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- EMDB-28666: Cryo-EM structure of SARS-CoV-2 Main protease C145S in complex wi... -

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Entry
Database: EMDB / ID: EMD-28666
TitleCryo-EM structure of SARS-CoV-2 Main protease C145S in complex with N-terminal peptide
Map data
Sample
  • Complex: Cryo-EM structure of SARS-CoV-2 Main protease C145S mutant
    • Protein or peptide: 3C-like proteinase
Keywordscovid19 / mpro / main protease / cryo-Em / 3cl / sars-cov-2 / 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 / 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 / symbiont-mediated suppression of host toll-like receptor signaling pathway / 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 / symbiont-mediated suppression of host NF-kappaB cascade / 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 / ubiquitinyl hydrolase 1 / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / cysteine-type deubiquitinase 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 / symbiont-mediated activation of host autophagy / copper ion binding / viral translational frameshifting / RNA-directed RNA polymerase / cysteine-type endopeptidase activity / viral RNA genome replication / 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 1ab
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsNoske GD / Song Y / Fernandes RS / Oliva G / Godoy AS
Funding support Brazil, 1 items
OrganizationGrant numberCountry
Sao Paulo Research Foundation (FAPESP)2013/07600-3 Brazil
CitationJournal: Nat Commun / Year: 2023
Title: An in-solution snapshot of SARS-COV-2 main protease maturation process and inhibition.
Authors: Gabriela Dias Noske / Yun Song / Rafaela Sachetto Fernandes / Rod Chalk / Haitem Elmassoudi / Lizbé Koekemoer / C David Owen / Tarick J El-Baba / Carol V Robinson / / Glaucius Oliva / Andre Schutzer Godoy /
Abstract: The main protease from SARS-CoV-2 (M) is responsible for cleavage of the viral polyprotein. M self-processing is called maturation, and it is crucial for enzyme dimerization and activity. Here we use ...The main protease from SARS-CoV-2 (M) is responsible for cleavage of the viral polyprotein. M self-processing is called maturation, and it is crucial for enzyme dimerization and activity. Here we use C145S M to study the structure and dynamics of N-terminal cleavage in solution. Native mass spectroscopy analysis shows that mixed oligomeric states are composed of cleaved and uncleaved particles, indicating that N-terminal processing is not critical for dimerization. A 3.5 Å cryo-EM structure provides details of M N-terminal cleavage outside the constrains of crystal environment. We show that different classes of inhibitors shift the balance between oligomeric states. While non-covalent inhibitor MAT-POS-e194df51-1 prevents dimerization, the covalent inhibitor nirmatrelvir induces the conversion of monomers into dimers, even with intact N-termini. Our data indicates that the M dimerization is triggered by induced fit due to covalent linkage during substrate processing rather than the N-terminal processing.
History
DepositionOct 26, 2022-
Header (metadata) releaseDec 7, 2022-
Map releaseDec 7, 2022-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28666.png

Downloads & links

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Map

FileDownload / File: emd_28666.map.gz / Format: CCP4 / Size: 44 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesX (Sec.)Y (Row.)Z (Col.)
0.65 Å/pix.
x 226 pix.
= 147.578 Å		0.65 Å/pix.
x 226 pix.
= 147.578 Å		0.65 Å/pix.
x 226 pix.
= 147.578 Å

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.653 Å
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Contour LevelBy AUTHOR: 6.44
Minimum - Maximum-13.286533 - 22.280235000000001
Average (Standard dev.)0.000000000011401 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions226226226
Spacing226226226
CellA=B=C: 147.578 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_28666_msk_1.map
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Sample components

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Entire : Cryo-EM structure of SARS-CoV-2 Main protease C145S mutant

EntireName: Cryo-EM structure of SARS-CoV-2 Main protease C145S mutant
Components
  • Complex: Cryo-EM structure of SARS-CoV-2 Main protease C145S mutant
    • Protein or peptide: 3C-like proteinase

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Supramolecule #1: Cryo-EM structure of SARS-CoV-2 Main protease C145S mutant

SupramoleculeName: Cryo-EM structure of SARS-CoV-2 Main protease C145S mutant
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 68 kDa/nm

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Macromolecule #1: 3C-like proteinase

MacromoleculeName: 3C-like proteinase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: SARS coronavirus main proteinase
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 34.567371 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GAMSAVLQSG FRKMAFPSGK VEGCMVQVTC GTTTLNGLWL DDVVYCPRHV ICTSEDMLNP NYEDLLIRKS NHNFLVQAGN VQLRVIGHS MQNCVLKLKV DTANPKTPKY KFVRIQPGQT FSVLACYNGS PSGVYQCAMR PNFTIKGSFL NGSSGSVGFN I DYDCVSFC ...String:
GAMSAVLQSG FRKMAFPSGK VEGCMVQVTC GTTTLNGLWL DDVVYCPRHV ICTSEDMLNP NYEDLLIRKS NHNFLVQAGN VQLRVIGHS MQNCVLKLKV DTANPKTPKY KFVRIQPGQT FSVLACYNGS PSGVYQCAMR PNFTIKGSFL NGSSGSVGFN I DYDCVSFC YMHHMELPTG VHAGTDLEGN FYGPFVDRQT AQAAGTDTTI TVNVLAWLYA AVINGDRWFL NRFTTTLNDF NL VAMKYNY EPLTQDHVDI LGPLSAQTGI AVLDMCASLK ELLQNGMNGR TILGSALLED EFTPFDVVRQ CSGVTFQ

UniProtKB: Replicase polyprotein 1ab

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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

Concentration0.25 mg/mL
BufferpH: 7.8
Details: 20 mM Tris pH 7.8, 150 mM NaCl, 1 mM EDTA, 1 mM DTT
VitrificationCryogen name: ETHANE / Chamber humidity: 100 %

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 13770 / Average electron dose: 43.38557582 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1956496
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7n6n

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 227533
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION
Final 3D classificationNumber classes: 4 / Software - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7n6n


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 218 / Target criteria: CC
Output model

PDB-8ey2:
Cryo-EM structure of SARS-CoV-2 Main protease C145S in complex with N-terminal peptide

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