGDSEVVLK KLKKSLNVAK SEFDRDAAMQ RKLEKMADQA MTQMYKQARS E DKRAKVTS ...String:
SKMSDVKCTS VVLLSVLQQL RVESSSKLWA QCVQLHNDIL LAKDTTEAFE KMVSLLSVLL SMQGAVDINK LCEEMLDNRA TLQAIASEF SSLPSYAAFA TAQEAYEQAV ANGDSEVVLK KLKKSLNVAK SEFDRDAAMQ RKLEKMADQA MTQMYKQARS E DKRAKVTS AMQTMLFTML RKLDNDALNN IINNARDGCV PLNIIPLTTA AKLMVVIPDY NTYKNTCDGT TFTYASALWE IQ QVVDADS KIVQLSEISM DNSPNLAWPL IVTALRANSA VKLQNNELSP VALRQMSCAA GTTQTACTDD NALAYYNTTK GGR FVLALL SDLQDLKWAR FPKSDGTGTI YTELEPPCRF VTDTPKGPKV KYLYFIKGLN NLNRGMVLGS LAATVRLQAG NATE VPANS TVLSFCAFAV DAAKAYKDYL ASGGQPITNC VKMLCTHTGT GQAITVTPEA NMDQESFGGA SCCLYCRCHI DHPNP KGFC DLKGKYVQIP TTCANDPVGF TLKNTVCTVC GMWKGYGCSC DQLREPMLQ

UniProtKB: Replicase polyprotein 1a

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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

Concentration1 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: OTHER / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
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:

7lkd

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.49 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 244544
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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