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- PDB-8k6d: Crystal structure of SARS-CoV-2 3CLpro M49K/S301P mutant in compl... -

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Basic information

Entry
Database: PDB / ID: 8k6d
TitleCrystal structure of SARS-CoV-2 3CLpro M49K/S301P mutant in complex with WU-04
Components3C-like proteinase nsp5
KeywordsVIRAL PROTEIN / SARS-CoV-2 3CLpro / mutant / WU-04 / inhibitor
Function / homology
Function and homology information


protein guanylyltransferase activity / RNA endonuclease activity producing 3'-phosphomonoesters, hydrolytic mechanism / mRNA guanylyltransferase activity / 5'-3' RNA helicase activity / Lyases; Phosphorus-oxygen lyases / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / ISG15-specific peptidase activity / TRAF3-dependent IRF activation pathway ...protein guanylyltransferase activity / RNA endonuclease activity producing 3'-phosphomonoesters, hydrolytic mechanism / mRNA guanylyltransferase activity / 5'-3' RNA helicase activity / Lyases; Phosphorus-oxygen lyases / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / ISG15-specific peptidase activity / TRAF3-dependent IRF activation pathway / Transcription of SARS-CoV-2 sgRNAs / Translation of Replicase and Assembly of the Replication Transcription Complex / snRNP Assembly / Replication of the SARS-CoV-2 genome / double membrane vesicle viral factory outer membrane / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / 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 ISG15-protein conjugation / G-quadruplex RNA binding / symbiont-mediated suppression of host toll-like receptor signaling pathway / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / omega peptidase 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 / symbiont-mediated suppression of host NF-kappaB cascade / DNA helicase / methyltransferase cap1 activity / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / mRNA 5'-cap (guanine-N7-)-methyltransferase 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 / regulation of autophagy / 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 suppression of host gene expression / copper ion binding / viral translational frameshifting / symbiont-mediated activation of host autophagy / 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 Nsp12 RNA-dependent RNA polymerase (RdRp) domain profile. / Coronavirus Nsp12 Interface domain profile. / : / : / Coronavirus Nonstructural protein 13, 1B domain / Coronavirus Non-structural protein 13, zinc-binding domain / Coronavirus Nonstructural protein 13, stalk domain / Nidovirus 2-O-methyltransferase / Nidovirus 2'-O-methyltransferase (2'-O-MTase) 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 / Coronavirus replicase NSP15, middle domain / Coronavirus replicase NSP15, N-terminal oligomerisation / Nidovirus 3'-5' exoribonuclease domain / Nidovirus 3'-5' exoribonuclease (ExoN) domain profile. / Non-structural protein NSP16, coronavirus-like / Non-structural protein 14, coronavirus / RNA polymerase, N-terminal, coronavirus / Coronavirus 2'-O-methyltransferase / Coronavirus proofreading exoribonuclease / Coronavirus RNA-dependent RNA polymerase, N-terminal / 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 / Coronavirus (CoV) Nsp2 middle domain profile. / NSP1, globular domain, alpha/betacoronavirus / 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 / Peptidase family C16 domain profile. / : / : / Coronavirus replicase NSP7 / Coronavirus 3Ecto domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp7 cofactor domain profile.
Similarity search - Domain/homology
Chem-J7R / Replicase polyprotein 1ab
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.65 Å
AuthorsZhang, L.J. / Hu, Q.
Funding support China, 1items
OrganizationGrant numberCountry
Other private China
CitationJournal: Cell Discov / Year: 2024
Title: Resistance mechanisms of SARS-CoV-2 3CLpro to the non-covalent inhibitor WU-04.
Authors: Zhang, L. / Xie, X. / Luo, H. / Qian, R. / Yang, Y. / Yu, H. / Huang, J. / Shi, P.Y. / Hu, Q.
History
DepositionJul 25, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jun 5, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: 3C-like proteinase nsp5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)34,3602
Polymers33,8341
Non-polymers5261
Water6,413356
1
A: 3C-like proteinase nsp5
hetero molecules

A: 3C-like proteinase nsp5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)68,7204
Polymers67,6672
Non-polymers1,0532
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_655-x+1,y,-z1
Buried area2650 Å2
ΔGint-10 kcal/mol
Surface area24680 Å2
MethodPISA
Unit cell
Length a, b, c (Å)51.571, 81.354, 89.567
Angle α, β, γ (deg.)90.000, 97.303, 90.000
Int Tables number5
Space group name H-MI121
Space group name HallC2y(x,y,-x+z)
Symmetry operation#1: x,y,z
#2: -x,y,-z
#3: x+1/2,y+1/2,z+1/2
#4: -x+1/2,y+1/2,-z+1/2
Components on special symmetry positions
IDModelComponents
11A-526-

HOH

21A-682-

HOH

31A-710-

HOH

41A-818-

HOH

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Components

#1: Protein 3C-like proteinase nsp5 / 3CL-PRO / 3CLp / Main protease / Mpro / Non-structural protein 5 / nsp5 / SARS coronavirus main proteinase


Mass: 33833.566 Da / Num. of mol.: 1 / Mutation: M49K,S301P
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: rep, 1a-1b / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P0DTD1, SARS coronavirus main proteinase
#2: Chemical ChemComp-J7R / ~{N}-[(1~{S},2~{R})-2-[[4-bromanyl-2-(methylcarbamoyl)-6-nitro-phenyl]amino]cyclohexyl]isoquinoline-4-carboxamide


Mass: 526.382 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C24H24BrN5O4 / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 356 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.75 Å3/Da / Density % sol: 55.34 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop
Details: 0.1 M sodium formate, 12% polyethylene glycol 3,350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU FR-X / Wavelength: 1.541 Å
DetectorType: RIGAKU HyPix-6000HE / Detector: PIXEL / Date: May 19, 2021
RadiationMonochromator: M / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.541 Å / Relative weight: 1
ReflectionResolution: 1.65→30 Å / Num. obs: 44111 / % possible obs: 99.98 % / Redundancy: 10.5 % / Biso Wilson estimate: 12.78 Å2 / CC1/2: 0.999 / CC star: 1 / Rmerge(I) obs: 0.05714 / Rpim(I) all: 0.01704 / Rrim(I) all: 0.05971 / Net I/σ(I): 33.21
Reflection shellResolution: 1.65→1.709 Å / Redundancy: 6.6 % / Rmerge(I) obs: 0.2446 / Mean I/σ(I) obs: 6.49 / Num. unique obs: 4387 / CC1/2: 0.976 / CC star: 0.994 / Rpim(I) all: 0.1029 / Rrim(I) all: 0.2658 / % possible all: 100

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Processing

Software
NameVersionClassification
CrysalisPro1.171.40.73adata collection
PHENIX1.19.1_4122refinement
CrysalisPro1.171.40.73adata reduction
CrysalisPro1.171.40.73adata scaling
PHENIX1.19.1_4122phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.65→30 Å / SU ML: 0.1331 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 17.0098
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.1808 1825 4.14 %
Rwork0.1625 42284 -
obs0.1633 44109 100 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 17.79 Å2
Refinement stepCycle: LAST / Resolution: 1.65→30 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2291 0 34 356 2681
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0092380
X-RAY DIFFRACTIONf_angle_d1.1493237
X-RAY DIFFRACTIONf_chiral_restr0.0796361
X-RAY DIFFRACTIONf_plane_restr0.0084419
X-RAY DIFFRACTIONf_dihedral_angle_d7.8131321
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.65-1.690.20461390.15963223X-RAY DIFFRACTION100
1.69-1.740.19681400.15743242X-RAY DIFFRACTION100
1.74-1.80.1821400.16323250X-RAY DIFFRACTION100
1.8-1.870.20761400.16543239X-RAY DIFFRACTION100
1.87-1.940.19991410.1693245X-RAY DIFFRACTION100
1.94-2.030.22921390.16873229X-RAY DIFFRACTION100
2.03-2.130.16821400.16033269X-RAY DIFFRACTION100
2.13-2.270.18581410.16343261X-RAY DIFFRACTION100
2.27-2.440.20291400.17383235X-RAY DIFFRACTION99.97
2.44-2.690.20671390.17853232X-RAY DIFFRACTION100
2.69-3.080.18891410.17453264X-RAY DIFFRACTION100
3.08-3.880.171420.15693278X-RAY DIFFRACTION100
3.88-300.13681430.14533317X-RAY DIFFRACTION99.97
Refinement TLS params.Method: refined / Origin x: 12.4633270464 Å / Origin y: -17.8697752581 Å / Origin z: 0.912146392933 Å
111213212223313233
T0.0670777962659 Å2-0.000372646586248 Å2-0.00752624981138 Å2-0.0585929637321 Å2-0.0131670413956 Å2--0.0489265044961 Å2
L0.613474080816 °2-0.0228653795758 °2-0.124386452498 °2-0.195863645156 °20.0727313451531 °2--0.156021376928 °2
S-0.00977847259263 Å °0.0201208254146 Å °0.00434609263308 Å °0.0215467587657 Å °-0.000766641840653 Å °0.00914034878749 Å °0.0137273599477 Å °-0.00918219697167 Å °-0.00396412577719 Å °
Refinement TLS groupSelection details: all

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